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Natural health products that inhibit angiogenesis: a potential source for investigational new agents to treat cancer—Part 1

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Posted 04 Jun 2012 — by James Street
Category antiangiogenesis, artemisinin, CURCUMIN, curcumin, feverfew (parthenolide), feverfew (parthenolide), Ginkgo Biloba (Kaempferol), Green Tea (Epigallocatechin-3-gallate), Green Tea (Epigallocatechin-3-gallate), Omega-3, quercetin, quercetin, Resveratrol, Silibinin

Curr Oncol. 2006 February; 13(1): 14–26.

PMCID: PMC1891166

S.M. Sagar, MD,* D. Yance, MH,† and R.K. Wong, MD*
This article has been cited by other articles in PMC.

Abstract

An integrative approach for managing a patient with cancer should target the multiple biochemical and physiologic pathways that support tumour development and minimize normal-tissue toxicity. Angiogenesis is a key process in the promotion of cancer. Many natural health products that inhibit angiogenesis also manifest other anticancer activities. The present article focuses on products that have a high degree of anti-angiogenic activity, but it also describes some of the many other actions of these agents that can inhibit tumour progression and reduce the risk of metastasis. Natural health products target molecular pathways other than angiogenesis, including epidermal growth factor receptor, the HER2/neu gene, the cyclooxygenase-2 enzyme, the nuclear factor kappa-B transcription factor, the protein kinases, the Bcl-2 protein, and coagulation pathways. The herbs that are traditionally used for anticancer treatment and that are anti-angiogenic through multiple interdependent processes (including effects on gene expression, signal processing, and enzyme activities) include Artemisia annua (Chinese wormwood), Viscum album (European mistletoe), Curcuma longa (curcumin), Scutellaria baicalensis (Chinese skullcap), resveratrol and proanthocyanidin (grape seed extract), Magnolia officinalis (Chinese magnolia tree), Camellia sinensis (green tea), Ginkgo biloba, quercetin, Poria cocos, Zingiber officinalis (ginger), Panax ginseng, Rabdosia rubescens hora (Rabdosia), and Chinese destagnation herbs. Quality assurance of appropriate extracts is essential prior to embarking upon clinical trials. More data are required on dose–response, appropriate combinations, and potential toxicities. Given the multiple effects of these agents, their future use for cancer therapy probably lies in synergistic combinations. During active cancer therapy, they should generally be evaluated in combination with chemotherapy and radiation. In this role, they act as modifiers of biologic response or as adaptogens, potentially enhancing the efficacy of the conventional therapies.

Keywords: Angiogenesis, anti-angiogenic, natural health products, herbal medicine, anticancer, clinical trials, integrative, molecular biology

1. INTRODUCTION

To progress, cancers require a source of nutrition and oxygen. Tumours that outgrow their oxygen supply cannot form masses greater than 1–2 mm without developing central necrosis. Neoplasms are genetically plastic and often adapt by switching on genes that increase their ability to invade and metastasize. A critical part of this process is the induction of local small blood vessels, termed “angiogenesis” 1,2.

Tumours do not grow progressively unless they induce a blood supply from the surrounding stroma. Cancers that lack angiogenesis remain dormant. Rapid logarithmic growth follows the acquisition of a blood supply. The tumour angiogenic switch seems to be activated when the balance shifts from angiogenic inhibitors to angiogenic stimulators.

The process of neovascularization is subtly controlled in normal tissues by a sequence of endogenous polypeptides that are secreted during growth, healing, and tissue renewal (Table I). Neoplasms are able to synthesize or induce some of these polypeptides, an activity that is partly achieved by the secretion of vascular endothelial growth factor (vegf) and angiopoietins (apns). Hypoxia stimulates these peptides; the result is a sprouting of endothelial cords. This sprouting creates profuse but immature networks of thin endothelial-lined channels, essential for tumour oxygenation. Although these networks permit progressive tumour growth, they are less efficient than the vascular supply to normal tissues. The apns recruit pericytes and initiate modelling of the vessel wall to more mature forms. Tumours often secrete a relative excess of vegf that results in disorganized and leaky vessels that cause local bleeding and edema.

Table I

Table I

Endogenous angiogenic polypeptides

Anti-angiogenic therapy has this theoretic attraction: it may be less susceptible to development of treatment resistance because it is directed to stroma rather than to genomically unstable tumour cells. Judah Folkman and his colleagues were the first to propose using inhibition of tumour vasculature formation as anticancer therapy 3. Their proposal led to the development and clinical trial of more than 20 drugs from groups that inhibit various steps in angiogenesis.

Recently, targeted therapies using monoclonal antibodies that antagonize the formation of new blood vessels have been developed. One example is bevacizumab (Avastin: Genentech, San Francisco, CA, U.S.A.). Bevacizumab is a genetically engineered humanized monoclonal immunoglobulin G antibody that blocks the vegf receptor in endothelial cells, thereby shutting off the tumour blood supply. When used in conjunction with chemotherapy, bevacizumab has been shown to extend life by a few months for some metastatic colorectal cancer patients 4. Preliminary evidence is available that adding bevacizumab to paclitaxel and carboplatin can improve survival by 2 months for non-squamous-cell lung cancer patients. Although bevacizumab increases survival for some patients, it increases the risk of adverse effects, including leucopenia, diarrhea, and hypertension. Use of bevacizumab is also associated with major risks of thrombosis (resulting in stroke and myocardial infarction), fatal hemorrhage (such as gastrointestinal bleeding or hemoptysis), and visceral perforation 5.

Anti-angiogenic therapies may also be combined with radiotherapy to improve local tumour control and reduce the risk of metastasis. During a course of radiotherapy, some tumours increase their angiogenic activity 6. Combined-modality therapy with anti-angiogenesis compounds induces a normal microvascular bed out of the disorganized tumour vessels. During the anti-angiogenic treatment, a critical time occurs when the vegf:apn ratio approximates normal. At that point, pericytes are recruited, the vascular basement membrane adopts a thinner morphology and tumour oxygenation temporarily increases. This is a favourable time to apply ionizing radiation, because such radiation is preferentially lethal to replicating and well-oxygenated cells. The combination of the anti-angiogenic agent and the radiation therapy is optimally effective if this window of opportunity is exploited 7–9.

So far, the evidence suggests that single anti-angiogenic agents have limited efficacy. Natural health products contain a range of complex organic chemicals that may have synergistic activity. They may inhibit angiogenesis by interacting with multiple pathways and by acting in other ways that can affect cell signalling, the apoptotic pathway, and the interaction of cancer cells with the immune system. Some anti-angiogenic agents also have anticoagulation activity that may also be associated with a reduction in metastasis. Heparin is a well-known example of a therapy with both anticoagulation and anti-angiogenic activities.

Rather than develop multiple monoclonal antibodies to target the multiple peptides and their receptors, an alternative approach might be to evaluate phytochemicals and certain animal-derived chemicals that influence multiple pathways. The science of pharmacognosy evaluates natural drugs derived from herbal remedies or phytomedicines. Minimal clinical research has been undertaken to evaluate the use of natural drugs as adjuvant therapy with conventional treatment using cytotoxic drugs and radiotherapy. Formal research is required on the timing of administration of natural health products with anti-cancer therapies. As noted in the earlier discussion of the administration of radiotherapy with anti-angiogenic treatment, timing may be critical. Anti-angiogenic natural health products may be most effective as maintenance therapy that impedes cancer recurrence following cytotoxic treatment. Human tumours can remain dormant for years because of a balance between cell proliferation and apoptosis.

2. WHAT IS ANGIOGENESIS?

Normal angiogenesis is the regulated formation of new blood vessels from existing ones. It is the basis of several physiologic processes, such as embryonic development, placenta formation, and wound healing. It is a good example of how a tumour can take control of normal processes and deregulate them to its own advantage.

In the normal and orderly formation of new blood vessels, endothelial cells receive a stimulatory signal from angiokinins and secrete specific enzymes such as matrix metalloproteinase (mmp) and heparinase that result in the dissolution of the extracellular matrix (ecm). The tight junctions between the endothelial cells are disrupted. The endothelial cells can then project through the newly created spaces and organize into fresh capillary tubes that grow toward the source of the blood supply 10,11.

The induction of new blood vessels provides tumours with a survival advantage. The survival and growth of cells depends on an adequate supply of oxygen and nutrients and on the removal of toxic products. Oxygen can diffuse radially from capillaries for only 150–200 μm. When distances exceed this maximum, cell death follows. Thus, the expansion of tumour masses beyond 1 mm in diameter depends on the development of a new blood supply—angiogenesis 12–14.

An increasing density of tumour vasculature raises the probability that the tumour will metastasize. Generally, increased microvascular density (“angiogenesis index”) is a significant indicator of poorer prognosis. Angiogenesis plays a central role in the progression of most solid tumours, including those of bladder, brain, breast, cervix, colon, lung, and prostate. Increased vascular density has also been found in the bone marrow of patients with acute myeloid leukemia and myeloma 15–24.

Cancer cells begin to promote angiogenesis quite early in the development of a tumour. The angiogenesis is characterized by oncogene-driven tumour expression of pro-angiogenic proteins (Table I).

The formation of new vasculature occurs in sequential steps. Endothelial cells must proliferate, migrate, and penetrate host stroma and the ecm. The endothelial cells must also undergo morphogenesis. The process of angiogenesis consists of activation and resolution phases. Activation requires initial degradation of the basement membrane, followed by endothelial cell migration, invasion of the surrounding ecm, endothelial cell proliferation, and capillary lumen formation. During resolution, the microvasculature matures and stabilizes by enclosure of the vessel by pericytes, inhibition of endothelial proliferation, reconstitution of basement membrane, and formation of gap junctions.

The vasculature of many solid tumours is not identical to that of normal tissue 25. The resolution phase is often incomplete in tumours, resulting in tumour microvessels that are highly irregular and tortuous and that are only partially lined with endothelium and basement membrane. Arteriovenous shunts and blind ends are common.

Failure of resolution may be a consequence of persistent overexpression of apn-2 in the tumour-associated vasculature. Differences are seen in cellular composition and permeability, in vessel stability, and in regulation of growth. The balance between factors that stimulate new blood vessel growth and those that inhibit it determines the vascular density. The inhibitory influence predominates in normal tissues; in tumours, many neoplastic cells switch from an angiogenesis-inhibiting to an angiogenesis-stimulating phenotype. That switch coincides with the loss of the wild-type allele of the TP53 tumour suppressor gene and is associated with reduced production of thrombospondin-1 (tsp-1), a controller of angiogenesis in fibroblasts 26–31.

The production of vegf is considered essential for most cancer cell migration and for angiogenesis. A high vegf expression level is associated with worse outcome in a wide array of malignancies. Expression of vegf messenger rna is upregulated by many oncogenes (including H-ras and K-ras, src, TP53, and c-jun) and growth factors including epidermal growth factor, transforming growth factors alpha and beta, insulin-like growth factor–1, and platelet-derived growth factor 32–38. Table II lists some cancer-associated genes implicated in angiogenesis.

Table II

Table II

Cancer-associated genes implicated in angiogenesis

3. THE ANGIOGENIC–METASTATIC PATHWAY AS A TARGET FOR ANTICANCER THERAPIES

The process of cancer metastasis consists of a series of interrelated sequential steps. Each step is rate-limiting and may be a target for therapy. The outcome of the process depends on both the intrinsic properties of the tumour cells and the responses of the host. The balance of these interactions varies from one tumour and patient to another. These are the major steps 39–41 in the formation of a metastasis:

  1. Transformation of normal cells into tumour cells, followed by growth. Initially depends on nutrients supplied by simple diffusion.
  2. Extensive vascularization (angiogenesis). Vascularization must occur if the tumour mass is to exceed 1 mm in diameter. The production and secretion of pro-angiogenic factors by tumour cells and host cells plays a major role in establishing a capillary network from the surrounding host tissue.
  3. Local invasion. Tumour cells use several mechanisms to invade the host stroma. Thin-walled venules, fragmented arterioles, and lymphatic channels offer little resistance to penetration and provide the most common pathways for entry of tumour cells into the circulation.
  4. Detachment and embolization. Single cells or clumps break away. Most circulating tumour cells are rapidly destroyed. Those that survive must arrest in the capillary beds of distant organs by adhering either to capillary endothelial cells or to the exposed subendothelial basement membrane.
  5. Extravasation into new host organ or tissue.
  6. Proliferation within the new host organ or tissue. To continue growing beyond the 1-mm diameter, the micrometastasis must develop a vascular network and evade destruction by host defences. The cells can then continue to invade blood vessels, enter the circulation, and produce additional metastases.

The growth of many cancers is associated with an absence of the endogenous inhibitors of angiogenesis—for example, interferon beta (infβ). A potent inhibitor of angiogenesis, infβ works by blocking interleukin-8 (IL-8), basic fibroblast growth factor (bfgf), and collagenase type v, which are all potent angiogenic factors that aid tumour development and invasiveness.

Vascular endothelial growth factor stimulates the proliferation and migration of endothelial cells and induces plasminogen activity and the expression of metalloproteinases. In several animal models, overexpression of vegf in tumour cells enhances tumour growth and metastasis by stimulating vascularization 12,42–49.

Some cytotoxic chemotherapy agents are being used at lower-than-normal doses, with the intent of inhibiting angiogenesis and minimizing toxicity 50,51. This strategy may permit advanced cancer patients to maintain a better quality of life. This low-dose therapy is termed “metronomic dosing” 52–54.

The metronomic model of conventional cytotoxic chemotherapy suggests that advantages may also accrue to the administration of combinations of phytochemicals that interact with the multistep process of angiogenesis 55. In other words, targeting the vascular endothelium with continuous low-dose noncytotoxic therapies may maintain tumour control without excessive toxicity. The potential role of such therapies for increasing overall survival (but not necessarily disease-free survival) and for maintaining quality of life requires evaluation in future clinical trials.

3.1 Role of the Tumour Microenvironment in Mediating the Response to Anti-angiogenic Therapy

Each individual tumour may display a different angiogenic phenotype because the expression of angiogenic factors in tumours is controlled both by intrinsic factors in the tumour cell and by the influence of the host microenvironment 56. The microenvironment can effect gene expression in tumours growing at various sites. The tumour cells themselves can alter the endothelial cell phenotype. Various sites of metastasis may express varying combinations of angiogenic factors and endothelial cell phenotypes 57. Interactions among the polypeptide angiogenic factors produced by the tumour are complex, functioning in a dynamic, reciprocal fashion with other factors present in the tumour microenvironment. Therefore, cytokine-targeted anti-angiogenic therapies or monoclonal antibodies against angiogenic growth factors must consider not only the angiogenic factors that are being released by tumour cells, but also the contribution of the tumour microenvironment to tumour angiogenesis.

The efficacy of anti-angiogenic compounds varies from one tumour to another. The more specific the intervention is to one domain of the angiogenic pathway, the less likely a beneficial reduction in tumour growth is to occur, because alternative pathways can compensate. If the angiogenic activity of a tumour is initiated primarily by only one or two factors, then blocking the activity of one factor may be enough to inhibit tumour growth. For example, expression of vegf and epidermal growth factor receptor (egfr) correlate with the metastatic characteristics of human colon cancer, and so targeting vegf or egfr may be beneficial 58. However, if several factors mediate the angiogenic activity in a particular tumour, an alternative intervention strategy is required.

Natural health products contain a cocktail of biologic chemicals that act on multiple pathways that initiate and maintain tumour angiogenesis. In addition, we hypothesize that angiogenesis within the tumour microenvironment may be more sensitive to a cocktail of natural health products administered continuously at relatively low doses than to single-agent pharmaceutical compounds administered intermittently at higher dose levels. In general, as compared with normal tissues, tumours contain very immature blood vessels that may be relatively more susceptible to anti-angiogenic therapies, permitting a therapeutic gain 59,60.

3.2 Screening Herbs for Anti-angiogenic Activity

One of the first anti-angiogenic agents to be isolated was a phytochemical. In 1990, Ingber et al. reported on the anti-angiogenic properties of fumagillin, a secreted antibiotic of the fungus Aspergillus fumigatus 61. Refined fumagillin produces excess toxicity, and so analogues of fumagillin were subsequently synthesized.

Various assays are used to screen natural health products for anti-angiogenic activity 51,62,63. Assays used for screening are briefly discussed in the next few subsections.

3.2.1 In Vitro Assays

The ability to maintain endothelial cells in culture has enabled the study of endothelial cell proliferation, migration, and function. For example, anti-angiogenic activity can be assessed by evaluating the potential of a substance to inhibit endothelial cell migration across a Boyden chamber. The bovine aortic endothelial cell assay is an established system.

In vitro assays are relatively inexpensive and give more rapid results. However, an ability to inhibit endothelial cell proliferation, migration, and tubule formation in vitro may not predict in vivo response. In vitro assays are a rapid method for initial screening of large numbers of agents. Definitive conclusions cannot be based on in vitro assays alone.

3.2.2 In vivo Assays

In vivo biologic assays are more specific for detecting anti-angiogenic activity. The chick embryo chorioallantoic membrane model is an extra-embryonic membrane that is commonly used to study agents that influence angiogenesis. An angiogenic response in the form of increased vessel density around the implant occurs 72–96 hours after stimulation with an angiogenic compound. An angiostatic compound will induce the vessels around the implant to become less dense and even to disappear. Other systems include animal cornea implantation, disc angiogenesis, Matrigel (Becton–Dickinson, Mountain View, CA, U.S.A.) systems, and tumour xenograft models.

In vivo assays provide a more complete physiologic assessment of angiogenesis, but are more time-consuming and expensive.

3.3 Criteria for Anti-angiogenic Activity

The degree of anti-angiogenic activity is dose-dependent. Most chemotherapy drugs have anti-angiogenic activity when administered at high doses. Clinicians are especially interested in compounds that, when administered at low doses, specifically interact and antagonize the steps involved in angiogenesis. These agents may have relatively low toxicity at low doses and may exhibit a higher therapeutic gain.

Most conventional chemotherapy drugs have some degree of anti-angiogenic activity as a consequence of their cytotoxic activity. Ideal botanical derivatives would specifically antagonize new vessel formation in tumours without significant toxicity to normal tissues and without major adverse reactions. The ideal agent would also inhibit tumour cell proliferation through other physiologic pathways, such as intracellular signalling pathways.

Multiple levels of anti-angiogenic activity may be required to overcome the development of resistance by tumour-associated endothelial cells. Survival factors—such as increased secretion of vegf and bfgf by the tumour cells—activate intracellular pathways that prevent apoptosis in tumour-associated endothelial cells.

Maximal anti-angiogenic activity usually requires prolonged exposure to low concentrations of the active agent. This approach contrasts with the concept of administering maximum-tolerated doses of cytotoxic drugs to maximize tumour-cell kill. Some reports have confirmed the utility of combining low, frequent–dose chemotherapy with an agent that specifically targets the endothelial cell compartment 52,53. The evidence suggests that an anti-angiogenic schedule can be more effective than the use of high-dose cytotoxic drugs alone. We hypothesize that concomitant scheduling of anti-angiogenic botanicals with low, frequent–dose cytotoxic therapies may have biologic advantages that can increase therapeutic gain.

4. NATURAL HEALTH PRODUCTS THAT INHIBIT ANGIOGENESIS

Further research is necessary to screen herbs that may be useful anti-angiogenic therapies. Tables III and ​andIVIV list natural health products with anti-angiogenic activity, and Table V lists herbs and their derivatives that inhibit vegf 55. A master herbalist can advise on potential herbal treatments derived from centuries of traditional observations and advanced traditional medical systems such as Traditional Chinese Medicine. It will be imperative to develop a new model of modern pharmacology based on traditional pharmacognosy.

Table III

Table III

Natural health products with potential direct and indirect anti-angiogenic activity a
Table IV

Table IV

Natural health products that inhibit cyclo-oxygenase-2 activity64
Table V

Table V

Herbs and their derivatives that specifically inhibit vascular endothelial growth factor and have direct activity against angiogenesis a

Our developing knowledge of cancer biology suggests that administering cytotoxic drug therapy at very high doses is not always appropriate. A new approach is to administer lower doses of synergistic organic chemicals. These complexes already exist in myriad botanicals. New laboratory techniques permit more specific assays of activity, enabling maintenance of quality assurance and consistency between batches of botanical preparations. Such quality standards will permit credible clinical trials of anti-angiogenic natural health products to be initiated. At the same time, the importance of a holistic approach to managing the patient with cancer should not be minimized. Anti-angiogenic therapies form only a small part of a complex management program. Attention to the patient’s overall health and ability to mount an immune response are subtle factors that may become more important in tipping the balance towards cancer control.

4.1 Herbs and Phytochemicals

4.1.1 Artemisia annua (Chinese Wormwood)

Artemisinin is the active constituent extracted from the plant Artemisia annua. Artemisinin has been used clinically as an anti-malaria drug 65. More recently, it was shown to be cytotoxic to cancer cells through induction of apoptosis 66.

Artesunate is a semi-synthetic derivative of artemisinin. Artesunate was tested in vitro in the human umbilical vein endothelial cell (huvec) model of angiogenesis and was shown to significantly inhibit angiogenesis in a dose-dependent manner 67. The inhibition of proliferation of huvecs was greater than that seen with cancer cells, fibroblast cells, and human endometrial cells. Those findings indicate that the anti-angiogenic activity of artesunate is greater than its cytotoxicity.

The anti-angiogenic effect of artemisinin in vivo was evaluated using transplanted human ovarian cancer (HO-891) cells in nude mice. Immunohistochemical staining for microvessel CD31 antigen, vegf, and the vegf receptor (KDR, formerly called FLK1) was performed. In treated mice, tumour growth was decreased and microvessel density was reduced without any toxicity to the host animals. Artemisinin also lowered vegf expression by tumour cells and KDR expression by endothelial cells. Artemisinin also has anticancer activity through other pathways. It inhibits the activation of nuclear factor kappa-B (nf-κb), an important activator protein in cancer development and progression 68.

4.1.2 Viscum album (European Mistletoe)

Viscum album is also known as Iscador (Weleda, Palisades, NY, U.S.A.). It is often used as an anticancer agent in anthroposophic and homeopathic medicine. Laboratory studies show that it is anti-angiogenic by downregulation of vegf; it also induces apoptosis of cancer cells 69,70. In a mouse model, lung metastases were reduced, and survival was increased 71. A clinical trial in human subjects showed an increase in survival in a variety of cancers, but the study was poorly controlled and no definitive conclusions could be drawn 72. Well-controlled clinical trials of V. album derivatives in combination with other anticancer therapies are warranted.

4.1.3 Curcuma longa (Curcumin)

Curcumin is the most active curcuminoid in turmeric. It interacts with cancer cells at a number of levels and can enhance the tumoricidal efficacy of cytotoxic chemotherapy and radiotherapy 73–75. Its anti-invasive effects are partly mediated by downregulation of matrix metalloproteinase-2 (MMP2) and upregulation of tissue inhibitor of metalloproteinase-1 (TIMP1) 76. These enzymes are involved in the regulation of tumour cell invasion.

Curcumin inhibits the transcription of two major angiogenesis factors, vegf and bfgf 77. It interacts with vegf- and nitric oxide–mediated angiogenesis in tumours 78,79. Elevated levels of nitric oxide correlate with tumour growth. Curcumin reduces nitric oxide generation in endothelial cells. The membrane-bound enzyme CD13 (aminopeptidase N) is found in blood vessels undergoing active angiogenesis. Curcumin binds to CD13 and blocks its activity, thereby inhibiting angiogenesis and invasion by tumour cells 80,81. Derivatives of curcumin may be developed to target CD13, providing a novel approach to reduce neoplastic angiogenesis 82,83.

Curcumin also downregulates the expression of the VEGF and MMP9 genes that are associated with angiogenesis. Demethoxycurcumin is a structural analogue of curcumin isolated from Curcuma aromatica. It specifically inhibits the expression of MMP9 84. Curcumin can interfere with the activity of both MMP2 and MMP9, the basis of the angiogenic switch, thereby reducing degradation of the ecm 85. It also interferes with the release of angiogenic factors that are stored in the ecm. It inhibits growth factor receptors such as egfr and vegf receptor and the intracellular signalling tyrosine kinases. This cell signalling system can promote further angiogenesis through gene activation that increases levels of cyclooxygenase-2 (cox-2), vegf, il-8, and the mmps86–88.

A phase i study of curcumin found no treatment-related toxicity at doses up to 8000 mg daily. Beyond 8000 mg daily, the bulky volume of the drug was unacceptable to the patients. Serum concentration of curcumin usually peaked at 1–2 hours after oral intake of curcumin and gradually declined within 12 hours 89. The study suggested that curcumin may prevent cancer progression. Derivatives of curcumin, such as copper chelates of curcuminoids, may have increased antitumour activity 83.

4.1.4 Scutellaria baicalensis (Chinese Skullcap)

Baicalin and baicalein are the main derivatives of the Chinese skullcap herb. They are potent anti-angiogenic compounds that reduce vegf, bfgf, 12-lipoxygenase activity, and mmp 90,91. Scutellaria baicalensis is one of the herbs found in pc-spes, a complex of Chinese herbs that is clinically active against advanced prostate cancer 92–94.

4.1.5 Resveratrol and Proanthocyanidin (Grape Seed Extract)

Resveratrol is a phytoalexin found in grapes and wine. It has anti-angiogenic activity demonstrated by its ability to inhibit division in huvecs and to decrease the lytic activity of mmp-2 95. Resveratrol inhibits vegf-induced angiogenesis by disruption of reactive oxygen species–dependent src kinase activation and subsequent ve-cadherin tyrosine phosphorylation 96,97. Resveratrol inhibits the growth of gliomas in rats by suppressing angiogenesis 98.

Edible berries contain high concentrations of proanthocyanidin. The latter inhibits vegf expression induced by tumour necrosis factor alpha (tnfα). Feeding proanthocyanidins to mice with tumour xenografts reduced vegf secretion, which resulted in reduced intratumoral microvasculature 99–101. On the other hand, one study showed that grape seed extract may upregulate oxidant-induced vegf expression, suggesting that proanthocyanidin can induce angiogenesis as part of normal tissue healing 102.

4.1.6 Magnolia officinalis (Chinese Magnolia Tree)

The seed cones of the Chinese magnolia tree contain substances that inhibit the growth of new blood vessels. Honokiol is the active ingredient. It may partly reduce angiogenesis through the regulation of platelet-derived endothelial cell growth factor and transforming growth factor beta (tgfβ) expression. It also inhibits nitric oxide synthesis and tnfα expression 103,104. In animal experiments, honokiol suppressed proliferation in blood vessel endothelial cells more than in other types of cells and thereby reduced tumour growth 105,106.

4.1.7 Silybum marianum (Milk Thistle)

Silibinin and silymarin are polyphenolic flavonoids isolated from the fruits or seeds of Silybum marianum. In the laboratory, silymarin demonstrates strong activity against a variety of tumours by downregulation of vegf and egfr 107,108. Silymarin suppresses vegf when used as a single agent against human ovarian cancer 109.

4.1.8 Camellia sinensis (Green Tea)

Tea contains polyphenols and catechins, mainly epigallocatechin-3 gallate (egcg) 110. These constituents inhibited proliferation of MDA-MB231 breast cancer cells and huvecs 111 and, in rodent studies, also suppressed breast cancer xenograft growth and reduced the density of tumour vessels 112. This activity was associated with a decrease in vegf, regulated at the level of transcription. In addition, egcg suppresses protein kinase C (pkc), another vegf transcription modulator.

Inhibition of vegf transcription is one of the molecular mechanisms involved in the anti-angiogenic effects of green tea that may contribute to its potential use for cancer treatment 113,114. Epigallocatechin-3 gallate may be administered as a powdered extract of green tea. An appropriate dose has been extrapolated from anti-angiogenic activity in rodent experiments115 as well as from a phase i study in humans 116. A dose of 1.0 g/m2 three times daily [equivalent to 7–8 Japanese cups (120 mL)] has been recommended. In practice, lower total daily doses of 2–4 g standardized green tea extract (95% polyphenols and 60% catechins) are usually prescribed. Each gram of this extract provides 400–500 mg of egcg. The dose-limiting adverse effects are the gastrointestinal and neurologic effects of caffeine. However, the caffeine may potentiate the anti-angiogenic effect of egcg 116.

4.1.9 Ginkgo biloba

Ginkgo biloba extract has anticancer effects that are related to its gene-regulatory and anti-angiogenic properties. The Ginkgo biloba extract used in most research is EGb 761, which contains about 25% flavonoids (ginkgo-flavone glycosides) and about 5% terpenoids (ginkgolides and bilobalides). The most potent flavonoid is ginkgolide B. The extract inhibits angiogenesis by downregulating vegf 117,118.

4.1.10 Quercetin

Quercetin is a flavone found in apples, onions, raspberries, red grapes, citrus fruit, cherries, broccoli, and leafy greens. It inhibits angiogenesis through multiple mechanisms, including interaction with the cox-2 and lipoxygenase-5 enzymes, egfr, the HER2 intracellular signalling pathway, and the nf-κb nuclear transcription protein 119–123. Quercetin may enhance the anticancer effects of tamoxifen through anti-angiogenesis 124.

4.1.11 Poria cocos

Poria cocos is a mushroom extract that, by tradition, has anticancer activity. It inhibits platelet aggregation and appears to be anti-angiogenic by down-regulating nf κb 125–129

4.1.12 Panax Ginseng

The lipophilic constituents of ginseng are called saponins (or ginsenosides). These extracts possess anticancer activities in tumours that include anti-angiogenesis and induction of tumour cell apoptosis 130.

4.1.13 Rabdosia rubescens Hara (Rabdosia)

Rabdosia is used in certain traditions to treat cancer. It is one constituent of the pc-spes formula that is active against prostate cancer. It contains ponicidin and oridonin, two diterpenoids that possess significant anti-angiogenic activity 131.

4.1.14 Extracts of Chinese Medicinal Herbs

Herbs that are used by tradition in China as anticancer agents have been screened for their anti-angiogenic activity 62. Table VI lists the most active herbs (those that exhibit more than 20% inhibition at 0.2 g herb/mL) by chorioallantoic membrane and bovine aortic endothelial cell assays.

Table VI

Table VI

Anti-angiogenesis activity of Chinese medicinal herbal extracts (exhibiting more than 20% inhibition at 0.2g herb/mL)62

4.2 Copper Antagonists

Some cancers are associated with high serum levels of copper. The role of copper in cancer promotion through pro-inflammatory cascades and angiogenesis induction is quite well established 132. Copper is essential for the function of many angiogenic growth factors. The angiogenic activity of bfgf, vegf, tnfα, and il-1 are copper-dependent.

Copper chelation with tetrathiomolybdate is a promising therapy for tumour control 133,134. The hypothesized mechanism of action for this substance is inhibition of angiogenic cytokines. Unlike certain current approaches to anti-angiogenic therapy that target single agents, tetrathiomolybdate inhibits multiple angiogenic cytokines. Part of the effect appears to stem from inhibition of nf-κb, which in turn controls transcription of many angiogenic factors and other cytokines.

Some angiogenic cytokines appear to have separate mechanisms of copper dependence. The inhibition of multiple angiogenic cytokines gives tetrathiomolybdate the potential to be a more global inhibitor of angiogenesis. Several aromatic herbs—such as Caryophylli flos, Cinnamomi cortex, Foeniculi fructus, and Zedoariae rhizoma—have inhibitory effects on lipid peroxidation or protein oxidative modification by copper 135. They may have a role to play in anti-angiogenesis, but further research is necessary for confirmation.

4.3 Animal Products

4.3.1 Shark and Bovine Cartilage

The resistance of cartilage to tumour formation has been correlated with its capacity to inhibit the formation of new blood vessels. A number of in vitro and in vivo studies have suggested the existence of anti-angiogenic compounds in shark and bovine cartilage 136. The clinical effectiveness of whole cartilage for the treatment of cancer was not confirmed in a recent phase iii randomized controlled trial 137. The main problem is lack of data correlating bioavailability with pharmacologic effects in the oral use of shark cartilage. Unsatisfactory outcomes in clinical trials may be secondary to inadequate bioavailability of the active constituents 138. Bioactive derivatives of shark cartilage are being extracted. The AE-941 derivative (Neovastat: Æterna Zentaris, Quebec, QC, Canada) is a standardized water-soluble extract that represents less than 5% of the crude cartilage. This multifunctional anti-angiogenic product contains several biologically active molecules 139. The mode of extraction developed by Æterna differs from that of many other preparations and may explain the preservation of the anti-angiogenic properties.

Neovastat is kept frozen until use to maximally preserve its biologic properties. Its anti-angiogenic activity may be attributable to the presence of a metalloproteinase inhibitor with a preferential inhibition of mmp-2 and to inhibition of serine elastase, of vegf binding to endothelial cells, and of tyrosine phosphorylation of the vegf receptor. Neovastat reduces the vegf-dependent increase in vascular permeability.

Paradoxically, shark cartilage extracts (including AE-941) also have fibrinolytic activity 140,141. However, fibrinolysis and anticoagulation may also reduce tumour cell metastasis 142,143. Shark cartilage extracts are pleiotropic, having multiple phenotypic activities.

No published phase iii randomized controlled trials have yet proven the utility of Neovastat for cancer treatment. Part of the funding for clinical studies of AE-941 comes from Technology Partnerships Canada (tpc), a research support program run by Canada’s federal government. The agreement is that Æterna will reimburse tpc upon commercialisation of AE-941–derived products. In January 2004, Æterna announced that development of AE-941 would be focused on non-small-cell lung cancer only 144.

4.3.2 Squalus acanthias (Dogfish Shark)

Squalamine is a cationic steroid isolated from the liver of the dogfish shark, Squalus acanthias 145. Squalamine significantly blocks vegf-induced activation of mitogen-activated protein kinase and cell proliferation in human vascular endothelial cells. Squalamine is anti-angiogenic for ovarian cancer xenografts, and it appears to enhance the cytotoxic effects of cisplatin chemotherapy, independent of HER2 status. Overexpression of HER2 is normally associated with resistance to cisplatin and promotion of tumour angiogenesis 146. In a phase ii trial of patients with advanced small-cell lung cancer, squalamine was administered at a dose of 300 mg/m2 by continuous infusion for 5 days, with paclitaxel and carboplatin given on day 1. Patient survival data and a satisfactory safety profile indicated that the combination should be explored further 147.

5. CONCLUSION

In vitro and in vivo studies are uncovering anti-angiogenic activity in many natural health products. Further preclinical research is required to define whether single compounds or complex mixtures will be optimal for clinical trials. A potential advantage of phytochemicals and other compounds derived from natural health products is that they may act through multiple cell-signalling pathways and reduce the development of resistance by cancer cells. Part 2 of this review will further discuss the latter issues.

6. REFERENCES

1. Fidler IJ. Regulation of neoplastic angiogenesis. J Natl Cancer Inst Monogr. 2001;28:10–14. [PubMed]
2. Fidler IJ. Angiogenesis and cancer metastasis. Cancer J. 2000;6(suppl 2):S134–41. [PubMed]
3. Folkman J. Angiogenesis inhibitors: a new class of drugs. Cancer Biol Ther. 2003;2(suppl 1):S127–33. [PubMed]
4. Kabbinavar FF, Hambleton J, Mass RD, Hurwitz HI, Bergsland E, Sarkar S. Combined analysis of efficacy: the addition of bevacizumab to fluorouracil/leucovorin improves survival for patients with metastatic colorectal cancer. J Clin Oncol. 2005;23:3706–12. [PubMed]
5. Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med. 2004;350:2335–42. [PubMed]
6. Ansiaux R, Baudelet C, Jordan BF, et al. Thalidomide radiosensitizes tumors through early changes in the tumor microenvironment. Clin Cancer Res. 2005;11:743–50. [PubMed]
7. Ergun A, Camphausen K, Wein LM. Optimal scheduling of radiotherapy and angiogenic inhibitors. Bull Math Biol. 2003;65:407–24. [PubMed]
8. Koukourakis MI, Giatromanolaki A, Sivridis E, et al. Squamous cell head and neck cancer: evidence of angiogenic regeneration during radiotherapy. Anticancer Res. 2001;21:4301–9. [PubMed]
9. Ma BB, Bristow RG, Kim J, Siu LL. Combined-modality treatment of solid tumors using radiotherapy and molecular targeted agents. J Clin Oncol. 2003;21:2760–76. [PubMed]
10. Folkman J. Fundamental concepts of the angiogenic process. Curr Mol Med. 2003;3:643–51. [PubMed]
11. Hendrix MJC, Seftor EA, Hess AR, Seftor RE. Vasculogenic mimicry and tumor-cell plasticity: lessons from melanoma. Nat Rev Cancer. 2003;3:411–21. [PubMed]
12. Folkman J. How is blood vessel growth regulated in normal and neoplastic tissue? G.H.A. Clowes Memorial Award Lecture. Cancer Res. 1986;46:467–73. [PubMed]
13. Auerbach W, Auerbach R. Angiogenesis inhibition: a review. Pharmacol Ther. 1994;63:265–311. [PubMed]
14. Fidler IJ, Ellis LM. The implications of angiogenesis for the biology and therapy of cancer metastasis. Cell. 1994;79:185–8. [PubMed]
15. Weidner N, Folkman J, Pozza F, et al. Tumor angiogenesis: a new significant and independent prognostic indicator in early stage breast carcinoma. J Natl Cancer Inst. 1992;84:1875–87. [PubMed]
16. Gasparini G, Harris AL. Clinical importance of the determination of tumor angiogenesis in breast carcinoma: much more than a new prognostic tool. J Clin Oncol. 1995;13:765–82. [PubMed]
17. Hall NR, Fish DE, Hunt N, Goldin RD, Guillou PJ, Monson JR. Is the relationship between angiogenesis and metastasis in breast cancer real? Surg Oncol. 1992;1:223–9. [PubMed]
18. Van Hoef ME, Knox WF, Dhesi SS, Howell A, Schor AM. Assessment of tumour vascularity as a prognostic factor in lymph node negative invasive breast cancer. Eur J Cancer. 1993;29A:1141–5. [PubMed]
19. Weidner N, Carroll PR, Flax J, Flumenfeld W, Folkman J. Tumor angiogenesis correlates with metastasis in invasive prostate carcinoma. Am J Pathol. 1993;143:401–9. [PMC free article] [PubMed]
20. Fregene TA, Khanuja PS, Noto AC, et al. Tumor-associated angiogenesis in prostate cancer. Anticancer Res. 1993;13:2377–81. [PubMed]
21. Graham CH, Rivers J, Kerbel RS, Stankiewicz KS, White WL. Extent of vascularization as a prognostic indicator in thin (<0.76 mm) malignant melanomas. Am J Pathol. 1994;145:510–14. [PMC free article] [PubMed]
22. Hollingsworth HC, Kohn EC, Steinberg SM, Rothenberg ML, Merino MJ. Tumor angiogenesis in advanced stage ovarian carcinoma. Am J Pathol. 1995;147:33–41. [PMC free article] [PubMed]
23. Maeda K, Chung YS, Takatsuka S, et al. Tumour angiogenesis and tumour cell proliferation as prognostic indicators in gastric carcinoma. Br J Cancer. 1995;72:319–23. [PMC free article] [PubMed]
24. Takahashi Y, Kitadai Y, Bucana CD, Cleary KR, Ellis LM. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res. 1995;55:3964–8. [PubMed]
25. Liotta LA, Steeg PS, Stetler–Stevenson WG. Cancer metastasis and angiogenesis: an imbalance of positive and negative regulation. Cell. 1991;64:327–36. [PubMed]
26. Kumar R, Yoneda J, Bucana CD, Fidler IJ. Regulation of distinct steps of angiogenesis by different angiogenic molecules. Int J Oncol. 1998;12:749–57. [PubMed]
27. Folkman J, Klagsbrun M. Angiogenic factors. Science. 1987;235:442–7. [PubMed]
28. Nagy JA, Brown LF, Senger DR, et al. Pathogenesis of tumor stroma generation: a critical role for leaky blood vessels and fibrin deposition. Biochim Biophys Acta. 1989;948:305–26. [PubMed]
29. Folkman J, Cotran R. Relation of vascular proliferation to tumor growth. Int Rev Exp Pathol. 1976;16:207–48. [PubMed]
30. Fidler IJ. Angiogenic heterogeneity: regulation of neoplastic angiogenesis by the organ microenvironment. J Natl Cancer Inst. 2001;93:1040–1. [PubMed]
31. Dameron KM, Volpert OV, Tainsky MA, Bouck N. Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science. 1994;265:1582–4. [PubMed]
32. Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors. Nat Rev Cancer. 2002;2:727–39. [PubMed]
33. Toi M, Matsumoto T, Bando H. Vascular endothelial growth factor: its prognostic, predictive, and therapeutic implications. Lancet Oncol. 2001;2:667–73. [PubMed]
34. Houck KA, Ferrara N, Winer J, Cachianes G, Li B, Leung DW. The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of rna. Mol Endocrinol. 1991;5:1806–14. [PubMed]
35. Houck KA, Leung DW, Rowland AM, Winer J, Ferrara N. Dual regulation of vascular endothelial growth factor bioavailability by genetic and proteolytic mechanisms. J Biol Chem. 1992;267:26031–7. [PubMed]
36. Shima DT, Deutsch U, D’Amore PA. Hypoxic induction of vascular endothelial growth factor (vegf) in human epithelial cells is mediated by increases in mrna stability. FEBS Lett. 1995;370:203–8. [PubMed]
37. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature. 1992;359:843–5. [PubMed]
38. Niklinska W, Burzykowski T, Chyczewski L, Niklinski J. Expression of vascular endothelial growth factor (vegf) in non-small cell lung cancer (nsclc): association with p53 gene mutation and prognosis. Lung Cancer. 2001;34(suppl 2):S59–S64. [PubMed]
39. Sugarbaker EV. Cancer metastasis: a product of tumor–host interactions. Curr Probl Cancer. 1979;3:1–59. [PubMed]
40. Hart IR, Goode NT, Wilson RE. Molecular aspects of the meta-static cascade. Biochim Biophys Acta. 1989;989:65–84. [PubMed]
41. Liotta LA, Stetler–Stevenson WG. Tumor invasion and metastasis: an imbalance of positive and negative regulation. Cancer Res. 1991;51(suppl 18):S5054–9.
42. Risau W. Mechanisms of angiogenesis. Nature. 1997;386:671–4. [PubMed]
43. Ferrara N, Carver–Moore K, Chen H, et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature. 1996;380:439–42. [PubMed]
44. Senger DR, Galli SJ, Dvorak AM, Perruzzi CA, Harvey VS, Dvorak HF. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science. 1983;219:983–5. [PubMed]
45. Kumar R, Yoneda J, Bucana CD, Fidler IJ. Regulation of distinct steps of angiogenesis by different angiogenic molecules. Int J Oncol. 1998;12:749–57. [PubMed]
46. Folkman J, Klagsbrun M. Angiogenic factors. Science. 1987;235:442–7. [PubMed]
47. Nagy JA, Brown LF, Senger DR, et al. Pathogenesis of tumor stroma generation: a critical role for leaky blood vessels and fibrin deposition. Biochim Biophys Acta. 1989;948:305–26. [PubMed]
48. Folkman J, Cotran R. Relation of vascular proliferation to tumor growth. Int Rev Exp Pathol. 1976;16:207–48. [PubMed]
49. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Med. 1995;1:27–31. [PubMed]
50. Oliff A, Gibbs JB, McCormick F. New molecular targets for cancer therapy. Sci Am. 1996;275:144–9. [PubMed]
51. Miller KD, Sweeney CJ, Sledge GW. Redefining the target: chemotherapeutics as antiangiogenics. J Clin Oncol. 2001;19:1195–206. [PubMed]
52. Hanahan D, Bergers G, Bergsland E. Less is more, regularly: metronomic dosing of cytotoxic drugs can target tumor angiogenesis in mice. J Clin Invest. 2000;105:1045–7. [PMC free article] [PubMed]
53. Maraveyas A, Lam T, Hetherington JW, Greenman J. Can a rational design for metronomic chemotherapy dosing be devised? Br J Cancer. 2005;92:1588–90. [PMC free article] [PubMed]
54. Hudis CA. Clinical implications of antiangiogenic therapies. Oncology. 2005;19(suppl 3):26–31. [PubMed]
55. Singh RP, Agarwal R. Tumor angiogenesis: a potential target in cancer control by phytochemicals. Curr Cancer Drug Targets. 2003;3:205–17. [PubMed]
56. Jung YD, Ahmad SA, Akagi Y, et al. Role of the tumor microenvironment in mediating response to anti-angiogenic therapy. Cancer Metastasis Rev. 2000;19:147–57. [PubMed]
57. Gutman M, Singh RK, Xie K, Bucana CD, Fidler IJ. Regulation of interleukin-8 expression in human melanoma cells by the organ environment. Cancer Res. 1995;55:2470–5. [PubMed]
58. Iqbal S, Lenz HJ. Integration of novel agents in the treatment of colorectal cancer. Cancer Chemother Pharmacol. 2004;54(suppl 1):S32–9. [PubMed]
59. Eberhard A, Kahlert S, Goede V, Hemmerlein B, Plate KH, Augustin HG. Heterogeneity of angiogenesis and blood vessel maturation in human tumors: implications for antiangiogenic tumor therapies. Cancer Res. 2000;60:1388–93. [PubMed]
60. Benjamin LE, Golijanin D, Itin A, Pode K, Keshet B. Selective ablation of immature blood vessels in established human tumors following vascular endothelial growth factor withdrawal. J Clin Invest. 1999;103:159–65. [PMC free article] [PubMed]
61. Ingber D, Fujita T, Kishimoto S, et al. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growth. Nature. 1990;348:555–7. [PubMed]
62. Wang S, Zheng Z, Weng Y, et al. Angiogenesis and anti-angiogenesis activity of Chinese medicinal herbal extracts. Life Sci. 2004;74:2467–78. [PubMed]
63. Kruger EA, Duray PH, Price DK, Pluda JM, Figg WD. Approaches to preclinical screening of antiangiogenic agents. Semin Oncol. 2001;28:570–6. [PubMed]
64. Wallace JM. Nutritional and botanical modulation of the inflammatory cascade: eicosanoids, cyclooxygenases, and lipoxygenases as an adjunct in cancer therapy. Integr Cancer Ther. 2002;1:7–37. [PubMed]
65. Mueller MS, Runyambo N, Wagner I, Borrmann S, Dietz K, Heide L. Randomized controlled trial of a traditional preparation of Artemisia annua L. (Annual Wormwood) in the treatment of malaria. Trans R Soc Trop Med Hyg. 2004;98:318–21. [PubMed]
66. Singh NP, Lai HC. Artemisinin induces apoptosis in human cancer cells. Anticancer Res. 2004;24:2277–80. [PubMed]
67. Chen HH, Zhou HJ, Wu GD, Lou XE. Inhibitory effects of artesunate on angiogenesis and on expressions of vascular endothelial growth factor and vegf receptor KDR/FLK-1. Pharmacology. 2004;7:1–9. [PubMed]
68. Aldieri E, Atragene D, Bergandi L, et al. Artemisinin inhibits inducible nitric oxide synthase and nuclear factor nf-κb activation. FEBS Lett. 2003;552:141–4. [PubMed]
69. Harmsma M, Gromme M, Ummelen M, Dignef W, Tusenius KJ, Ramaekers FC. Differential effects of Viscum album extract, IscadorQu on cell cycle progression and apoptosis in cancer cells. Int J Oncol. 2004;25:1521–9. [PubMed]
70. Park WB, Lyu SY, Kim JH, et al. Inhibition of tumor growth and metastasis by Korean mistletoe lectin is associated with apoptosis and antiangiogenesis. Cancer Biother Radiopharm. 2001;16:439–47. [PubMed]
71. Zarkovic N, Vukovic T, Loncaric I, et al. An overview on anticancer activities of the Viscum album extract Isorel. Cancer Biother Radiopharm. 2001;16:55–62. [PubMed]
72. Grossarth–Maticek R, Kiene H, Baumgartner SM, Ziegler R. Use of Iscador, an extract of European Mistletoe (Viscum album), in cancer treatment: prospective nonrandomized and randomized matched-pair studies nested within a cohort study. Altern Ther Health Med. 2001;7:57–76. [PubMed]
73. Narayan S. Curcumin, a multi-functional chemopreventive agent, blocks growth of colon cancer cells by targeting beta-catenin–mediated transactivation and cell-cell adhesion pathways. J Mol Histol. 2004;35:301–7. [PubMed]
74. Sen S, Sharma H, Singh N. Curcumin enhances vinorelbine mediated apoptosis in nsclc cells by the mitochondrial pathway. Biochem Biophys Res Commun. 2005;331:1245–52. [PubMed]
75. Khafif A, Hurst R, Kyker K, Fliss DM, Gil Z, Medina JE. Curcumin: a new radio-sensitizer of squamous cell carcinoma cells. Otolaryngol Head Neck Surg. 2005;132:317–21. [PubMed]
76. Shao ZM, Shen ZZ, Liu CH, et al. Curcumin exerts multiple suppressive effects on human breast carcinoma cells. Int J Cancer. 2002;98:234–40. [PubMed]
77. Arbiser JL, Klauber N, Rohan R, et al. Curcumin is an in vivo inhibitor of angiogenesis. Mol Med. 1998;4:376–83. [PMC free article] [PubMed]
78. Sreejayan Rao MN. Nitric oxide scavenging by curcuminoids. J Pharm Pharmacol. 1997;49:105–7. [PubMed]
79. Garcia–Cardena G, Folkman J. Is there a role for nitric oxide in tumor angiogenesis? J Natl Cancer Inst. 1998;90:560–1. [PubMed]
80. Gururaj AE, Belakavadi M, Venkatesh DA, Marme D, Salimath BP. Molecular mechanisms of antiangiogenic effect of curcumin. Biochem Biophys Res Commun. 2002;297:934–42. [PubMed]
81. Shim JS, Kim JH, Cho HY, et al. Irreversible inhibition of CD13/aminopeptidase N by the antiangiogenic agent curcumin. Chem Biol. 2003;10:695–704. [PubMed]
82. Hahm ER, Gho YS, Park S, Park C, Kim KW, Yang CH. Synthetic curcumin analogs inhibit activator protein-1 transcription and tumor-induced angiogenesis. Biochem Biophys Res Commun. 2004;321:337–44. [PubMed]
83. John VD, Kuttan G, Krishnakutty K. Anti-tumour studies of metal chelates of synthetic curcuminoids. J Exp Clin Cancer Res. 2002;21:219–24. [PubMed]
84. Kim JH, Shim JS, Lee SK, et al. Microarray-based analysis of anti-angiogenic activity of demethoxycurcumin on human umbilical vein endothelial cells: crucial involvement of the down-regulation of matrix metalloproteinase. Jpn J Cancer Res. 2002;93:1378–85. [PubMed]
85. Chen HW, Yu SL, Chen JJ, et al. Anti-invasive gene expression profile of curcumin in lung adenocarcinoma based on a high throughput microarray analysis. Mol Pharmacol. 2004;65:99–110. [PubMed]
86. Dorai T, Cao YC, Dorai B, Buttyan R, Katz AE. Therapeutic potential of curcumin in prostate cancer. iii: Curcumin inhibits proliferation, induces apoptosis and inhibits angiogenesis of LNCaP prostate cancer cells in vivo. Prostate. 2001;47:293–303. [PubMed]
87. Reddy S, Aggarwal BB. Curcumin is a non-competitive and selective inhibitor of phosphorylase kinase. FEBS Lett. 1994;341:19–22. [PubMed]
88. Leu TH, Su SL, Chuang YC, Maa MC. Direct inhibitory effect of curcumin on src and focal adhesion kinase activity. Biochem Pharmacol. 2003;66:2323–31. [PubMed]
89. Cheng AL, Hsu CH, Lin JK, et al. Phase i clinical trial of curcumin, a chemopreventive agent, in patients with high-risk pre-malignant lesions. Anticancer Res. 2001;21:2895–900. [PubMed]
90. Liu JJ, Huang TS, Cheng WF, Lu FJ. Baicalein and baicalin are potent inhibitors of angiogenesis: inhibition of endothelial cell proliferation, migration and differentiation. Int J Cancer. 2003;106:559–65. [PubMed]
91. Miocinovic R, McCabe NP, Keck RW, Jankun J, Hampton JA, Selman SH. In vivo and in vitro effect of baicalein on human prostate cancer cells. Int J Oncol. 2005;26:241–6. [PubMed]
92. Oh WK, George DJ, Hackmann K, Manola J, Kantoff PW. Activity of the herbal combination, pc-spes, in the treatment of patients with androgen-independent prostate cancer. Urology. 2001;57:122–6. [PubMed]
93. Small EJ, Frohlich MW, Bok R, et al. Prospective trial of the herbal supplement pc-spes in patients with progressive prostate cancer. J Clin Oncol. 2000;18:3595–603. [PubMed]
94. Hsieh TC, Lu X, Chea J, Wu JM. Prevention and management of prostate cancer using pc-spes: a scientific perspective. J Nutr. 2002;132(suppl 11):S3513–17.
95. Cao Y, Fu ZD, Wang F, Liu HY, Han R. Anti-angiogenic activity of resveratrol, a natural compound from medicinal plants. J Asian Nat Prod Res. 2005;7:205–13. [PubMed]
96. Igura K, Ohta T, Kuroda Y, Kaji K. Resveratrol and quercetin inhibit angiogenesis in vitro. Cancer Lett. 2001;171:11–16. [PubMed]
97. Lin MT, Yen ML, Lin CY, Kuo ML. Inhibition of vascular endothelial growth factor-induced angiogenesis by resveratrol through interruption of src dependent vascular endothelial cadherin tyrosine phosphorylation. Mol Pharmacol. 2003;64:1029–36. [PubMed]
98. Tseng SH, Lin SM, Chen JC, et al. Resveratrol suppresses the angiogenesis and tumor growth of gliomas in rats. Clin Cancer Res. 2004;10:2190–202. [PubMed]
99. Roy S, Khanna S, Alessio HM, et al. Anti-angiogenic property of edible berries. Free Radic Res. 2002;36:1023–31. [PubMed]
100. Bagchi D, Bagchi M, Stohs SJ, et al. Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention. Toxicology. 2000;148:187–97. [PubMed]
101. Singh RP, Tyagi AK, Dhanalakshmi S, Agarwal R, Agarwal C. Grape seed extract inhibits advanced human prostate tumor growth and angiogenesis and upregulates insulin-like growth factor binding protein-3. Int J Cancer. 2004;108:733–40. [PubMed]
102. Khanna S, Roy S, Bagchi D, Bagchi M, Sen CK. Upregulation of oxidant-induced vegf expression in cultured keratinocytes by a grape seed proanthocyanidin extract. Free Radic Biol Med. 2001;31:38–42. [PubMed]
103. Lee BC, Doo HK, Lee HJ, et al. The inhibitory effects of aqueous extract of Magnolia officinalis on human mesangial cell proliferation by regulation of platelet-derived growth factor-bb and transforming growth factor-β1 expression. J Pharmacol Sci. 2004;94:81–5. [PubMed]
104. Son HJ, Lee HJ, Yun–Choi HS, Ryu JH. Inhibitors of nitric oxide synthesis and tnf-α expression from Magnolia obovata in activated macrophages. Planta Med. 2000;66:469–71. [PubMed]
105. Chen F, Wang T, Wu YF, et al. Honokiol: a potent chemotherapy candidate for human colorectal carcinoma. World J Gastroenterol. 2004;10:3459–63. [PubMed]
106. Bai X, Cerimele F, Ushio–Fukai M, et al. Honokiol, a small molecular weight natural product, inhibits angiogenesis in vitro and tumour growth in vivo. J Biol Chem. 2003;278:35501–7. [PubMed]
107. Jiang C, Agarwal R, Lu J. Antiangiogenic potential of a cancer chemopreventive flavonoid antioxidant, silymarin: inhibition of key attributes of vascular endothelial cells and angiogenic cytokine secretion by cancer epithelial cells. Biochem Biophys Res Commun. 2000;276:371–8. [PubMed]
108. Singh RP, Sharma G, Dhanalakshmi S, Agarwal C, Agarwal R. Suppression of advanced human prostate tumor growth in athymic mice by silibinin feeding is associated with reduced cell proliferation, increased apoptosis, and inhibition of angiogenesis. Cancer Epidemiol Biomarkers Prev. 2003;12:933–9. [PubMed]
109. Gallo D, Giacomelli S, Ferlini C, et al. Antitumor activity of the silybin-phosphatidylcholine complex, IdB 1016, against human ovarian cancer. Eur J Cancer. 2003;39:2403–10. [PubMed]
110. Lee MJ, Maliakal P, Chen L, et al. Pharmacokinetics of tea catechins after ingestion of green tea and (–)-epigallocatechin-3-gallate by humans: formation of different metabolites and individual variability. Cancer Epidemiol Biomarkers Prev. 2002;11:1025–32. [PubMed]
111. Sartippour MR, Shao ZM, Heber D, et al. Green tea inhibits vascular endothelial growth factor (vegf) induction in human breast cancer cells. J Nutr. 2002;132:2307–11. [PubMed]
112. Cao Y, Cao R. Angiogenesis inhibited by drinking tea. Nature. 1999;398:381. [PubMed]
113. Tang FY, Nguyen N, Meydani M. Green tea catechins inhibit vegf-induced angiogenesis in vitro through suppression of ve-cadherin phosphorylation and inactivation of akt molecule. Int J Cancer. 2003;106:871–8. [PubMed]
114. Kojima–Yuasa A, Hua JJ, Kennedy DO, Matsui–Yuasa I. Green tea extract inhibits angiogenesis of human umbilical vein endothelial cells through reduction of expression of vegf receptors. Life Sci. 2003;73:1299–313. [PubMed]
115. Fassina G, Vene R, Morini M, et al. Mechanisms of inhibition of tumor angiogenesis and vascular tumor growth by epigallocatechin-3-gallate. Clin Cancer Res. 2004;10:4865–73. [PubMed]
116. Pisters KM, Newman RA, Coldman B, et al. Phase i trial of oral green tea extract in adult patients with solid tumors. J Clin Oncol. 2001;19:1830–8. [PubMed]
117. Zhang L, Rui YC, Yang PY, Qiu Y, Li TJ, Liu HC. Inhibitory effects of Ginkgo biloba extract on vascular endothelial growth factor in rat aortic endothelial cells. Acta Pharmacol Sin. 2002;23:919–23. [PubMed]
118. DeFeudis FV, Papadopoulos V, Drieu K. Ginkgo biloba extracts and cancer: a research area in its infancy. Fundam Clin Pharmacol. 2003;17:405–17. [PubMed]
119. Banerjee T, Van der Vliet A, Ziboh VA. Down regulation of cox-2 and inos by amentoflavone and quercetin in A549 human lung adenocarcinoma cell line. Prostaglandins Leukot Essent Fatty Acids. 2002;66:485–92. [PubMed]
120. O’Leary KA, de Pascual–Tereasa S, Needs PW, Bao YP, O’Brien NM, Williamson G. Effect of flavonoids and vitamin E on cyclooxygenase-2 (cox-2) transcription. Mutat Res. 2004;551:245–54. [PubMed]
121. Huynh H, Nguyen TT, Chan E, Tran E. Inhibition of ErbB-2 and ErbB-3 expression by quercetin prevents transforming growth factor alpha (tgf-α)- and epidermal growth factor (egf)-induced human pc-3 prostate cancer cell proliferation. Int J Oncol. 2003;23:821–9. [PubMed]
122. Igura K, Ohta T, Kuroda Y, Kaji K. Resveratrol and quercetin inhibit angiogenesis in vitro. Cancer Lett. 2001;171:11–16. [PubMed]
123. Tan WF, Lin LP, Li MH, et al. Quercetin, a dietary-derived flavonoid, possesses antiangiogenic potential. Eur J Pharmacol. 2003;459:255–62. [PubMed]
124. Ma ZS, Huynh TH, Ng CP, Do PT, Nguyen TH, Huynh H. Reduction of CWR22 prostate tumor xenograft growth by combined tamoxifen-quercetin treatment is associated with inhibition of angiogenesis and cellular proliferation. Int J Oncol. 2004;24:1297–304. [PubMed]
125. Lee KY, You HJ, Jeong HG, et al. Polysaccharide isolated from Poria cocos sclerotium induces nf-κb/rel activation and inos expression through the activation of p38 kinase in murine macrophages. Int Immunopharmacol. 2004;4:1029–38. [PubMed]
126. Mizushina Y, Akihisa T, Ukiya M, et al. A novel dna topoisomerase inhibitor: dehydroebriconic acid; one of the lanostane-type triterpene acids from Poria cocos. Cancer Sci. 2004;95:354–60. [PubMed]
127. Park WH, Joo ST, Park KK, Chang YC, Kim CH. Effects of the Geiji–Bokryung–Hwan on carrageenan-induced inflammation in mice and cyclooxygenase-2 in hepatoma cells of HepG2 and Hep3B. Immunopharmacol Immunotoxicol. 2004;26:103–12. [PubMed]
128. Chen YY, Chang HM. Antiproliferative and differentiating effects of polysaccharide fraction from fu-ling (Poria cocos) on human leukemic U937 and HL-60 cells. Food Chem Toxicol. 2004;42:759–69. [PubMed]
129. Jin Y, Zhang L, Zhang M, et al. Antitumor activities of heteropolysaccharides of Poria cocos mycelia from different strains and culture media. Carbohydr Res. 2003;338:1517–21. [PubMed]
130. Sato K, Mochizuki M, Saiki I, Yoo YC, Samukawa K, Azuma I. Inhibition of tumor angiogenesis and metastasis by a saponin of panax ginseng, ginsenoside-Rb2. Biol Pharm Bull. 1994;17:635–9. [PubMed]
131. Meade–Tollin LC, Wijeratne EM, Cooper D, et al. Ponicidin and oridonin are responsible for the antiangiogenic activity of Rabdosia rubescens, a constituent of the herbal supplement pc spes. J Nat Prod. 2004;67:2–4. [PubMed]
132. Senesse P, Meance S, Cottet V, Faivre J, Boutron–Ruault MC. High dietary iron and copper and risk of colorectal cancer: a case–control study in Burgundy, France. Nutr Cancer. 2004;49:66–71. [PubMed]
133. Lowndes SA, Harris AL. Copper chelation as an antiangiogenic therapy. Oncol Res. 2004;14:529–39. [PubMed]
134. Brewer GJ. Copper lowering therapy with tetrathiomolybdate as an antiangiogenic strategy in cancer. Curr Cancer Drug Targets. 2005;5:195–202. [PubMed]
135. Toda S. Inhibitory effects of aromatic herbs on lipid peroxidation and protein oxidative modification by copper. Phytother Res. 2003;17:546–8. [PubMed]
136. Barber R, Delahunt B, Grebe SK, Davis PF, Thornton A, Slim GC. Oral shark cartilage does not abolish carcinogenesis but delays tumor progression in a murine model. Anticancer Res. 2001;21:1065–9. [PubMed]
137. Loprinzi CL, Levitt R, Barton DL, et al. Evaluation of shark cartilage in patients with advanced cancer. Cancer. 2005;104:176–82. [PubMed]
138. Gonzalez RP, Leyva A, Moraes MO. Shark cartilage as a source of antiangiogenic compounds: from basic to clinical research. Biol Pharm Bull. 2001;24:1097–101. [PubMed]
139. Gingras D, Renaud A, Mousseau N, Beliveau R. Shark cartilage extracts as antiangiogenic agents: smart drinks or bitter pills? Cancer Metastasis Rev. 2000;19:83–6. [PubMed]
140. Ratel D, Glazier G, Provencal M, et al. Direct-acting fibrinolytic enzymes in shark cartilage extract: potential therapeutic role in vascular disorders. Thromb Res. 2005;115:143–52. [PubMed]
141. Gingras D, Labelle D, Nyalendo C, et al. The antiangiogenic agent Neovastat (AE-941) stimulates tissue plasminogen activator activity. Invest New Drugs. 2004;22:17–26. [PubMed]
142. Bobek V, Boubelik M, Fiserova A, et al. Anticoagulant drugs increase natural killer cell activity in lung cancer. Lung Cancer. 2005;47:215–23. [PubMed]
143. Altinbas M, Coskun HS, Er O, et al. A randomized clinical trial of combination chemotherapy with and without low-molecular-weight heparin in small cell lung cancer. J Thromb Haemost. 2004;2:1266–71. [PubMed]
144. AE 941. Drugs R D. 2004;5:83–9. [PubMed]
145. Brunel JM, Salmi C, Loncle C, Vidal N, Letourneux Y. Squalamine: a polyvalent drug of the future? Curr Cancer Drug Targets. 2005;5:267–72. [PubMed]
146. Li D, Williams JI, Pietras RJ. Squalamine and cisplatin block angiogenesis and growth of human ovarian cancer cells with or without HER-2 gene overexpression. Oncogene. 2002;21:2805–14. [PubMed]
147. Herbst RS, Hammond LA, Carbone DP, et al. A phase i/ii trial of continuous five-day infusion of squalamine lactate (MSI-1256F) plus carboplatin and paclitaxel in patients with advanced non-small cell lung cancer. Clin Cancer Res. 2003;9:4108–15. [PubMed]

The Antiangiogenic Agent Neovastat (Æ-941) Induces Endothelial Cell Apoptosis 1

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Posted 04 Jun 2012 — by James Street
Category Antiagiogenesis, antiangiogenesis
  1. Dominique Boivin,
  2. Sébastien Gendron,
  3. Édith Beaulieu,
  4. Denis Gingras and
  5. Richard Béliveau2

+ Author Affiliations


  1. Laboratoire de médecine moléculaire, Hôpital Ste-Justine-Université du Quebec à Montréal, Centre de cancérologie Charles-Bruneau, Centre de Recherche de l’Hôpital Ste-Justine, Montreal, Quebec H3T 1C5, Canada

Abstract

Neovastat (Æ-941), a naturally occurring multifunctional antiangiogenic agent (derived from shark cartilage,) has been shown to inhibit key components of the angiogenic process, including matrix metalloproteinases and vascular endothelial growth factor-mediated signaling events. In this study, we report the presence of a proapoptotic activity within this compound. Neovastat treatment of bovine aortic endothelial cells caused cell death with characteristics of apoptosis, including chromatin condensation and DNA fragmentation. Neovastat markedly induced caspase-3, caspase-8, and caspase-9 activities, at similar levels to those measured in cells treated with tumor necrosis factor-α. Activation of caspases by Neovastat appears to be essential for its proapoptotic effects because all apoptotic features were blocked by zVAD-fmk, a broad-spectrum caspase inhibitor. The activation of caspases was correlated with the cleavage of the nuclear substrate poly(ADP-ribose) polymerase, and by a concomitant release of cytochrome c from mitochondria to the cytoplasm. Neovastat-induced apoptosis appears to be specific to endothelial cells because treatment of other cell types such as U-87, COS-7, NIH-3T3, and SW1353 did not result in increased caspase-3 activity. These results demonstrate that Neovastat contains a proapoptotic factor that specifically induces the activation of caspases in endothelial cells and the resulting apoptosis of these cells.

Introduction

There is compelling evidence that tumor-induced neovascularization, i.e., tumor angiogenesis, represents a central process involved in the aggressive growth of tumors and of their metastases (1). This strict requirement for angiogenesis for sustained tumor growth has led to the development of alternative strategies for treating cancer based on the selective interference with the growth of tumor microvessels (1). The usefulness of this approach was exemplified in recent studies showing that the treatment of mice bearing tumors with antiangiogenic molecules such as angiostatin and endostatin results in tumor regression (2–4). These observations have thus driven considerable interest in identifying novel angiostatic proteins and molecules that particularly inhibit endothelial cell proliferation, migration, and vessel formation (5–8). However, notwithstanding the specific inhibitory effects of these compounds toward endothelial cell functions, the mechanisms involved in their antiangiogenic effects remain poorly understood.

Apoptotic cell death is a complex, tightly regulated process that involves drastic structural changes in cell morphology, including chromatin condensation, disassembly of the nuclear and cytoplasmic networks, DNA fragmentation, and membrane blebbing (9), resulting in the fragmentation of the cell into apoptotic bodies that are rapidly phagocytosed by neighboring cells (10). It is now well accepted that the activation of a unique family of cysteine proteases named caspases plays a central role in the signaling and execution phases of apoptosis induced by a variety of stimuli, including cytokines like TNF3-α, radiation, and chemotherapeutic drugs (11). Caspases exist in cells as inactive zymogens that are activated by proteolytic cleavage on appropriate apoptotic signals, leading to the degradation of a variety of critical target proteins, thereby disabling important cellular processes and promoting cell death (12).

Caspase activation during apoptosis occurs through a caspase cascade that is initiated by a subset of caspases containing long NH2-terminal domains that mediate interaction with caspase-activating factors. For instance, pro-caspase-8 becomes activated after its recruitment to death receptor complexes (13, 14), whereas caspase-9 is activated through its interaction with Apaf-1 and cytochrome c, in the presence of ATP or dATP (15). Activated caspase-8 and caspase-9 in turn cleave and activate other caspases such as caspase-3, -6, and -7, which represent the main caspase activity in apoptotic cells (15, 16). Caspase-8 induces downstream activation of executioner caspases either directly or indirectly, through activation of the BH3-domain-only subset of proapoptotic members of the bcl-2 family, leading to cytochrome c release from mitochondria and the activation of caspase-9 (17).

Several recent reports have suggested that the induction of endothelial cell apoptosis may represent a common feature of antiangiogenic molecules. Indeed, the incubation of endothelial cells with thrombospondin (18–20), angiostatin (21, 22), endostatin (23), canstatin (24), antithrombin (25), or kringle 5 (26) all resulted in the generation of a number of apoptotic figures such as phosphatidylserine translocation and DNA fragmentation. However, except for thrombospondin-induced endothelial apoptosis, in which the involvement of kinase-dependent caspase activation was suggested (19), very little information is available regarding the apoptotic pathways activated by antiangiogenic molecules nor on the involvement of caspase activities in their effects.

Cartilage was the first tissue reported to contain biological inhibitor(s) of angiogenesis. Moreover, it has long been recognized as an abundant source of angiostatic molecules (27), such as TIMP-1 and TIMP-2 (28, 29), troponin-1 (30), SCF-2 (31), thrombospondins (32), and metastatin (33). Recently, Neovastat, a naturally occurring inhibitor of angiogenesis derived from marine cartilage (dogfish), has been tested in Phase II clinical trials in non-small cell lung cancer and in renal cell carcinoma (34).4 It is currently undergoing Phase III clinical trials for the treatment of refractory renal cell carcinoma and nonresectable small cell lung cancer in addition to a Phase II pivotal clinical trial for the treatment of recurrent multiple myeloma (35).

There is now considerable evidence that the clinical benefits observed upon Neovastat treatment rely on the presence of multiple angiogenesis inhibitors within the compound (34, 36). Neovastat inhibits chick embryo vascularization and Matrigel-induced angiogenesis in vivo (37) as well as tumor growth of the DA3 breast adenocarcinoma (38, 39), the HGD human glioblastoma (40), and Lewis lung carcinoma metastasis in mice (37). At the molecular level, the antiangiogenic activity of Neovastat has been correlated with the inhibition of metalloproteinases (MMP-2, MMP-9, MMP-12) and serine elastases (41, 42). Neovastat also inhibits several functions of endothelial cells that are mediated by VEGF, including capillary sprouting, tubulogenesis, and hyperpermeability, possibly through interference with VEGF receptor-2 function (43). Neovastat has also been found to induce the activity of the tissue-type plasminogen activator, which generates the accumulation of angiostatin, an endogenous inhibitor of angiogenesis (44).

In this work, we sought to determine whether the inhibitory effect of Neovastat on cell proliferation could be related to a possible induction of endothelial cell apoptosis. Our results indicate that this compound indeed contains an endothelial-specific proapoptotic inducer that stimulates cell death through the activation of caspase-3, -8, and -9. To the best of our knowledge, these results represent the first demonstration of a proapoptotic activity in cartilage and strengthen the notion that extracts from this tissue contain multiple angiostatic activities.

Materials and Methods

Materials and Antibodies.

Neovastat (Æ-941) was obtained from Æterna Laboratories (Quebec City, Quebec, Canada; 45). zVAD-fmk, etoposide, and Casputin reagent were from BIOMOL Research Laboratories (Plymouth Meeting, PA). Acetyl-Asp-Glu-Val-Asp-7-AFC (Ac-DEVD-AFC), acetyl-Ile-Asp-Thr-Asp-7-AFC (Ac-IETD-AFC), and acetyl-Leu-Glu-His-Asp-7-AMC (Ac-LEHD-AMC) were purchased from BIOSOURCE International (Camarillo, CA). Recombinant human TNF-α was purchased from Calbiochem (La Jolla, CA). DAPI nucleic acid stain was purchased from Molecular Probes (Eugene, OR). Anti-PARP monoclonal antibody (clone C-2–10) was purchased from Clontech (Palo Alto, CA). Anti-β-actin was purchased from Sigma (St. Louis, MO). Anti-Bax monoclonal antibody (clone 6A7) was from BIOSOURCE International. Anti-Bcl-2 monoclonal antibody (clone 7) was purchased from Transduction Laboratories (San Diego, CA). Anti-cytochrome c was purchased from PharMingen (San Diego, CA). Anti-COX IV antibody was purchased from Molecular Probes.

Cell Culture.

BAECs obtained from Clonetics (San-Diego, CA) were cultured in low glucose DMEM containing 10% heat-inactivated calf serum (MediCorp, Montreal, Quebec, Canada), 100 units/ml penicillin G, 100 μg/ml streptomycin, and 1 ng/ml basic fibroblast growth factor (Upstate Biotechnology, Lake Placid, NY). U-87 MG cell line (American Type Culture Collection) was cultured in MEM containing 10% heat-inactivated FCS (MediCorp) and antibiotics. HUVECs, obtained from Clonetics, were cultured in M199 medium containing 20% heat-inactivated FCS, 40 μg/ml endothelial cell growth supplement (Upstate Biotechnology), 90 μg/ml heparin (Life Technologies, Inc., Burlington, Ontario, Canada), and antibiotics. Human dermal microcapillary endothelial cells transformed with SV40 large T antigen (Ref. 46; HMEC-1) were cultured in MCDB 131 medium containing 10% heat-inactivated FCS, 10 ng/ml EGF, 1 ng/ml hydrocortisone, and antibiotics. COS-7, NIH-3T3, and SW1353 cell lines (all from American Type Culture Collection) were cultured in DMEM containing 10% heat-inactivated calf serum and antibiotics. All of the media and antibiotics were from Life Technologies, Inc.

Treatment of Cells with Neovastat, Etoposide, and TNF-α.

Cells were grown to about 80% confluence and treated with Neovastat (85 μg of protein/ml) or with other inducers of apoptosis, such as etoposide (10 μm) or TNF-α (25 ng/ml), and cycloheximide (10 μg/ml). HUVEC and human microcapillary endothelial cells were treated with Neovastat in the presence of cycloheximide (10 μg/ml). When required, zVAD-fmk was added 1 h before treatment at a final concentration of 25 μm.

Cell Viability Assays.

Cells, grown to ∼80% confluence in 12-well plates, were treated for various periods of time. Adherent and nonadherent cells were collected and viability was assessed by mixing aliquots of cell suspensions with an equal volume of 0.4% trypan blue (Life Technologies, Inc.). Cells that picked up the dye were considered to be dead.

Fluorimetric Caspase-3, Caspase-8, and Caspase-9 Assays.

Cells treated with Neovastat, etoposide, or TNF-α/CHX were collected and washed in cold PBS. Cells were lysed in Apo-Alert lysis buffer (Clontech, Palo Alto, CA) for 20 min at 4°C, and the lysates were clarified by centrifugation at 16,000 × g for 20 min. Caspase activities were determined by incubation with 50 μm fluorogenic peptide substrates acetyl-Asp-Glu-Val-Asp-7-AFC (Ac-DEVD-AFC; caspase-3-specific) or acetyl-Ile-Asp-Thr-Asp-7-AFC (Ac-IETD-AFC; caspase-8-specific) or 250 μm acetyl-Leu-Glu-His-Asp-7-AMC (Ac-LEHD-AMC, caspase 9 specific) in assay buffer {50 mm HEPES-NaOH (pH 7.4), 100 mm NaCl, 10% sucrose, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 5 mm DTT, and 1 mm EDTA} on 96-well plates. The release of AFC (λex = 400 nm, λem = 505 nm) or AMC (λex = 380 nm, λem = 460 nm) was monitored for at least 20 min at 37°C on a SpectraMAX Gemini fluorescence plate reader (Molecular Devices). Caspase activities were expressed as rfu/s/μg of protein used in the assay.

TUNEL and DAPI Staining.

BAECs were grown on glass coverslips and were treated for 18 h. After the treatment, coverslips were washed twice with PBS, fixed for 30 min in 3.7% paraformaldehyde/PBS at room temperature, washed again twice in PBS, and then permeabilized in 0.2% Triton X-100/PBS for 5 min at 4°C. Permeabilized cells were washed in PBS, and the TUNEL assay was performed as described in the In Situ Cell Death Detection kit (Roche Diagnostic, Laval, Quebec, Canada). The nuclear morphology of cells was analyzed by the staining of DNA with DAPI (Molecular Probes).

Immunoblot Analysis.

Cells were harvested and lysed as described for caspase assays, and the protein concentration was measured using bicinchoninic acid protein assay reagent (Pierce, Rockford, IL) and BSA as the standard. Equal amounts of protein samples in sample buffer [62.5 mm Tris-HCl (pH 6.8), 100 mm DTT, 10% glycerol, 2% SDS, and 0.1% bromophenol blue] were heated at 100°C for 3 min and separated on 0.75-mm-thick SDS-polyacrylamide gels with a MINI-PROTEAN II apparatus (Bio-Rad). Proteins were electroblotted onto 0.45-μm-pore diameter polyvinylidene difluoride membranes (Roche Diagnostics) with a semi-dry apparatus (Millipore) in transfer buffer (96 mm glycine, 10 mm Tris, and 20% methanol) for 1 h at 80 mA/gel. Membranes were blocked overnight at 4°C in Tris-buffered saline [20 mm Tris-HCl (pH 7.5), 137 mm NaCl] containing 0.1% (v/v) Tween 20 and 3% BSA. Blots were incubated with primary antibodies in blocking buffer for 2 h at room temperature, followed by a 1-h incubation with a 1:10,000 dilution of horseradish peroxidase-conjugated donkey antirabbit IgG or goat antimouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) in the same incubation medium. Immunoreactive bands were revealed with enhanced chemiluminescence Western blotting kit (Renaissance, NEN Life Science, Boston, MA) and SuperRX films (Fuji).

DNA Degradation Assay.

Low-molecular-weight DNA was isolated from cells by a modified Hirt extraction procedure (47). BAECs were grown in 100-mm-diameter plates, treated for 16–20 h, harvested, and washed in cold PBS. Cells were lysed in 0.5 ml of lysis buffer [10 mm Tris-HCl (pH 8.0), 5 mm EDTA, 100 mm NaCl, and 0.5% (w/v) SDS] containing 1 mg of Pronase/ml. Lysates were incubated at 37°C for 2 h, and 150 μl of 5 m NaCl was added (1 m final concentration). Samples were incubated overnight at 4°C and centrifuged at 16 000 × g for 30 min. DNA was precipitated from the supernatant with ethanol. Samples were centrifuged at 16 000 × g for 30 min and pellets were resuspended in Tris/EDTA [10 mm Tris-HCl (pH 8.0), 0.1 mm EDTA]. Nucleic acids were treated with 10 μg of RNase A (Sigma) for 1 h at 37°C and then were analyzed on 1% agarose gels stained with ethidium bromide.

Preparation of Mitochondria and Cytosol for Measurement of Cytochrome c.

Cells were harvested and washed in cold PBS. Cell pellets were resuspended in homogenization buffer, and mitochondrial and cytosolic extracts were prepared by the method described previously (48).

Results

Neovastat Induces Endothelial Cell Death.

Neovastat is a natural, multifunctional antiangiogenic agent produced from cartilage extracts. In the course of studies aimed at identifying activities contributing to its anticancer properties, Neovastat was tested for its ability to induce cell death in endothelial cells. Endothelial and nonendothelial cells were incubated with Neovastat (85 μg/ml), and, at various times, cell viability was measured by exclusion of trypan blue. Fig. 1 shows that Neovastat induced 50% cell death of BAECs at about 24 h of treatment and that by 48 h, virtually all of the cells were dead. The viability of nonendothelial cell lines U-87 (glioblastoma), HT-1080 (fibroblasts), and NIH-3T3 (fibroblasts), however, was not altered by Neovastat, which suggested an endothelial cell specificity. In contrast, all four of the cell lines were highly sensitive to TNF-α/CHX, a well-characterized inducer of death-receptor-mediated cell apoptosis (11, 49). Importantly, Neovastat-induced BAEC cell death was greatly reduced by the presence of zVAD-fmk, a broad-spectrum inhibitor of caspases, which indicated that the cell death is caspase dependent. Endothelial-specific and caspase-dependent induction of cell death by Neovastat was also observed using other methods (WST-1 cell proliferation assay, fluorescence-activated cell sorting (FACS) analysis using annexin V), which further supported its proapoptotic action on endothelial cells (data not shown).

Neovastat Induces DNA Fragmentation and Chromatin Condensation in the Nucleus of Endothelial Cells.

To determine whether Neovastat-induced endothelial cell death was caused by programmed cell death rather than by necrosis, we first looked at fundamental characteristics of apoptotic cells: DNA condensation and fragmentation. We used two complementary methods to examine DNA fragmentation in the nuclei of apoptotic cells: TUNEL assay, in which DNA fragmentation is visualized after incorporation of Fluorescein-dUTP; and the DNA ladder assay, in which low-molecular-weight DNA is extracted from cells and visualized on agarose gels in the presence of ethidium bromide. DAPI staining was used to show chromatin condensation, another hallmark of apoptosis. As shown in Fig. 2A, BAECs treated with Neovastat were stained by the TUNEL assay, whereas labeling was not observed when Neovastat-treated cells were cultured in the presence of zVAD-fmk. Nuclei from untreated cells were weakly and uniformly stained with DAPI, whereas the shape of nuclei from Neovastat-treated cells appeared irregular. Moreover, the nucleus of Neovastat-treated cells contained bright fluorescent spots that are characteristic of condensed chromatin. Low-molecular-weight DNA extracted from cells treated with Neovastat, as well as that from cells treated with etoposide or TNF-α/CHX, showed degradation patterns typical of cells undergoing apoptosis (Fig. 2B). This degradation was diminished when cells were treated in the presence of zVAD-fmk. These results show that Neovastat treatment induced apoptosis in endothelial cells and indicate that caspases are involved in the process.

Neovastat Increases Intracellular Activities of Caspase-3 and Caspase-8.

Most cell-death pathways involve the activation of caspases, leading to the degradation and inactivation of key cellular proteins such as DNA repair, signaling, and structural proteins. We used fluorogenic peptide substrates to measure caspase-3 activities (Fig. 3A) and caspase-8 activities (Fig. 3B) after 0, 3, 6, and 24 h of incubation with Neovastat. BAECs were also treated with TNF-α/CHX as a positive control for activation of these caspases. Comparable levels of caspase-3 and -8 activities were detected in cell extracts after 24 h of treatment with Neovastat or TNF-α/CHX. However, although caspase-3 and -8 activities were readily detectable after 3 h of treatment with TNF-α/CHX, the activation of these caspases after treatment with Neovastat was much slower, which suggested a different mechanism of induction. No caspase activity was detected in extracts from cells treated in the presence of 25 μm zVAD-fmk, confirming that the protease activity measured was attributable to caspases and not to other types of proteases.

Neovastat-induced Activation of Caspases Is Specific to Endothelial Cells.

The effect of Neovastat on caspase-3 activity was measured in several nonendothelial and endothelial cell lines (Table 1). Neovastat treatment induced caspase-3 activity in BAECs, HUVECs, and HMEC-1 cells but failed to induce caspase-3 in U-87 glioblastoma, NIH-3T3 fibroblast, MCF-7 human breast cancer, SW1353 chondrosarcoma, and COS-7 simian kidney (Table 1). Neovastat did not induce caspase activities in U-87 (Fig. 4, C and D), whereas these cells were highly responsive to TNF-α/CHX. The specificity of action of Neovastat and TNF-α/CHX on caspase activities was verified with Casputin, a chimeric protein containing domains from human X-linked inhibitor of apoptosis that inhibits caspase-3, -7 and, weakly, caspase-10 but not caspase-1, -2, -6, and -8 (50, 51). DEVDase activity (caspase-3) was strongly inhibited by Casputin. In contrast, IETDase activity (caspase-8) was only slightly reduced in the presence of Casputin, which confirmed that caspase-8 activity was measured under our experimental conditions.

Neovastat Induces PARP Cleavage but Does Not Change Bcl-2 and Bax Expression Levels in BAEC.

Activated caspase-3 is able to cleave numerous cellular substrates including PARP. As shown in Fig. 5, Neovastat, etoposide, and TNF-α/CHX induced cleavage of PARP into the characteristic Mr 85,000 fragment. Moreover, coincubation with 25 μm zVAD-fmk completely inhibited PARP cleavage induced by Neovastat and etoposide. However, PARP cleavage induced by TNF-α was only weakly inhibited by zVAD-fmk at this concentration (not shown), possibly because of the very strong apoptotic response induced by this cytokine. However, the addition of 50 μm zVAD-fmk significantly reduced TNF-mediated PARP cleavage (Fig. 5). Expression levels of Bcl-2 and Bax remained unchanged by treatment with any of the apoptosis inducers. In agreement with the previous results showing that caspases are not activated by Neovastat in U-87 cells (Fig. 4, C and D), PARP cleavage was not observed in these cells treated with Neovastat. In contrast, PARP was cleaved in U-87 cells treated with TNF-α/CHX (Fig. 5, lower panel).

Neovastat Treatment Induces Cytochrome c Release in BAEC.

To identify which mechanisms are involved in Neovastat-induced activation of caspase-3, we examined the effect of Neovastat treatment on cytochrome c release. Cytochrome c released in the cytoplasm forms a complex with Apaf-1, dATP, and pro-caspase-9, leading to the activation of caspase-9 followed by downstream activation of effector caspases such as caspase-3 (15, 16). BAECs were treated with Neovastat and, at various time intervals, cells were subjected to subcellular fractionation. Then, equal amounts of proteins from mitochondria and cytosol fractions were probed for cytochrome c by immunoblotting. β-actin and COX subunit IV were used as a control for the amount of protein loaded in the cytosol and mitochondrial fractions, respectively. Neither their levels nor their translocation were modulated by Neovastat (Fig. 6A). As shown in Fig. 6, A and B, cytochrome c was released in the cytosol of cells treated with Neovastat. Moreover, the levels of cytochrome c detected in the mitochondrial fraction from treated cells were greatly reduced. Because release of cytochrome c is known to result in the activation of caspase-9, we used the fluorogenic substrate LEHD-AMC to detect such an activity in extracts from cells treated with Neovastat and TNF-α/CHX. As shown in Fig. 6C, caspase-9 activity was increased in both Neovastat- and TNF-α/CHX-treated cells. Moreover, the activation of caspase-9 is prevented in cells treated in the presence of zVAD-fmk. Furthermore, this activity was strongly inhibited by the caspase-9 inhibitor LEHD-CHO. These results, thus, strongly suggest that the induction of caspase activities by Neovastat may involve the mitochondrial pathway.

Discussion

Neovastat is an antiangiogenic drug that has reached Phase III clinical trial evaluation. In contrast to other inhibitors of angiogenesis derived from natural sources, there is significant information available about the mechanism of action of Neovastat. We have recently reported that it contains inhibitors of MMPs as well as of serine elastases (41). It has also been reported that Neovastat inhibits endothelial cell proliferation, whereas it had no significant effect on the proliferation of fibroblast and muscle cells nor on several tumor cell lines (37), which suggests that Neovastat acts directly at the endothelial cell level.

In this work, we provide evidence that Neovastat exerts at least some of its endothelial-specific effects through the induction of apoptosis. Consistent with the presence of a proapoptotic activity within Neovastat as reported for many antiangiogenic molecules (20–24, 26), incubation of endothelial cells with the compound promotes the generation of a number of apoptotic hallmarks, including chromatin condensation and DNA fragmentation. Our results indicate that these alterations in endothelial cell structure are related to the activation of at least three key members of the caspase family, namely caspase-3, caspase-8, and caspase-9. First, we observed that Neovastat induced a marked increase in the activity of these enzymes toward fluorogenic substrates, the extent of activation being similar to that induced by TNF-α/CHX, a prototypic apoptotic inducer. This increase in caspase activities is crucial for the propaptotic activity of Neovastat because DNA fragmentation, chromatin condensation, and PARP cleavage were abolished by coincubation with zVAD-fmk, a broad-spectrum caspase inhibitor. By contrast, Neovastat did not induce detectable apoptosis nor increased caspase activities in many other cell types, which indicated that its action is specific to endothelial cells.

The mechanisms by which Neovastat induces caspase-3 and -8 activities remain to be established. Caspase-8 is an initiator caspase that is activated on stimulation of death receptors and subsequently activates the executioner caspases either directly or through a Bid-dependent cytochrome c release from mitochondria (17, 52). It is, thus, tempting to speculate that a similar pathway may be involved in Neovastat-induced apoptosis. In this respect, we observed that the incubation of cells with Neovastat resulted in a time-dependent release of cytochrome c from mitochondria, in a manner similar to that observed after incubation with TNF-α. However, in a number of cell types, caspase-8 has been suggested to function as an executioner caspase, being activated by caspase-9 (53). The exact apoptotic pathways involved in Neovastat-induced apoptosis are currently under investigation.

The presence within Neovastat of an endothelial-specific proapoptotic factor may, thus, significantly contribute to the antiangiogenic activity of the compound. Given the recent observations of antiprotease as well as anti-VEGF activities in Neovastat (41), the activity of Neovastat appears to act through multiple targets. Its efficiency against several crucial steps of the angiogenic cascade closely linked to tumor progression contrast from other natural or chemical antiangiogenic agents, in which only one activity was targeted. Moreover, Neovastat’s multiple mechanism of action could reflect the presence of several components that could synergistically act together to control neovascularization. The findings reported in this paper may, thus, be of significant importance to our understanding of the mechanisms by which Neovastat elicits its antiangiogenic, antitumoral, and antimetastatic effects.

Fig. 1.

Neovastat treatment induces endothelial cell-specific cell death. Subconfluent cells were either left untreated (○) or treated with 85 μg/ml Neovastat (▪) or with 25 ng/ml TNF-10 μg/ml CHX (▴) for 0, 24, 48, and 72 h. BAECs were also treated with Neovastat in the presence of 25 μm zVAD-fmk (□). Adherent and nonadherent cells were collected, and viability was assessed by trypan blue exclusion. Cell death is expressed as the percentage of cells incorporating the dye relative to the total amount of cells.

Fig. 2.

Neovastat treatment induces DNA fragmentation. A, BAECs grown on coverslips were either left untreated (Control) or treated with 85 μg/ml Neovastat in the absence or in the presence of 25 μm zVAD-fmk. Fragmented DNA was detected by the TUNEL assay, and DNA condensation was detected by staining with DAPI, as described in “Materials and Methods.” B, BAECs were either left untreated or treated with 85 μg/ml Neovastat (Neov) or 10 μm etoposide (Etop) or 25 ng/ml TNF-α-10 μg/ml CHX, in the absence or in the presence of zVAD-fmk. Serum concentration was 10% during etoposide and TNF-α/CHX treatments or 0.5% during Neovastat treatment. After 16 h, low-molecular-weight DNA was isolated and then analyzed on 1% agarose gels stained with ethidium bromide.

Fig. 3.

Time course of caspase-3 and caspase-8 induction in BAECs. Subconfluent cells were either left untreated (▴, ▵) or treated with 85 μg/ml Neovastat (□, ▪) or with 25 ng/ml TNF-10 μg/ml CHX (•, ○) for 0, 3, 6, and 24 h in the absence (filled symbols) or in the presence (open symbols) of 25 μm zVAD-fmk. Extracts from control and treated cells were used to determine DEVDase (A, caspase-3) and IETDase (B, caspase-8) activities.

Fig. 4.

Neovastat treatment increases caspase-3 and caspase-8 activities in BAECs but not in U-87 glioblastoma cells. Subconfluent cells were either left untreated or treated with 85 μg/ml Neovastat or 10 μm etoposide or 25 ng/ml TNF-α-10 μg/ml CHX for 18 h in the absence or in the presence of 25 μm zVAD-fmk. Extracts from control and treated cells were used to determine DEVDase (caspase-3) and IETDase (caspase-8) activities in the absence (filled bars) or in the presence (open bars) of 40 μm Casputin, as described in “Materials and Methods.”

Fig. 5.

Western blot analysis of BAECs and U-87 cells for proteins involved in apoptosis. BAECs and U-87 cells were either left untreated or treated with 85 μg/ml Neovastat (Neov) or 10 μm etoposide (Etop) or TNF-α/CHX, in the absence or in the presence of 25 μm (for Neovastat and etoposide) or 50 μm zVAD-fmk (for TNF-α/CHX). After 18 h, cells were harvested, and cell extracts were prepared. Twenty μg of protein was subjected to SDS-PAGE followed by electrotransfer onto polyvinylidene difluoride. Immunoblot analysis was performed with antibodies directed against PARP, Bcl-2, Bax, and β-actin.

Fig. 6.

Neovastat treatment induces release of mitochondrial cytochrome c and caspase 9 activity in BAEC. A, BAECs were either left untreated or treated with 85 μg/ml Neovastat. At the indicated periods of time, cells were harvested, fractionated into mitochondrial (Mito) and cytosolic (Cytosol) fractions, and then analyzed by Western blot for cytochrome c, COX IV, and β-actin. B, cytochrome c detected in mitochondrial (○) and cytosolic fractions (•) was quantified using IPlab Gel Image Processing Software (Signal Analytic), and the results were expressed as percentage. C, BAECs were either left untreated or treated with Neovastat or TNF-α/CHX for 18 h in the absence or in the presence of zVAD-fmk. Extracts from control and treated cells were used to determine LEHDase (caspase-9) activity in the absence (filled bars) or in the presence (open bars) of 100 μm LEHD-CHO, as described in “Materials and Methods.”

Table 1

Induction of caspase-3 in various cell lines

Cells were untreated or treated with Neovastat for 18 h in DMEM containing 0.5% serum. Extracts from control and treated cells were used to determine DEVDase (caspase-3) activity as described in “Materials and Methods.”

Acknowledgments

We thank Drs. Patrick Poyet and Muriel Steel for their critical reading of the manuscript.

Footnotes

  • ↵1 Supported by Æterna Laboratories, Québec City, Québec, Canada.

  • ↵2 To whom requests for reprints should be addressed, at Laboratoire de médecine moléculaire Ste-Justine-UQAM, Centre de cancérologie Charles-Bruneau, 3175, Chemin Côte-Ste-Catherine, Montréal, Québec (Canada) H3T 1C5. Fax: (514) 345-2359; Email: molmed@justine.umontreal.ca

  • ↵3 The abbreviations used are: TNF, tumor necrosis factor; BAEC, bovine aortic endothelial cell; DAPI, 4′,6-diamino-2-phenylindole; HUVEC, human umbilical vein endothelial cell; MMP, matrix metalloproteinase; PARP, poly(ADP-ribose) polymerase; rfu, relative fluorescence unit; TIMP, tissue inhibitor of MMP; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; EGF, epidermal growth factor; VEGF, vascular endothelial growth factor; zVAD-fmk, z-Val-Ala-Asp-fluoromethylketone; AFC, amino-4-trifluoromethylcoumarin; AMC, amino-4-methylcoumarin; CHX, cycloheximide; COX, cytochrome oxidase.

  • ↵4 J. Latreille, G. Batist, F. Laberge, P. Champagne, D. Croteau, P. Falardeau, C. Levinton, C. Hariton, W. K. Hariton, and É. Dupont. Phase I/II trial on the safety and efficacy of Neovastat (Æ-941) in the treatment of non-small cell lung cancer, submitted for publication.

    • Accepted July 1, 2002.
    • Received February 13, 2002.
    • Revision received May 23, 2002.

References

  1. ↵
    Folkman, J. Angiogenesis in cancer, vascular, rheumatoid and other disease.Nat. Med. , 1: 27 –31,1995 .
  2. ↵
    Hanahan, D., and Folkman, J. Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.Cell , 86: 353 –364,1996 .
  3. Brem, S. Angiogenesis and cancer control: from concept to therapeutic trial.Cancer Control , 6: 436 –458,1999 .
  4. ↵
    Boehm, T., Folkman, J., Browder, T., and O’Reilly. M. S. Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance.Nature (Lond.) , 390: 404 –407,1997 .
  5. ↵
    Pluda, J. M. Tumor-associated angiogenesis: mechanisms, clinical implications, and therapeutic strategies.Semin. Oncol. , 24: 203 –218,1997 .
  6. Rak, J., and Kerbel, R. S. Treating cancer by inhibiting angiogenesis: new hopes and potential pitfalls.Cancer Metastasis Rev. , 15: 231 –236,1996 .
  7. Taraboletti, G., and Margosio, B. Antiangiogenic and antivascular therapy for cancer.Curr. Opin. Pharmacol. , 1: 378 –384,2001 .
  8. ↵
    Cao, Y. Endogenous angiogenesis inhibitors and their therapeutic implications.Int. J. Biochem. Cell Biol. , 33: 357 –369,2001 .
  9. ↵
    Kerr, J. F., Wyllie, A. H., and Currie, A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.Br. J. Cancer , 26: 239 –257,1972 .
  10. ↵
    Wyllie, A. H. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation.Nature (Lond.) , 284: 555 –556,1980 .
  11. ↵
    Strasser, A., O’Connor, L., and Dixit, V. M. Apoptosis signaling.Annu. Rev. Biochem. , 69: 217 –245,2000 .
  12. ↵
    Nicholson, D. W. Caspase structure, proteolytic substrates, and function during apoptotic cell death.Cell Death Differ. , 6: 1028 –1042,1999 .
  13. ↵
    Muzio, M., Chinnaiyan, A. M., Kischkel, F. C., O’Rourke, K., Shevchenko, A., Ni, J., Scaffidi, C., Bretz, J. D., Zhang, M., Gentz, R., Mann, M., Krammer, P. H., Peter, M. E., and Dixit, V. M. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex.Cell , 85: 817 –827,1996 .
  14. ↵
    Boldin, M. P., Goncharov, T. M., Goltsev, Y. V., and Wallach, D. Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death.Cell , 85: 803 –815,1996 .
  15. ↵
    Li, P., Nijhawan, D., Budihardjo, I., Srinivasula, S. M., Ahmad, M., Alnemri, E. S., and Wang, X. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade.Cell , 91: 479 –489,1997 .
  16. ↵
    Slee, E. A., Harte, M. T., Kluck, R. M., Wolf, B. B., Casiano, C. A., Newmeyer, D. D., Wang, H. G., Reed, J. C., Nicholson, D. W., Alnemri, E. S., Green, D. R., and Martin, S. J. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner.J. Cell Biol. , 144: 281 –292,1999 .
  17. ↵
    Li, H., Zhu, H., Xu, C. J., and Yuan, J. Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis.Cell , 94: 491 –501,1998 .
  18. ↵
    Nor, J. E., Mitra, R. S., Sutorik, M. M., Mooney, D. J., Castle, V. P., and Polverini, P. J. Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by activating the caspase death pathway.J. Vasc. Res. , 37: 209 –218,2000 .
  19. ↵
    Jimenez, B., Volpert, O. V., Crawford, S. E., Febbraio, M., Silverstein, R. L., and Bouck, N. Signals leading to apoptosis-dependent inhibition of neovascularization by thrombospondin-1.Nat. Med. , 6: 41 –48,2000 .
  20. ↵
    Guo, N., Krutzsch, H. C., Inman, J. K., and Roberts, D. D. Thrombospondin 1 and type I repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells.Cancer Res. , 57: 1735 –1742,1997 .
  21. ↵
    Claesson-Welsh, L., Welsh, M., Ito, N., Anand-Apte, B., Soker, S., Zetter, B., O’Reilly, M., and Folkman, J. Angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif RGD.Proc. Natl. Acad. Sci. USA , 95: 5579 –5583,1998 .
  22. Lucas, R., Holmgren, L., Garcia, I., Jimenez, B., Mandriota, S. J., Borlat, F., Sim, B. K., Wu, Z., Grau, G. E., Shing, Y., Soff, G. A., Bouck, N., and Pepper, M. S. Multiple forms of angiostatin induce apoptosis in endothelial cells.Blood , 92: 4730 –4741,1998 .
  23. ↵
    Dhanabal, M., Ramchandran, R., Waterman, M. J., Lu, H., Knebelmann, B., Segal, M., and Sukhatme, V. P. Endostatin induces endothelial cell apoptosis.J. Biol. Chem. , 274: 11721 –11726,1999 .
  24. ↵
    Kamphaus, G. D., Colorado, P. C., Panka, D. J., Hopfer, H., Ramchandran, R., Torre, A., Maeshima, Y., Mier, J. W., Sukhatme, V. P., and Kalluri, R. Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth.J. Biol. Chem. , 275: 1209 –1215,2000 .
  25. ↵
    Larsson, H., Sjoblom, T., Dixelius, J., Ostman, A., Ylinenjarvi, K., Bjork, I., and Claesson-Welsh, L. Antiangiogenic effects of latent antithrombin through perturbed cell-matrix interactions and apoptosis of endothelial cells.Cancer Res. , 60: 6723 –6729,2000 .
  26. ↵
    Lu, H., Dhanabal, M., Volk, R., Waterman, M. J., Ramchandran, R., Knebelmann, B., Segal, M., and Sukhatme, V. P. Kringle 5 causes cell cycle arrest and apoptosis of endothelial cells.Biochem. Biophys. Res. Commun. , 258: 668 –673,1999 .
  27. ↵
    Brem, H., and Folkman, J. Inhibition of tumor angiogenesis mediated by cartilage.J. Exp. Med. , 141: 427 –439,1975 .
  28. ↵
    Moses, M. A., Sudhalter, J., and Langer, R. Identification of an inhibitor of neovascularization from cartilage.Science (Wash. DC) , 248: 1408 –1410,1990 .
  29. ↵
    Murray, J. B., Allison, K., Sudhalter, J., and Langer, R. Purification and partial amino acid sequence of a bovine cartilage-derived collagenase inhibitor.J. Biol. Chem. , 261: 4154 –4159,1986 .
  30. ↵
    Moses, M. A., Wiederschain, D., Wu, I., Fernandez, C. A., Ghazizadeh, V., Lane, W. S., Flynn, E., Sytkowski, A., Tao, T., and Langer, R. Troponin I is present in human cartilage and inhibits angiogenesis.Proc. Natl. Acad. Sci. USA , 96: 2645 –2650,1999 .
  31. ↵
    Liang, J. H., and Wong, K. P. The characterization of angiogenesis inhibitor from shark cartilage.Adv. Exp. Med. Biol. , 476: 209 –223,2000 .
  32. ↵
    Miller, R. R., and McDevitt, C. A. Thrombospondin is present in articular cartilage and is synthesized by articular chondrocytes.Biochem. Biophys. Res. Commun. , 153: 708 –714,1988 .
  33. ↵
    Liu, N., Lapcevich, R. K., Underhill, C. B., Han, Z., Gao, F., Swartz, G., Plum, S. M., Zhang, L., and Gree, S. J. Metastatin: a hyaluronan-binding complex from cartilage that inhibits tumor growth.Cancer Res. , 61: 1022 –1028,2001 .
  34. ↵
    Gingras, D., Batist, G., and Béliveau, R. Æ-941 (Neovastat®): a novel multifunctional antiangiogenic compound.Expert Rev. Anticancer Ther. , 1: 341 –347,2001 .
  35. ↵
    Falardeau, P., Champagne, P., Poyet, P., Hariton, C., and Dupont, É. Æ-941 (Neovastat), a naturally occurring multifunctional antiangiogenic product in Phase III clinical trials.Semin. Oncol. , 28: 620 –625,2001 .
  36. ↵
    Gingras, D., Renaud, A., Mousseau, N., and Béliveau, R. Shark cartilage extracts as antiangiogenic agents: smart drinks or bitter pills?Cancer Metastasis Rev. , 19: 83 –86,2000 .
  37. ↵
    Dupont, É., Falardeau, P., Mousa, S. A., Dimitriadou, V., Pépin, M-C., Wang, T., and Alaoui-Jamali, M. A. Antiangiogenic and antimetastatic properties of neovastat (Æ-941), an orally active extract derived from cartilage tissue.Clin Exp Metastasis , 19: 145 –153,2002 .
  38. ↵
    Dupont, É., Alaoui-Jamali, M., Wang, T., Doillon, C., Ross, N., Thibodeau, A., Alpert, L., Savard, P., and Falardeau, P. Angiostatic and antitumoral activity of Neovastat (Æ-941), a molecular fraction derived from shark cartilage.Proc. Am. Assoc. Cancer Res. , 38: 227 ,1997 .
  39. ↵
    Castronovo, V., Dimitriadou, V., Savard, P., Rivière, M., and Dupont, E. Cartilage as a source of natural inhibitors of angiogenesis. In: B. Teicher (ed.),Antiangiogenic Agents in Cancer Therapy , pp. 175 –183. Totowa, NJ: Humana Press Inc,1999 .
  40. ↵
    Berger, F., Jourde, P., and Benabid, A.-L. Æ-941 (Neovastat) shows a beneficial effect in experimental glioma and is associated with high angiostatin level in treated tumors.Proc. Am. Assoc. Cancer Res. , 42: 724 ,2001 .
  41. ↵
    Gingras, D., Renaud, A., Mousseau, N., Beaulieu, É., Kachra, Z., and Béliveau, R. Matrix proteinase inhibition by Æ-941, a multifunctional antiangiogenic compound.Anticancer Res. , 21: 145 –155,2001 .
  42. ↵
    Dupont, É., Savard, P. E., Jourdain, C., Juneau, C., Thibodeau, A., Ross, N., Marenus, K., Maes, D. H., Pelletier, G., and Sauder, D. N. Antiangiogenic properties of a novel shark cartilage extract: potential role in the treatment of psoriasis.J. Cutan. Med. Surg. , 2: 146 –152,1998 .
  43. ↵
    Béliveau, R., Gingras, D., Kruger, E. A., Lamy, S., Sirois, P., Simard, B., Sirois, M. G., Tranqui, L., Baffert, F., Beaulieu, É., Dimitriadou, V., Pépin, M-C., Courjal, F., Ricard, I., Poyet, P., Falardeau, P., Figg, W. D., and Dupont, É. The antiangiogenic agent Neovastat (Æ-941) inhibits VEGF-mediated biological effects.Clin. Cancer Res. , 8: 1242 –1250,2002 .
  44. ↵
    Berger, F., Jourdes, P., and Benabid, A-L. Æ-941 (Neovastat) shows a beneficial effect in experimental glioma and is associated with high angiostatin level in treated tumors. Proc. Am. Assoc.Cancer Res. , 42: 724 ,2001 .
  45. ↵
    Dupont, É., Brazeau, P., and Juneau, C. Extracts of shark cartilage having an antiangiogenic activity and an effect on tumor progression: process of making thereof. United States Patent, 5,618,925. Washington, D.C.: Government Printing Office,1997 .
  46. ↵
    Ades, E. W., Candal, F. J., Swerlick, R. A., George, V. G., Summers, S., Bosse, D. C., and Lawley, T. J. HMEC-1: establishment of an immortalized human microvascular endothelial cell line.J. Investig. Dermatol. , 99: 683 –690,1992 .
  47. ↵
    Hirt, B. Selective extraction of polyoma DNA from infected mouse cell cultures.J. Mol. Biol. , 26: 365 –369,1967 .
  48. ↵
    Tang, D. G., Li, L., Zhu, Z., and Joshi, B. Apoptosis in the absence of cytochrome c accumulation in the cytosol.Biochem. Biophys. Res. Commun. , 242: 380 –384,1998 .
  49. ↵
    Schulze-Osthoff, K., Ferrari, D., Los, M., Wesselborg, S., and Peter, M. E. Apoptosis signaling by death receptors.Eur. J. Biochem. , 254: 439 –459,1998 .
  50. ↵
    Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. X-linked IAP is a direct inhibitor of cell-death proteases.Nature (Lond.) , 388: 300 –304,1997 .
  51. ↵
    Takahashi, R., Deveraux, Q., Tamm, I., Welsh, K., Assa-Munt, N., Salvesen, G. S., and Reed, J. C. A single BIR domain of XIAP sufficient for inhibiting caspases.J. Biol. Chem. , 273: 7787 –7790,1998 .
  52. ↵
    Luo, X., Budihardjo, I., Zou, H., Slaughter, C., and Wang, X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors.Cell , 94: 481 –490,1998 .
  53. ↵
    Engels, I. H., Stepczynska, A., Stroh, C., Lauber, K., Berg, C., Schwenzer, R., Wajant, H., Janicke, R. U., Porter, A. G., Belka, C., Gregor, M., Schulze-Osthoff, K., and Wesselborg, S. Caspase-8/FLICE functions as an executioner caspase in anticancer drug-induced apoptosis.Oncogene , 19: 4563 –4573,2000 .

Normalizing tumor blood vessels improves delivery of only the smallest nanomedicines

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Posted 09 Apr 2012 — by James Street
Category Antiagiogenesis, antiangiogenesis, NanoTechnology, Physics and Engineering
Posted: Apr 9th, 2012
(Nanowerk News) Combining two strategies designed to improve the results of cancer treatment – antiangiogenesis drugs and nanomedicines – may only be successful if the smallest nanomedicines are used. A new study from Massachusetts General Hospital (MGH) researchers, appearing in Nature Nanotechnology (“Normalization of tumour blood vessels improves the delivery of nanomedicines in a size-dependent manner”), finds that normalizing blood vessels within tumors, which improves the delivery of standard chemotherapy drugs, can block the delivery of larger nanotherapy molecules.
“We found that vascular normalization only increases the delivery of the smallest nanomedicines to cancer cells,” says Vikash P. Chauhan, of the Steele Laboratory of Tumor Biology in the MGH Radiation Oncology Department, lead author of the report. “We also showed that the smallest nanomedicines are inherently better than larger nanomedicines at penetrating tumors, suggesting that smaller nanomedicines may be ideal for cancer therapy.”
Tumors need to generate their own blood supply to continue growing, but vessels supplying tumors tend to be disorganized, oversized and leaky. Not only does this prevent the delivery of chemotherapy drugs to cells not close to tumor vessels, but the leakage of plasma out of blood vessels increases pressure within the tumor, further reducing the ability of drugs to penetrate tumors. Treatment with drugs that inhibit angiogenesis – the process by which new vessels are generated – reduces some of these abnormalities, a process called vascular normalization that has been shown to improve treatment of some cancers with standard chemotherapy drugs.
Nanomedicines are actually designed to exploit tumor vessel abnormality. While the molecules of standard chemotherapy drugs are about one nanometer – a billionth of a meter – nanomedicine molecules are from 10 to 100 times larger, too large to penetrate the pores of blood vessels in normal tissues but small enough to pass through the oversized pores of tumor vessels. Since the size of nanomedicines should keep them out of normal tissues, they are prescribed to reduce the negative side effects of chemotherapy.
The current study was designed to investigate whether the use of antiangiogenesis drugs to normalize tumor vasculature would improve or impede delivery of nanomedicines to tumor cells. In studies using a mouse model of breast cancer, the investigators first confirmed that treatment with DC101, an antibody to a molecule essential to blood vessel growth, temporarily decreased the diameter of enlarged tumor blood vessels. They then showed that this vascular normalization improved the penetration into tumors of 12-nanometer particles but not of 60- or 125-nanometer molecules.
A mathematical model prepared by the MGH team predicted that, while the abnormally large pores in the walls of tumor blood vessels lead to increased pressure within the tumor that impedes the entry of drugs, reducing pore size by antiangiogenesis treatment would relieve intratumor pressure, allowing the entry of those molecules that fit through the smaller pores. To test this prediction, they treated mice with implanted breast tumors either with DC101 and Doxil, a 100-nanometer version of the chemotherapy drug doxorubicin, or with DC101 and Abraxane, a 10-nanometer version of paclitaxel. Although treatment with both chemotherapeutics delayed tumor growth, vascular normalization with DC101 improved the effectiveness only of Abraxane and had no effect on Doxil treatment.
“A variety of anticancer nanomedicines are currently in use or in clinical trials,” says Chauhan, who is a graduate student at the Harvard School of Engineering and Applied Sciences (SEAS). “Our findings suggest that combining smaller nanomedicines with antiangiogenic therapies may have a synergistic effect and that smaller nanomedicines should inherently penetrate tumors faster than larger nanomedicines, due to the physical principles that govern drug penetration. While it looks like future development of nanomedicines should focus on making them small – around 12 nanometers in size – we also need to investigate ways to improve delivery of the larger nanomedicines that are currently in use.”
“Antiangiogenic agents are prescribed to a large number of cancer patients in combination with conventional therapeutics,” explains Rakesh K. Jain, PhD, director of the Steele Lab and senior and corresponding author of the Nature Nanotechnology report. “Our study provides guidelines on how to combine the antiangiogenic drugs with nanotherapeutics.” Jain is Cook Professor of Radiation Oncology (Tumor Biology) at Harvard Medical School.

Brain Cancer Blood Vessels Not Substantially Tumor-Derived

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Posted 11 Mar 2012 — by James Street
Category antiangiogenesis, Avastin, Brain, Stem Cell Research

ScienceDaily (Mar. 8, 2012) — Johns Hopkins scientists have published laboratory data refuting studies that suggest blood vessels that form within brain cancers are largely made up of cancer cells. The theory of cancer-based blood vessels calls into question the use and value of anticancer drugs that target these blood vessels, including bevacizumab (Avastin).

“We don’t question whether brain cancer cells have the potential to express blood vessel markers and may occasionally find their way into blood vessels, but we do question the extent to which this happens,” says Charles Eberhart, M.D., Ph.D., chief of neuropathology at the Johns Hopkins University School of Medicine. “In general, we find no evidence in our study that these vessels contain substantial amounts of cancer cells.”

Eberhart, professor of pathology, ophthalmology and oncology at Johns Hopkins, said he first encountered claims about the cancerous nature of tumor blood vessels about a year ago when he was invited to join students at a journal club meeting, a forum for discussing studies published in medical journals. “My first reaction to this research was ‘How could this be true?’” says Eberhart. “Our clinical experience examining tissue from brain cancers does not support it.”

Studies have long demonstrated that malignant brain tumors contain large numbers of blood vessels to feed their growing demand for nutrients. The blood vessels are formed when tumors pump out growth factors that increase vessel production. Such studies opened the door to treatment strategies that specifically targeted blood-vessel growth and the vessel cells themselves.

More recently, scientists in Italy and the Memorial Sloan Kettering Cancer Center in New York published results of studies suggesting that these tumor blood vessels are made by primitive types of brain cancer cells that are a form of stem cells. In their studies, they found tumor markers on blood vessel cells in 20 to 90 percent of their brain cancer samples. The U.S./Italian research teams said their findings also suggested that the cancerlike blood vessels were more prone to drug resistance, potentially explaining why drugs like bevacizumab yield tumor-shrinking responses, but only for short periods. Bevacizumab is currently approved by the U.S. Food and Drug Administration for use in patients with colorectal, lung, kidney and brain cancers.

Eberhart said pathologists, including those who work on brain tissue, use certain tissue-based techniques to distinguish cancer cells from normal ones. When evaluating specimens of brain tissue removed during surgery for suspected cancer, he said, most pathologists agree that blood vessel cells in these specimens consistently lack the molecular changes associated with cancer cells, according to Eberhart. In fact, they often use these blood vessel cells as “normal controls” to compare with potentially cancerous ones.

After the journal club experience, Eberhart teamed up with fellow neuropathologist Fausto Rodriguez, M.D., and colleagues at the Dana Farber Cancer Institute and Harvard Medical School in Boston to look more closely at the molecular features of blood vessel cells in brain cancer samples. They tested more than 100 samples from patients at Johns Hopkins and Dana Farber for EGFR and IDH1 markers, two common genes altered in brain cancer.

“We also used a marker called CD34 to differentiate vascular [blood vessel] cells from other types of cells,” says Rodriguez, assistant professor of pathology at Johns Hopkins. The research teams found no more than 10 percent of their samples contained vascular cells with EGFR or IDH1 cancer markers, and in those rare tumor samples, only a few cells exhibited those markers. The Johns Hopkins-Dana Farber-Harvard team tested all parts of the vessel walls for presence of the cancer markers.

Results of the team’s laboratory experiments were published in the online journal Oncotarget in January.

Although the two groups used different markers to identify vessel cells, Rodriguez says “there is no marker that is absolute for each cell.”

Eberhart and Rodriguez noted that the U.S./Italian research teams focused mainly on cell-by-cell research techniques in dissociated specimens to evaluate cancer markers, losing associations that can be made by looking at a cell’s shape and physical relationship within clusters of cells. The Johns Hopkins and Dana Farber researchers conducted studies examining cells in intact tissue.

“Pathologists with extensive experience in examining cells become accustomed to quickly identifying a blood vessel cell from a normal cell, and we can gain a lot of information when we look at how cells connect with other cells in real-life examples,” notes Rodriguez, who says that his team’s findings could potentially apply to any cancer thought to contain stem cells.

In addition to Eberhart and Rodriguez, the research team included Brent Orr from Johns Hopkins and Keith Ligon from the Dana Farber Cancer Institute/Harvard Medical School.

Funding for the study was provided by a National Institutes of Health postdoctoral fellowship (T32CA067751) to Orr and a grant (5R01NS055089) to Eberhart.

Dual Inhibition of MET and VEGF Signaling With Cabozantinib Blocks Tumor Invasiveness and Metastasis

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Posted 28 Feb 2012 — by James Street
Category antiangiogenesis, cabozantinib, Cabozantinib, metastases, VGEF

press release

Feb. 24, 2012, 10:00 a.m. EST

– Preclinical data published in Cancer Discovery support clinical development program evaluating cabozantinib’s potential in multiple oncology indications

 

SOUTH SAN FRANCISCO, Calif., Feb 24, 2012 (BUSINESS WIRE) — Exelixis, Inc. EXEL -.00% today announced the company’s lead compound, cabozantinib, is highlighted in a new peer-reviewed publication demonstrating that simultaneous inhibition of MET and VEGF signaling reduces tumor invasiveness and metastasis in preclinical models of pancreatic cancer. The research, led by Dr. Donald M. McDonald at the University of California, San Francisco (UCSF), showed that selective inhibition of VEGF signaling with a neutralizing antibody against VEGF or with a small molecule kinase inhibitor resulted in more invasive and metastatic tumors than from placebo-treated mice. Importantly, this effect was accompanied by increased expression of MET. The researchers went on to show that treatment with cabozantinib (which targets both MET and VEGF signaling), or with a combination of selective inhibitors targeting both pathways, reduced these malignant processes. The researchers also reported that cabozantinib prolonged survival compared with all other treatment combinations examined.

The preclinical data will be published in the March 1, 2012 issue of Cancer Discovery and are also discussed in a press release issued by the American Association for Cancer Research, the journal’s publisher. Starting today, the article will be available at http://cancerdiscovery.aacrjournals.org . Researchers at Exelixis collaborated on the studies with UCSF.

“These data provide important insights into the potential clinical benefits of simultaneously inhibiting the MET and VEGF signaling pathways with cabozantinib, and add to the scientific rationale for our ongoing clinical investigation of the compound,” said Michael M. Morrissey, Ph.D., president and chief executive officer at Exelixis. “To date, cabozantinib has shown activity in 12 of 13 tumor types studied, including particularly encouraging interim results in castration-resistant prostate, medullary thyroid, renal, liver, ovarian, non-small cell lung, and breast cancers, as well as melanoma. These results suggest that, in many types of tumors, cabozantinib may have a potentially differentiated activity profile as compared to compounds that inhibit only VEGF or MET.”

In the research described in Cancer Discovery, tumor-bearing mice were treated with an anti-VEGF antibody or with sunitinib, which inhibits multiple tyrosine kinases including VEGF receptors. These treatments were tested alone or in combination with an inhibitor of MET. Separate groups of animals were treated with cabozantinib. Key findings include:

– Cabozantinib reduced tumor invasiveness compared with VEGF inhibition alone, through a mechanism consistent with MET inhibition.

– Liver metastases were completely absent in animals treated with cabozantinib.

– Overall survival was longest in cabozantinib-treated animals. All animals treated with cabozantinib survived until the end of the study, whereas most or all animals in all other treatment groups did not survive until the end of the study.

“Inhibition of VEGF signaling has become a mainstay of cancer therapy, and its ability to delay disease progression and prolong survival in certain cancers has been extensively documented. However, there is a growing body of evidence suggesting that VEGF inhibition on its own can lead to increased tumor aggressiveness in some preclinical models and in at least one human cancer,” said Donald M. McDonald, M.D., Ph.D., a member of the Helen Diller Comprehensive Cancer Center and the Cardiovascular Research Institute and professor of anatomy at UCSF. “These new preclinical findings suggest that upregulation of MET contributes to the evasive response of tumors to anti-VEGF therapy, and that simultaneous inhibition of MET and VEGF signaling can confer the benefits associated with VEGF inhibition while significantly reducing, and in some cases reversing, invasion and metastasis. Additional preclinical and clinical evaluation of combined MET and VEGF inhibition are clearly warranted.”

About Cabozantinib

Cabozantinib is a potent, dual inhibitor of MET and VEGFR2. Cabozantinib is an investigational agent that provides coordinated inhibition of metastasis and angiogenesis to kill tumor cells while blocking their escape pathways. The therapeutic role of cabozantinib is currently being investigated across several tumor types. MET is upregulated in many tumor types, thus facilitating tumor cell escape by promoting the formation of more aggressive phenotypes, resulting in metastasis. MET-driven metastasis may be further stimulated by hypoxic conditions in the tumor environment, which are often exacerbated by selective VEGF-pathway inhibitors. In preclinical studies, cabozantinib has shown powerful tumoricidal, antimetastatic and antiangiogenic effects, including:

– Extensive apoptosis of malignant cells

– Decreased tumor invasiveness and metastasis

– Decreased tumor and endothelial cell proliferation

– Blockade of metastatic bone lesion progression

– Disruption of tumor vasculature

About Exelixis

Exelixis, Inc. is a biotechnology company committed to developing small molecule therapies for the treatment of cancer. Exelixis is focusing its proprietary resources and development efforts exclusively on cabozantinib (XL184), its most advanced product candidate, in order to maximize the therapeutic and commercial potential of this compound. Exelixis believes cabozantinib has the potential to be a high-quality, broadly-active, differentiated pharmaceutical product that can make a meaningful difference in the lives of patients. Exelixis has also established a portfolio of other novel compounds that it believes have the potential to address serious unmet medical needs, many of which are being advanced by partners as part of collaborations. For more information, please visit the company’s web site at www.exelixis.com .

Forward-Looking Statements

This press release contains forward-looking statements, including, without limitation, statements related to: the continued development and clinical, therapeutic and commercial potential of, and opportunities for, cabozantinib; the belief that the referenced research and data support the cabozantinib clinical development program; the belief that interim results in various cancers are encouraging and suggest that cabozantinib may have a potentially differentiated activity profile compared to compounds that inhibit only VEGF or MET; the potential benefits of simultaneous inhibition of MET and VEGF; and the belief that additional preclinical and clinical evaluation of combined MET and VEGF inhibition are clearly warranted. Words such as “support,” “potential,” “ongoing,” “encouraging,” “suggest,” “may,” “can,” “warranted,” “believes,” and similar expressions are intended to identify forward-looking statements. These forward-looking statements are based upon Exelixis’ current plans, assumptions, beliefs and expectations. Forward-looking statements involve risks and uncertainties. Exelixis’ actual results and the timing of events could differ materially from those anticipated in such forward-looking statements as a result of these risks and uncertainties, which include, without limitation: risks related to the potential failure of cabozantinib to demonstrate safety and efficacy in clinical testing; Exelixis’ ability to conduct clinical trials of cabozantinib sufficient to achieve a positive completion; the availability of data at the referenced times; the sufficiency of Exelixis’ capital and other resources; the uncertain timing and level of expenses associated with the development of cabozantinib; the uncertainty of the FDA approval process; market competition; and changes in economic and business conditions. These and other risk factors are discussed under “Risk Factors” and elsewhere in Exelixis’ annual report on Form 10-K for the fiscal year ended December 30, 2011 and Exelixis’ other filings with the Securities and Exchange Commission. Exelixis expressly disclaims any duty, obligation or undertaking to release publicly any updates or revisions to any forward-looking statements contained herein to reflect any change in Exelixis’ expectations with regard thereto or any change in events, conditions or circumstances on which any such statements are based.

SOURCE: Exelixis, Inc.

        
        Exelixis, Inc. 
        Charles Butler, 650-837-7277 
        Vice President 
        Investor Relations and Corporate Communications 
        cbutler@exelixis.com

Cancer Therapy More Potent When It Hits Two Targets

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Posted 28 Feb 2012 — by James Street
Category antiangiogenesis, cabozantinib, crizotinib, MET, sunitinib, VGEF

ScienceDaily (Feb. 24, 2012) — Simultaneous targeting of two different molecules in cancer is an effective way to shrink tumors, block invasion, and stop metastasis, scientists at the University of California, San Francisco (UCSF) have found — work that may improve the effectiveness of combination treatments that include drugs like Avastin.

 

The two-target approach, tested in mice with a type of cancer known as neuroendocrine pancreatic tumors, may have broad application for treating a wide variety of cancers, the UCSF team said. The drugs used in the tests belong to classes of pharmaceuticals that are either on the market or under development in clinical trials.

Clinical trials also are already underway to gauge effectiveness of the approach in humans with prostate cancer, breast cancer, and other tumor types. The UCSF study, described in the journal Cancer Discovery this week, is the first to show how the drug combination works in the laboratory.

The results are promising, said Donald McDonald, MD, PhD, a member of the UCSF Helen Diller Comprehensive Cancer Center and the Cardiovascular Research Institute and professor of anatomy, who led the research.

In the study, treating mice with the dual-target approach turned aggressive tumors with invasive fingers penetrating surrounding tissues and many metastases into tiny balls with few or no metastases.

“It’s the combination of approaches — there’s a synergy between the two,” McDonald said. “You add two and two, and you get 10.”

How each target works

The two targets are both proteins that scientists have known for years are involved in cancer. Both play important roles in malignant tumors.

The first, called c-MET, is involved in two processes associated with the most deadly cancers. A clinical marker of cancer aggressiveness, c-MET drives tumor invasion into surrounding tissues. It is also involved in metastasis — the spread of cancer cells to other parts of the body where they can establish new tumors.

The second target is a protein known as vascular endothelial cell growth factor (VEGF). VEGF is a protein that promotes the growth of new blood vessels. Growing tumors hijack this process to expand their network of blood vessels to provide nutrients. Drugs blocking VEGF have been developed based on the simple assumption that tumors cannot grow if you choke off their blood supply.

Drugs that target these molecules are in development, and a few are already on the market. The U.S. Food and Drug Administration (FDA) approved the first of these in 2004 to treat metastatic colon cancer. That drug, called Avastin, is manufactured by the South San Francisco-based company Genentech. Avastin was approved for metastatic breast cancer in 2008 under the FDA’s accelerated approval program.

The FDA revoked approval of Avastin for breast cancer last year after further assessing the relative risks and benefits to women taking it. Blocking VEGF seemed to slow tumor growth for awhile, but the FDA determined that it did not significantly improve or extend the lives of most women taking it.

“It was not clear why some tumors responded and others did not. It was also unclear why some tumors would respond initially and then would stop responding,” said McDonald, who has studied blood vessels in tumors and the effect of cancer drugs for years in his UCSF laboratory.

Two years ago former UCSF professor Douglas Hanahan and colleagues found in laboratory experiments that Avastin-like drugs would shrink tumors but unexpectedly did something else as well. The drugs also morphed tumors from roundish blobs into highly irregular growths with tendrils that penetrated surrounding tissues and even spread to other organs — suggesting that the VEGF blockade could also make tumors more aggressive, invasive and metastatic.

McDonald’s group confirmed Hanahan’s findings and discovered that c-MET was involved. In their latest research, Barbara Sennino, PhD, with other investigators in his group set out to determine whether c-MET drove tumor aggressiveness during anti-VEGF therapy. What their paper shows is that blocking c-MET and VEGF together in mice is more powerful than blocking either alone because it not only slows tumor growth but also reduces invasion and metastasis.

They tested two inhibitors of VEGF — a neutralizing antibody and sunitinib — and three inhibitors of c-MET — crizotinib, PF-04217903, and cabozantinib (XL184). Unlike the other agents, cabozantinib simultaneously inhibits both c-MET and VEGF. Inhibition of c-MET and VEGF together with a drug combination or with cabozantinib had more profound effects on tumors than any of the agents that blocked only one of the targets.

These promising laboratory results still need more tests of safety and effectiveness in the clinic, McDonald said, and it may be a year or more before the drugs are routinely available to patients.

Russians find new way of curing cancer

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Posted 03 Jan 2012 — by James Street
Category antiangiogenesis, Osteosarcoma
Jan 2, 2012 21:57 Moscow Time

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Scientists from the Russian Central Institute of Orthopedics and Traumatic Surgery have created a new method of treating cancer.

The method still has been used only as an experiment, but has already won the approval of foreign experts.

Every tumor has its own “network” of blood vessels, and to stop its growth, these vessels must be blocked so that the tumor would not be feed with blood anymore. Thus, the inventors of the new method have suggested to block them with special granules. For vessels of a larger diameter, instead of granules, metal spirals are used. When introduced into a patient’s body, these spirals go through his or her arteries and unfold themselves when they reach the place of their “destiny”.

To introduce these granules and spirals into a patient’s body, only an injection is needed.

“In fact, the procedure is no less traumatic than a prick of a syringe,” one of the creators of the new method, Professor Alexander Balberkin, says. “But it is very effective – at least, according to preliminary data. However, it still cannot be recommended as a universal method – it is effective only if combined with other methods.”

Unfortunately, the new method is ineffective against some kinds of tumors. For example, it is hard to stop the growth of a gristle tumor by it. Still, the method has proven to be effective against bone or bone marrow tumors and can be recommended to patients for whom an operation is contraindicated.

“However, the new method still needs a more thorough approbation,” Professor Balberkin says.

“We have conducted about 600 operations,” he says, “but we still don’t have reliable statistic data. It is too early to sum up the results – some time must pass so that we are able to watch the condition of our patients. At the same time, we can already say that, at least in some cases, the results may be positive. For example, a new method may be useful while preparing a patient for an operation – it enables to lower the blood losses several times.”

Professor Balberkin says that doctors from Kazakhstan and other former Soviet republics already show great interest in the new method. Moreover, German oncologists are already using it quite successfully for curing uterine cancer, kidney cancer and spine tumors – although, at first, German doctors were very skeptical about the method suggested by their Russian colleagues.

Lactoferrin–is immunoregulatory, inhibits angiogenesis, and binds iron

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Posted 21 Nov 2011 — by James Street
Category Antiagiogenesis, antiangiogenesis, lactoferrin

Lactoferrin–is immunoregulatory, inhibits angiogenesis, and binds iron
Perhaps one of the most promising therapeutic uses of lactoferrin, a milk protein with bacteriostatic properties, may be as a nontoxic, anticancer agent. Lactoferrin, a minor fraction of whey, results in a significant reduction in the incidence of esophageal, lung, bladder, and colon cancer in laboratory rats (Ushida et al. 1999; Masuda et al. 2000; Tsuda et al. 2002).

Since evidence indicates milk products protect against colon cancer, researchers speculate that bovine lactoferrin, a natural ingredient in milk, may be the chemoprotective agent (Tsuda et al. 2000b). Rats treated with a carcinogen and supplemented with 2% bovine lactoferrin for 36 weeks had a reduced incidence of colon cancer (27% of that observed in a control group; rats receiving 0.2% bovine lactoferrin reduced incidence to 46%). A remarkable 43% reduction in spontaneous lung metastasis (compared to controls) occurred after implanting colon carcinoma 26 (Co 26 Lu) in lactoferrin-treated laboratory animals (Tsuda et al. 2000a).

In addition to inhibiting angiogenesis (the vascular network that sustains the tumor), lactoferrin maintains the integrity of the immune system (Yoo et al. 1997; Tsuda et al. 2002). Typically, bovine lactoferrin prompts an increase in the number of natural killer cells, as well as the cytotoxicity of white blood cells (Tsuda et al. 2000a). The antibiotic, anti-inflammatory, and immune-modulating properties of lactoferrin appear active against the gastritis-, ulcer-, and cancer-inducing bacterium Helicobacter pylori (Dial et al. 2002).

Lactoferrin, a natural iron-binding protein, scavenges free radicals in fluids and inflamed areas, suppressing free radical mediated damage. It decreases the availability of iron in neoplastic cells, depriving them of an iron supply (Khan et al. 2001; Weinberg 2001).

The suggested dosage is 300-900 mg a day of the superior apolactoferrin (iron-depleted) form of lactoferrin. Lactoferrin is a natural component of cows’ and human mothers’ milk, but is also found in the milk of sheep, goats, and pigs.

Less Is More for Common Cancer Drug, Study Suggests

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Posted 01 Oct 2011 — by James Street
Category Alternative Therapies, Prostate Cancer, topotecan, topotecan

ScienceDaily (Sep. 30, 2011) — University of Georgia scientists have found that smaller, less toxic amounts of chemotherapy medicine given frequently to mice with human prostate cancer noticeably slowed tumor growth. The mice suffered fewer side effects compared with traditional cancer treatment relying on heavy doses that can cause hair and bone loss.

While chemotherapy given repeatedly in small portions, called metronomic dosing, is not new, the study’s authors say that the dosing appears to alter the cellular activity of the drug topotecan. This finding offers promising new ways to use topotecan-which is widely used and approved by the Food and Drug Administration for cervical and other cancers-to combat slowly growing prostate tumors. The findings appear this month in the journal Cancer Biology and Therapy.

“At these lower doses, there isn’t enough topotecan to follow a classic cell death pathway,” said study co-author Robert D. Arnold, a Georgia Cancer Coalition Distinguished Scholar and assistant professor in the UGA College of Pharmacy. “Our research suggests that metronomic dosing altered topotecan’s behavior.”

Scientists have known that topotecan given to patients in large, traditional doses kills cancer cells by deactivating proteins known as enzymes that are necessary for cell growth, Arnold explained. By contrast, metronomic dosing of topotecan prevents new blood vessels-which are necessary for growth-from forming in the tumor. Arnold and his colleagues discovered that topotecan did not change the amount of blood vessels formed, but significantly decreased tumor size and altered genes critical for controlling cell growth.

Brian S. Cummings, a co-author and associate professor at the pharmacy college, compared topotecan’s process of killing tumor cells to the everyday task of running an errand.

“Let’s assume you’re going to go to the grocery store and you could walk, ride your bike or take the car,” he said. “Those are different mechanisms of action. You will still get to the same place.”

He added that researchers try to determine which pathway, or transportation choice, cells take after different amounts of exposure to topotecan. Their results suggest that when topotecan is given frequently in low doses, the drug could be changing the type of genes turned on in the tumor. These changes may be related to the structure or architecture of a gene-not a change in gene sequence. Such changes could be considered epigenetic, but more research is needed, Arnold said.

The study suggests that metronomic dosing of topotecan can reduce prostate cancer growth at drug concentrations far below those that can be toxic to healthy cells in the body.

Given the limited treatment options for late-stage prostate cancer and clinical use of topotecan, new clinical trials could occur in the near future, Arnold said. The same research team is now studying the dosing effects of topotecan in breast cancer models.

Co-authors of the study are Ibrahim Aljuffali, currently at King Saud University, Saudi Arabia, and Jason Mock, Leah Costyn, Ha Nguyen and Dr. Tamas Nagy of UGA.

The research was funded in part by an UGA Faculty Research Grant, an Interdisciplinary Toxicology Program equipment grant, a Georgia Cancer Coalition Distinguished Scholar grant, as well as a King Saud University fellowship and graduate stipend support from the National Institutes of Health

Model for in vivo progression of tumors based on co-evolving cell population and vasculature

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Posted 08 Aug 2011 — by James Street
Category antiangiogenesis, Biomedical Engineering, Local Recurrence, Lung Metastases, Mathematical and Physical Modelling, Metastases, metastases

Scientific Reports
1,
Article number:
31
doi:10.1038/srep00031
Received
Accepted
Published

With countless biological details emerging from cancer experiments, there is a growing need for minimal mathematical models which simultaneously advance our understanding of single tumors and metastasis, provide patient-personalized predictions, whilst avoiding excessive hard-to-measure input parameters which complicate simulation, analysis and interpretation. Here we present a model built around a co-evolving resource network and cell population, yielding good agreement with primary tumors in a murine mammary cell line EMT6-HER2 model in BALB/c mice and with clinical metastasis data. Seeding data about the tumor and its vasculature from in vivo images, our model predicts corridors of future tumor growth behavior and intervention response. A scaling relation enables the estimation of a tumor’s most likely evolution and pinpoints specific target sites to control growth. Our findings suggest that the clinically separate phenomena of individual tumor growth and metastasis can be viewed as mathematical copies of each other differentiated only by network structure.

Figures at a glance

Introduction

A multitude of biological processes ranging from genetic and epigenetic mutations, DNA damage, to complex intra- and intercellular signaling dynamics undoubtedly play key roles in triggering cancer in a given patient1, 2, 3, 4, 5, 6. However, for many of these biological processes the various detailed biochemical reactions that take place are unknown. Similarly, the exact interplay between processes can also be ambiguous. Rather, it is the qualitative effect of varying a particular reactant or altering the environmental conditions in a systematic fashion that we observe, without necessarily understanding all of the underlying processes involved. For example, once formed, tumors seem to evolve in a fairly generic way: They either lie dormant, or grow, fed by the underlying network vasculature, capable of generating new vessels via angiogenesis when needed7. Generally, an absence of nutrients will tend to reduce growth, while sufficient supply leads to a progression in tumor cell behavior from differentiation and proliferation to migration7. Metastasis of cancer to lymph nodes and other organs, thought to be the most lethal aspect of the disease, likewise may depend on myriad patient-specific factors concerning the lymphatic system, immune response, micro-environmental factors and general patient health8. However, once again, the actual process is fairly generic – involving the spread of cancer cells from the primary tumor through the lymphatic and circulatory systems. Most fundamentally, at the heart of all these processes is the essential interplay between an evolving population of cancer cells which is fed by – and feeds back on – an underlying blood vessel network structure which supplies nutrients to the tumor and tissue, but simultaneously provides a transport network through which cancer cells can metastasize to other parts of the body and drugs are delivered to the tumor. Yet, the blood vessel structure is typically highly irregular in tumors, and further complicated by the highly dynamic structural growth and degradation interplay with the evolving tumor mass, making an averaged description for modeling purposes insufficient. These factors clearly emphasize the importance of incorporating relevant network structures not only for tumor progression prognosis, but also for the analysis of effective treatments. For these reasons, to model the progression of tumor growth behavior it may be more productive and informative to implement universally observed, and biologically derived, qualitative behavior in the model dynamics. Such qualitative mechanisms have proved to be useful in building models which correlate well with experimental findings, deepening our understanding of the basic underlying processes and making practical predictions possible9.

Early tumor models often resembled a theoretical exercise, looking at averaged behavior whilst neglecting the importance of environmental heterogeneities at various length-scales – or were computationally too expensive due to the ambitiously detailed nature of the model setup and sheer number of cells necessary to investigate long term behavior9, 10, 11. All models by design are simplifications and approximations based on assumptions of the true biological system. Cancer models, regardless of mathematical rigor and modeling complexity, are typically criticized as too simplistic for complex tumor-related phenomena9. However, promisingly, a rapidly growing number of models have seen a close symbiotic collaboration between theoreticians, biologists, oncologists and clinicians, which has lead to novel predictions emerging from the model results, which were subsequently experimentally verified9, 10, 11, 12, 13, 14, 15, 16.

We believe that the greatest shortcoming is the current lack of implementation of clinical images into models as initial conditions for patient specific prognosis9, 10, 11, 12, 13, 14, 15, 16. Most are seeded with artificial initial conditions of cancer size, shape and density, as well as environmental parameters, struggling to combine the model with data gathered from clinical images17, 18, 19. Great advances in imaging techniques have enabled more and more accurate visualization of the problem zone, allowing for a wide range in length scales, and time resolution, particularly at the molecular level. However, these have not been successfully implemented into tissue level cancer modeling mainly due to multi-scale compatibility issues. Indeed, the majority of models are inflexible to even the simplest extensions, modifications or re-scaling. A direct one-to-one mapping of all cells is unfeasible20, 21, 22, 23, 24, 25, 26, 27, 28, 29, whilst modeling the global spatially-averaged behavior fails to describe important cellular and environmental heterogeneities in the system which may be particularly important during early tumor growth12, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39.

Here we present a simple multi-scale model which addresses two fundamental issues. First, the uncertainties in the details of the biological processes are accounted for by describing behavior in local regions by a well established averaged behavior growth equation, whilst preserving the heterogeneities in each region; second, the ability to take advantage of invaluable data gathered from patient images, to be used as seed for the model for comparison and further development, taking an appropriately coarse-grained length scale for the model to adapt to the image resolution. We realize that many more details could be included in a model of primary cancer growth or metastasis. Many models and methodologies exist, ranging in length and time scale, capturing the biology of intra and intercellular signaling up to tissue level dynamics, each successfully mimicking some part of the complex emerging phenomena such as tumor growth, angiogenesis and metastasis. Yet each also comes with limitations. The primary purpose of the presented model is a complementary one to existing models, seeing how far one can go in explaining a wide range of clinical data using a simplified and minimal, adaptive and multi-scalable, yet most crucially, data driven approach to understand and predict a patient’s highly personalized tumor progression from early growth through to metastasis and even treatment strategy analysis using a single model.

Our model seeded with in vivo data predicts robust growth corridors of future tumor growth behavior in good agreement with a murine mammary cell line EMT6-HER2 model in BALB/c mice, as well as reproducing clinical human patient data of metastasis. Moreover, the model predicts a hidden scaling relation between the underlying nutrient supplying vessel structure and cancer co-evolution, a finding which estimates a tumor’s most likely evolution, and more importantly, pinpoints specific vessel target sites to optimally control tumor growth.

Results

In vivo data implementation in mathematical model

A mathematical model was developed to purposefully seed up-to-date patient information for a personalized prognosis, and to bridge the gap between the length scale extremes of current mathematical modeling efforts (see Methods section for model description). A minimal-mechanism of a co-evolving nutrient network and cancer population is applied to the growth of a single tumor. Key to the individualized prognosis is the implementation of in vivo images as initial conditions. Later in the paper, we show how exactly the same multi-scale mathematical model can be applied at the level of systemic metastasis simply by making a change in the biological interpretation of its network features.

Figure 1 illustrates the methodology of extracting and coarse-graining information from immunofluorescent stained in vivo images whilst preserving the heterogeneity of initial vessel density, and hence nutrient supply, of a tumor. The cellular activity inside local regions, created by the boxes of the imposed grid, is described by the biologically ubiquitous discrete logistic map, which is a good approximation of the universally observed Gompertzian growth behavior of cancer40, which preserves the biological importance of the unit cell, and accounts for the local cell-cell and cell-microenvironment interactions41, 42, 43. From a mathematical modeling point of view, previous applications of the logistic equation to cancer either apply the logistic equation to the entire tumor44 or form a continuous time spatial diffusion equation which allows the unrealistic transfer of arbitrarily small amounts of cancer across space. The presented application is novel since the discretization into smaller regions, forming a grid of coupled autonomous logistic equations, allows universal growth behavior in each region to be applied using appropriate growth rates extracted from an image (regional vessel density), whilst coupling allows inter-regional diffusion, migration and communication. Our model specifically accounts for the fact that cancer consists physically of discrete units (cells) and hence there is a lower bound below which a continuous formulation of cell density becomes incorrect, yet above which the changing size and mass of cells deems a continuous description valid.

Figure 1: Model setup.
Model setup.

Schematic of implementing in vivo immunofluorescent image data into the mathematical model as initial condition. The right half illustrates the step-by-step procedure of extracting and coarse graining information from the in vivo images. The tumor growth behavior in each box is modeled as Logistic growth, and the model equations capture the fundamental interplay between an evolving population of cancer cells which is fed by – and feeds back on – an underlying nutrient network, and its spreading through transport processes. The blue inset shows the time ordering of events at each time step of the mathematical model.

Experimental in vivo growth data fitting to model

To test the agreement of the model results to in vivo growth data, the model was seeded with in vivo images of muscle vasculature from the flanks of untreated BALB/c mice as initial condition, representing potential regions of primary tumor growth (implantation zone in mouse model). Figure 2 shows a growth corridor (blue shaded area) predicted from our model. The corridor is formed by the blue dashed line, which is the average growth curve of 2000 model simulations, where the tumor seed was virtually implanted at different locations on the image for each run, and the solid blue lines are the standard deviation. Hence, Fig. 2 predicts the most likely growth behavior if a cancer were to originate somewhere in the environment depicted by the image. Assuming BALB/c mice generally have similar initial conditions (image in Fig. 2), the flanks of 5 mice were implanted with murine mammary cell line EMT6-HER2, whose growth data (dark blue points) fall inside the growth corridor with good agreement. Similar analysis was repeated with many more images of various other regions of the flanks in BALB/c mice confirming robust behavior in the predicted growth corridors.

Figure 2: Model substantiation.
Model substantiation.

Fit of in vivo experimental growth data to a growth corridor determined by the model seeded with an image of initial muscle vascular structure in BALB/c mice. The growth corridor (shaded region) is formed by the average growth curve of virtual tumor implantations (blue dashed line), and standard deviation (solid blue lines), showing good agreement with growth data. The yellow circle and box are representative regions with fast and slow growth curves respectively. The three insets show sample growth patterns of the virtual tumor with (a) necrotic core and proliferating ring46 (b) diffusive growth in nutrient rich environments47 (c) multiple source growth. The purple data points show growth data of EMT6-HER2 tumors treated with an endostatin-antibody fusion protein, and the dashed purple line model results, where we mimicked the decreasing effect of the protein on the vasculature. The model used the same number of injections and time interval as in the in vivo experiments.

The yellow circle and box in Fig. 2 are representative regions with fast and slow growth curves respectively. The three insets Fig. 2a–2c show sample growth patterns of the virtual tumor. Interestingly, the distance between high density vessel sources is of vital importance (as analyzed Fig. 3). In the absence of angiogenesis, should the maximum radius, l, to which a tumor can grow from a single source be smaller than the distance to the next vessel, d, then the tumor will remain a finite size, and eventually starve and die, as shown in Fig. 2b. However, if l > d, Fig. 2c depicts how a neighboring source can facilitate continued growth.

Figure 3: Growth behavior in finite source environments.
Growth behavior in finite source environments.

Finite number of sources may be due to anti-angiogenic treatment. For example, (a) is an immunofluorescent image of short, scattered vessels inside an EMT6-HER2 tumor treated with an endostatin-antibody fusion protein, (b) is the coarse-grained result for model implementation, and (c) shows the remaining sources if a threshold is applied. Model results suggest that small clusters of cancer cells remaining around vessels can lead to more aggressive re-growth (inset of (c)). (d) – (g) illustrate the collapse of data points onto a linear relationship by accounting for appropriate average distance between sources and final radius of tumor (see inset illustrations). The distance between sources is calculated of (d) all sources when maximally distributed, (e) all sources in the system at actual position, (f) sources inside the tumor (g) sources inside the tumor yet neglecting the sources on the perimeter of the tumor where the tumor cell density is too small to result in growth.

Systematic treatment strategy analysis

Also, we briefly illustrate the efficacy of the model to systematically analyze all possible treatment strategies (dosage, interval, frequency of which drug/treatment combination), to predict personalized treatment effectiveness. The dashed purple line in Fig. 2 shows that our model’s predictions of treatment are also consistent with in vivo experiments. Results are shown for BALB/c mice implanted with cell line EMT6-HER2, and subsequently injected with αHER2-huEndo fusion proteins45 which is an endostatin-antibody fusion protein specifically engineered to target the HER2 receptor and limit the growth of adjacent blood vessels through the action of a fused anti-angiogenic endostatin domain. By measuring the biological effect of a single injection of the endostatin-antibody fusion protein on the tumor, the model subsequently simulated the same number of injections and same time interval as in the in vivo experiments with good agreement. A systematic analysis of all possible treatment strategies of varying dosage, frequency, and schedule will be presented elsewhere.

Universal growth behavior scaling from vessel location

Given the dynamic interplay of the growing tumor with the underlying vessel structure, Fig. 3 analyses the growth behavior in finite source environments (Fig. 3a–3c), where the distance between vessels, alluded to in Fig. 2a–2c, becomes important to the tumor’s progression. For example, Fig. 3a shows an immunofluorescent image of vessels inside an EMT6-HER2 tumor that has been treated with an endostatin-antibody fusion protein45 resulting in a finite number of short, small-clustered and scattered vessels. Model results suggest that in cases where small clusters of cancer cells survive around remaining vessels (even after anti-angiogenic treatment) islands of re-growth can occur, as shown in the inset of Fig. 3c, leading to a more aggressive re-growth rate than before treatment. The heterogeneous nature of remaining vessel locations not only presents the problem of indefinite re-growth of cancer by movement beyond the finite radius each vessel can sustain individually, but also the additional challenge of optimizing and analyzing drug delivery strategies for efficacy and efficiency. Specifically, the vasculature in a tumor is highly irregular in structure creating regions completely void of vessels and regions densely packed with vessels. This implies that drug delivery will be highly disproportional not reaching all areas of the tumor48. Even with the advent of a genetically targeted approach where a drug is specifically designed for a patient, there still exists a need for delivery analysis locally in primary tumors as well as globally via metastatic spread. Our model is ideally suited to systematically analyze the effect of vascular structure on delivery, in addition to the countless possible multiple drug therapies48, to help optimize experimental design by taking into account the heterogeneities of the system which usually cause variation and hence unpredictability.

Much like forest fires49 or nutrient source manipulation in conservation corridor analysis50, the distance between vessel sources is key in determining the most likely progression of a tumor. Hence, in Fig. 3, we identify a measure based on the distance between sources to predict its evolution, and hence identify the key targets which allow control and limitation of the final tumor growth size. The results of Fig. 3g show that as long as the initial vasculature heterogeneity can be quantified, the diversity in final tumor size disappears under an universal scaling. The initial vasculature structure can be used to assess where a particular patient’s tumor sits on this scaled curve thereby providing a prediction of its final size.

Figures 3d–3g show the same data using different measures of average distance between sources and each dot is one realization of the model simulations. Central to the universal scaling of Fig. 3g is identifying which sources to include, as illustrated in the insets of Figs. 3d–3g. In Fig. 3d, we calculated the average distance between all sources, where the sources were assumed to be maximally separated, and plotted against the final radius of the tumor, rmax. Figure 3e calculates the average distance between all sources using the actual position of the sources within the system. Yet, as argued in Fig. 2a–2c sources only become significant if their distance is smaller than the potential radius of the growing tumor. Hence, in Fig. 3f, only the distances between sources on or inside the final tumor boundary were included. This resulted in the clusters of points below the red line of Fig. 3e to be pushed closer to the red line, as indicated by the blue arrow. Finally, the scatter below the red line of Fig. 3f can be explained by circumstances where the growing tumor does reach another source, yet the cancer cell density pushed into them is below a critical threshold, too little to result in cell proliferation. Hence, eliminating such cases resulted in the final plot Fig. 3g.

The results of Fig. 3 illustrate the important possibility of systematically targeting specific vessels. For example, in Fig 3c, say a cancer seed originating from the three sources in the centre is predicted to result in a final tumor radius depicted by the red circle determined from Fig. 3f. Inside the radius is a fourth source, highlighted by the green arrow in Fig. 3c, which would facilitate further growth to a new radius. Hence, one could minimally target the single source (green arrow) to prevent further growth, rather than taking more invasive measure, and thus, perhaps preserve functionality of the affected system. This analysis has a powerful consequence, in that, it gives the surgeon an exact size of tumor to remove, or which vessel sources to block in order to control the final size of the tumor.

Multi-scalability of local model to predict global metastasis data

Finally, we explore the extendibility and multi-scalability of the model to the global phenomenon of metastasis. Metastasis is usually treated as an entirely separate topic in modeling since the underlying biology is different. However, as illustrated in Fig. 4, we successfully apply the same model equations to both single tumors and metastasis, simply by changing the interpretation of the terms: Instead of the cancer cell diffusion to neighboring boxes on a regular lattice representing free space for growth, the boxes represent lymph nodes and the underlying inter-box connections the lymphatic system. As shown in the lower panel of Fig. 4, the growth within each box is now a macro-level version of the single tumor model in which we use the logistic growth map to apply to the entire space in which a tumor may grow. In other words, we simply apply our exact same mathematical equations (Eqns. (1)–(3) in Methods) on a different scale, and with a different network for diffusion (Fig. 4). As discussed in Ref. (8), cancer cells can spread to other organs at every time step from the beginning of the primary tumor’s growth.

Figure 4: Model implementation and results of metastasis.
Model implementation and results of metastasis.

Metastasis uses the same model as for single tumor growth. The upper panel shows average cumulative distribution plotted for different underlying networks: random (blue solid) and scale-free (orange solid). The clinical data (red circles) lies somewhere between the two types of networks suggesting that the precise network structure does not matter to make a first-order prediction. The red dashed line is a fit to the clinical data by varying the r distribution51. Finally, the green dashed line shows the Poisson complementary cumulative distribution function with mean equal to the mean number of affected sites from the clinical data. It is the expected curve based on the assumption that nodes get infected independently (i.e. random), and illustrates that the empirical and theory are fat-tailed compared to purely random. The lower panel shows a schematic of the similarities of single tumor growth and metastasis using the same model.

Interestingly, as shown in Fig. 4, the results do not depend sensitively on the choice of network – as long as it is irregular (e.g. random or scale-free). The upper panel shows metastasis on different underlying networks: random (blue solid) and scale-free (orange solid). Clearly, the clinical data (red circles) lies somewhere between the two types of networks. Generally, diffusion on networks is reasonably insensitive to the network structure as long as the distribution of links is fairly broad, and the distance over which the diffusion takes place is short. In other words, the cancer does not spread far enough into the network to feel the difference between a random network and scale-free network – at least, to first order. This implies that knowledge of people’s precise lymphatic network details are not required in order to make a first-order prediction of the probability that n nodes will be positive. The red dashed line in Fig. 4 is a fit to the clinical data53. The green dashed line shows the Poisson complementary cumulative distribution function with mean equal to the mean number of affected sites from the clinical data, which demonstrates that the empirical data and theory are fat-tailed compared to purely random.

Discussion

The ever increasing number of discoveries about the biological processes underlying tumor progression, set against the many aspects which still remain unknown or ambiguous, has led to the creation of many extremely complex mathematical descriptions (perhaps motivated by the desire to include as many biological details as possible) which are computationally intensive and include many unknown parameters. These models can be generally categorized into two extremes: The molecular level, trying to understand the intra and intercellular signaling dynamics of individual or small clusters of cells, and the tissue level, modeling the emergence of phenomena such as angiogenesis and metastasis. Yet, the molecular models are difficult to scale up to enough cells to comprise a full organ, whilst the tissue level models often lack the heterogeneities vital to an accurate, and personalized, prediction.

In this paper, we presented a model which aims to bridge this gap, and provide a practical, multi-scale model capable to be seeded with in vivo images to predict the most likely tumor growth behavior through prediction corridors, as well as subsequent spreading behavior of metastasis. For both length scales, the model results show good agreement to in vivo growth data of a cell line EMT6-HER2 model in BALB/c mice, as well as clinical human patient data of metastasis. Furthermore, we outline the use of the model for systematic treatment analysis, focusing on the effect of vascular structure on drug delivery. A novel scaling relationship between the tumor and the underlying nutrient sources not only predicts the most likely progression of the tumor, but also identifies key vessel target sites to optimally control tumor growth.

Despite its quantitative accuracy and simplicity, our model’s neglect of the wealth of known biological details associated with cell biology and physiology, may attract criticism of our minimal-model approach as resembling the ‘Consider a spherical cow…’ cliché typically levied at physicists. However, the existing gap between model sophistication and clinical need demands the exploration of such an approach in our opinion. The unique coupling of image data with the mathematical model allows information about the heterogeneity of the system to be preserved, and more importantly, be utilized for individualized prognosis. Hence, the model cancer growth is directly driven by in vivo information and demonstrates a new approach to modeling cancer growth using patient specific data, showing good agreement at multiple length scales for a variety of phenomena. As such it complements existing theoretical approaches rather than replacing them, and can be integrated with them in the future.

Methods

Mathematical model

The blue inset of Fig.1 shows the time ordering of events at each time step of the mathematical model, and corresponds to two coupled, discrete equations applied within each box of the grid. The first equation is:

where and are the cancer concentrations at the beginning and end of time interval Δt. The tumor growth rate, ri,n, at time step n in box i is assumed to be directly proportional to the vessel density in box i extracted from the image. As described below (see Image information extraction), the initial cancer, , and endothelial cell densities, ri,n = 0, are extracted from in vivo images stained for both types of cells at time t = 0.

At this stage only vessel density is considered as the primary driving force of growth rate. Nutrients determine individual cell behavior and thus population response. Yet, rather than applying a single r as was done in previous models44, we split the system for maximal heterogeneity, making the model highly non-deterministic.

Furthermore, the model equations capture the tendency of any overcrowding of cancer cells to crush the vasculature or cause it to regress, leading to lower nutrient supply52 and thus slower growth. Hence, the equation for vessel density (i.e. cancer growth rate) is given by:

Following our methodology of implementing a coarse grained view of an universally observed growth behavior, the single parameter α incorporates all details which may contribute to the vessel density such as vessel stabilizing and/or destabilizing factors, (anti) angiogenic growth factors, as well as any therapeutic agents. This may be crude and biologically unsatisfying, yet due to its observation driven nature, in short, this setup captures the co-evolving, dynamic, feedback-driven interplay between cancer and the underlying nutrient network52.

Finally, cancer cell mobility to neighboring boxes is modeled via simple diffusion:

where again, similar to α, β represents all properties of the environment, which could influence the ease of cancer cell diffusion53, as well as other local gradients such as chemotaxis and haptotaxis. More specifically, α is some function of growth promotion (negative α) and inhibition (positive α) factors which influence angiogenesis and nutrient deprivation conditions via the adaptive and feedback-driven value of ri,n at all time steps. Despite a long list of possible influences, we expect as a first approximation that the values of α and β will take on similar values for patients from similar risk groups. In the future, we will make α and β functions of specific factors, making the model more biologically accurate and hence more patient specific. For example as a first proof-of-principle, we show in Fig. 2 that the effect of an anti-angiogenic endostatin-antibody fusion protein which breaks down the vessel structure and halts angiogenesis (as verified by in vivo images), can be successfully mimicked by reflecting the fusion proteins destructive effect on the vessels by means of a positive value of α in the model.

Image information extraction

Without loss of information the image colors are converted to grayscale for easier manipulation. A grid is imposed, where each box size of the grid is chosen to correspond to approximately 100 cells. The box size can be adapted according to the system and type of image. Finally, the individual pixel values contained in each grid are added and averaged, to represent the average vessel density in each box. These values then provide the initial condition for the tumor’s evolution, making the model as patient-specific as desired. This procedure can be repeated for any property of interest.

In vivo imaging procedure

In vivo immunofluorescent images of the muscle vascular structure in the flanks of BALB/c mice were taken prior to implantation s.c. contra-laterally of murine mammary tumor cell line EMT6-HER2 (1×106 cells per mouse). Two mice were sacrificed for blood vessel analysis. Histologic sections of muscle from the sacrificed mice were analyzed using immunofluorescent staining for DAPI (red color; example image has 10× magnification).

Growth corridor analysis

We only seeded blood vessel structure for Fig. 2 since an analysis was done prior to implantation of the tumor seed. Hence, we virtually implanted a tumor in the mathematical model, recorded the resulting growth curve, and repeated this procedure 2000 times (corresponding to approximately a 10% sample size), each time using a different location. Furthermore, this procedure was repeated with images from various locations in the flanks of the BALB/c mice. Similar initial conditions can be seeded into the model concerning the size and location of an already growing tumor. Immunofluorescent images can be taken of the growing tumor, and hence, a similar procedure can be performed. The chosen parameter values for the presented results are at this stage arbitrary, yet our general findings are robust to variations in α and β. A table of parameter values for various cell line types will be presented elsewhere.

Endostatin-antibody fusion protein treatment

BALB/c mice (n = 4 per group) were implanted s.c. contralaterally with EMT6 and EMT6-HER2 (1×106 cells per mouse), followed on day 4 by equimolar injections every other day (7 time treatments) of αHER2-huEndo-P125A (42 μg), or PBS. On day 12, two mice were sacrificed for the blood vessel analysis after four treatments. We analyzed histologic sections of tumors from the sacrificed mice using immunofluorescent staining for PECAM (vessels) and DAPI for counter-staining of the nucleus. Although still a preliminary result, the dashed purple line in Fig. 2 is an average of 1000 model results where we mimicked the inhibitory effect of the protein on the vasculature formation.

Metastasis network analysis

For each of the 100 sites (or nodes), we drew ri from a normal distribution N(µ = 1, σ = 0.2) and took α, β for non-primary tumor sites to be β = 0.8, α = 0.2. Furthermore, for each trial we seeded the tumor at a randomly picked primary site with C0 = 0.5, and β‘ = 0.6, α‘ = 0.4. The average cumulative distribution of 3000 trials is plotted for both types of networks, where a new network was generated for each trial. The clinical data was fitted by varying the r distribution; in this case a skewed distribution with peak close to r = 0.01. However, the same fit can be achieved by starting from a random network and simply adding more and more links, slowly tending towards a scale-free network.

Author information

Affiliations

  1. Division of Theoretical Bioinformatics (B080), German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany

    • Sehyo C. Choe

  2. Department of Bioinformatics and Functional Genomics, Institute of Pharmacy and Molecular Biotechnology (IPMB) and Bioquant, Heidelberg University, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany

    • Sehyo C. Choe

  3. Department of Physics, University of Miami, 1320 Campo Sano Ave., Coral Gables, Florida 33146, USA

    • Guannan Zhao,
    • Zhenyuan Zhao &
    • Neil F. Johnson

  4. Sylvester Comprehensive Cancer Center, University of Miami, 1475 NW 12th Ave., Florida 33136, USA

    • Joseph D. Rosenblatt,
    • Hyun-Mi Cho &
    • Seung-Uon Shin

  5. Division of Hematology/Oncology, University of Miami Miller School of Medicine, NW 12th Ave., Florida 33136, USA

    • Joseph D. Rosenblatt,
    • Hyun-Mi Cho &
    • Seung-Uon Shin

Contributions

S.C.C., N.F.J. and G.Z. worked on the data and data analysis. S.C.C. and N.F.J. worked on the model development. All authors participated in the write up and associated discussions, giving detailed feedback in all areas of the project.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

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