Defeat Osteosarcoma » Molecular Osteosarcoma Studies

High serum alkaline phosphatase cooperating with MMP-9 predicts metastasis and poor prognosis in patients with primary osteosarcoma in Southern China

James Street — Fri, 17 Feb 2012 02:39:41 +0000

Background Osteosarcoma is a malignant tumor with high ability to form invasion and metastasis. Identifying prognostic factor in osteosarcoma is helpful to select those patients for more aggressive management.

Our study evaluated serum alkaline phosphatase (ALP) cooperating with matrix metalloproteinase-9 (MMP-9) as an important prognostic predictor for local recurrence and distant metastasis of osteosarcoma.Methods 177 cases were included from the osteosarcoma patients treated at 1st Affiliated Hospital of Sun Yat-sen University (1999-2008). Pre-chemotherapy serum ALP (pre-ALP) were studied and correlated with tumor recurrence, lung metastasis and patient survival.

MMP-9 protein in tumor tissues was detected by immunohistochemistry and correlated with pre-ALP level.Results Pre-ALP were partitioned into normal, high, and very high groups, in each group the incidence of metastases was 12.2%, 21.2% and 34.6%, respectively (p=0.007). In the three groups the mean disease-free survival (DFS) was 57+/-3.15, 28+/-3.57 and 14+/-3.35 months, respectively (p<0.001); overall survival (OS) was 92+/-26.89, 39+/-8.61 and 17+/-5.07 months, respectively (p<0.001).

By multivariate analysis, elevated serum pre-ALP were associated with shorter DFS (p=0.018) and OS (p=0.031). If elevated ALP levels decreased after clinical treatment, the incidence of lung metastasis rate decreased (p=0.028); DFS and OS were both prolonged (p<0.001).

Pre-ALP was also positively correlated with MMP-9 expression (p=0.015) in tumor tissue.Conclusions Pre-ALP was an independent prognostic factor for the survival of osteosarcoma patients in south China, and correlated with MMP-9 expression and lung metastasis. ALP can also serve as a prognostic marker for treatment, and merit large-scale validation studies.

Author: Ju HanBicheng YongCanqiao LuoPingxian TanTingsheng PengJingnan Shen
Credits/Source: World Journal of Surgical Oncology 2012, 10:37

Researchers find surprising role for enzyme in tumor cell division and new drug to combat it

James Street — Fri, 18 Nov 2011 08:03:22 +0000

November 13, 2011

Researchers at the University of California, San Diego School of Medicine and the UC San Diego Moores Cancer Center have identified a new drug discovery approach enabling the destruction of the most highly proliferative tumors. The discovery, published in the Nov. 13 online issue of the journal Nature Medicine, points to an effective, alternative method for killing fast-growing cancer cells without causing some of the negative effects of current therapies.

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The scientists, led by David A. Cheresh, PhD, professor of pathology and associate director for translational research at the Moores Cancer Center, used an innovative chemical and biological approach to design a new class of drugs that arrests division in virtually all tumor cells by binding to and altering the structure of an enzyme called RAF.

RAF has been long-studied, but its role in cell division – critical to cell proliferation and tumor growth – was a surprise. “By designing a new class of drugs that changes the shape of RAF, we were able to reveal this previously undiscovered role for RAF in a wide range of highly proliferative tumors,” Cheresh said.

Current cancer drugs that target enzymes like RAF are generally designed to interact with the active site of the enzyme. Unfortunately, these drugs often lack specificity, Cheresh said. “They hit many different targets, meaning they can produce undesired side effects and induce dose-limiting toxicity.” More of a concern is that tumor cells often develop resistance to this class of drugs rendering them inactive against the cancer.

Cheresh and colleagues pursued development of a new class of RAF inhibitors that do not bind to the active site of the enzyme and so avoid the limitations of current drugs. Instead, this new class, called allosteric inhibitors, changes the shape of the target enzyme and in doing so, renders it inactive. The specific drug tested, known as KG5, singles out RAF in proliferating cells, but ignores normal or resting cells. In affected tumor cells, RAF is unable to associate with the mitotic apparatus to direct cell division, resulting in cell cycle arrest leading to apoptosis or programmed cell death. KG5 in a similar manner effectively interferes with proliferating blood vessels, a process called angiogenesis.

“It’s an unusual discovery, one that really challenges current dogma,” said Cheresh. “Before this drug was designed, we had no idea RAF could promote tumor cell cycle progression. This may be only one example of how, by designing drugs that avoid the active site of an enzyme, we can identify new and unexpected ways to disrupt the growth of tumors. In essence, we are attacking an important enzyme in a whole new way and thereby discovering new things this enzyme was intended for.”

KG5 produced similar results in tests on cancer cell lines, in animal models and in tissue biopsies from human cancer patients. The research team has since developed variants of KG5 that are 100-fold more powerful than the original drug. They hope one of these more powerful compounds will soon enter clinical trials at Moores Cancer Center.

The new RAF targeted compounds are being developed by Amitech Therapeutic Solutions, Inc a start-up company in San Diego.

Provided by University of California – San Diego

Niiki Pharma Reports on Synergistic Activity of Novel Anti-Cancer Agent NKP-1339 with Other Anti-Cancer Agents

James Street — Wed, 16 Nov 2011 21:55:32 +0000

Abstract Presented at 2011 AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics: Discovery, Biology and Clinical Applications

HOBOKEN, N.J. and PHILADELPHIA, Nov. 15, 2011 /PRNewswire/ — Niiki Pharma Inc. presented the results of preclinical combination studies of its lead product, NKP-1339, at the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics held in San Francisco, CA, November 12-16, 2011.

The data were presented as a poster, titled “NKP-1339 synergistic activity in both in vitro and in vivo preclinical models highlights the therapeutic opportunity for NKP-1339 combination trials with a broad range of anti-tumor agents.”

Data summary:

In vitro combination studies were performed in breast, colon, lung, gastric, prostate, pancreatic and liver tumor cell lines. NKP-1339 was tested in combination with cisplatin, oxaliplatin, 5-FU, docetaxel, doxorubicin, gemcitabine, erlotinib or sorafenib as appropriate for each tumor cell type. Synergistic cytotoxicity was seen with all combinations tested.
In vivo gastric carcinoma xenograft model was tested with NKP-1339 in combination with cisplatin. The combination showed significant (P<0.05) tumor growth delay and extended survival when compared to the single agent activity for either compound.

The broad synergism of NKP-1339 across the tested anti-neoplastic agents is consistent with its proposed mechanism of action through inhibition of the GRP78 pathway.

“The synergy we have demonstrated in both in vitro and in vivo settings suggests that NKP-1339 will be effective in combination with these anti-cancer agents in the clinical setting. The favorable efficacy and safety profile observed to date in our ongoing single agent NKP-1339 Phase I trial warrants its investigation in combination studies for our next round of clinical trials,” noted Angela Ogden MD, Chief Medical Officer at Niiki Pharma.

About NKP-1339

NKP-1339 is a first-in-class small molecule anti-cancer compound. NKP-1339 down-regulates the GRP78 pathway, a key regulator of mis-folded protein processing and a tumor survival factor. Up-regulation of GRP78 is associated with intrinsic and chemotherapy-induced resistance in many tumor types. In preclinical studies, single agent NKP-1339 has demonstrated activity against multiple tumor types, including those resistant to other anti-cancer agents.

NKP-1339 was discovered by Professor Bernhard K. Keppler, Dean of the Faculty of Chemistry at University of Vienna, Austria and President of Austrian Association of University Professors.

About Niiki Pharma Inc.

Niiki Pharma is a development focused oncology company specializing in first-in-class cancer treatments directed at novel cellular targets and related companion diagnostics.

Links
Niiki Pharma Inc.	www.niikipharma.com
Professor Bernhard K. Keppler, University of Vienna	http://anorg-chemie.univie.ac.at

UK Regulator Gives Takeda Bone Cancer Drug (Mifamurtide) Final Backing

James Street — Wed, 26 Oct 2011 07:11:30 +0000

By Sten Stovall

Published October 25, 2011

| Dow Jones Newswires

Published October 25, 2011 | Dow Jones Newswires

LONDON -(Dow Jones)- Takeda Pharmaceutical Co.’s (4502.TO) drug mifamurtide has secured final backing from the U.K.’s health-care cost-effectiveness regulator for use on the state-funded National Health Service in treating bone cancer in children and young people.

The National Institute for Health and Clinical Excellence, or NICE, said more patients are effectively cured of osteosarcoma, the most common form of bone cancer, when taking mifamurtide along with the usual treatment of surgery and chemotherapy.

“For the small number of patients who benefit from mifamurtide, the health benefits continue over the rest of their lives, effectively being a cure,” NICE Chief Executive Andrew Dillon said in a statement.

In September, NICE reversed a previous position opposing use of mifamurtide, which has the brand name Mepact, after Takeda offered to reduce its price for supplying the medicine. Under a revised patient access scheme from the Japanese firm, mifamurtide’s incremental cost-effectiveness ratio was cut to GBP56,000 from GBP67,000. NICE Wednesday said it consequently considers the medicine a cost-effective use of NHS resources.

“Today’s recommendation of mifamurtide will help children and young people with this very painful and distressing disease,” Dillon added.

Proteasome inhibition with bortezomib suppresses growth and induces apoptosis in osteosarcoma

James Street — Thu, 11 Aug 2011 17:27:38 +0000

Issue

International Journal of Cancer

Volume 127, Issue 1, pages 67–76, 1 July 2010

Yuriy Shapovalov,
David Benavidez,
Daniel Zuch,
Roman A. Eliseev^,†

Article first published online: 5 NOV 2009

DOI: 10.1002/ijc.25024

Keywords:

osteosarcoma;
proteasome inhibition;
bortezomib;
apoptosis;
Runx2

Abstract

Osteosarcomas are primary bone tumors of osteoblastic origin that mostly affect adolescent patients. These tumors are highly aggressive and metastatic. Previous reports indicate that gain of function of a key osteoblastic differentiation factor, Runx2, leads to growth inhibition in osteosarcoma. We have previously established that Runx2 transcriptionally regulates expression of a major proapoptotic factor, Bax. Runx2 is regulated via proteasomal degradation, and proteasome inhibition has a stimulatory effect on Runx2. In this study, we hypothesized that proteasome inhibition will induce Runx2 and Runx2-dependent Bax expression sensitizing osteosarcoma cells to apoptosis. Our data showed that a proteasome inhibitor, bortezomib, increased Runx2 and Bax in osteosarcoma cells. In vitro, bortezomib suppressed growth and induced apoptosis in osteosarcoma cells but not in nonmalignant osteoblasts. Experiments involving intratibial tumor xenografts in nude mice demonstrated significant tumor regression in bortezomib-treated animals. Immunohistochemical studies revealed that bortezomib inhibited cell proliferation and induced apoptosis in osteosarcoma xenografts. These effects correlated with increased immunoreactivity for Runx2 and Bax. In summary, our results indicate that bortezomib suppresses growth and induces apoptosis in osteosarcoma in vitro and in vivo suggesting that proteasome inhibition may be effective as an adjuvant to current treatment regimens for these tumors. Published 2009 UICC. This article is a US Government work and, as such, is in the public domain in the United States of America.

Osteosarcoma is a devastating primary bone cancer of osteoblastic origin that affects children and young adults.1, 2 It is highly aggressive, and has a propensity for early distant spread with metastases involving the lung in more than 80% of cases.3, 4 At present, the treatment of osteosarcoma includes chemotherapy with doxorubicin/adriamycin or methotrexate and large-scale surgery; however, current chemotherapeutic regimens do not significantly increase the postsurgical 5-year survival rate of 50–60%.5 There is, therefore a need for new treatment modalities in osteosarcoma that would complement current treatments and improve overall survival.

According to clinico-pathological data, 80% of osteosarcomas exhibit undifferentiated phenotype6 and express low amounts of bone-specific osteoblastic markers, such as osteocalcin.7, 8 Expression of osteocalcin and many other osteoblastic differentiation markers is regulated by the bone-specific member of the Runx family of transcription factors, Runx2.9 Runx factors are the key mediators of the TGFβ- and BMP-dependent signaling.10 Runx1, Runx2 and Runx3 regulate cell growth and differentiation in hematopoietic,11 skeletal,12 and nerve and gut13 tissues, respectively. During malignant transformation, function of the Runx family factors is frequently disrupted. Runx1 is mutated and present as a nonfunctional fusion protein in hematologic malignancies, such as acute myeloid leukemia14; Runx2 function is suppressed in osteosarcoma15; and Runx3 is mutated in gastric cancers.16 The fact that structurally and functionally similar Runx factors are inactivated in various types of cancer suggests that they may act as tumor suppressors. However, various reports also indicate an oncogenic role of Runx factors in different tumors as reviewed by Blyth et al.17 As regards with osteosarcoma, expression of a late differentiation marker, osteocalcin, is significantly decreased in these tumors7, 8 indicating that Runx2 function is compromised. Moreover, Thomas et al. have recently established that Runx2 activity is suppressed in all studied osteosarcoma cell lines and Runx2 protein levels are dramatically reduced in phenotypically aggressive osteosarcoma cell lines, such as 143B, U2OS, G292 and some others.15 We and others have shown that gain of Runx2 function inhibits proliferation and induces apoptosis in osteosarcoma cells.15, 18 We have also identified a possible mechanism of tumor suppressor action of Runx2. We showed that Runx2 transcriptionally activates expression of a major proapoptotic gene, Bax,19 thus decreasing the Bcl2 to Bax ratio and sensitizing cells to apoptosis.18 Therefore treatments that induce Runx2 may be effective in suppression of tumor growth in osteosarcoma.

Runx2 is known to be regulated via proteasomal degradation.20–22 Recent studies by Mukherjee et al.23 and Giuliani et al.24 have demonstrated that in osteoblasts, proteasome inhibition induces Runx2 protein level and activity. We therefore hypothesized that in osteosarcoma, proteasome inhibition will induce Runx2 and Bax leading to growth inhibition and induction of apoptosis. We studied the effect of a clinically approved proteasome inhibitor, bortezomib (Velcade®), in osteosarcoma cell lines in vitro and in orthotopic osteosarcoma xenografts in mice.

Material and Methods

Materials

Most chemicals were obtained from Sigma (St. Louis, MO) unless otherwise noted. Cell culture media, media ingredients and antibiotics were from Invitrogen (Carlsbad, CA). Bortezomib was from Millennium Pharmaceuticals (Cambridge, MA). Oligonucleotides were custom made by IDT (Coralville, IA). Primary antibodies for Runx2 (mouse monoclonal) and ubiquitin (rabbit polyclonal) were from Santa Cruz (Santa Cruz, CA), for Bax (rabbit monoclonal) from Epitomics (Burlingame, CA), and for β-actin (mouse monoclonal) from Sigma. The Ki-67/active caspase-3 antibody mix for immunohistochemistry was from Biocare (Concord, CA). Secondary antibodies, Precision Plus molecular weight markers, dry milk and buffer ingredients for immunoblotting were from Bio-Rad (Hercules, CA). Secondary antibodies for immunohistochemistry were from Rockland (Gilbertsville, PA). Retroviral shRNA vectors against Runx2 and a control vector, pSM2c, were from Open Biosystems (Huntsville, AL). FuGENE® HD transfection reagent was from Roche (Basel, Switzerland)

Cell culture and treatment

Human fetal osteoblasts transformed with SV40 T antigen, hFOB 1.19, and osteosarcoma cells, HOS and 143B, were obtained from ATCC (Manassas, VA). Luciferase-expressing 143B-luc cells were a kind gift of Dr. T.C. He. OS187 cells were a kind gift of Dr. R. Gorlick. HFOB cells were cultured in DMEM/F12 medium at 37°C. HOS, 143B, 143B-luc and OS187 cells were cultured in DMEM medium at 37°C. All media were supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin mixture. Seeding densities were kept constant and adjusted for different plating surface areas. All cells were cultured for 24 hr before treatments. Either bortezomib dissolved in PBS or PBS alone was added to cell media, and various assays were performed at indicated time-points as described below. Subsets of 143B cells were infected with either pSM2c control retrovirus or with 1 of 4 retroviruses carrying different shRNAs against Runx2. Stable clones were selected for 2 weeks using puromycin at 2 μg ml⁻¹. Runx2 expression was assayed with immunoblotting and the 2 clones showing strongest knock-down of Runx2 were chosen for our study along with the stable pSM2c-transfected control.

Cellular growth assay

Cells plated on 6-well plates at indicated time-points were trypsinized, resuspended in 1 ml of PBS and counted using Cellometer® automated cell counter from Nexcelom Bioscience (Lawrence, MA).

Real-time RT-PCR

Total cellular RNA was isolated using RNeasy Mini Kit by Qiagen (Valencia, CA) and reverse transcribed into cDNA using iScript cDNA synthesis kit by Bio-Rad according to the manufacturer’s instructions. One microgram of cDNA was subjected to real-time PCR using following sets of primers: Runx2 (5-CCG GAA TGC CTC TGC TGT TAT GA-3′ and 5′-ACT GAG GCG GTC AGA GAA CAA ACT-3′), alkaline phosphatase (5′-TGC AGT ACG AGC TGA ACA GGA ACA-3′ and 5′-TCC ACC AAA TGT GAA GAC GTG GGA-3′), Bax (5′- CAC CAG CTC TGA GCA GAT CAT GAA G -3′ and 5′- GCG GCA ATC ATC CTC TGC AG -3′) and GAPDH (5′-GAG TCA ACG GAT TTG GTC GT-3′ and 5′-GAC AAG CTT CCC GTT CTC AG-3′). Real-time PCR was performed using the RotorGene real-time DNA amplification system (Qiagen). SYBR Green reagent produced by Abgene (Rockford, IL) was used to monitor DNA synthesis. The expression of proteins of interest was normalized to the expression of GAPDH.

Dual luciferase promoter-reporter assay

Cells plated on 12-well plates were transfected with the 6xOSE-luc firefly luciferase reporter9 at 0.5 μg per well. The renilla luciferase promoter-less reporter, pRL-TK, produced by Promega (Madison, WI), was cotransfected at 0.05 μg per well as a reference. FuGene HD reagent by Roche was used for transfections. After 24 hr cells were lyzed and firefly and renilla luciferase activities were measured using a Dual Luciferase Reporter Assay System (Promega) in an Optocomp 1 luminometer according to the manufacturer’s instructions. The firefly luciferase signal was normalized to the renilla luciferase signal and expressed as relative luminescence units (RLU).

Western blotting

Cells were lyzed, and the protein concentration in lysates was measured using the Bradford assay. Twenty five to forty micrograms of total protein per sample was mixed 1:1 with 2xLaemmli buffer; boiled and subjected to electrophoresis using NuPage precast 4–12% gradient polyacrylamide gels (Invitrogen) followed by electroblotting onto polyvinylidene difluoride membranes (Bio-Rad). Blots were blocked in 5% dry milk dissolved in PBS containing 0.01% of Tween-20 (PBST), probed with primary antibody resuspended in 2.5% dry milk dissolved in PBST at 2 (Runx2), 1 (ubiquitin) or 0.5 (Bax) μg ml⁻¹, incubated with horseradish peroxidase-conjugated secondary antibody resuspended in 2.5% dry milk dissolved in PBST at 0.2 μg ml⁻¹, developed using SuperSignal WestPico chemiluminescent substrate produced by Thermo Scientific (Rockford, IL), and photographed. To verify equal loading, blots were stripped in Re-Blot Plus stripping buffer produced by Millipore (Billerica, MA), reprobed with anti-β-actin antibody resuspended in 2.5% dry milk in PBST at 0.5 μg ml⁻¹ and developed as described above. Band intensities were measured using densitometry and Adobe® software.

Apoptotic morphology and nuclear condensation assay

Cells plated on 24-well plates were treated with bortezomib as indicated in the text. At 24 and 48 hr thereafter, cellular morphology was examined using Zeiss AxioVert inverted microscope under visible light illumination. A nuclear fluorescent stain, Hoechst 33342, was added to the media at 1 μM. Fluorescent nuclear signal was visualized using the above microscope under UV light illumination.

Caspase-3 activity assay

Caspase-3 activity in cell lysates was measured using fluorogenic substrate cleavage assay.25 The reaction mixture contained caspase-3 fluorogenic substrate Ac-DEVD-AMC produced by Calbiochem (San Diego, CA) resuspended at 20 μM in PBS and 10 μg of the cell lysate. The reactions were loaded into 96-well plates and incubated at 37°C for 30 min. Fluorescence of the -AMC tag cut off by active caspase-3 was measured at 440 nm (excitation 380 nm) using a Hitachi plate reader.

In vivo osteosarcoma xenograft model

To model osteosarcoma in vivo we used human osteosarcoma 143B cells expressing luciferase (143B-luc).26 Cells were grown to confluency and resuspended in sterile Hanks buffered saline (HBSS) at 5 × 10⁶ per ml. Ten microliters of the cell suspension containing 5 × 10⁴ cells were injected orthotopically into the medullar cavity of right tibiae of 5-week-old immunocompromised nude Nu/Nu mice (Crl:Nu/Nu-FoxN1^nu) purchased from Charles River Laboratories (Cambridge, MA). The injection was performed using a Hamilton syringe into the opening in tibial tuberosity pre-made with a 25-gauge needle. Left tibias were sham injected. Tumor growth was monitored longitudinally using bioluminescence imaging (BLI). For bioluminescent imaging the mice were anesthetized and injected intraperitoneally with 100 μl of luciferin substrate. Bioluminescence was measured using a Xenogen imager. A total of 16 mice were injected of which 12 developed tumors as detected with BLI.

Treatment of mice with bortezomib

On Day 8 after the injection of tumor cells, 12 tumor-bearing mice were randomly divided in 2 groups—the control group (−Bzm) and the treated group (+Bzm). Animals in the treated group received 1 mg kg⁻¹ of bortezomib dissolved in sterile PBS intraperitoneally (IP) every 3 days for 3 weeks. Mice in the control group received sterile PBS IP injections. All animals were sacrificed on Day 28 after the injection of tumor cells, and both hind limbs were excised. Tumor volume was calculated from the 2D caliper measurements using the following formula: tumor volume = length × (width)² × π/6.27

Immunohistochemistry

Both tumor-bearing and sham-injected tibias were fixed in 10% neutral buffered formalin for 3 days, decalcified in 14% EDTA for 10 days, and subsequently embedded in paraffin. Serial 4 μm sections were cut and mounted on glass slides. Sections were either stained with H&E or processed for immunohistochemistry as follows: sections were deparaffinized using xylene and ethanol, rehydrated and permeabilized with 0.1% Triton in a TRIS-citrate retrieval buffer at 95°C for 15 min. To block endogenous peroxidase activity, sections were treated with methanol/1% H₂O₂ for 30 min. After the wash, sections were blocked with TBS/0.1% triton/10% normal serum for 1 hr, followed by incubation with the primary antibody for 1 hr. The immunolabeling performed included Ki-67/active caspase-3 double labeling, Runx2 and Bax. After subsequent rinse in PBS, sections were incubated with the corresponding HRP-conjugated secondary antibody, washed and developed with DAB (Ki-67, Runx2 and Bax) and/or Vulcan Red (active caspase-3) substrates. After the development reaction had been terminated, sections were rinsed, dehydrated, mounted and counterstained with either hematoxylin (Ki-67/active caspase-3 and Bax) or FastGreen (Runx2) stains.

In situ proliferation and apoptosis assay

To assess the effect of bortezomib on cell proliferation and apoptosis in xenografted osteosarcoma tumors in mice, formalin-fixed, paraffin-embedded tissue sections were probed with the Ki-67/active caspase-3 antibody cocktail. The tissue sections were visualized using a Zeiss Axioplan light microscope equipped with a DAGE video camera that was connected to a Ludl XYZ motorized stage, a color monitor, and a personal computer, and the number of Ki-67 and active caspase-3 positive cells was counted. Quantitative data were obtained utilizing a software program Stereologer 1.3.1 developed by Systems Planning and Analysis (Alexandria, VA), which provided us with an unbiased optical dissector approach for accurate cell counting. The entire tissue section was chosen as a reference area. In addition, spacing of 1,100 μm for Ki-67 and 400 μm for active caspase-3 was established as an optimal interval that would allow us to sample the population of positively stained cells within the reference area. Placement of counting grids provided us with random areas to be analyzed. Results were obtained as the number of positively stained cells per field of view. The treated group was compared to the control group and the results were expressed as the fold change over control (active caspase-3) or percent of control (Ki-67).

Quantitative analysis of Runx2 and Bax immunostaining

Tissue sections probed for Runx2 or Bax were visualized using Zeiss Axioscop 40 microscope and 40× objective. The random fields (10 per section) were photographed under constant illumination and exposure using a high resolution digital camera attached to the microscope. The images were blindly analyzed for nuclear Runx2 or cytosolic Bax staining intensities using the ImageJ software. The treated group was compared to the control group and the results were expressed as the fold change over control.

Statistical analysis

Experiments were repeated 3 to 5 times. Mean values and standard deviations were calculated, and the statistical significance was determined using a Student’s t-test or ANOVA. Data with p < 0.05 were considered statistically significant.

Results

Characterization of osteosarcoma cell lines, HOS, 143B and OS187

The majority of osteosarcomas are aggressive and metastatic.6 Human osteosarcoma cell lines, 143B and OS187, have been reported to have an aggressive and invasive phenotype and form tumors and metastases when injected into mice.15, 26, 28 We therefore chose 143B and OS187 cell lines for our study as representatives of an aggressive osteosarcoma. The 143B cells are Ki-Ras-transformed derivatives of HOS cells.26 For this reason, HOS cell line was used as a nontransformed control for 143B cells. HOS cells show limited tumorigenic and metastatic potential.26 OS187 cells were included to expand our study and to exclude a possibility of cell-line specific effects in related HOS and 143B cells. OS187 cell line was produced directly from an osteosarcoma patient specimen.29 It has been described as an aggressive cell line that forms tumors when injected orthotopically into mouse bone and is capable of lung metastasis.28 Immortalized human osteoblastic cell line, hFOB,30 was included as a nonmalignant control. HFOB cells are transformed with a temperature-sensitive SV40 T large antigen which is active at 33°C leading to unrestricted cell growth, but inactivated at 37°C.30 To avoid potential artifacts caused by active SV40 T large antigen, we used hFOB cells at a nonpermissive temperature of 37°C. These conditions allow adequate growth of hFOB cells and have been used in previous studies.31

To assess the phenotype of HOS, 143B and OS187 cells, we first measured their cell growth rate in comparison to hFOB cells. As shown in Figure 1a, the growth rate of HOS cells was slightly higher than the growth rate of nonmalignant hFOB osteoblasts while the growth rate of 143B and OS187 cells was significantly increased. The 143B and OS187 cells had similar doubling time of ∼24 hr. Next, we measured expression of osteoblastic differentiation markers, Runx2 and alkaline phosphatase (ALP) using real-time RT-PCR approach. Figure 1b (top panel) shows that all studied osteosarcoma cells expressed Runx2 confirming their commitment to osteoblastic lineage. Runx2 mRNA levels in HOS and 143B cells were higher whereas in OS187 cells they were 50% lower than in nonmalignant controls. ALP mRNA level (Fig. 1b, bottom panel) in HOS cells was similar to that in hFOB cells while in 143B and OS187 cells, it was significantly decreased, confirming their undifferentiated phenotype. Western blot analysis demonstrated that total Runx2 protein levels were slightly decreased in HOS cells and significantly downregulated in 143B and OS187 cells when compared to hFOB cells (Fig. 1c, top blot). The most pronounced decrease was observed in the faster migrating MASN isoform of Runx2 (lower band) while the slower migrating MRIPV isoform (upper band) was less affected.32 We then assessed Runx2 transcriptional activity using the 6xOSE-luc promoter-reporter construct.9 The basal activity of this reporter in the absence of forced expression of Runx2 was relatively low and ranged from 1,000 to 7,000 RLU or 5- to 20-fold over the background shown as a dotted line in Figure 1d. Figure 1d shows that 6xOSE-luc activity was significantly decreased in all studied osteosarcoma cells when compared to nonmalignant hFOB cells. In our previous work, we have described a regulatory role of Runx2 in Bax expression.18 We therefore investigated whether Runx2 protein levels in the studied cells correlate with Bax protein levels. Figure 1c (middle blot) shows that Bax protein levels correlated with Runx2 levels and were slightly decreased in HOS cells and significantly decreased in 143B and OS187 cells when compared to hFOB cells. In summary, these data indicate that tumorigenic and metastatic osteosarcoma cells, 143B and OS187, have a highly proliferative and undifferentiated phenotype whereas less aggressive HOS cells are less proliferative and more differentiated. Runx2 protein levels correlate with Bax levels and inversely correlate with proliferative capacity in the studied cells.

Figure 1. Cell growth and expression of differentiation markers in osteosarcoma cells and in immortalized non-malignant osteoblasts. (a) Cell growth assay. HFOB, HOS, 143B and OS187 cells were seeded in 6-well plates at a density of 200,000 per well. After 24 or 48 hr cells were harvested and counted using an automated cell counter; (b) Assay of mRNA expression of osteoblastic differentiation markers. Real-time RT-PCR analysis was performed to measure relative expression of Runx2 (top panel) and alkaline phosphatase (ALP, bottom panel) as described in Methods; (c) Assay of protein expression of Runx2 and Bax. Immunoblotting was performed to assess Runx2 (top blot) protein levels in cell lysates. Blots were then reprobed for Bax (middle blot) and for β-actin to verify equal loading (bottom blot). Blots are representatives of 3; (d) Assay of Runx2 transcriptional activity. Dual luciferase assay and the 6xOSE-luc reporter were used to measure Runx2 transcriptional activity in the studied cells. Dotted line represents a Background signal. Data in (a), (b), and (d) are Means ± SD (n = 3 to 5). Asterisk (*) in (d) indicates p < 0.05 when compared to the levels found in hFOB cells.

Effect of proteasome inhibition with bortezomib on Runx2 and Bax in osteosarcoma cells

Our data in Figure 1 illustrate a discrepancy between mRNA and protein levels of Runx2 in the studied osteosarcoma cells. Relative to hFOB cells, in HOS cells Runx2 mRNA was at 260% while Runx2 protein measured with densitometry was at 80%; in 143B cells Runx2 mRNA was at 150% while Runx2 protein was at 46%; and in OS187 cells Runx2 mRNA was at 50% while Runx2 protein was at 38%. This discrepancy between mRNA and protein levels likely indicates increased Runx2 protein degradation. Runx2 is known to be regulated by proteasome20–22; and proteasome inhibition increases Runx2 levels and function.23, 24 We therefore studied effects of a proteasome inhibitor, bortezomib, on Runx2 protein level and function in HOS, 143B and OS187 osteosarcoma cells. For our experiments we chose a concentration of bortezomib of 25 nM because, according to the pharmacokinetic study by Attar et al.,33 this is a physiologically relevant intermediate plasma concentration of bortezomib in patients after a bolus injection. Figure 2a (top blot) shows that 24-hr incubation with bortezomib at 25 nM increased the amount of ubiquitinated proteins confirming the inhibitory effect of bortezomib on proteasomes in the studied cells. Runx2 protein levels were also significantly increased in bortezomib-treated cells (middle blot). Runx2 transcriptional activity as measured with the 6xOSE-luc reporter, was upregulated in bortezomib-treated cells with the most pronounced effect observed in OS187 cells (Fig. 2b). As has been reported by our group before,18 Runx2 regulates Bax expression. Therefore, we examined whether the bortezomib-mediated increase in Runx2 protein level and function correlates with Bax expression. Bax expression in bortezomib-treated osteosarcoma cells was measured using real-time RT-PCR and showed, on average, a 3-fold induction when compared to cells treated with vehicle control (Fig. 2c). The above experiments demonstrate that proteasome inhibition with bortezomib increases Runx2 protein level and activity as well as Bax expression in the studied osteosarcoma cells.

Figure 2. Proteasome inhibition with bortezomib restores Runx2 and Bax in osteosarcoma cells. Cells were seeded at the same density, cultured for 24 hr, and then treated with bortezomib at 25 nM for another 24 hr. Cells were collected and total RNA or protein was extracted. (a) Effect of proteasome inhibition on Runx2 protein level. Immunoblotting was performed to assess ubiquitin (top blot) protein levels in cell lysates. Blots were re-probed for Runx2 (middle blot) and for β-actin (bottom blot). Blots are representatives of 3; (b) Effect of proteasome inhibition on Runx2 activity. Dual luciferase assay and the 6xOSE-luc reporter were used to measure Runx2 transcriptional activity in the studied cells; (c) Effect of proteasome inhibition on Bax expression. Bax mRNA levels were measured using real-time RT-PCR analysis and normalized to GAPDH mRNA levels. Data in (b) and (c) are Means ± SD (n = 4). Asterisk (*) indicates p < 0.05 when compared to the levels found in corresponding control cells (−Bzm).

Proteasome inhibition selectively suppresses growth and induces apoptosis in osteosarcoma cells

As mentioned above, Runx2 has been shown to function as a tumor suppressor in osteosarcoma. Therefore, by restoring Runx2 protein level and activity, bortezomib may exert an inhibitory effect on osteosarcoma cells. In addition, as a general proteasome inhibitor, bortezomib may affect other molecular pathways regulated by proteasome and thus confer an additional suppression of cell proliferation and functioning. To determine the effect of bortezomib in the studied cells, we performed a dose-response and time-course analysis by evaluating growth rates of nonmalignant hFOB cells and of HOS, 143B and OS187 osteosarcoma cells in vitro. Figure 3a shows that increasing doses of bortezomib did not have any inhibitory effect on the growth of hFOB cells. However, in HOS, 143B and OS187 osteosarcoma cell lines bortezomib suppressed cell growth in a dose-dependent manner. To expand our study and confirm the effect of proteasome inhibition, we used another proteasome inhibitor, MG132, and found that after 24 hr it decreased osteosarcoma cell number on average by 70% and hFOB cell number by 30% (Data not shown). Because MG132 is not an FDA approved compound and because it shows higher toxicity in nonmalignant hFOB cells, we continued our studies using only bortezomib. Bortezomib-treated osteosarcoma cells exhibited morphological features of apoptosis, such as shrinkage and rounding of cell bodies along with condensation of chromatin (Fig. 3b). A specific apoptotic marker protease, caspase-3, was activated in bortezomib-treated osteosarcoma cells (Fig. 3c). These data indicate that proteasome inhibition with bortezomib selectively suppresses growth and induces apoptosis in osteosarcoma cells but not in nonmalignant control cells in culture.

Figure 3. Bortezomib suppresses growth and induces apoptosis in osteosarcoma cells. (a) Effect of proteasome inhibition on growth of osteosarcoma cells. Cells were plated at similar density in 6-well plates and after 24-hr incubation, were treated with the indicated doses of bortezomib or with PBS as a vehicle control. After 24 and 48 hr, cells were harvested and counted in an automated cell counter; (b) Bortezomib-treated osteosarcoma cells show apoptotic morphology. Cells cultured for 24 hr were treated with 25 nM bortezomib (+Bzm) or PBS (−Bzm) for another 24 hr. Cellular morphology was examined under the microscope and photographed (top panels). Chromatin condensation and nuclear shrinkage was detected by staining the cells with Hoechst33342 and visualizing them under the UV illumination using a fluorescence microscope (lower panels). Panels are representative of 12; (c) Activation of caspase-3 in bortezomib-treated osteosarcoma cells. Cells cultured for 24 hr were treated with 25 nM bortezomib (+Bzm) or PBS (−Bzm) for another 24 hr. Cells were lyzed and caspase-3 activity in cell lysates was measured using a fluorogenic substrate, Ac-DEVD-AMC, as described in detail in Methods. Data in (a) and (c) are Means ± SD (n = 4–6). Asterisk (*) indicates p < 0.05 when compared to vehicle control-treated cells (−Bzm). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Knock-down of Runx2 abolishes the inhibitory effect of bortezomib in osteosarcoma cells

By inhibiting protein degradation, bortezomib can have a wide range of effects on various cell signaling systems other than Runx2. To evaluate a specific contribution of Runx2 in the inhibitory effect of bortezomib on osteosarcoma cells, we performed a stable knock-down of Runx2 in 143B cells using 2 different retroviral shRNA constructs. We then treated these cells as well as control cells transfected with the empty pSM2c retroviral vector with bortezomib at 25 nM for 24 hr. Figure 4a shows that cells transfected with either shRNA1 or shRNA2 against Runx2 contained ∼30% of the amount of Runx2 found in cells transfected with the control vector (pSM). Moreover, while treatment with bortezomib significantly increased Runx2 protein levels in control transfectants (Fig. 4a, lane 2), it did not increase Runx2 protein levels in shRNA-transfected cells (Fig. 4a, lane 4 and 6). Bax expression, as measured with real-time RT-PCR correlated with Runx2 protein levels and significantly increased in bortezomib-treated control transfectants but remained unchanged in bortezomib-treated shRNA1- and 2-transfected cells. Figure 4c shows that the knock-down of Runx2 abolished the inhibitory effect of bortezomib in 143B osteosarcoma cells. These results demonstrate that the inhibitory effect of bortezomib in osteosarcoma cells is at least partially dependent on Runx2.

Figure 4. Knockdown of Runx2 decreases the effect of bortezomib in osteosarcoma cells. The 143B cells stably transfected with either a control vector, pSM2c (pSM), or 2 different shRNA constructs against Runx2, shRNA1 and 2, were cultured for 24 hr, treated with bortezomib at 25 nM for 24 hr and harvested. (a) Knock-down of Runx2 in 143B cells. Runx2 protein levels in stably transfected cells were assayed with immunoblotting. Bortezomib induced Runx2 levels in control cells but not in shRNA1- or 2-transfected cells. Blots were re-probed for β-actin to verify equal loading. Blots are representative of 3; (b) Effect of proteasome inhibition on Bax expression in control and Runx2 shRNA-transfected cells. Bax expression was assayed with real-time RT-PCR; (c) Effect of proteasome inhibition on growth of control and Runx2 shRNA-transfected cells. Cells were counted in an automated cell counter. Data in (b) and (c) are Means ± SD (n = 4). Asterisk (*) indicates p < 0.05 when compared to vehicle control-treated cells (−Bzm).

Inhibition of proteasome with bortezomib suppresses growth of osteosarcoma xenografts in vivo

To study the effect of bortezomib on osteosarcoma development in vivo, we performed xenografting of osteosarcoma cells into nude Nu/Nu mice. Luciferase-expressing 143B (143B-luc) cells26 were injected orthotopically into right tibiae of mice. Development of xenografted tumors was followed longitudinally using bioluminescent imaging (BLI). Figure 5a demonstrates the presence of a strong bioluminescent signal in tumor-bearing mice (top right panel) and absence of a signal in uninjected animals (top left panel). By Day 8, tumor-injected tibiae in 12 out of 16 animals developed consistent BLI signal which increased on average 4-fold when compared to the signal at Day 1, indicating successful engraftment of the tumors. Tumor engraftment was later confirmed histologically (Fig. 5a, lower panels). At Day 8, we divided animals in 2 groups so that each group had equal average BLI signal, and started intraperitoneal PBS (control group) or bortezomib (treatment group) injections at a dose of 1 mg kg⁻¹ every 3 days. In the absence of detailed pharmacokinetic studies of bortezomib in mice, it is difficult to correlate the in vivo and in vitro doses. Previously bortezomib was used at 0.5–2 mg kg⁻¹ in mouse models and gave consistent responses without any noticeable toxicity.34, 35 We, therefore, chose an intermediate dose of 1 mg ml⁻¹ for our animal studies. We have not noticed any toxic effects, i.e., weight loss, in our treated animals. Animals were sacrificed at Day 28, and tumor sizes were determined. Figure 5b shows that bortezomib had a pronounced inhibitory effect on growth of osteosarcoma xenografts in mice. The average size of osteosarcoma tumors in the bortezomib-treated group was only 30% of that in the control group.

Figure 5. Osteosarcoma xenograft model and effect of bortezomib on tumor progression in vivo. Athymic nude mice were injected intratibially with 143B-luc cells at Day 0 and development of tumors was monitored by measuring bioluminescence. At Day 8, mice were divided in 2 groups and received either PBS or bortezomib at 1 mg kg⁻¹ every 3 days. Mice were sacrificed at Day 28. (a) Engrafted tumors emit strong bioluminescent signal (top panels). Bioluminescent imaging (BLI) was performed before (top left panel) and 8 days after (top right panel) the injection of tumor cells. Histochemical analysis confirms tumor engraftment (bottom panels). Both tumor-bearing (bottom right panel) and non-tumor bearing (bottom left panel) limbs were excised, processed for histochemistry and stained with H&E. Femoral heads (marked with open arrowheads) are shown for orientation. When compared to normal tibiae (marked with solid arrowhead), 143B-luc injected tibiae develop tumors (marked with “X”). The panels are representative of 6; (b) Bortezomib suppresses growth of osteosarcoma in vivo. At sacrifice tumors were measured with calipers and tumor sizes calculated as described in Methods; (c) Bortezomib inhibits proliferation and induces apoptosis in osteosarcoma xenografts. Shown is the representative H&E staining (left panel) and immunostaining for Ki-67/active caspase-3 (right panel) of bortezomib-treated tumors. White arrows in the left panel and black arrows in the right panel mark apoptotic cells. The yellow arrow in the right panel marks proliferating, Ki-67-positive cells; (d) Runx2 (top panels) and Bax (bottom panels) immunostaining intensities are increased in tumor samples from bortezomib-treated animals. Tissue sections from osteosarcoma xenografts in mice were probed for Runx2 or Bax and photographed as described in Methods. Data in (b) are Means ± SD (n = 6). Asterisk (*) indicates p < 0.05 when compared to the vehicle control-treated group (−Bzm).

To elucidate a possible mechanisms of bortezomib-induced inhibition of osteosarcoma tumor growth, we performed histochemical studies and immunostaining of formalin-fixed paraffin-embedded tumor sections. Cytological analysis of H&E stained sections revealed presence of cells exhibiting apoptotic morphology in bortezomib-treated group, such as shrinkage and rounding of cell bodies along with condensation and fragmentation of nuclei (Fig. 5c, left panel, marked by white arrows). To assess changes in tumor cell proliferation and confirm induction of apoptosis in the bortezomib-treated group in vivo, sections were subjected to double immunostaining for Ki-67 and the active form of caspase-3 respectively (Fig. 5c, right panel). The assay showed decreased number of cells positive for Ki-67 (brown staining marked by yellow arrow) and increased presence of cells positive for active caspase-3 (pink staining marked by black arrows). To determine whether these effects of bortezomib correlated with the increase in Runx2 and Bax levels in xenografted tumors, tumor sections were probed with Runx2- and Bax-specific antibodies (Fig. 5d). The assay showed that both Runx2 (top panels) and Bax (bottom panels) staining intensities were significantly increased in tumors from the bortezomib-treated group. Quantitative analysis of immunostaining for Ki-67, active caspase-3, Runx2 and Bax demonstrated that Ki-67 immunoreactivity decreased by 60% (Fig. 6a); active caspase-3 immunoreactivity increased 3-fold (Fig. 6b); Runx2 immunoreactivity increased 2.7-fold (Fig. 6c) and Bax immunoreactivity increased 2-fold (Fig. 6d) in the bortezomib-treated animals when compared to the control animals. Taken together, these experiments indicate that proteasome inhibition with bortezomib suppresses growth, induces apoptosis, and increases Runx2 and Bax levels in xenografted osteosarcoma tumors in mice.

Figure 6. Bortezomib inhibits proliferation, induces apoptosis, and increases Runx2 and Bax levels in osteosarcoma xenografts in mice. Quantitative analysis of Ki-67 (a), active caspase-3 (b), Runx2 (c) and Bax (d) immunostaining in osteosarcoma xenografts. The number of Ki-67 positive or active caspase-3 positive cells was determined using stereological approach and Runx2 and Bax staining intensities were measured as described in Methods. Immunostaining in the control group (−Bzm) was compared to the immunostaining in the treated group (+Bzm) and results were plotted. Data are Means ± SD (n = 6). Asterisk (*) indicates p < 0.05 when compared to the vehicle control-treated group (−Bzm).

Discussion

A summary of our key findings is as follows: (i) when compared to nonmalignant hFOB cells, osteosarcoma cell lines, HOS, 143B and OS187 have decreased protein levels of Runx2 and Bax as well as suppressed Runx2 activity; (ii) proteasome inhibition with bortezomib restores Runx2 level and function and increases Bax expression in these osteosarcoma cells; (iii) bortezomib induces apoptosis in HOS, 143B and OS187 cells in vitro; and (iv) bortezomib induces tumor regression and apoptosis in orthotopic osteosarcoma xenografts in mice that correlates with increased Runx2 and Bax levels. On the basis of these findings, we conclude that bortezomib may be effective when used as a therapeutic adjunct in management of osteosarcoma and that its effect may be related to restoration of the level and function of Runx2, which acts as a tumor suppressor in this type of malignancy.

These data complement our previous findings that gain of Runx2 function in osteosarcoma cells sensitizes these cells to apoptosis via transcriptional upregulation of a major proapoptotic factor, Bax.19 Our results also provide an explanation for a recently described suppression of Runx2 level and function in a variety of osteosarcoma cell lines.15 Increased proteasomal degradation may play a major role in this suppression of Runx2 and, as suggested by our findings, inhibition of proteasome may effectively restore Runx2 level and function in osteosarcoma cells. Proteasomal degradation has been previously implicated in the regulation of Runx220–22, 36 however its role in down-regulation of Runx2 level in cancerous cells has not been described. The mechanism underlying the observed increased degradation of Runx2 in osteosarcoma is a subject of future studies and is currently under investigation by our group. Targeting for proteasomal degradation by Smurf1 or by Cyclin D1/Cdk4 has been shown to destabilize Runx2 and suppress Runx2 protein levels.20, 21 We have found that Smurf1 is significantly upregulated in the studied osteosarcoma cell lines and that siRNA-mediated inhibition of Smurf1 restores Runx2 levels in these cells (data not shown) implicating Smurf1 as a ubiquitin ligase responsible for Runx2 degradation in osteosarcoma. Overall, our data confirm previous findings15, 18 that Runx2 can act as a tumor suppressor in osteosarcoma and suggest that in such instances osteosarcoma cells develop mechanisms to suppress Runx2 leading to arrested differentiation and desensitization to pro-apoptotic signals. It should be noted that substantial evidence has also been accumulated on the oncogenic role of Runx2 and other 2 Runx factors in cancer. As suggested in an elegant review by Blyth et al., this ambiguous role of Runx factors in cancer may be due to their dependence on various co-factors, presence of functional p53 protein, and other regulatory signals.17 The fact that Runx2 increases the potential for bone metastasis in breast and prostate cancer cells37, 38 may be a tissue specific effect. Active Runx2 might be necessary for breast and prostate cancer cells to mimic bone-like phenotype and to home effectively in bone, while the described pro-apoptotic function of Runx2 may be suppressed via some mechanism that is yet to be elucidated.

Our most clinically relevant finding is that bortezomib, a proteasome inhibitor approved for clinical use and proven effective in treating lymphoma and multiple myeloma,39 may be equally effective in suppressing osteosarcoma. Our data indicate that bortezomib selectively induces apoptosis in osteosarcoma cells in vitro and in osteosarcoma xenografts in vivo. Inhibition of proteasome may have an effect on a wide range of cellular mechanisms. However, regardless of the pathways involved, suppression of tumor growth and selective induction of apoptosis by bortezomib in osteosarcoma is a promising and potentially important finding. It suggests a novel treatment option for this devastating disease. Pre- or postoperative bortezomib regimen may be an effective complement to current chemotherapies.

Acknowledgements

The 143B-luc cells were a kind gift of Dr. T.C. He of the University of Chicago to Dr. E. Sampson. OS187 cells were a kind gift of Dr. R. Gorlick of the Memorial Sloan-Kettering Cancer Center. The authors thank R. Tierney, L. MacMahon and Dr. L. Flick of the University of Rochester for their help with immunohistochemistry; Dr. E. Sampson of the University of Rochester for his help with osteosarcoma xenograft model and shRNA experiments; and Dr. D. Hicks, Dr. R. Rosier and Dr. R. O’Keefe of the University of Rochester for their expertise and fruitful discussions.

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Mitaplatin, a potent fusion of cisplatin and the orphan drug dichloroacetate

James Street — Thu, 11 Aug 2011 06:22:36 +0000

Shanta Dhar ^a and
Stephen J. Lippard ^a,^b,¹

+ Author Affiliations

^aDepartment of Chemistry and
^bKoch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139

Contributed by Stephen J. Lippard, October 29, 2009 (received for review August 30, 2009)

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Abstract

The unique glycolytic metabolism of most solid tumors, known as the Warburg effect, is associated with resistance to apoptosis that enables cancer cells to survive. Dichloroacetate (DCA) is an anticancer agent that can reverse the Warburg effect by inhibiting a key enzyme in cancer cells, pyruvate dehydrogenase kinase (PDK), that is required for the process. DCA is currently not approved for cancer treatment in the USA. Here, we present the synthesis, characterization, and anticancer properties of c,t,c-[Pt(NH₃)₂(O₂CHCl₂)₂Cl₂], mitaplatin, in which two DCA units are appended to the axial positions of a six-coordinate Pt(IV) center. The negative intracellular redox potential reduces the platinum to release cisplatin, a Pt(II) compound, and two equivalents of DCA. By a unique mechanism, mitaplatin thereby attacks both nuclear DNA with cisplatin and mitochondria with DCA selectively in cancer cells. The cytotoxicity of mitaplatin in a variety of cancer cell lines equals or exceeds that of all known Pt(IV) compounds and is comparable to that of cisplatin. Mitaplatin alters the mitochondrial membrane potential gradient (Δψ_m) of cancer cells, promoting apoptosis by releasing cytochrome c and translocating apoptosis inducing factor from mitochondria to the nucleus. Cisplatin formed upon cellular reduction of mitaplatin enters the nucleus and targets DNA to form 1,2-intrastrand d(GpG) cross-links characteristic of its own potency as an anticancer drug. These properties of mitaplatin are manifest in its ability to selectively kill cancer cells cocultured with normal fibroblasts and to partially overcome cisplatin resistance.

Normal cells typically use mitochondrial oxidative phosphorylation to metabolize glucose and switch over to glycolysis only when there is little or no oxygen, producing lactate as a byproduct. Cancer cells avidly consume glucose for energy by glycolysis to survive in the hypoxic environment of malignant lesions (1), a phenomenon known as the Warburg effect (2). The dependence of cancer cells on glycolysis comes not only from oxygen deprivation, but also partly from their inability to synthesize ATP in response to the mitochondrial membrane potential gradient (Δψ_m) (3). This unique glucose metabolic pathway of cancer cells has identified the mitochondrion as a prime target for cancer therapy (4–7). In addition, cancer cells develop the ability to avoid apoptosis by various pathways that ignore the command to commit cellular suicide (8, 9). Compounds that trigger apoptosis through selective action on mitochondrial target sites of cancer cells bypass defective upstream mechanisms and trigger apoptosis in tumor cells that are otherwise resistant (10).

Dichloroacetate (DCA) is used in humans to treat lactic acidosis (11). DCA inhibits the activity of pyruvate dehydrogenase kinase (PDK), thereby stimulating the mitochondrial enzyme pyruvate dehydrogenase (PDH). When turned off, PDH no longer converts pyruvate to acetyl-CoA required for mitochondrial respiration and glucose-dependent oxidative phosphorylation (12). DCA thus shifts cellular metabolism from glycolysis to glucose oxidation, decreasing Δψ_m (13) and helping to open mitochondrial transition pores (MTPs). This metabolic switch facilitates translocation of proapoptotic mediators like cytochrome c (cyt c) and apoptosis inducing factor (AIF), both of which stimulate apoptosis. DCA thereby drives cancer cells to commit suicide by apoptosis (13). Unlike most other anticancer agents, DCA does not appear to have any deleterious effect on normal cells. DCA reverses mitochondrial changes in a wide range of cancers, making malignant cells more vulnerable to normal cell death programs (14). Being an orphan drug, DCA is both nonpatentable and readily available, but it is not yet approved for use in cancer therapy (15). There is substantial preclinical evidence from both in vitro and in vivo models that DCA might be useful to treat cancer in humans, and a translation to early- phase clinical trials would be of interest (16–18). Funding for such trials would be a challenge because DCA is a generic drug. However, because it withdraws cancer cells from a state of apoptosis resistance, DCA is an attractive sensitizer that could be given concurrently with chemotherapy or radiation therapy. Alternatively, a formulation could be synthesized that incorporates DCA.

Platinum(II) compounds are used in 50% of all cancer therapies (19). Among these, cisplatin, carboplatin, and oxaliplatin have Food and Drug Administration approval and are in the clinic worldwide (20, 21). The use of platinum(II) drugs, cisplatin in particular, to treat malignancies is limited because of side effects and acquired resistance (22). Resistance can emerge from failure to execute apoptosis despite initiation of the apoptotic cascade caused by either the predominance of anti-apoptotic factors or defects in downstream effectors. Cisplatin resistance in ovarian carcinoma cells is associated with a reduced apoptotic response (23). To overcome tumor cell resistance and toxicity to normal tissues, we have been exploring strategies to target platinum constructs to cancer cells. Our tactic has been to employ substitutionally inert platinum(IV) compounds (24), which serve as prodrugs and release clinically effective levels of platinum(II) compounds, such as cisplatin, following cellular uptake (25–27). Appropriately designed platinum(IV) complexes are less likely to be deactivated before reaching their cancer cell destination target. The activity of platinum(IV) complexes generally involves reduction with loss of the axial ligands, affording an active platinum(II) complex that readily binds to DNA. Satraplatin is one such Trojan horse platinum(IV) compound that is currently under investigation for the treatment of patients with advanced prostate cancer (28).

We therefore designed a Pt(IV) compound (mitaplatin, 1) having two DCA moieties (Fig. 1) in the axial positions. We hypothesized that DCA released inside the cells by reduction of the platinum would simultaneously alter mitochondrial metabolism and deliver a dose of cisplatin (Fig. 1). Mitaplatin was thereby expected to have dual killing modes toward cancer cells, one in which cisplatin interacts with its key target, nuclear DNA, and the other, DCA released upon reduction, following a pathway to induce mitochondria-dependent apoptosis by mitochondrial membrane depolarization and efflux of proapoptotic mediators. Here, we describe the synthesis, characterization, and dual-action cell killing ability of mitaplatin as well as its remarkable ability to selectively destroy cancer cells in a coculture with normal fibroblasts.

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Fig. 1.

Chemical structures and mechanism of action of mitaplatin (1). After crossing the plasma membrane, mitaplatin becomes reduced to release of the active drugs cisplatin and DCA. DCA inhibits mitochondrial PDK, which leads to PDH activation and increased glucose oxidation by promoting influx of acetyl-CoA into the mitochondria. DCA decreases the mitochondrial membrane potential (Δψ_m). Opening of the Δψ_m-sensitive mitochondrial transition pores (MTPs) leads to efflux of cyt c and AIF. Cisplatin formed in the reduction process interacts with its key target, nuclear DNA.

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Results and Discussion

Synthesis and Characterization of Mitaplatin (1).

Mitaplatin (1), a formulation of DCA, was prepared by reaction of c,c,t-[Pt(NH₃)₂Cl₂(OH)₂] with dichloroacetic anhydride in >50% yield. Its formation was evidenced by disappearance of the O–H stretching band of the starting compound and the presence of a C=O stretch at 1651 cm⁻¹ in the infrared spectra. The structure was confirmed by ¹H, ¹³C, and ¹⁹⁵Pt NMR spectroscopy, by HRMS, and by elemental analysis. ESI-HRMS (M–H) Calcd. = 554.8145, Found = 554.8138. ¹H NMR (DMSO-d₆) δ 7.95 (s, 2H), 6.52 (br, 6H); ¹³C NMR (DMSO-d₆) δ 170.41, 65.27; ¹⁹⁵Pt NMR (DMSO-d₆): δ = 1205.28 ppm. Anal: Calcd for C₄H₈Cl₆N₂O₄Pt: C, 8.64; H, 1.45; N, 5.04. Found: C, 8.13; H, 1.65; N, 4.88. Mitaplatin is redox-active and displays an irreversible reduction revealed by cyclic voltammetric analysis. The Pt(IV)/Pt(II) couple is near −0.173 V vs. Ag/AgCl at pH 7.4 and the value at pH 6.0 is −0.152 vs. Ag/AgCl. Voltammograms are given in Figs. S1 and S2. These reduction potentials indicate that mitaplatin will be readily reduced in cells. The cathodic reduction potential depends on the electron-withdrawing power and the bulkiness of the axial and auxiliary ligands. The low reduction potential of mitaplatin is influenced by the presence of chlorine atoms from the DCA molecules near the platinum center (29).

In Vitro Cellular Cytotoxicity Assays.

The ability of mitaplatin to promote cell death was evaluated by the MTT assay and the results were compared against those for cisplatin or DCA using NTera-2, HeLa, U2OS, A549, and MCF-7 cancer cells as well as MRC-5 normal fibroblasts (Fig. S3). Results are presented in Table 1. Mitaplatin has an IC₅₀ value of 0.051 μM, comparable to that of cisplatin (IC₅₀, 0.043 μM), in cisplatin-sensitive testicular NTera-2 cells and is more toxic than DCA alone. In U2OS osteosarcoma cells, cisplatin has an IC₅₀ of 3.9 μM, whereas that of mitaplatin is 6.4 μM. Similarly, in HeLa cervical cancer cells, comparable IC₅₀ values for mitaplatin and cisplatin were observed, 2.0 and 1.20 μM, respectively. Control experiments with the well known platinum(IV) compound c,c,t-[Pt(NH₃)₂Cl₂(O₂CCH₃)₂], revealed it to be several-fold less active than mitaplatin in all cells (Table S1). Mitaplatin was also established to have cytotoxicity comparable to that of cisplatin in the NCI/DTP 60 cell line growth inhibition assay, exceeding almost all known Pt(IV) compounds. This enhanced potency of mitaplatin is consistent with the expected dual killing mechanism.

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Table 1.

Cell killing ability of mitaplatin

Mitaplatin Promotes Apoptosis in Cancer Cells.

To investigate the ability of mitaplatin to promote apoptosis in cancer cells by a mitochondrial-regulated mechanism, changes in the mitochondrial transmembrane potential (Δψ_m) of cancerous NTera-2 and healthy normal fibroblast cells before and after mitaplatin treatment were investigated by two assays. Mitochondrial attack is associated with a drop in Δψ_m. For this reason Δψ_m is an important parameter of mitochondrial function and has been used to monitor mitochondrial death. 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) is a lipophilic cationic dye which, depending on Δψ_m, accumulates as a green monomer in the cytoplasm or as red-emitting aggregates in hyperpolarized mitochondria of cancer cell (30). The negative charge established by the mitochondrial membrane potential allows the lipophilic dye, bearing a delocalized positive charge, to enter mitochondria where it accumulates. When a critical concentration is exceeded, J-aggregates form, which fluoresce red. In apoptotic cells, Δψ_m collapses, and JC-1 cannot accumulate in mitochondria. In these cells, JC-1 remains in the cytoplasm in a green fluorescent monomeric form. Control NTera-2 cells exhibited heterogeneous staining of the cytoplasm with both red and green fluorescence in the same cells (Fig. 2A). Treatment of these cells with mitaplatin for 4 h decreased the red fluorescence. Mitochondrial membrane depolarization was detected by a shift in fluorescence emission of JC-1 from red to green. There was no significant effect of mitaplatin on Δψ_m of normal fibroblasts or of cisplatin on Δψ_m of NTera-2 cells. The detailed results are given in Fig. S4.

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Fig. 2.

Disruption of mitochondrial function and induction of apoptosis in cancer cells by mitaplatin. (A) Changes in the mitochondrial membrane potential as revealed by the JC-1 assay. Treatment with 100 μM mitaplatin dramatically caused the collapse of mitochondrial membrane potentials in NTera-2 cells. In live cells, JC-1 exists either as a green fluorescent monomer at depolarized membrane potentials (positive to −100 mV) or as an orange-red fluorescent J-aggregate at hyperpolarized membrane potentials (negative to −140 mV). The shift in membrane charge was observed by disappearance of fluorescent red-orange-stained mitochondria (large negative Δψ_m) and an increase in fluorescent green-stained mitochondria (loss of Δψ_m). (B) Reversal of mitochondrial membrane potential by tetramethyl rhodamine methyl ester (TMRM) assay. Mitaplatin significantly depolarized the NTera-2 cells but had no effect on the healthy normal fibroblast cells. Mitochondria were stained with mitotracker red. (C) Cytochrome c release visualized by fluorescence microscopy. Immunolocalization of cytochrome c (green) and mitochondrial morphology (red) shown in untreated NTera-2 cells and in mitaplatin treated NTera-2 cells. Cells were grown for 24 h on glass coverslips and treated with mitaplatin, fixed after treatment, and immunostained with anti-cytochrome c monoclonal antibodies. Mitochondria were stained with mitotracker red. (D) Translocation of AIF in mitaplatin treated cells. Staining of AIF (Ab) and nuclei (Hoechst) in NTera-2 cells before and after 12 h treatment with mitaplatin. Arrows indicate cells with particularly evident presence of AIF in the nucleus.

To investigate whether DCA released from mitaplatin can restore the hyperpolarization of cancer cells to the level of normal cells, we carried out a TMRM assay (31). All cancer cell lines have significantly more hyperpolarized Δψ_m compared to normal cells and therefore exhibit increased fluorescence of the Δψ_m-sensitive positive dye tetramethyl rhodamine methyl ester, TMRM. Incubation of the NTera-2 cells with mitaplatin for 48 h reversed the hyperpolarization and returned the Δψ_m to the level of normal cells (Fig. 2B). In contrast, mitaplatin did not alter the Δψ_m of the normal fibroblasts. Because dichloroacetate activates pyruvate dehydrogenase, which increases delivery of pyruvate into mitochondria, DCA released upon reduction of mitaplatin (Fig. 1) increased glucose oxidation, depolarizing the mitochondria and returning the membrane potential to levels of the noncancer cells.

Mitochondrial cyt c, which functions as an electron carrier in the respiratory chain, translocates to the cytosol in cells undergoing apoptosis, where it participates in activation of apoptotic proteins (32). The mechanism responsible for this process is unknown. Cyt c release from mitochondria is an early event in the apoptotic process induced by mitaplatin treatment in NTera-2 cells, as visualized by using a FITC-conjugated antibody for the protein and fluorescence microscopy. The cytosol from untreated cells showed no detectable cyt c (Fig. 2C). In contrast, cytosolic cyt c accumulated significantly after 4 h of treatment with mitaplatin.

AIF is a proapoptotic mitochondrial protein (33). Like cyt c, AIF is a bifunctional protein having both electron transfer and apoptogenic functions. AIF is released from mitochondria and translocated to nuclei, stimulating chromatin condensation and DNA fragmentation. We were interested to determine whether mitochondrial outer membrane permeabilization by mitaplatin induces apoptosis only by release of caspase-dependent factors, such as cyt c, or whether caspase-independent processes, such as that mediated by AIF, might be operative. We therefore investigated the location of AIF in the mitaplatin-treated cells. As shown in Fig. 2D, mitaplatin treatment led to translocation of AIF from the mitochondria to nuclei of NTera-2 cells.

To quantify mitaplatin-induced apoptosis in cancer cells, an annexin-V assay was performed by using flow cytometry. With this analysis, we determined the percentage of apoptotic cells at 48 h after exposure to mitaplatin, cisplatin, or DCA. Apoptosis was detected in cancerous U2OS, HeLa, and A549 cells with 10 μM mitaplatin and cisplatin. Cisplatin at 10 μM concentration evoked apoptosis in normal MRC-5 cells whereas mitaplatin did not produce any detectable apoptosis with these normal cells (Table 2).

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Table 2.

Quantification of apoptosis induced by mitaplatin, cisplatin, and DCA using an annexin V assay

Visualization of Pt-1,2-d(GpG) Adduct Formation by Mitaplatin.

Because the anticancer activity of cisplatin derives from the formation of intrastrand 1,2-d(GpG) cross-links on nuclear DNA (34), we investigated whether cisplatin released by reduction of mitaplatin leads to this signature event by using a monoclonal antibody R-C18 (35) specific for this adduct (SI Text). After 12 h incubation of NTera-2 cells with mitaplatin, formation of 1,2-d(GpG) intrastrand cross-links was observed by antibody-derived green fluorescence in the nuclei of these cells (Fig. S5). These results confirm that mitaplatin has dual cell-killing modes involving DCA, which destroys mitochondrial function, and cisplatin, which simultaneously impedes DNA-mediated processes in the nucleus.

Selective Killing of Cancer Cells by Mitaplatin.

One of the main obstacles to cancer therapy is the inability to successfully target cancer cells, while not harming normal cells. Even when therapeutic agents are delivered locally to a primary tumor, systemic toxicities still arise. Modern medicine desperately needs anti-cancer molecules that kill cancer cells and leave healthy cells alone. Most cancer therapies today are very toxic to tumor and healthy cells alike, and the patient can succumb to treatment rather than the disease. Furthermore, cells develop a resistance to external agents, so chemotherapy may only work for a short time period. Because DCA has selective toxicity toward cancer cells by targeting PDK, we investigated whether mitaplatin would also display specificity for cancer. We therefore treated a coculture of normal fibroblasts and cancerous NTera-2 cells with mitaplatin, cisplatin, or a mixture of one equivalent of cisplatin and two equivalents of DCA, the stoichiometric composition released upon intracellular mitaplatin reduction. The morphology of these cells at different time points was examined by using bright field microscopy (Fig. 3A). The two types of cells in the coculture are clearly visible because of differences in their morphology. In the coculture, cisplatin and the mixture of cisplatin and DCA killed both the fibroblasts and NTera-2 cancer cells, whereas mitaplatin selectively killed the cancer cells. The results obtained in this study provide compelling evidence that mitaplatin can selectively kill cancer cells, leaving normal cells untouched.

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Fig. 3.

Mitaplatin selectively induced apoptosis of human cancer cells and not normal cells. (A) Treatment of a coculture of normal fibroblast cells (elongated) and NTera-2 cells (round) with cisplatin, mitaplatin, and a mixture of one equivalent of cisplatin and two equivalents of DCA. (B) Selective killing of cancerous A549 (round) cells by mitaplatin in a coculture with normal MRC-5 (elongated) cells assayed using LIVE/DEAD staining. After mitaplatin or cisplatin exposure for 24 h, cells were stained with calcein AM (green fluorescence) and ethidium homodimer-1 (red fluorescence) to differentiate between live and dead cells, respectively.

To verify that preferential cancer cell killing occurs with mitaplatin, this study was further extended to a coculture of human lung cancer A549 cells and normal human lung fibroblasts MRC-5.

The selective killing of normal cells by mitaplatin was demonstrated by using a LIVE/DEAD viability assay, which allowed for the simultaneous determination of live and dead cells in a coculture by labeling live cells with calcein AM dye, which fluoresces only when cleaved by intracellular esterase enzymes, and ethidium heterodimer (EthD-1), which only enters dead cells with disrupted cell membranes (Fig. 3B). Fig. 3B confirms that, unlike cisplatin, a conventional chemotherapeutic agent, mitaplatin selectively induced cell death in human cancer A549 cells, but not in normal MRC-5 cells under the similar treatment conditions. To address whether the selective killing of cancer cells by mitaplatin might be a consequence of its selective uptake, we measured the nuclear and cytosolic concentrations of platinum by atomic absorption spectroscopy (AAS) after mitaplatin or cisplatin treatment of normal and cancer cells. Cytosolic and nuclear extracts were prepared from normal MRC-5 and cancerous A549 cells after incubation with 10 μM mitaplatin or cisplatin for 24 h. Platinum concentrations determined by AAS (Table S2) confirm the uptake of mitaplatin by both cells types.

Mitaplatin Action on Cisplatin-Resistant Cells.

Although very little is known about the effects of cisplatin on the mitochondria of tumor cells (36), a recent study showed that it might have direct impact on mitochondria in head and neck cancer (37). Mitochondrial defects are associated with the cisplatin resistance phenotype (38), and several hypotheses have been suggested to explain this observation. A more negative membrane potential might promote translocation of the active, cationic form of cisplatin from the cytoplasm to mitochondria, thus diminishing platination of nuclear DNA. This effect would suggest that a combination of cisplatin with a mitochondrial targeting moiety would be an attractive therapeutic strategy for attacking cisplatin-resistant tumors. We therefore studied a pair of cisplatin sensitive A2780 and resistant A2780/CP70 ovarian cancer cells (Table 1 and Fig. S6). As controls we used cisplatin and c,c,t-[Pt(NH₃)₂Cl₂(O₂CCH₃)₂]. The cells displayed a low level of resistance to mitaplatin (IC₅₀ for A2780, 1.1 μM; IC₅₀ for A2780/CP70, 3.34 μM) compared to cisplatin (corresponding IC₅₀ values of 0.56 and 6.0 μM). Results for the A2780/CP70 cells indicate that DCA plays a role in making cisplatin-resistant cells susceptible toward mitaplatin treatment. A2780/CP70 cells were much more resistant to the control platinum(IV) compound c,c,t-[Pt(NH₃)₂Cl₂(O₂CCH₃)₂]. These data suggest mitaplatin as a promising candidate for further development in the treatment of cisplatin-resistant cells.

Summary.

In conclusion, mitaplatin displays a dual-killing mode that can only be effective in cancer cells. The platinum center interacts with its own target, nuclear DNA, and DCA released upon reduction attacks mitochondria. These results support the utility of mechanisms targeting cancer cell-specific pathways as an avenue for developing selective anticancer agents. Mitaplatin offers a formulation for future studies incorporating the orphan drug DCA to further its use in the clinic.

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Materials and Methods

The complexes cis-[Pt(NH₃)₂Cl₂] (39) and c,c,t-[Pt(NH₃)₂Cl₂(OH)₂] (40) were synthesized as described. Distilled water was purified by passage through a Millipore Milli-Q Biocel water purification system (18.2 MΩ) containing a 0.22-μm filter. Anti-cytochrome c (Ab-1) sheep polyclonal antibody was procured from Calbiochem. Alexa Fluor 488-labeled secondary antibody donkey anti-(sheep IgG) was obtained from Invitrogen for cytochrome c detection. For AIF detection, we used a rabbit polyclonal IgG antibody from Santa Cruz Biotechnology, Inc. Alexa Fluor 546-labeled secondary antibody goat anti-(rabbit IgG) was purchased from Invitrogen. The detection of the cisplatin 1,2-d(GpG) intrastrand adduct was carried out using a monoclonal adduct-specific antibody R-C18 which was kindly provided by Jürgen Thomale (University of Duisburg-Essen). FITC labeled secondary antibody rabbit anti-(rat Ig) was obtained from Invitrogen. Specific adhesion slides for immunofluoresecence were purchased from Squarix Biotechnology. JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbo-cyanine iodide) was obtained from Cayman Chemicals. ¹H, ¹³C, and ¹⁹⁵Pt NMR spectra were recorded on a Bruker AVANCE-400 NMR spectrometer with a Spectro Spin superconducting magnet in the Massachusetts Institute of Technology Department of Chemistry Instrumentation Facility (MIT DCIF). Atomic absorption spectroscopic measurements were taken on a Perkin-Elmer AAnalyst 300 spectrometer. HRMS analysis was carried out on a Bruker Daltonics APEXIV 4.7 Tesla Fourier Transform Ion Cyclotron Resonance mass spectrometer in the MIT DCIF. Fluorescence imaging studies were performed with an Axiovert 200M inverted epifluorescence microscope (Zeiss) equipped with an EM-CCD digital camera C9100 (Hamamatsu). An X-Cite 120 metal-halide lamp (EXFO) was used as the light source. The microscope was operated with Volocity software (Improvision).

Synthesis of Mitaplatin c,c,t-[Pt(NH₃)₂Cl₂(O₂CCHCl₂)₂] (1).

To a solution of c,c,t-[Pt(NH₃)₂Cl₂(OH)₂] (0.2 g, 0.6 mmol) in DMF (5 mL) was added dichloroacetic anhydride (0.28 g, 1.5 mmol) and the reaction mixture was stirred at room temperature for 4 h. Diethyl ether was added to the mixture to precipitate a light yellow solid, which was washed several times with diethyl ether and dried. Mitaplatin (1) was isolated in 55% (0.29 g) yield. IR (KBr): ν_max 3178, 3076, 3012, 1651, 1568, 1435, 1333, 1214, 1103, 1021, 819, 789, 723, 666, 582 cm⁻¹; ESI-HRMS (M–H) Calcd. = 554.8145, Found = 554.8138. ¹H NMR (DMSO-d₆) δ 7.95 (s, 2H), 6.52 (br, 6H); ¹³C NMR (DMSO-d₆) δ 170.41, 65.27; ¹⁹⁵Pt NMR (DMSO-d₆): δ = 1205.28 ppm. Anal: Calcd for C₄H₈Cl₆N₂O₄Pt: C, 8.64; H, 1.45; N, 5.04. Found: C, 8.13; H, 1.65; N, 4.88.

Detection of Cyt c and AIF.

NTera-2 cells were seeded on microscope coverslips (1 cm) at a confluence of 1,600 cells per slip and incubated overnight at 37 °C in DMEM. The medium was changed and mitaplatin was added to a final concentration of 100 μM. The cells were incubated for 4, 12, or 24 h at 37 °C. The medium was then removed and the cells were incubated with fixing solution for 1 h at room temperature followed by three washes with PBS (pH 7.4). Cells were then fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) at room temperature for 1 h, then washed twice with PBS. Cells were permeabilized with 0.1% Triton X-100 in PBS for 30 min, followed by six washes with PBS. Cells were then rinsed with blocking buffer (PBS, 0.1% goat serum, 0.075% glycin), incubated for 1 h at 37 °C with the anti-cytochrome c [anti-cytochrome c (Ab-1) sheep pAb, Calbiochem] antibody or AIF [AIF (H-300), Santa Cruz Biotechnology Inc.] antibody, both diluted 1:50 in blocking buffer, washed twice with blocking buffer, and incubated at 37 °C with Alexa Fluor 488 donkey anti-(sheep IgG) (Invitrogen) antibody for cyt c release and Alexa Fluor 546 goat anti-rabbit IgG (Invitrogen) antibody for AIF (dilution 1:50 in blocking buffer) for 1 h. After two washes with blocking buffer and four washes with water, Mitrotacker Red for cyt c release and Hoechst bis-benzamide for AIF release were used to stain mitochondria and nuclei, respectively. Microscope coverslips were mounted on microscope slides using mounting solution for imaging.

JC-1 Assay.

GM61869 and NTera-2 cells were cultured on cover slips to a density of 1 × 10⁶ cells/mL and incubated overnight at 37 °C. Cells were then treated with 100 μM mitaplatin for 4 and 48 h at 37 °C. A solution of JC-1 reagent (Cayman Chemicals; 10 μg/mL in DMEM) was added and incubation was carried out at 37 °C for 30 min. The cells were washed with PBS five times, fixed in 4% paraformaldehyde, and mounted onto glass slides using the procedure described above.

TMRM Assay.

Analysis of mitochondrial membrane potential (Δψ_m) was carried out by using TMRM. A similar procedure as mentioned above for the JC-1 assay was followed. Before fixing the cells, they were treated with 2 μM TMRM for 30 min at 37 °C.

LIVE/DEAD Assay.

In vitro selective killing was performed using the LIVE/DEAD Viability/Cytoxicity Assay (Molecular Probes). A549 and MRC-5 cells were cultured on sterile glass coverslips as subconfluent monolayers for 24 h at 37 °C in 5% CO₂ and grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. The cells were then treated with 100 μM cisplatin or mitapltin for 24 h at 37 °C in 5% CO_2. The cells were washed with Dulbecco’s PBS (D-PBS) to remove serum esterase activity generally present in serum-supplemented growth media before the assay. Calcein AM (4 mM in anhydrous dimethyl sulfoxide, DMSO) and EthD-1 (2 mM in DMSO/water, 1:4 vol/vol) were added to PBS (1:1,000 ratio) to produce a LIVE/DEAD working solution as recommended by the manufacturer. The samples were first washed in three changes of PBS and 100 μL LIVE/DEAD working solution was added on the coverslip and incubated at room temperature for 30 min. Subsequently, the samples were placed in PBS before being examined with a fluorescence microscope.

Annexin-V Assay.

Flow cytometry with a Vybrant Apoptosis Assay kit (annexin V conjugated to allophycocyanin, Invitrogen) was used to determine whether treatment specifically induces apoptosis. Briefly, 5 × 10⁵ cells for each cell line were seeded into six-well tissue culture plates and incubated overnight to 60–70% confluence under standard growth conditions. Media for the cell lines were then replaced with fresh growth media with and without a 10 μM dose of cisplatin, mitaplatin, and a 20 μM dose of DCA. Treatment groups for each cell line were replicated three times. The cells were then incubated for 48 h at 37 °C and harvested with 0.25% trypsin-EDTA. Cells were washed with PBS and subsequently stained by annexin V as per the manufacturer’s protocol. Flow cytometry was performed on a BD LSR II flow cytometer (BD Biosciences) and data were analyzed on BD FACSDiva (BD Biosciences).

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Acknowledgments

This work was supported by the National Cancer Institute Grant CA034992 (to S.J.L.) and the Koch Institute for Integrative Cancer Research (S.D.).

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Footnotes

¹To whom correspondence should be addressed at:

Department of Chemistry, Room 18–498, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307.

E-mail: lippard@mit.edu
Author contributions: S.D. and S.J.L. designed research; S.D. performed research; S.D. and S.J.L. analyzed data; and S.D. and S.J.L. wrote the paper.
The authors declare no conflict of interest.
This article contains supporting information online at www.pnas.org/cgi/content/full/0912276106/DCSupplemental.

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Kaempferol (found in Ginkgo Biloba) induced apoptosis via endoplasmic reticulum stress and mitochondria-dependent pathway in human osteosarcoma U-2 OS cells

James Street — Thu, 21 Jul 2011 06:34:19 +0000

Mol Nutr Food Res. 2010 Nov;54(11):1585-95.

Huang WW, Chiu YJ, Fan MJ, Lu HF, Yeh HF, Li KH, Chen PY, Chung JG, Yang JS.

Source

Department of Biological Science and Technology, China Medical University, Taichung, Taiwan.

Abstract

Kaempferol is a natural flavonoid. Previous studies have reported that kaempferol has anti-proliferation activities and induces apoptosis in many cancer cell lines. However, there are no reports on human osteosarcoma. In this study, we investigate the anti-cancer effects and molecular mechanisms of kaempferol in human osteosarcoma cells. Our results demonstrate that kaempferol significantly reduces cell viabilities of U-2 OS, HOB and 143B cells, especially U-2 OS cells in a dose-dependent manner, but exerts low cytotoxicity on human fetal osteoblast progenitor hFOB cells. Comet assay, DAPI staining and DNA gel electrophoresis confirm the effects of DNA damage and apoptosis in U-2 OS cells. Flow cytometry detects the increase of cytoplasmic Ca(2+) levels and the decrease of mitochondria membrane potential. Western blotting and fluorogenic enzymatic assay show that kaempferol treatment influences the time-dependent expression of proteins involved in the endoplasmic reticulum stress pathway and mitochondrial signaling pathway. In addition, pretreating cells with caspase inhibitors, BAPTA or calpeptin before exposure to kaempferol increases cell viabilities. The anti-cancer effects of kaempferol in vivo are evaluated in BALB/c(nu/nu) mice inoculated with U-2 OS cells, and the results indicate inhibition of tumor growth. In conclusion, kaempferol inhibits human osteosarcoma cells in vivo and in vitro.

Family forced to fund life-saving (Mepact) treatment

James Street — Sun, 01 May 2011 04:31:07 +0000

Published Date: 01 May 2011

By Lyndsay Buckland

THE family of a teenager forced to pay tens of thousands for a cancer drug denied to him by the NHS in Scotland has criticised the “postcode lottery” faced by patients trying to access life-prolonging treatments.

Jamie Marshall Macdonald was diagnosed with osteosarcoma, a rare type of bone cancer, last September. After surgery to remove the cancer, the 18-year-old had the regular course of chemotherapy for patients with this disease.

But when the family’s own research suggested another drug – Mepact - could improve his chances of survival, their attempts to have it prescribed by the NHS were denied.

The same drug – which costs £114,000 for a 36-week course – has been given to patients in England through a special fund set up last year to pay for expensive cancer treatments.

Jamie’s parents, Simon Marshall and Ann Macdonald, have now attacked a system in which patients in some parts of the UK are given drugs denied to others.

The family’s case has reignited debate about how to fund expensive drugs from increasingly tight NHS budgets, with some politicians saying there should be no differences within the UK in terms of access to NHS resources.

Jamie’s family, from Glasgow, discovered he had cancer after he suffered symptoms that included a painful lump on one of his legs.

The cancer was caught at an early stage and he had chemotherapy followed by surgery to remove a tumour. Since then he has had standard chemotherapy treatment.

Then, while researching the disease, the family found out about Mepact, which can be used alongside chemotherapy to reduce the chances of cancer returning. Studies have found that 68 per cent of patients on Mepact survive without the disease coming back, compared with 61 per cent of patients who did not receive it. Crucially, it also reduces the risk of dying from the disease by 28 per cent.

“Our reading and research on various websites drew our attention to Mepact as an additional drug treatment. So we opened discussions (with doctors] about that,” Marshall said. “We thought that, in addition to the other treatment, this might be beneficial.”

The family were told that the drug had not been recommended for NHS use by the Scottish Medicines Consortium (SMC), which assesses drugs, and they would have to make an application for exceptional funding through their health board. Although their doctor supported their application, NHS funding was refused.

The family has now used savings and donations from relatives to pay for the drug but are concerned other patients may not be able to afford it.

“Very few people would be in the fortunate circumstances we have found ourselves in,” Macdonald said.

The couple praised the care given to Jamie, who hopes to study sociology at university, by the Beatson Cancer Centre in Glasgow. But they said they had concerns about the process of assessing drugs for rare diseases and also the systems in place for exceptional funding. Marshall said that given the rarity of osteosarcoma – which affects just 150 people in the UK each year – Mepact should never have been rejected by the SMC in Scotland or its English equivalent, the National Institute for Health and Clinical Excellence.

The £250 million Cancer Drug Fund was introduced by the coalition last October to fund drugs rejected by the assessment bodies but recommended by clinicians.

Campaigners are concerned that inequalities of care will persist unless money is put aside for cases where general funding is not available.

The Scottish Conservatives are proposing the introduction of a Scottish Cancer Drugs Fund, of up to £10m a year.Health spokesman Murdo Fraser said: “Sadly, Jamie’s plight is not unique in Scotland. We believe that it is unacceptable there are expensive cancer drugs, recommended by doctors, available to patients in England and Wales, but not available in Scotland.”

Scottish Labour’s health spokeswoman Jackie Baillie said: “It is vital that those suffering from rare conditions get access to the medicines they require and we must ensure that patients in one part of Scotland are not disadvantaged compared with patients living in England and Wales.”

An SNP spokesperson said: “Everyone recognises the importance of decisions on medication and treatment being made by health professionals and not politicians. When consultants recommend access to medicines we expect health boards to respond flexibly and favourably to requests.”

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Page 2 of 2

Last Updated: 30 April 2011 6:48 PM
Source: Scotland On Sunday
Location: Scotland

Targeting Inflammatory Kinase as an Adjuvant Treatment for Osteosarcomas

James Street — Fri, 22 Apr 2011 07:04:10 +0000

Kyucheol Noh, MD¹, Kyung-Ok Kim, PhD¹, Neel R. Patel, BS¹, J. Robert Staples, BS¹, Hiroshi Minematsu, PhD¹, Kumar Nair, BA¹ and Francis Young-In Lee, MD, PhD¹

¹ Center for Orthopaedic Research, Department of Orthopaedic Surgery, Columbia University, 650 West 168th Street, New York, NY 10032. E-mail address for F.Y.-I. Lee: fl127@columbia.edu

Investigation performed at the Center for Orthopaedic Research,Department of Orthopaedic Surgery, Columbia University, NewYork, NY

A commentary by John H. Healey, MD, is available at www.jbjs.org/commentaryand is linked to the online version of this article.

Disclosure: In support of their research for or preparationof this work, one or more of the authors received, in any oneyear, outside funding or grants in excess of $10,000 from theOrthopaedic Science and Research Foundation. Neither they nora member of their immediate families received payments or otherbenefits or a commitment or agreement to provide such benefitsfrom a commercial entity.

Background A subset of patients with aggressive osteosarcomas respondspoorly to conventional cytotoxic chemotherapy. Recent evidencefrom studies involving the liver, skin, stomach, and colon suggeststhat carcinogenesis is associated with inflammation. Mitogen-activatedprotein kinase (MAPK)/extracellular signal-regulated kinase1/2 (ERK1/2) has diverse roles in cancer and inflammation. Thehypothesis of the present study is that targeted ERK1/2 inhibitionwill demonstrate anti-cancer effects in osteosarcoma both invitro and in vivo.

Methods The therapeutic effect of PD98059, a MAPK/ERK pathway inhibitor,was examined with respect to cell death, survival, and anti-apoptoticprotein expression by means of flow cytometry and immunoblottingin vitro. Additionally, we transplanted green fluorescent proteinand luciferase-tagged 143B osteosarcoma cells into the proximalpart of the tibia of nude mice. Mice were randomly assignedto treatment with doxorubicin, PD98059, or both. Vehicle-treatedmice served as controls. Treatment outcome was assessed by measuringbioluminescence and by monitoring survival.

Results In vitro, ERK1/2 blockage increased the expression of pro-apoptoticproteins and increased cell death in 143B osteosarcoma cells.Doxorubicin treatment increased the expression of Bcl-2, ananti-apoptotic protein, but this upregulation was blocked bycombined treatment with PD98059, suggesting a role for ERK1/2in conferring drug resistance. In osteosarcoma-bearing mice,targeting ERK1/2 with PD98059 resulted in prolonged survivalin comparison with vehicle-treated control mice (median survivaltime, sixty-seven days compared with seventy-four days; p =0.0272; survival ratio = 0.9122; 95% confidence interval = 0.4354to 1.389). Standalone doxorubicin treatment yielded similaranimal morbidity (median survival time, sixty-seven days comparedwith seventy-six days; p = 0.0170; survival ratio = 0.8882;95% confidence interval = 0.4181 to 1.358). Combined PD98059and doxorubicin treatment further prolonged survival (mediansurvival time, sixty-seven days compared with eighty-two days;p = 0.0023; survival ratio = 0.8232; 95% confidence interval= 0.3606 to 1.286).

Conclusions Inhibiting ERK1/2 signaling resulted in osteosarcoma cell deathby upregulating pro-apoptotic genes and inhibiting the Bcl-2-mediatedresistance to doxorubicin. In osteosarcoma-bearing mice, ERK1/2targeting alone or in combination with doxorubicin prolongedsurvival as compared with untreated mice.

Clinical Relevance Our study highlights the anti-cancer effect of the inflammatorykinase inhibitor PD98059 on osteosarcoma cells by inducing celldeath and by inhibiting a potential drug-resistance mechanism.Taken together, these results suggest that ERK signaling blockade(targeted therapy) may be considered as a new targeted adjuvanttherapy for osteosarcoma.

New study finds compounds show promise in blocking STAT3 signaling as treatment for osteosarcoma

James Street — Tue, 12 Apr 2011 06:17:02 +0000

Contact: Erin Pope
Erin.Pope@NationwideChildrens.org
614-355-0495
Nationwide Children’s Hospital

A study appearing in the journal Investigational New Drugs and conducted by researchers at Nationwide Children’s Hospital, discovered that two new small molecule inhibitors are showing promise in blocking STAT3, a protein linked to the most common malignant bone tumor, osteosarcoma. These small molecule inhibitors – one derived from a portion of the turmeric spice – may serve as a new, non-toxic treatment for these deadly tumors.

Osteosarcoma is aggressive and its treatment outlook has not changed significantly over the last 20 years. Treatment consists of a combination of toxic chemotherapy and aggressive surgical resection. Yet, despite these options, patients have at most a 50-to-60 percent five-year disease-free survival rate.

“The outcome for patients with advanced or metastatic osteosarcoma continues to be dismal, emphasizing the need for new therapies,” said the study’s lead author Jaiyuh Lin, PhD, principal investigator in the Center for Childhood Cancer in The Research Institute at Nationwide Children’s Hospital. “Directly targeting STAT3 signaling represents a potential therapeutic approach to treating this type of cancer.”

STAT3 is a member of a protein family that plays a role in relaying signals from cytokines and growth factors. The abnormal activation of STAT proteins is becoming more commonly associated with unrestricted cell growth and transformation of normal cells into malignant cells. Abnormal STAT3 activation has been seen in human and canine osteosarcoma cell lines and shows cancer-causing-capabilities in cultured cells and mouse models.

“Recent experiments aimed at blocking STAT3 signaling have been successful, resulting in the inhibition of growth and the induction of death in tumors,” said Dr. Lin, also a faculty member at The Ohio State University College of Medicine. “They have also shown that blocking STAT3 in normal cells is neither harmful nor toxic.”

Dr. Lin and his team evaluated two newly developed compounds, LLL12 and FLLL32, to determine their ability to inhibit STAT3 activity in human osteosarcoma cells. FLLL32 is derived from the dietary agent curcumin, the principal compound in the popular Indian spice turmeric.

Findings showed that both agents were able to inhibit STAT3 activity and suppressed tumor growth in the mouse model that was developed using human osteosarcoma cells, and primary osteosarcoma xenograft provided by Nationwide Children’s Hospital scientist, Peter Houghton, PhD, directly from a patient.

“This study suggests that LLL12 and FLLL32 should be suitable for targeting osteosarcoma and possibly certain types of cancer cells with persistently activated STAT3,” said Dr. Lin. “This approach deserves further exploration as a potential treatment of osteosarcoma.”

Defeat Osteosarcoma » Molecular Osteosarcoma Studies

High serum alkaline phosphatase cooperating with MMP-9 predicts metastasis and poor prognosis in patients with primary osteosarcoma in Southern China

Researchers find surprising role for enzyme in tumor cell division and new drug to combat it

Niiki Pharma Reports on Synergistic Activity of Novel Anti-Cancer Agent NKP-1339 with Other Anti-Cancer Agents

Abstract Presented at 2011 AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics: Discovery, Biology and Clinical Applications

UK Regulator Gives Takeda Bone Cancer Drug (Mifamurtide) Final Backing

Proteasome inhibition with bortezomib suppresses growth and induces apoptosis in osteosarcoma

International Journal of Cancer

Keywords:

Abstract

Material and Methods

Materials

Cell culture and treatment

Cellular growth assay

Real-time RT-PCR

Dual luciferase promoter-reporter assay

Western blotting

Apoptotic morphology and nuclear condensation assay

Caspase-3 activity assay

In vivo osteosarcoma xenograft model

Treatment of mice with bortezomib

Immunohistochemistry

In situ proliferation and apoptosis assay

Quantitative analysis of Runx2 and Bax immunostaining

Statistical analysis

Results

Characterization of osteosarcoma cell lines, HOS, 143B and OS187

Effect of proteasome inhibition with bortezomib on Runx2 and Bax in osteosarcoma cells

Proteasome inhibition selectively suppresses growth and induces apoptosis in osteosarcoma cells

Knock-down of Runx2 abolishes the inhibitory effect of bortezomib in osteosarcoma cells

Inhibition of proteasome with bortezomib suppresses growth of osteosarcoma xenografts in vivo

Discussion

Acknowledgements

References

Mitaplatin, a potent fusion of cisplatin and the orphan drug dichloroacetate

Abstract

Results and Discussion

Synthesis and Characterization of Mitaplatin (1).

In Vitro Cellular Cytotoxicity Assays.

Mitaplatin Promotes Apoptosis in Cancer Cells.

Visualization of Pt-1,2-d(GpG) Adduct Formation by Mitaplatin.

Selective Killing of Cancer Cells by Mitaplatin.

Mitaplatin Action on Cisplatin-Resistant Cells.

Summary.

Materials and Methods

Synthesis of Mitaplatin c,c,t-[Pt(NH3)2Cl2(O2CCHCl2)2] (1).

Detection of Cyt c and AIF.

JC-1 Assay.

TMRM Assay.

LIVE/DEAD Assay.

Annexin-V Assay.

Acknowledgments

Footnotes

References

Kaempferol (found in Ginkgo Biloba) induced apoptosis via endoplasmic reticulum stress and mitochondria-dependent pathway in human osteosarcoma U-2 OS cells

Source

Abstract

Family forced to fund life-saving (Mepact) treatment

Targeting Inflammatory Kinase as an Adjuvant Treatment for Osteosarcomas

New study finds compounds show promise in blocking STAT3 signaling as treatment for osteosarcoma

Synthesis of Mitaplatin c,c,t-[Pt(NH₃)₂Cl₂(O₂CCHCl₂)₂] (1).