Archive for the ‘Cisplatin’ Category

New drug candidate shows promise against cancer

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Posted 25 Jul 2012 — by James Street
Category Cisplatin
Platinum compound may offer an alternative to cisplatin, a widely used chemotherapy agent.
Anne Trafton, MIT News Office

July 11, 2012

Drugs containing platinum are among the most powerful and widely used cancer drugs. However, such drugs have toxic side effects, and cancer cells can eventually become resistant to them.

MIT chemistry professor Stephen J. Lippard, who has spent much of his career studying platinum drugs, has now identified a compound that kills cancer cells better than cisplatin, the most commonly used platinum anticancer drug. The new compound may be able to evade cancer-cell resistance to conventional platinum compounds.

“I’ve long believed that there’s something special about platinum and its ability to treat cancer,” Lippard says. Using new variants, “we might have a chance of applying platinum to a broader range of cancer types, more successfully,” he says.

Mildred Dresselhaus, Ann Graybiel and Jane Luu
From left to right: Postdoc Ying Song, MIT chemistry professor Stephen J. Lippard and postdoc Ga Young Park.
Photo: M. Scott Brauer

Lippard is senior author of a paper describing the new drug candidate, known as phenanthriplatin, in the Proceedings of the National Academy of Sciences (PNAS). Lead author is postdoc Ga Young Park; other authors are graduate student Justin Wilson and postdoc Ying Song.

Cisplatin, first approved to treat cancer in 1978, is particularly effective against testicular cancer, and is also used to treat ovarian and some lung tumors, as well as lymphoma and other cancers. At its center is a platinum atom bound to two ammonia molecules and two chloride ions. When the compound enters a cancerous cell, it becomes positively charged because water molecules replace its chloride ions. The resulting positive ion can attack negatively charged DNA, forming cross-links with the DNA strands and making it difficult, if not impossible, for the cell to read that section of DNA. Too much of this damage, if not repaired, kills the cell.

For many years, Lippard has studied the mechanism of cisplatin’s action and has pursued similar drugs that could be more powerful, work against more types of cancer, have fewer side effects and evade cancer-cell resistance.

One way to do that is to vary the structure of the platinum compound, altering its activity. In this case, the researchers studied compounds that are similar to cisplatin, but have only one replaceable chlorine atom. Such a compound can bind to DNA at only one site instead of two.

From early research on platinum compounds done in the 1970s, researchers thought that platinum compounds needed two DNA binding sites to have an effect on cancer cells. However, in the 1980s, it was discovered that certain positively charged platinum compounds that can only bind to DNA at one site have anti-cancer activity, rekindling interest in them.

In 2008, Lippard’s group investigated a compound called pyriplatin, in which one of the chlorine atoms of cisplatin is replaced by a six-membered pyridine ring that includes five carbon atoms and one nitrogen atom. This compound had some anti-cancer activity, but was not as powerful as cisplatin or oxaliplatin, another FDA-approved platinum-based cancer drug.

Lippard then set out to create similar compounds with larger rings, which he theorized might be more effective at blocking DNA transcription. One of those was phenanthriplatin, the compound described in the new PNAS paper.

Phenanthriplatin was tested against 60 types of cancer cells as part of the National Cancer Institute’s cancer-drug screening program, and it was found to be four to 40 times more potent than cisplatin, depending on the cancer type. It also showed a different pattern of activity than that of cisplatin, suggesting that it could be used to treat types of cancer against which cisplatin is ineffective.

One reason for the efficacy of phenanthriplatin is that it can get into cancer cells more easily than cisplatin, Lippard says. Previous studies have shown that platinum compounds containing carbon can pass through specific channels, found in abundance on cancer cells, that allow positively charged organic compounds to enter. Another reason is the ability of phenanthriplatin to inhibit transcription, the process by which cells convert DNA to RNA in the first step of gene expression.

Another advantage of phenanthriplatin is that it seems to be able to evade some of cancer cells’ defenses against cisplatin. Sulfur-containing compounds found in cells, such as glutathione, can attack platinum and destroy it before it can reach and bind to DNA. However, phenanthriplatin contains a bulky three-ring attachment that appears to prevent sulfur from inactivating the platinum compounds as effectively.

Luigi Marzilli, a professor of chemistry at Louisiana State University, says the new compound appears to be very promising. “It expands the utility of platinum drugs and avoids some of the problems that existing drugs have,” says Marzilli, who was not part of the research team.

The researchers are now conducting animal tests to determine how the drug is distributed throughout the body, and how well it kills tumors. Depending on the results, they may be able to modify the compound to improve those properties, Lippard says.

Live imaging shows response to cancer drugs can be boosted by altering tumor microenvironment

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Posted 16 Apr 2012 — by James Street
Category Chemotherapy, Cisplatin, doxorubicin
Posted On: April 16, 2012 – 4:30pm

Cold Spring Harbor, NY – It should be possible to significantly improve the response of common cancers to existing “classical” chemotherapy drugs, say scientists at Cold Spring Harbor Laboratory (CSHL), by introducing agents that alter the interaction of cancer cells with their immediate surroundings, called the tumor microenvironment.

In research published online today in the journal Cancer Cell, CSHL Assistant Professor Mikala Egeblad and her team report using “live” microscopy to observe how cancer cells in mouse tumors react to the widely used chemotherapeutic agent doxorubicin. They found that selective inhibition of two factors that regulate the tumor microenvironment — enzymes called matrix metalloproteinases (MMPs) and a class of immune signaling molecules called chemokines — made breast tumors in mice more responsive to the drug.

It is well known that genetic mutations and epigenetic changes in cancer cells contribute to a tumor’s capacity to resist treatment. But tumors contain many other cells besides cancer cells and surprisingly little is known about how factors secreted from these non-cancerous cells — “stromal” cells, which constitute the tumor microenvironment – influence drug resistance. Such cells include white blood cells, some of which are inflammatory.

Egeblad’s team used real-time microscopic imaging to scrutinize how cancer cells react to doxorubicin in the context of different tumor microenvironments. The resulting time-lapse movies revealed how drugs flowed through – and leaked out of – blood vessels feeding tumors; the manner and rate at which drugs killed cancer cells in tumors of different stages of advancement; and dynamics of the interactions between cells of the tumor and those of the surrounding stromal tissue, before, during and after drug administration.

“We were able to see clearly that the microenvironment contributes critically to drug response,” Egeblad says, “specifically via regulation of the permeability, or ‘leakiness,’ of blood vessels running through and around the tumor, and also by impacting the local recruitment of inflammatory cells.”

When viewed at the microscopic level, resistance to doxorubicin was found to be associated with tumor stage. Observing tumors continuously following drug administration led to the discovery that this response correlated with the ability of blood vessels to transport doxorubicin to the cancer cells, which was comparatively greatest not early or late, but at intermediate stages of tumor development.

Mice engineered to lack the gene that encodes the MMP9 enzyme, which helps regulate the permeability of blood vessels, “had significantly leakier blood vessels, and this corresponded strikingly with a better response to doxorubicin,” according to Egeblad.

Existing chemical inhibitors of MMP enzymes have failed in clinical trials, she noted. “But our data suggest that these or other drugs that affect vascular permeability could be used to achieve better responses to chemotherapies.”

Another important discovery gleaned from the CSHL team’s real-time imaging was that myeloid cells — inflammatory cells that are one of the most common kinds of non-cancerous cells in tumors — were consistently recruited to the tumor site during chemotherapy [video clip to be posted]. Myeloid cells tend to be drawn to places where cells have died. The team found that this attraction, called chemotaxis, is the result of the activation of signaling by CCL2, a member of a class of immune cell recruiting molecules called chemokines.

By knocking out the gene encoding the receptors (called CCR2) for this chemokine, the team was able to diminish myeloid cell recruitment to the tumor. Importantly, this also resulted in a significantly improved response to doxorubicin and to another commonly used chemotherapy drug, cisplatin. This observation is important because it points to a novel way of potentially boosting the cancer cell-killing effectiveness of chemotherapeutic drugs.

The CSHL team now has the goal of finding additional ways to boost the response chemotherapy by determining how the myeloid cells that are recruited to tumors during chemotherapy contribute to the response of cancer cells to drug treatment.

 

 

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

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Posted 27 Dec 2011 — by James Street
Category Cisplatin, DCA (Dichloroacetate), Mitaplatin

Shanta Dhara and Stephen J. Lipparda,b,1
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 (sent for review August 30, 2009)

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 (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
ownpotency 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.
Full Article Here

Transtympanic Injections of N-acetylcysteine for the Prevention of Cisplatin-induced Ototoxicity: A Feasible Method With Promising Efficacy

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Posted 02 Dec 2011 — by James Street
Category Cisplatin, N-acetylcisteine

Riga, Maria G. MD, PhD; Chelis, Leonidas MD; Kakolyris, Stylianos MD, PhD; Papadopoulos, Stergios MD; Stathakidou, Sofia MD; Chamalidou, Eleni MD; Xenidis, Nikolaos MD, PhD; Amarantidis, Kyriakos MD; Dimopoulos, Prokopios MD; Danielides, Vasilios MD, PhD

Abstract

Objectives: Ototoxicity is a common and irreversible adverse effect of cisplatin treatment with great impact on the patients’ quality of life. N-acetylcysteine is a low-molecular-weight agent which has shown substantial otoprotective activity. The role of transtympanic infusions of N-acetylcysteine was examined in a cohort of patients treated with cisplatin-based regimens.

Patients and Methods: Twenty cisplatin-treated patients were subjected, under local anesthesia, to transtympanic N-acetylcysteine (10%) infusions in 1 ear, during the hydration procedure preceding intravenous effusion of cisplatin. The contralateral ear was used as control. The number of transtympanic infusions was respective to the number of administered cycles. Hearing acuity was evaluated before each cycle with pure tone audiometry by an audiologist blinded to the treated ear.

Results: A total of 84 transtympanic infusions were performed. In treated ears, no significant changes in auditory thresholds were recorded. In the control ears cisplatin induced a significant decrease of auditory thresholds at the 8000 Hz frequency band (P=0.008). At the same frequency (8000 Hz), the changes in auditory thresholds were significantly larger for the control ears than the treated ones (P=0.005). An acute pain starting shortly after the injection and lasting for a few minutes seemed to be the only significant adverse effect.

Conclusions: Transtympanic injections of N-acetylcysteine seem to be a feasible and effective otoprotective strategy for the prevention of cisplatin-induced ototoxicity. Additional studies are required to further clarify the efficiency of this treatment and determine the optimal dosage and protocol.

Research Finds Cancer Drug Cisplatin Binds Like Glue in Cellular RNA

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Posted 25 Nov 2011 — by James Street
Category Cisplatin, RNA, RNAi

University of Oregon researchers have revealed that an anti-cancer drug used extensively in chemotherapy binds pervasively to RNA up to 20-fold more than it does to DNA. This is a surprise finding that suggests new targeting approaches might be useful.

Medical researchers have long known that cisplatin, a platinum compound used to fight tumors in nearly 70 percent of all human cancers, attaches to DNA. Its attachment to RNA had been assumed to be a fleeting thing, says UO chemist Victoria J. DeRose, who decided to take a closer look due to recent discoveries of critical RNA-based cell processes.

“We’re looking at RNA as a new drug target,” she said. “We think this is an important discovery because we know that RNA is very different in tumors than it is in regular healthy cells. We thought that the platinum would bind to RNA, but that the RNA would just degrade and the platinum would be shunted out of the cell. In fact, we found that the platinum was retained on the RNA and also bound quickly, being found on the RNA as fast as one hour after treatment.”

The National Institutes of Health-supported research is detailed in a paper placed online ahead of regular publication in ACS Chemical Biology, a journal of the American Chemical Society. Co-authors with DeRose, a member of the UO chemistry department and Institute of Molecular Biology, were UO doctoral students Alethia A. Hostetter and Maire F. Osborn.

The researchers applied cisplatin to rapidly dividing and RNA-rich yeast cells (Saccharomyces cerevisiae, a much-used eukaryotic model organism in biology). They then extracted the DNA and RNA from the treated cells and studied the density of platinum per nucleotide with mass spectrometry. Specific locations of the metal ions were further hunted down with detailed sequencing methods. They found that the platinum was two to three times denser on DNA but that there was a much higher whole-cell concentration on RNA. Moreover, the drug bound like glue to specific sections of RNA.

DeRose is now pursuing the ramifications of the findings. “Can this drug be made to be more or less reactive to specific RNAs?” she said. “Might we be able to go after these new targets and thereby reduce the drug’s toxicity?”

While cisplatin is effective in reducing tumor size, its use often is halted because of toxicity issues, including renal insufficiency, tinnitus, anemia, gastrointestinal problems and nerve damage.

The extensive roles of RNA have come under intense scrutiny since completion of the human genome opened new windows on DNA, life’s building blocks. It had been assumed that RNA was simply a messenger that coded for protein activity. New technologies, DeRose said, have shown that a vast amount of RNA performs an amazing level of different functions in gene expression, controlling it in specific ways during development or disease, particularly in cancer cells.

In this project, DeRose’s team only explored cisplatin’s binding on two forms of RNA: ribosomes, where the highest concentration of the drug was found; and messenger RNA. There are more areas to be looked at, said DeRose, whose group initially developed experience using and mapping platinum’s activity as a mimic for other metals in her research on RNA enzymes.

DeRose is now planning work with UO colleague Hui Zong, a biologist studying how cancer emerges, to extend the research into mouse cells to see if the findings in yeast RNA hold up. An additional collaboration with UO chemist Michael Haley involves the creation of new platinum-based drugs with “reaction handles” that will allow researchers to easily pull the experimental drugs out of cells, while still attached to their biological targets. New developments in ‘deep’ RNA sequencing, available through the UO’s Genomic Core Facilities, could then provide a much broader view of platinum’s preferred resting sites in the cell.

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

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Posted 11 Aug 2011 — by James Street
Category Cisplatin, DCA (Dichloroacetate), Dog Osteosarcoma, Glucose, Human osteosarcoma research, Molecular, Molecular Osteosarcoma Studies, Osteosarcoma, Warburg Hypothesis
  1. Shanta Dhara and
  2. Stephen J. Lipparda,b,1

+ Author Affiliations


  1. aDepartment of Chemistry and

  2. bKoch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139

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

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 (Δψm) 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 (Δψm) (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 Δψm (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.

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 (Δψm). Opening of the Δψm-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.

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.

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 (Δψm) 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 Δψm. For this reason Δψm 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 Δψm, 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, Δψm 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 Δψm of normal fibroblasts or of cisplatin on Δψm of NTera-2 cells. The detailed results are given in Fig. S4.

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 Δψm) and an increase in fluorescent green-stained mitochondria (loss of Δψm). (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 Δψm compared to normal cells and therefore exhibit increased fluorescence of the Δψm-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 Δψm to the level of normal cells (Fig. 2B). In contrast, mitaplatin did not alter the Δψm 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).

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.

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.

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): νmax 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 (Δψm) 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% CO2. 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).

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.).

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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Curcumin compound improves effectiveness of head and neck cancer treatment, U-M study finds

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Posted 29 Jul 2011 — by James Street
Category Cisplatin, CURCUMIN, Head and Neck

Compound sensitizes resistant cells, allowing lower doses of chemotherapy

IMAGE: This is Thomas Carey, Ph.D., of the University of Michigan Health System.

Click here for more information.

ANN ARBOR, Mich. — A primary reason that head and neck cancer treatments fail is the tumor cells become resistant to chemotherapy drugs. Now, researchers at the University of Michigan Comprehensive Cancer Center have found that a compound derived from the Indian spice curcumin can help cells overcome that resistance.

When researchers added a curcumin-based compound, called FLLL32, to head and neck cancer cell lines, they were able to cut the dose of the chemotherapy drug cisplatin by four while still killing tumor cells equally as well as the higher dose of cisplatin without FLLL32.

The study appears this week in the Archives of Otolaryngology – Head and Neck Surgery.

“This work opens the possibility of using lower, less toxic doses of cisplatin to achieve an equivalent or enhanced tumor kill. Typically, when cells become resistant to cisplatin, we have to give increasingly higher doses. But this drug is so toxic that patients who survive treatment often experience long-term side effects from the treatment,” says senior study author Thomas Carey, Ph.D., professor of otolaryngology and pharmacology at the U-M Medical School and co-director of the Head and Neck Oncology Program at the U-M Comprehensive Cancer Center.

That tumors become resistant to cisplatin is a major reason why head and neck cancer patients frequently see their cancer return or spread. It also plays a big role in why five-year survival for head and neck cancer has not improved in the past three decades.

FLLL32 is designed to sensitize cancer cells at a molecular level to the antitumor effects of cisplatin. It targets a key type of protein called STAT3 that is seen at high levels in about 82 percent of head and neck cancers. High levels of STAT3 are linked to problems with normal cell death processes, which allow cancer cells to survive chemotherapy treatment. STAT3 activation has been associated with cisplatin resistance in head and neck cancer.

Curcumin is known to inhibit STAT3 function, but it is not well-absorbed by the body. FLLL32 was developed by researchers at Ohio State University to be more amenable to use in people. The current study used the compound only in cell lines in the laboratory.

In the current study, researchers compared varying doses of cisplatin alone with varying doses of cisplatin plus FLLL32 against two sets of head and neck cancer cells: one line that was sensitive to cisplatin and one line that was resistant.

They found that FLLL32 decreased the activation levels of STAT3, sensitizing both resistant and sensitive tumor cells to cisplatin. Further, lower doses of cisplatin with FLLL32 were equally effective at killing cancer cells as the higher doses of cisplatin alone.

Separate studies suggest FLLL32 may not be well-absorbed by the body and researchers are developing a next generation compound that they hope improves on that. The U-M team plans to further study this newer compound for its potential as part of head and neck cancer treatment. Clinical trials using this compound are not currently available.

###

Head and neck cancer statistics: 36,540 Americans will be diagnosed with head and neck cancer this year and 7,880 will die from the disease, according to the American Cancer Society

Additional authors: Waleed M. Abuzeid, M.B.B.S.; Samantha Davis, B.S., M.D.; Alice L. Tang, B.A., M.D.; Lindsay Saunders, B.S.; J. Chadwick Brenner, M.S.E.; Emily Light, M.S.; Carol R. Bradford, M.D.; and Mark E.P. Prince, M.D., all from U-M; Jiayuh Lin, Ph.D., from the Nationwide Children’s Hospital, Columbus, Ohio; James R. Fuchs, Ph.D., from The Ohio State University

Funding: National Institute of Dental and Craniofacial Research, Head and Neck Specialized Program of Research Excellence (SPORE) grant, National Cancer Institute, American Cancer Society

Disclosure: None

Reference: Archives of Otolaryngology – Head and Neck Surgery, Vol. 137, No. 5, pp. 499-507

Resources:
U-M Cancer AnswerLine, 800-865-1125
U-M Comprehensive Cancer Center, www.mcancer.org
Clinical trials at U-M, www.UMClinicalStudies.org

Alpha Lipoic Acid and Frataxin: A New Indication for an Old Antioxidant?

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Posted 15 Jun 2011 — by James Street
Category Alpha Lipoic Acid, Alpha Lipoic Acid, antioxidants, Chemotherapy, Cisplatin, docetaxel, Drug Resistance
Alpha Lipoic Acid and Frataxin: A New Indication for an Old Antioxidant?
James W. Russell
James W. Russell, Department of Neurology, University of Maryland School of Medicine & Maryland VA Medical Center, Baltimore, MD 21201, USA;

Correspondence: James W. Russell, M.D., M.S., Department of Neurology, University of Maryland, School of Medicine, 22 South Greene Street, Box 175, Baltimore, MD 21201-1595, Tel:             410-706-6689 begin_of_the_skype_highlighting 410-706-6689 end_of_the_skype_highlighting ; Fax: 410-706-4949 begin_of_the_skype_highlighting 410-706-4949 end_of_the_skype_highlighting, E-mail: JRussell@som.umaryland.edu

Cisplatin is an effective treatment for breast, ovarian, testicular, and small cell lung malignancies, however its use leads to a dose-limiting and cumulative sensory neuronopathy. The effects of cisplatin neurotoxicity can persist for decades (Strumberg et al., 2002). The mechanism of cisplatin toxicity is uncertain, although it has been shown in vitro to reduce fast axonal transport, and induces apoptosis in dorsal root ganglion cells (DRG) by forming high affinity adducts between cisplatin and either genomic or mitochondrial DNA (McDonald et al., 2005; Peltier and Russell 2002). Adduct formation is associated with translocation of the proapoptotic protein Bax to the mitochondrion and release of cytochrome c into the cytosol. This series of events leads to a fas receptor-independent form of programmed cell death (McDonald and Windebank 2002). Cisplatin is frequently administered in combination with paclitaxel and the effect of combination therapy has recently been tested in an animal model (Carozzi et al., 2009). Current data are insufficient to conclude if any tested neuroprotective agents, for example amifostine, diethyldithiocarbamate, glutathione, Org 2766, or Vitamin E prevent or reduce the neurotoxicity of platin drugs (Albers et al., 2007).

Dr. Melli and colleagues in “Alpha-lipoic Acid Prevents Mitochondrial Damage and Neurotoxicity in Experimental Chemotherapy Neuropathy” present an intriguing new mechanism for cisplatin neuronal injury. Using an embryonic day (E-15) DRG culture system, neurons exposed to cisplatin showed a significant reduction in frataxin expression (Melli et al., 2008). Human frataxin is a ~17kDa protein whose deficiency has been associated with Friedreich’s ataxia. Friedreich’s ataxia is a progressive neurodegenerative disease that affects both central and peripheral axons. However, like cisplatin neuropathy, Friedreich’s ataxia is associated with significant degeneration of DRG sensory neurons. Frataxin is intimately associated with several aspects of intracellular iron metabolism and detoxification including iron binding/storage and iron chaperone activity (Campanella et al., 2009). Frataxin also interacts with the electron transport chain proteins, activates glutathione peroxidase, and increases the mitochondrial membrane potential. Frataxin deficiency is associated with a severe deficiency in mitochondrial DNA, an event that results in reduced oxidative phosphorylation and altered antioxidant defenses. Furthermore, a major consequence of the severe depletion of mitochondrial DNA would be mitochondrial bioenergetic failure in the peripheral nervous system (Koch and Britton 2008).
Another observation in the present study is the formation of autophagosomes in DRG treated with cisplatin. Typical double membrane bound vacuoles containing degenerative mitochondria were observed in DRG neurons. Autophagy is an important process involved in the degradation of cytoplasmic organelles and in particular mitochondria. Recent research shows that autophagosomes form on the surface of the mitochondria and they then peel off from mitochondria. Autophagic programmed cell death (type II) is characterized by the accumulation of autophagic vesicles (autophagosomes and autophagolysosomes) and is often observed when massive cell elimination is demanded or when phagocytes do not have easy access to the dying cells (Shintani and Klionsky 2004). It is unclear if autophagy causes neuronal or axonal pathology or is a result of the injury. However despite this uncertainty, the current observations by Melli and colleagues provide a rational explanation for the pathophysiological changes that occur in cisplatin neuropathy.
In the present study, paclitaxel reduced the number of functioning mitochondria in DRG neurons and Schwann cells, induced apoptosis in both cells, and impaired neurite growth. Paclitaxel is a common adjunctive therapy in women with node positive breast cancer. It is frequently used in combination with cisplatin and other chemotherapeutic drugs and is also used for other solid tumors such as ovarian and non-small cell lung cancer. A length-dependent sensorimotor axonal neuropathy is a common dose-dependent side effect of treatment. It can also rarely cause cranial neuropathies, motor involvement and autonomic dysfunction (Peltier and Russell 2006). Paclitaxel binds to tubulin and hyperstabilizes microtubules thus promoting the assembly and reducing the disassembly of microtubules in unmyelinated and myelinated axons. These changes reduce normal axonal transport. Several potential therapies have been assessed in taxol-induced neuropathy including glutamine and calpain inhibitors (Peltier and Russell 2006). However, these potential neuroprotective therapies have not been tested in large randomized clinical trials.
An important observation in the study by Melli et al is the finding that alpha-lipoic acid (α-lipoic acid) prevented mitochondrial damage and that this was dependent on expression of frataxin. α-lipoic acid had neuroprotective effects with both cisplatin and paclitaxel toxicity in cell culture. In contrast to the data with cisplatin, the effect of α-lipoic acid on paclitaxel induced apoptosis was less significant, which is not surprising as apoptosis is not the main toxic mechanism of paclitaxel. In DRG cultures transfected with anti-frataxin siRNA, there was reduced axonal outgrowth. Cisplatin and paclitaxel showed increased neurotoxicity in frataxin knockdown cultures and α-lipoic acid did not prevent the axonal damage as it did in non-transfected cultures. In contrast, α-lipoic acid increased the expression of frataxin in sensory neurons. A further observation was that whereas cisplatin significantly reduces the expression of frataxin, paclitaxel does not. This is despite an increased neurotoxicity in the anti-frataxin siRNA cultures. The implication of this is not clear. Importantly, the α-lipoic acid had to be administered prior to exposure to cisplatin or paclitaxel in order to prevent neurotoxicity. It should be clearly noted that these are cell culture studies and may not be clinically relevant. However, α-lipoic acid has been shown in a small study to improve neuropathy when used post docetaxel/cisplatin treatment in subjects who had already developed peripheral neuropathy (Gedlicka et al., 2003). Patients were treated with 600 mg intravenous α-lipoic acid once a week for 3–5 weeks followed by 1800 mg orally daily for up to 6 months. These results will need to be confirmed in a larger randomized controlled study.
α-lipoic acid is one of the most extensively studied antioxidants. Oxidative stress has been associated with several types of neuropathy including diabetic and chemotherapy-induced neuropathy (Russell and Kaminsky 2005). In the peripheral nerve, α-lipoic acid reduces oxidative stress and the generation of peroxinitrites, inhibits activation of caspases, and improves peripheral nerve endoneurial blood flow. α-lipoic acid in vivo is reduced to active dihydrolipoate and is able to regenerate other antioxidants such as vitamin C, vitamin E, and reduced glutathione through redox cycling. The antioxidant potential of α-lipoic acid has been used to treat several neurological diseases including multiple sclerosis and stroke. However, it has been used most extensively for the treatment of neuropathy, and in particular in diabetic neuropathy. Most experimental diabetic neuropathy studies have shown variable degrees of improvement with α-lipoic acid treatment. Clinical trials have shown mixed results. However, in one of the larger, multicenter, randomized, double-blind, placebo-controlled studies of diabetic neuropathy, there was a small but significant improvement in the neuropathy symptom score but not in other endpoint measures (Ziegler et al., 1999). In general, short term treatment with α-lipoic acid mildly improves both neuropathic symptoms and deficits and the treatment has relatively few side effects.
The observation that α-lipoic acid prevents neuronal and Schwann cell injury in an experimental cell culture model of cisplatin and paclitaxel-induced toxicity and that this is dependent on levels of frataxin, is a novel finding. It remains to be seen whether these observations will prove to be true in toxic neuropathy in humans and if α-lipoic acid will prevent this neurotoxicity. Further basic science studies to examine alternative mechanism/s of action of α-lipoic acid in chemotherapy-induced neuropathy and more robust clinical trials with α-lipoic acid are needed.
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Alpha-lipoic acid prevents mitochondrial damage and neurotoxicity in experimental chemotherapy neuropathy

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Posted 15 Jun 2011 — by James Street
Category Alpha Lipoic Acid, Chemotherapy, Cisplatin, docetaxel, Neuropathy

Giorgia Mellia, Corresponding Author Contact Information, E-mail The Corresponding Author, Michela Taianaa, Francesca Camozzia, Daniela Trioloc, Paola Podinic, Angelo Quattrinic, Franco Taronib and Giuseppe Lauriaa

aNeuromuscular Diseases Unit, IRCCS Foundation Neurological Institute “Carlo Besta”, Via Celoria, 11 20133, Milan, Italy

bBiochemistry and Genetics Units, IRCCS Foundation Neurological Institute “Carlo Besta”, Milan, Italy

cSan Raffaele Vita e Salute University, Milan, Italy

Received 27 June 2008;
revised 8 August 2008;
accepted 21 August 2008.
Available online 9 September 2008.

 

Abstract

The study investigates if alpha-lipoic acid is neuroprotective against chemotherapy induced neurotoxicity, if mitochondrial damage plays a critical role in toxic neurodegenerative cascade, and if neuroprotective effects of alpha-lipoic acid depend on mitochondria protection.

We used an in vitro model of chemotherapy induced peripheral neuropathy that closely mimic the in vivo condition by exposing primary cultures of dorsal root ganglion (DRG) sensory neurons to paclitaxel and cisplatin, two widely used and highly effective chemotherapeutic drugs. This approach allowed investigating the efficacy of alpha-lipoic acid in preventing axonal damage and apoptosis and the function and ultrastructural morphology of mitochondria after exposure to toxic agents and alpha-lipoic acid. Our results demonstrate that both cisplatin and paclitaxel cause early mitochondrial impairment with loss of membrane potential and induction of autophagic vacuoles in neurons. Alpha-lipoic acid exerts neuroprotective effects against chemotherapy induced neurotoxicity in sensory neurons: it rescues the mitochondrial toxicity and induces the expression of frataxin, an essential mitochondrial protein with anti-oxidant and chaperone properties. In conclusion mitochondrial toxicity is an early common event both in paclitaxel and cisplatin induced neurotoxicity. Alpha-lipoic acid protects sensory neurons through its anti-oxidant and mitochondrial regulatory functions, possibly inducing the expression of frataxin. These findings suggest that alpha-lipoic acid might reduce the risk of developing peripheral nerve toxicity in patients undergoing chemotherapy and encourage further confirmatory clinical trials.

Keywords: Neurotoxicity; Mitochondria; Chemotherapy; Alpha-lipoic acid; Frataxin

Article Outline

Introduction
Materials and methods
Neuronal cells and Schwann cells cultures
Neuroprotection assay: axonal outgrowth measurement
Neuroprotection assay: neuronal apoptosis
Mitochondrial function assay
Mitochondrial membrane potential changes assay
Electron microscope analysis
Real-time PCR assay
Small interfering RNA experiments
Results
Alpha-lipoic acid protect sensory neurons against paclitaxel and cisplatin induced axonal damage and apoptosis
Paclitaxel and cisplatin neurotoxicity is associated with a reduction of functioning mitochondria in DRG cultures
Alpha-lipoic acid prevents the early loss of membrane potential differential in mitochondria exposed to paclitaxel and cisplatin
Alpha-lipoic acid rescues mitochondrial morphological abnormalities induced by paclitaxel and cisplatin
Alpha-lipoic acid induces frataxin expression in sensory neurons
Discussion
Acknowledgements
References

The impact of quercetin on cisplatin-induced clastogenesis and apoptosis in murine marrow cells

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Posted 09 Jun 2011 — by James Street
Category Chemotherapy, Cisplatin, quercetin, quercetin
  1. Sabry M. Attia*

+ Author Affiliations


  1. Department of Pharmacology, College of Pharmacy, King Saud University, PO 2457, Riyadh 11451, Saudi Arabia
  1. *To whom correspondence should be addressed. Tel:             +966 542927708 begin_of_the_skype_highlighting +966 542927708 end_of_the_skype_highlighting ; Fax: +966 14677200; Email: attiasm@yahoo.com
  • Received December 5, 2009.
  • Revision received January 10, 2010.
  • Accepted January 14, 2010.

Abstract

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The aim of the present investigation is to determine whether the quercetin in combination with cisplatin can ameliorate cisplatin-induced clastogenesis and apoptosis in the bone marrow cells of mice. The scoring of chromosomal aberrations, micronuclei and mitotic activity were undertaken in the current study as markers of clastogenicity. Apoptosis was analysed by the Annexin V–propidium iodide assay and the occurrence of a hypodiploid DNA peak. Oxidative stress markers such as bone marrow lipid peroxidation and reduced glutathione were assessed as a possible mechanism underlying this amelioration. Quercetin was neither clastogenic nor apoptogenic in mice at doses equivalent to 50 or 100 mg/kg for 2 days. Pre-treatment of mice with quercetin significantly reduced cisplatin-induced clastogenesis and apoptosis in the bone marrow cells and these effects were dose and time dependent. Prior administration of quercetin ahead of cisplatin challenge ameliorated oxidative stress markers. Overall, this study provides for the first time that quercetin has a protective role in the abatement of cisplatin-induced clastogenesis and apoptosis in the bone marrow cells of mice that resides, at least in part, in its antioxidant effects. Therefore, quercetin can be a good candidate to decrease the deleterious effects of cisplatin in the bone marrow cells of cancer patients treated with this drug.

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