Archive for the ‘DCA (Dichloroacetate)’ Category

Cool science, but is it news?

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Posted 04 Jun 2012 — by James Street
Category DCA (Dichloroacetate)

By Kelly Crowe, CBC News

Posted: May 28, 2012 4:45 PM ET

Last Updated: May 28, 2012 4:42 PM ET

The other day a news release with this headline landed in my email: “McMaster University researchers discover drug destroys human cancer stem cells but not healthy ones.” Of course, the writers of the press release know, that I know, that the discovery didn’t just happen in the last few days. The headline is press release code for: “our researchers have been working on this for a few years and they are about to be published in a prominent medical journal, and if their work is deemed significant enough for Cell, it might also be interesting enough for the daily news.” Then it’s up to the folks here at the CBC Health Unit to decide, “is it news?”

It’s something we struggle with every day, trying to decide when to report on raw, technical and basic scientific discoveries, and how to avoid exaggerating the impact of these early, incremental developments.

It’s tempting to get excited. After all, killing cancer cells is one of the major goals of medical research. The problem is, most of the time, in these early stages, scientists are killing cancer cells in petri dishes or mice, not in living, breathing humans. Still, if the discovery is published in a prestigious journal, that, by itself, is an impressive achievement. We are flooded with advance media notices, from the journals, the research institutes and others, all wanting to make sure we know about upcoming publications. But if a Canadian scientist ends up in Nature, or Science or Cell, is that, by itself, newsworthy? Even if the discovery is so early and incremental that it will be a decade, or more, before it makes any contribution to human health, if ever?

Right now, new technology is allowing researchers to throw all kinds of molecules at cancer cells, to see what happens. If a molecule cripples the cancer cells’ ability to proliferate, or causes some other disabling effect, it could, one day, after much research, and many clinical trials, become a new drug, another weapon to add to the cancer arsenal. For the scientists, and others in the drug industry, the race begins to try to answer that question, to push the discovery into the crowded pipeline, to leap into the fight for limited resources to find money for further research, for clinical trials on humans, to see if the molecule works the same way on people as it did in the test tube or in mice. But for most of us, the discovery won’t change anything, except to spark hope that a new cancer breakthrough is imminent, a potentially hazardous side effect to the headline that I will talk about in a moment.

Many things can go wrong along the way, things that will never be reported in the news. The potential drug might fail to show any effect in humans, or it might be toxic, or it might not capture the interest of a drug company and thus might never be pushed through the system. And at the end of it all, if it survives, it might turn out to be no better than drugs already in use. If all goes well, and it clears all the hurdles, at best, it will join the other drugs in the pharmaceutical toolbox and help to turn the 200 or more diseases that we call ‘cancer’ into chronic, treatable conditions. All important work in the business of advancing medicine, but is every one of the those discoveries, if-everything-goes-well-over-the-next-ten-years, news? And does reporting on that very early glimmer of potential end up doing more harm, by raising the desperate hopes of anxious cancer patients?

It has happened before.

Flash back to 2007 and this headline, “Long-used drug shows new promise for cancer.” The drug, in that case, was dichloroacetate, or DCA, a generic drug used to treat rare metabolic disorders. When researchers at the University of Alberta in Edmonton tested it on cancer cells, they discovered that it seemed to flip a switch, turning on a mechanism that causes abnormal cells to die. Again, really cool science. But was it news, when no one knew if it would work?

The media decided, back in 2007, that it was news, and headlines flew around the world, describing a “drug that shrinks cancer cells and can even make tumours disappear.” “No nasty side effects,” the stories said, just dissolve the powder in water, for a mere $2 a dose. One story suggested the discovery could “soon be used to treat many forms of cancer.”

Patients began demanding the drug, even before a single human trial had happened. They started buying it from unscrupulous dealers selling powder that in some cases didn’t even contain the effective ingredient. A U.S. court convicted an Edmonton man for selling corn starch over the internet and calling it DCA.

Meanwhile, the hope generated by the media coverage inspired a local Alberta community to raise the money for a tiny clinical trial, that under other circumstances might have been funded by a drug company. But this was an abandoned drug, long off patent, and not attractive from a profit perspective. Still, committed researchers at the University of Alberta did the hard work, using donated funds, and completed the first clinical trial on five patients with terminal brain cancer. DCA seemed to have an effect, slightly shrinking the tumors in a few of the patients. We reported on this new development in December 2010, three years after the first story. I asked lead researcher Dr. Evangelos Michelakis if the trial results were as exciting as he thought they would be. He said, “it is exciting for me, but I don’t think anybody could claim this drug works based on this trial because there were too few patients.”

So the story ended the same way as it had begun in 2007. The drug shows promise, but it’s a long way from being something patients can use.

At around the same time, a group of researchers at the University of Guelph reported that DCA might not kill cancer cells, but, in fact, might make things worse, by protecting them. It was a preliminary finding, and needed further study, but it’s an example of the kind of complicating questions that arise as early discoveries are tested.

So where does DCA sit now, five years after the original excitement? Stalled, due to lack of interest, according to Dr. Michelakis. “We have not initiated another clinical trial with DCA in cancer,” he told me in an email this week, “It was my hope that other centres, independent of us, will be inspired to do similar trials, but I have not seen any signs that this is the case.”

“I am also disappointed that other investigators have not been interested to test this drug with proper trials on their patients,” he added, “but I understand that without funding (although DCA itself is very cheap) this is very difficult. As I had said in the beginning of this work, taking a generic drug to patients with a deadly disease is as difficult a task as one can imagine in modern medicine, and it requires many people to participate and push the agenda. One person in one centre cannot do it.”

But he has not abandoned research on DCA. Just this week, he has had another paper published in the Journal Oncology, online ahead of print. The paper describes another discovery about DCA, that suggests it can inhibit angiogenesis, (the development of blood vessels), and possibly cut off a tumor’s blood supply, a goal of drugs like Avastin, that have so far failed to live up to their early, and much publicized, promise.

“Our work points to the fact that to inhibit angiogenesis long term, the Avastin-like drugs are not enough because they inhibit the pathway quite downstream allowing the tumor to escape upstream,” Dr. Michelakis said in his email, adding that the mechanism he has identified might work better because it happens earlier in the process.

Once again, we’re still at the beginning of the story. After all the trials, and the set-backs, DCA might or might not become another weapon in the fight against cancer.

And what about the stem cell killer from McMaster? It might also lead to a future cancer therapy, one day. Or it might not. And what about the dozens of other intriguing discoveries that are jamming up my email right now? It’s all really cool science, but is it news?

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

Possible Anti-Cancer Target: Enzyme That Flips Switch on Cells’ Sugar Cravings

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Posted 26 Dec 2011 — by James Street
Category DCA (Dichloroacetate), FGFR1, FGFR1, Glucose, PDHK (pyruvate dehydrogenase kinase), PDHK (pyruvate dehydrogenase kinase), Warburg Hypothesis

research has shown that cancer cells tend to take up more glucose than healthy cells.

Researchers are increasingly interested in exploiting this tendency with drugs that target cancer cells’ altered metabolism.

Cancer cells’ sugar cravings arise partly because they turn off their mitochondria, power sources that burn glucose efficiently, in favor of a more inefficient mode of using glucose. They benefit because the byproducts can be used as building blocks for fast-growing cells.

Scientists at Winship Cancer Institute of Emory University have shown that many types of cancer cells flip a switch that diverts glucose away from mitochondria. Their findings suggest that tyrosine kinases, enzymes that drive the growth of several types of cancer, play a greater role in mitochondria than previously recognized.

The results also highlight the enzyme PDHK (pyruvate dehydrogenase kinase) as an important point of control for cancer cell metabolism.

The results were published online Thursday by the journal Molecular Cell.

“We and others have shown that PDHK is upregulated in several types of human cancer, and our findings demonstrate a new way that PDHK activity is enhanced in cancer cells,” says Jing Chen, PhD, associate professor of hematology and medical oncology at Emory University School of Medicine and Winship Cancer Institute. “PDHK is a very attractive target for anticancer therapy because of its role in regulating cancer metabolism.”

Chen and Sumin Kang, PhD, assistant professor of hematology and medical oncology at Emory University School of Medicine, are co-corresponding authors. Postdoctoral fellows Taro Hitosugi, Jun Fan and Tae-Wook Chung are co-first authors of the paper. Co-authors at Emory include Georgia Chen, PhD, Sagar Lonial, MD, Haian Fu, PhD, and Fadlo Khuri, MD. Collaborators at Yale University, Novartis and Cell Signaling Technology contributed to the paper.

Chen and his colleagues started out studying the tyrosine kinase FGFR1, which is activated in several types of cancer. Tyrosine kinases attach a phosphate to other proteins, making them more or less active. They found that FGFR1 activates the enzyme PDHK, which has a gatekeeper function for mitochondria.

“We used FGFR1 as a platform to look at how metabolic enzymes are modified by oncogenic tyrosine kinases,” Chen says. “We discovered that several oncogenic tyrosine kinases activate PDHK, and we found that many of those tyrosine kinases are found within mitochondria.”

This was a surprise because tyrosine kinases are usually thought to drive growth by being active next to the cell membrane, Chen says.

Introducing a form of PDHK that is insensitive to tyrosine kinases into human cancer cells forces the cells to grow more slowly and form smaller tumors in mice, they found. This indicates that PDHK could be a target for drugs that specifically target cancer cells’ altered metabolism.

The experimental drug dichloroacetate (DCA), which inactivates PDHK, is being used in new clinical trials for cancer. Chen is collaborating with Haian Fu, professor of pharmacology and director of the Emory Chemical Biology Discovery Center, to find other, more potent inhibitors of PDHK.

Dichloroacetate metabolically targeted therapy defeats cytotoxicity of standard anticancer drugs

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Posted 11 Aug 2011 — by James Street
Category Chemotherapy, DCA (Dichloroacetate)

Dirk Heshe, Stephanie Hoogestraat, Christine Brauckmann, Uwe Karst, Joachim Boos and Claudia Lanvers-Kaminsky

Abstract

Purpose

The observation that the orphan drug dichloroacetate (DCA) selectively promotes mitochondria-regulated apoptosis and inhibits tumour growth in preclinical models by shifting the glucose metabolism in cancer cells from anaerobic to aerobic glycolysis attracted not only scientists’, clinicians’ but also patients’ interests and prompted us to further evaluate DCA effects against paediatric malignancies.

Methods

The effects of DCA on mitochondrial membrane potential (ΔΨm), cell viability and induction of apoptosis were evaluated in paediatric tumour cell lines and the non-malignant cell line HEK293. In addition, combinations of DCA with the standard anticancer drugs cisplatin, doxorubicin, and temozolomide were tested and intra- and extra-cellular platinum species analysed.

Results

DCA selectively induced phosphatidylserine externalisation and reduced ΔΨm in paediatric tumour cells compared to HEK293 cells, but DCA concentrations ≤10 mmol/L only moderately inhibited the growth of 18 paediatric tumour cell lines. DCA neither influenced the in vitro stability of cisplatin nor the cellular cisplatin uptake, but it abrogated the cytotoxicity of cisplatin in 7 out of 10 cell lines. DCA also affected the cytotoxicity of doxorubicin but did not influence the cytotoxicity of temozolomide. Despite phosphatidylserine externalisation, DCA failed to activate caspase 3/7 and, moreover, suppressed caspase 3/7 activation by cisplatin and doxorubicin.

Conclusions

Our results indicate that apart from the intriguing effects of DCA on the glucose metabolism of cancer cells, the use of DCA for cancer treatment has to be evaluated carefully. Moreover, compassionate use of the orally available drug by patients with cancer themselves without medical supervision is strongly discouraged at present.

Keywords DCA – Warburg effect – Cancer – Cisplatin – Cytotoxicity

This work fulfils the requirements for the medical doctoral thesis of Dirk Heshe.

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

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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Dichloroacetate (DCA) as a potential metabolic-targeting therapy for cancer

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Posted 11 Aug 2011 — by James Street
Category DCA (Dichloroacetate)

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British Journal of Cancer (2008) 99, 989–994. doi:10.1038/sj.bjc.6604554 www.bjcancer.com
Published online 2 September 2008

Dichloroacetate (DCA) as a potential metabolic-targeting therapy for cancer

E D Michelakis¹, L Webster¹ and J R Mackey²

¹Department of Medicine, University of Alberta, Edmonton, Canada
²Department of Oncology, University of Alberta, Edmonton, Canada

Correspondence: Dr ED Michelakis, Department of Medicine, University of Alberta Hospital, 8440-112 Street, Edmonton, AB, Canada T6G 2B7; E-mail: evangelos.michelakis@capitalhealth.ca

Received 18 December 2007; Revised 28 April 2008; Accepted 4 July 2008; Published online 2 September 2008.

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Abstract

The unique metabolism of most solid tumours (aerobic glycolysis, i.e., Warburg effect) is not only the basis of diagnosing cancer with metabolic imaging but might also be associated with the resistance to apoptosis that characterises cancer. The glycolytic phenotype in cancer appears to be the common denominator of diverse molecular abnormalities in cancer and may be associated with a (potentially reversible) suppression of mitochondrial function. The generic drug dichloroacetate is an orally available small molecule that, by inhibiting the pyruvate dehydrogenase kinase, increases the flux of pyruvate into the mitochondria, promoting glucose oxidation over glycolysis. This reverses the suppressed mitochondrial apoptosis in cancer and results in suppression of tumour growth in vitro and in vivo. Here, we review the scientific and clinical rationale supporting the rapid translation of this promising metabolic modulator in early-phase cancer clinical trials.

Keywords:

mitochondria, metabolism, apoptosis, potassium channels, positron emission tomography, glycolysis

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A paradigm shift is needed in cancer therapeutics

Although some battles have been won since the declaration of the ‘war on cancer’ in 1971 in the United States, the war is ongoing. Despite enormous investments from industry and the public, oncology has an impressively poor success rate in the clinical development of effective investigational drugs; less than a third of that in cardiovascular or infectious diseases (Kamb et al, 2007). Drug development in oncology has typically focused on targets essential for the survival of all dividing cells, leading to narrow therapeutic windows. Non-essential targets offer more selectivity but little efficacy. It is extremely rare to find an essential target that is unique to cancer cells; the dependence of CML cells on Ableson kinase is only induced by a chromosomal translocation in the malignant clone, making the efficacy and selectivity of imatinib for CML an exception in cancer therapy (Kamb et al, 2007). The most important reason for the poor performance of cancer drugs is the remarkable heterogeneity and adaptability of cancer cells. The molecular characteristics of histologically identical cancers are often dissimilar and molecular heterogeneity frequently exists within a single tumour. The view that ‘there are many different types of cancers’ is increasingly shared by scientists and clinical oncologists. This has important implications, including the realisation that specific drugs have to be developed and tested for molecularly defined tumours and effects in one might not necessarily be relevant to another cancer.

The biggest challenge remains the selective induction of cell death (mainly apoptosis) in cancer but not normal cells. Pragmatically, an ideal anticancer therapy would be easily administered (possibly an orally available small molecule) and affordable. Most new anticancer drugs are prohibitively expensive not only for millions of patients from developing countries, but also for many patients without strong medical insurance in developed countries.

One way that the problem of heterogeneity of ‘proximal’ molecular pathways in cancer can be addressed is by targeting more ‘distal’ pathways that integrate several proximal signals, as long as the common distal pathways remain essential and specific to cancer cells. The unique metabolism of most solid tumours integrates many proximal pathways and results in a remodeling of mitochondria (where the regulation of energy production and apoptosis converge), to produce a glycolytic phenotype and a strong resistance to apoptosis. There is now growing evidence that the mitochondria might be primary targets in cancer therapeutics instead of simple bystanders during cancer development. This cancer-specific metabolic remodeling can be reversed by dichloroacetate (DCA), a mitochondria-targeting small molecule, that penetrates most tissues after oral administration (Bonnet et al, 2007; Pan and Mak, 2007). The molecular and direct metabolic response to DCA can also be followed by measuring glucose uptake in tumours by positron emission tomography (PET) imaging, non-invasively and prospectively. Such metabolic strategies might be able to shift the paradigm of experimental therapeutics in oncology.

The preclinical work on DCA (showing effectiveness in a variety of tumours and relatively low toxicity) (Bonnet et al, 2007), its structure (a very small molecule), the low price (it is a generic drug) and the fact that DCA has already been used in humans for more than 30 years, provide a strong rationale for rapid clinical translation. Here, we expand the scientific rationale and discuss several practical points that will be important in the clinical evaluation of DCA as anticancer therapy.

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The metabolism of cancer cells

Most cancers are characterised by aerobic glycolysis (GLY), that is, they use glycolysis for energy production, despite the fact that oxygen is present. In 1929, Warburg first observed this (i.e., the Warburg effect) and suggested it resulted from mitochondrial dysfunction, preventing the mitochondria-based glucose oxidation (GO) (Warburg, 1930). Because GO is far more efficient in generating ATP compared with GLY (producing 36 vs 2 ATP per glucose molecule), cancer cells upregulate glucose receptors and significantly increase glucose uptake in an attempt to ‘catch up’. Positron emission tomography imaging has now confirmed that most solid tumours have significantly increased glucose uptake and metabolism, compared with non-cancerous tissues (Figure 1). This bio-energetic difference between cancer and normal cells, might offer a very selective therapeutic target, as GLY is not typically seen in normal tissues apart from skeletal muscle during strenuous exercise. However, this area of experimental oncology has remained controversial; the glycolytic profile has traditionally been viewed as a result of cancer progression, not a cause and therefore the interest in targeting tumour metabolism has been low. Furthermore, at first glance, the glycolytic profile of cancer is difficult to understand, using an evolutionary model of carcinogenesis. First, why would these highly proliferating and energy-demanding cells rely on GLY rather than the much more efficient GO? Second, GLY results in significant lactic acidosis, which might cause significant toxicity to the surrounding tissues and the cancer cells themselves. Recent advances have caused a rekindling of the metabolic hypothesis of cancer suggesting that these facts are not as conflicting as they appear at first (Gatenby and Gillies, 2004):

Figure 1.

Brain MRI showing a large glioblastoma tumour with areas of necrosis within the tumour and significant brain oedema. On the right, a corresponding FDG-Glucose PET from the same patient shows much higher glucose uptake within the tumour, compared with the surrounding brain tissue.

Full figure and legend (49K)

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Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

Gatenby and Gillies (2004) recently proposed that as early carcinogenesis often occurs in a hypoxic microenvironment, the transformed cells have to rely on anaerobic GLY for energy production. Hypoxia-inducible factor (HIF) is activated in hypoxic conditions and it has been shown to induce the expression of several glucose transporters and most of the enzymes required for GLY (Semenza et al, 1994). For example, HIF induces the expression of pyruvate dehydrogenase kinase (PDK) (Kim et al, 2006), a gate-keeping enzyme that regulates the flux of carbohydrates (pyruvate) into the mitochondria. In the presence of activated PDK, pyruvate dehydrogenase (PDH) is inhibited, limiting the entry of pyruvate into the mitochondria, where GO can take place. In other words, activated PDK promotes completion of GLY in the cytoplasm with metabolism of pyruvate into lactate; inhibited PDK ensures an efficient coupling between GLY and GO.

Initially, tumours compensate by increasing glucose uptake into the cells. Furthermore, Gatenby and Gillies (2004) list a number of mechanisms through which lactic acidosis facilitates tumour growth: breakdown of extra-cellular matrix allowing expansion, increased cell mobility/metastatic potential and (along with HIF) activation of angiogenesis. Although tumours eventually become vascularised and are not significantly hypoxic anymore (although some tumours remain hypoxic at the core because the quality of the neo-vessel formation is poor) the aerobic glycolytic profile persists. This suggests that the (initially adaptive) metabolic remodeling confers a survival advantage to cancer cells. Indeed, recent evidence suggests that transformation to a glycolytic phenotype offers resistance to apoptosis (Plas and Thompson, 2002) (Kim and Dang, 2005, 2006).

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Glycolysis is associated with resistance to apoptosis

Several of the enzymes involved in glycolysis are also important regulators of apoptosis and gene transcription, suggesting that links between metabolic sensors, cell death and gene transcription are established directly through the enzymes that control metabolism (Kim and Dang, 2005). For example, hexokinase activation leads to a significant suppression of apoptosis; activated hexokinase translocates from the cytoplasm to the mitochondrial membranes where it interacts with and suppresses several key components of mitochondria-dependent apoptosis (Pastorino et al, 2005). It is therefore not surprising that hexokinase is upregulated and activated in many cancers (Kim and Dang, 2006). How does this occur? The promoter of hexokinase contains both p53 and HIF response elements and both mutated p53 and activated HIF increase hexokinase expression (Mathupala et al, 1997). In addition, the oncogenic protein Akt is upregulated in many cancers and induces a glycolytic metabolic profile through a number of mechanisms (Elstrom et al, 2004). Akt increases both the expression and activity of hexokinase (Gottlob et al, 2001; Elstrom et al, 2004). The gene that normally antagonises Akt, PTEN, is mutated (loss of function mutation) in a large number of cancers. Very recent data revealed even more links between p53 and metabolism: p53 regulates the expression of a critical enzyme of GLY through the production of TIGAR and is also directly regulating the expression of a subunit of cytochrome c oxidase, an important element of complex IV of the electron transport chain in mitochondria (reviewed in (Pan and Mak, 2007)). In other words, the most common molecular abnormality in cancer, that is, the loss of p53 function, induces metabolic and mitochondrial changes, compatible with the glycolytic phenotype. Likewise, the c-myc transcription factor increases the expression of many enzymes of GLY and can induce this same metabolic phenotype (Kim and Dang, 2005, 2006).

To conclude, an evolutionary theory of carcinogenesis identifies metabolism and GLY as a critical and early adaptive mechanism of cancer cells against hypoxia, that persist because it offers resistance to apoptosis in cancer cells (Gatenby and Gillies, 2004). The genetic theory on carcinogenesis, also identifies GLY and metabolism as an end result of activation of many diverse oncogenes, including c-myc, Akt/PTEN and p53 (Pan and Mak, 2007). Therefore, it is possible that this metabolic phenotype is centrally involved in the pathogenesis of cancer and is not simply a ‘by-product’ of carcinogenesis. Although it is not clear whether this metabolic phenotype directly induces malignancy, it certainly ‘facilitates’ carcinogenesis (Kim and Dang, 2006). In addition, this metabolic signature is the common denominator of multiple and diverse pathways; which means that if it is therapeutically targeted it might offer selectivity for malignant cells of diverse cellular and molecular origins.

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Mitochondria and apoptosis

Shifting metabolism away from mitochondria (GO) and towards the cytoplasm (GLY), might suppress apoptosis, a form of cell death that is dependent on mitochondrial energy production (Figure 2). Pro-apoptotic mediators, like cytochrome c and apoptosis-inducing factor, are protected inside the mitochondria. When the voltage- and redox-sensitive mitochondrial transition pore (MTP) opens, they are released in the cytoplasm and induce apoptosis, although it is possible that this can occur without MTP opening (Halestrap, 2005). Mitochondrial depolarisation and increased ROS are associated with opening of the MTP (Zamzami and Kroemer, 2001). Mitochondrial membrane potential and ROS production are dependent on the flux of electrons down the electron transport chain (ETC), which in turn are dependent on the production of electron donors (NADH, FADH₂) from the Krebs’ cycle. Suppressing the entry of pyruvate into the mitochondria and thus the production of acetyl-CoA, will suppress both Krebs’ cycle and the ETC and thus MTP opening and apoptosis.

Figure 2.

A glycolytic environment is associated with an antiapoptotic and pro-proliferative state, characterizing most solid tumours. Increase entry of pyruvate into the mitochondria by either DCA or inhibition of LDH, promotes glucose oxidation, increased apoptosis and decreased proliferation and tumour growth (see text for discussion).

Full figure and legend (148K)

Mitochondria can also affect downstream mechanisms involved in proliferation and apoptosis. For example, mitochondria uptake can directly regulate intracellular Ca⁺⁺, the increase of which is associated with increased proliferation and activation of many transcription factors. Also, the mitochondria-produced superoxide can be dismutated to H₂O₂ through the manganese superoxide dismutase and diffuse freely, activating plasma membrane K⁺ channels, thereby regulating the influx of Ca⁺⁺ and the activity of caspases. K⁺ channels are transmembrane proteins allowing the passage of K⁺ ions through the plasma membrane. Closing of K⁺ channels or decreasing their expression results in an increase in [K⁺]_i which, in turn, increases the tonic inhibition that cytosolic K⁺ exerts on caspases (Remillard and Yuan, 2004). The voltage-gated family of K⁺ channels (Kv) is redox-sensitive and therefore can be regulated by the mitochondria. For example, mitochondria-derived H₂O₂ can activate certain Kv channels, like Kv1.5 (Bonnet et al, 2007).

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DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

We recently showed that several cancer cell lines (non-small cell lung cancer, breast cancer and glioblastoma) had hyperpolarised mitochondria, compared with non-cancer cell lines (Bonnet et al, 2007), a finding that was first described by Dr Chen at the Dana Farber Institute in the 1980s (Chen, 1988). This was associated with suppressed levels of mitochondria-derived ROS and decreased activity and expression of Kv channels. The Ca⁺⁺-sensitive transcription factor NFAT was also active (i.e., nuclear) in the cancer cells. NFAT is a transcription factor that has been shown to increase the levels of the antiapoptotic bcl-2 and decrease the levels of the Kv channel Kv1.5. All of these features are compatible with an antiapoptotic state and could be secondary to a suppressed mitochondrial activity: decrease entry of pyruvate would eventually result in decrease flux of electrons in the ETC and therefore decreased ROS production, closing of the existing redox-sensitive Kv channels and increased intracellular Ca⁺⁺. The decreased ROS could also contribute to closure of the redox-sensitive MTP and mitochondrial hyperpolarisation. The decreased entry of pyruvate into the mitochondria (and therefore the decreased GO) would result in compensatory GLY. Increased hexokinase levels would contribute to the hyperpolarisation of the mitochondria; increased hexokinase in a glycolytic environment is known to be translocated to the mitochondrial membrane, inhibiting the voltage-dependent anion channel (a component of the MTP), resulting in hyperpolarisation and suppression of apoptosis (Pastorino et al, 2005) (Figure 2).

Dichloroacetate activated the pyruvate dehydrogenase, which resulted in increased delivery of pyruvate into the mitochondria. As predicted, DCA increased GO and depolarised the mitochondria, returning the membrane potential towards the levels of the non-cancer cells, without affecting the mitochondria of non-cancerous cells (Figure 2). Remarkably, all the above features of the cancer cells were ‘normalised’ following the increase in GO and the mitochondrial depolarisation: ROS increased, NFAT was inactivated and function/expression of Kv channels was increased. Most importantly, apoptosis was induced in the cancer cells with both cytochrome c and apoptosis-inducing factor efflux from the mitochondria. This resulted in a decrease in tumour growth both in vitro and in vivo in xenotransplant models (Bonnet et al, 2007) (Figure 3). In addition to the induction of apoptosis by DCA in non-small cell lung cancer, breast cancer and glioblastoma cell lines reported in our original publication (Bonnet et al, 2007), very recently DCA was shown to induce apoptosis in endometrial (Wong et al, 2008) and prostate (Cao et al, 2008) cancer cells by largely the same mechanism, independently confirming our results. Furthemore, as predicted, activating mitochondria by DCA increases O₂ consumption in the tumour and dramatically enhances the effectiveness of hypoxia-specific chemotherapies in animal models (Cairns et al, 2007).

Figure 3.

Dichloroacetate depolarises mitochondria and suppresses tumour growth in vivo. On the left, non-small cell lung cancer cells are loaded with TMRM before and after treatment with DCA (the higher the red fluorescence the higher the mitochondrial membrane potential; nuclei in blue). The same cells were injected in the flank of nude rats. On the right these rats are imaged with a rodent PET-CT (GammaMedica). Simultaneous CT and FDG-Glucose PET imaging shows that DCA therapy decreases both the size and the glucose uptake in the tumour.

Full figure and legend (149K)

It is important here to clarify that simply inhibiting GLY, will not promote pyruvate entry into the mitochondria, that is, it will not re-activate mitochondria. It will also be toxic to several non-cancerous tissues that depend on GLY for energy production. Inhibiting GLY (which has previously been tested as a potential treatment for cancer) results in ATP depletion and necrosis, not apoptosis, because apoptosis is an energy-consuming process, requiring active mitochondria (Xu et al, 2005). The ‘trick’ is to enhance the GLY to GO coupling, not just inhibit GLY. One of the ways that this can happen is by activating PDH, or inhibiting LDH, bringing pyruvate into the mitochondria and enhancing GO (Figure 2). This hypothesis is also supported by the recently published work that inhibition of LDH (by siRNA), which promotes the transfer of pyruvate into the mitochondria (in that sense mimicking DCA), also promotes cancer apoptosis and decreases tumour growth in vitro and in mice xenotransplants (Fantin et al, 2006).

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DCA: mechanism of action and clinical experience

Dichloroacetate is a small molecule of 150 Da (see structure in Figure 3) explaining in part the high bioavailability of this drug and the fact that it can penetrate into the traditional chemotherapy sanctuary sites, including the brain. In vitro, DCA activates PDH by inhibition of PDK at concentration of 10–250 μM or 0.15–37.5 μg ml⁻¹ in a dose-dependent fashion (Stacpoole, 1989). To date, four different isoforms of PDK have been identified that have variable expression and sensitivity to the inhibition by DCA (Sugden and Holness, 2003). The isozyme constitutively expressed in most tissues and with the highest sensitivity to DCA is PDKII; in our published preclinical work we showed that PDK2 inhibition with siRNA completely mimicked DCA effects (Bonnet et al, 2007).

Oral DCA can achieve 100% bioavailability. Many studies using IV and oral DCA aimed to identify the optimal dose for DCA. The end point measured was the decrease in lactate levels in both the blood and the cerebrospinal fluid. A decrease in lactate levels is the immediate result of the inhibition of PDK (and thus activation of PDH) by DCA. Several studies treated patients with DCA and directly measured PDH activity in muscle biopsies. Dichloroacetate administered at 35–50 mg kg⁻¹ decreases lactate levels by more than 60% and directly activates PDH by 3–6 fold (Howlett et al, 1999; Parolin et al, 2000).

Although the pharmacokinetics of DCA in healthy volunteers follow a simple one-compartment model, they are more complex in severely abnormal states like severe lactic acidosis or cirrhosis. Dichloroacetate inhibits its own metabolism by an unknown mechanism, and the clearance of DCA decreases after multiple doses (Stacpoole et al, 2003). Although the initial half-life with the first dose is less than one hour, this half-life increases to several hours with subsequent doses. However, there is a plateau of this effect and DCA serum levels do not continue to rise with chronic use. This is also true for DCA metabolites (which do not have any biologic effect, at least on PDH). For example, the serum DCA levels after 5 years of continued treatment with oral DCA at 25 mg kg⁻¹ are only slightly increased compared with the levels after the first several doses (and remain in the range of approximately 100 μg ml⁻¹) (Mori et al, 2004). The effects on lactate levels are sustained and persist after the DCA levels decrease, because the inhibition of PDK is not immediately reversible; DCA ‘locks’ PDK in a sustained inactive state.

A large number of children and adults have been exposed to DCA over the past 40 years, including healthy volunteers and subjects with diverse disease states. Since its first description in 1969 (Stacpoole, 1969), DCA has been studied to alleviate the symptoms or the haemodynamic consequences of the lactic acidosis complicating severe malaria, sepsis, congestive heart failure, burns, cirrhosis, liver transplantation and congenital mitochondrial diseases. Single-arm and randomised trials of DCA used doses ranging from 12.5 to 100 mg kg⁻¹ day⁻¹ orally or intravenously (reviewed in (Stacpoole et al, 2003)). Although DCA was universally effective in lowering lactate levels, it did not alter the course of the primary disease (for example sepsis).

More than 40 nonrandomised trials of DCA in small cohorts of patients have been reported, but the first two randomised control trials of chronic oral therapy with DCA in congenital mitochondrial diseases were reported in 2006. In the first, a blinded placebo-controlled study was performed with oral DCA administered at 25 mg kg⁻¹ day⁻¹ in 30 patients with MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) (Kaufmann et al, 2006). Most patients enrolled in the DCA arm developed symptomatic peripheral neuropathy, compared with 4 out of 15 in the placebo arm, leading to the termination of the study. Seventeen out of 19 patients had at least partial resolution of peripheral neurological symptoms by 9 months after discontinuation of DCA. This neurotoxicity resembled the pattern of length-dependent, axonal, sensorimotor polyneuropathy without demyelination. No other toxicities were reported. It is important to note that peripheral neuropathy often complicates MELAS because of primary or secondary effects on peripheral nerves; for example these patients also have diabetes and diabetes-related peripheral neuropathy.

In contrast, another randomised placebo-controlled double-blinded study failed to show any significant toxicity of DCA, including peripheral neuropathy. In this study only one of 21 children with congenital lactic acidosis treated with DCA orally at 25 mg kg⁻¹ day⁻¹ for 6 months demonstrated mild peripheral neuropathy. Serial nerve conduction studies failed to demonstrate any difference in incidence of neuropathy in the 2 arms (placebo vs DCA). Sleepiness and lethargy, muscular rigidity of the upper extremity and hand tremor were reported in one patient in each group (Stacpoole et al, 2006).

The higher incidence of peripheral neuropathy in adult MELAS patients may represent an intrinsic predisposition to this complication in MELAS or its associated conditions, that is, diabetes mellitus; this toxicity might also be age-dependent. In summary, peripheral neuropathy is a potential side effect of DCA that appears to be largely reversible. As peripheral neuropathy is a frequent complication of taxane, platinum and vinca-alkaloid chemotherapies, the risk for DCA-associated peripheral neuropathy may depend on whether cancer patients have prior or concurrent neurotoxic therapy.

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DCA: clinical testing in cancer?

There is substantial evidence in preclinical in vitro and in vivo models that DCA might be beneficial in human cancer (Bonnet et al, 2007; Cairns et al, 2007; Cao et al, 2008; Wong et al, 2008). The concept is strengthened by the fact that LDH inhibition in mice with human cancer xenotransplants, also induced apoptosis and inhibited growth, improving survival (Fantin et al, 2006). There is also 40 years of human experience with mechanistic studies of DCA in human tissues after oral use, pharmacokinetic and toxicity data from randomised studies for 6 months, and 5-year use case reports. This supports an easy translation to early-phase clinical trials.

Dichloroacetate could be tested in a variety of cancer types. The realisation that (i) a diverse group of signalling pathways and oncogenes result in resistance to apoptosis and a glycolytic phenotype, (ii) the majority of carcinomas have hyperpolarised/remodeled mitochondria, and (iii) most solid tumours have increased glucose uptake on PET imaging, suggest that DCA might be effective in a large number of diverse tumours. However, direct preclinical evidence of anticancer effects of DCA has been published only with non-small cell lung cancer, glioblastoma and breast, endometrial and prostate cancer. In addition, the lack of mitochondrial hyperpolarisation in certain types of cancer, including oat cell lung cancer, lymphomas, neuroblastomas and sarcomas (Chen, 1988), suggest that DCA might not be effective in such cases. Cancers with limited or no meaningful therapeutic options like recurrent glioblastoma or advanced lung cancer should be on top of the list of cancers to be studied.

No patient with cancer has received DCA within a clinical trial. It is unknown whether previously studied dose ranges will achieve cytotoxic intra-tumoral concentrations of DCA. In addition, the overall nutritional and metabolic profile of patients with advanced cancer differs from those in the published DCA studies. Furthermore, pre-exposure to neurotoxic chemotherapy may predispose to DCA neurotoxicity. Carefully performed phase I dose escalation and phase II trials with serial tissue biopsies are required to define the maximally tolerated, and biologically active dose. Clinical trials with DCA will need to carefully monitor neurotoxicity and establish clear dose-reduction strategies to manage toxicities. Furthermore, the pharmacokinetics in the cancer population will need to be defined.

The preclinical experience with DCA monotherapy warrants clinical trials with DCA as a single agent or in direct comparison with other agents. However, as it ‘unlocks’ cancer cells from a state of apoptosis resistance, DCA might be an attractive ‘apoptosis-sensitizer’ agent. In that sense, DCA could both precede and be given concurrently with chemotherapy or radiation therapy, in an attempt to increase their effectiveness, decrease the required doses and limit the toxicity of standard therapies (Cairns et al, 2007).

The ability to approach metabolism as an integrator of many diverse signalling pathways, prompts consideration of the imaging and diagnostic studies that might track metabolic modulation. As discussed above, important questions that need to be answered in clinical trials using DCA include: (i) can PET be used as a predictor of clinical response or as a means of documenting non-invasively a reversal of the glycolytic phenotype in response to DCA? (ii) can mitochondrial membrane potential or the acute effects on DCA in fresh tumour biopsies, predict clinical response to DCA and facilitate patient selection?

Funding for such trials would be a challenge for the academic community as DCA is a generic drug and early industry support might be limited. Fundraising from philanthropies might be possible to support early phase I–II or small phase III trials. However, if these trials suggest a favourable efficacy and toxicity, the public will be further motivated to directly fund these efforts and national cancer organisations like the NCI, might be inspired to directly contribute to the design and structure of larger trials. It is important to note that even if DCA does not prove to be the ‘dawn of a new era’ (Pan and Mak, 2007), initiation and completion of clinical trials with a generic compound will be a task of tremendous symbolic and practical significance. At this point the ‘dogma’ that trials of systemic anticancer therapy cannot happen without industry support, suppresses the potential of many promising drugs that might not be financially attractive for pharmaceutical manufacturers. In that sense, the clinical evaluation of DCA, in addition to its scientific rationale, will be by itself another paradigm shift.

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Note to proof

Since the acceptance of this review two important papers have confirmed the novel anticancer effects of DCA in prostate and endometrial cancers: Wong JY et al, Dichloroacetate induces apoptosis in endometrial cancer cells. Gynecol Oncol June 2008; 109(3): 394–402 and Gao et al, Dichloroacetate (DCA) sensitizes both wild-type and over expressing Bcl-2 prostate cancer cells in vitro to radiation. Prostate 1 August 2008; 68(11): 1223–1231.

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References

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Archive for the ‘DCA (Dichloroacetate)’ Category

By Kelly Crowe, CBC News

Posted: May 28, 2012 4:45 PM ET

Last Updated: May 28, 2012 4:42 PM ET

Abstract

Purpose

Methods

Results

Conclusions

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

Minireview

Dichloroacetate (DCA) as a potential metabolic-targeting therapy for cancer

Abstract

Keywords:

A paradigm shift is needed in cancer therapeutics

The metabolism of cancer cells

Glycolysis offers an early adaptation to the hypoxic microenvironment in carcinogenesis

Glycolysis is associated with resistance to apoptosis

Mitochondria and apoptosis

DCA reverses the mitochondrial remodeling, unlocking the cancer cells from a state of apoptosis resistance: preclinical work

DCA: mechanism of action and clinical experience

DCA: clinical testing in cancer?

Note to proof

References

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