Pierre J. Dilda

A peptide trivalent arsenical inhibits tumor angiogenesis by perturbing mitochondrial function in angiogenic endothelial cells

Mitochondria are the powerhouse of the cell and their disruption leads to cell death. We have used a peptide trivalent arsenical, 4-(N-(S-glutathionylacetyl)amino) phenylarsenoxide (GSAO), to inactivate the adenine nucleotide translocator (ANT) that exchanges matrix ATP for cytosolic ADP across the inner mitochondrial membrane and is the key component of the mitochondrial permeability transition pore (MPTP). GSAO triggered Ca(2+)-dependent MPTP opening by crosslinking Cys(160) and Cys(257) of ANT. GSAO treatment caused a concentration-dependent increase in superoxide levels, ATP depletion, mitochondrial depolarization, and apoptosis in proliferating, but not growth-quiescent, endothelial cells. Endothelial cell proliferation drives new blood vessel formation, or angiogenesis. GSAO inhibited angiogenesis in the chick chorioallantoic membrane and in solid tumors in mice. Consequently, GSAO inhibited tumor growth in mice with no apparent toxicity at efficacious doses.

Mitochondrial Metabolism Inhibitors for Cancer Therapy

ABSTRACTCancer cells catabolise nutrients in a different way than healthy cells. Healthy cells mainly rely on oxidative phosphorylation, while cancer cells employ aerobic glycolysis. Glucose is the main nutrient catabolised by healthy cells, while cancer cells often depend on catabolism of both glucose and glutamine. A key organelle involved in this altered metabolism is mitochondria. Mitochondria coordinate the catabolism of glucose and glutamine across the cancer cell. Targeting mitochondrial metabolism in cancer cells has potential for the treatment of this disease. Perhaps the most promising target is the hexokinase-voltage dependent anion channel-adenine nucleotide translocase complex that spans the outer- and inner-mitochondrial membranes. This complex links glycolysis, oxidative phosphorylation and mitochondrial-mediated apoptosis in cancer cells. This review discusses cancer cell mitochondrial metabolism and the small molecule inhibitors of this metabolism that are in pre-clinical or clinical development.

Noninvasive Imaging of Cell Death Using an Hsp90 Ligand

Cell death plays a central role in normal physiology and in disease. Common to apoptotic and necrotic cell death is the eventual loss of plasma membrane integrity. We have produced a small organoarsenical compound, 4-(N-(S-glutathionylacetyl)amino)phenylarsonous acid, that rapidly accumulates in the cytosol of dying cells coincident with loss of plasma membrane integrity. The compound is retained in the cytosol predominantly by covalent reaction with the 90 kDa heat shock protein (Hsp90), the most abundant molecular chaperone of the eukaryotic cytoplasm. The organoarsenical was tagged with either optical or radioisotope reporting groups to image cell death in cultured cells and in murine tumors ex vivo and in situ. Tumor cell death in mice was noninvasively imaged by SPECT/CT using an (111)In-tagged compound. This versatile compound should enable the imaging of cell death in most experimental settings.

Glutathione S-conjugates as prodrugs to target drug-resistant tumors

Living organisms are continuously exposed to xenobiotics. The major phase of enzymatic detoxification in many species is the conjugation of activated xenobiotics to reduced glutathione (GSH) catalyzed by the glutathione-S-transferase (GST). It has been reported that some compounds, once transformed into glutathione S-conjugates, enter the mercapturic acid pathway whose end products are highly reactive and toxic for the cell responsible for their production. The cytotoxicity of these GSH conjugates depends essentially on GST and gamma-glutamyl transferases (γGT), the enzymes which initiate the mercapturic acid synthesis pathway. Numerous studies support the view that the expression of GST and γGT in cancer cells represents an important factor in the appearance of a more aggressive and resistant phenotype. High levels of tumor GST and γGT expression were employed to selectively target tumor with GST- or γGT-activated drugs. This strategy, explored over the last two decades, has recently been successful using GST-activated nitrogen mustard (TLK286) and γGT-activated arsenic-based (GSAO and Darinaparsin) prodrugs confirming the potential of GSH-conjugates as anticancer drugs.

Abstract 3228: Dichloroacetate reverses the Warburg effect, inhibiting growth and sensitizing breast cancer cells towards apoptosis

The Warburg effect occurs in 90% of tumours and causes a high rate of glycolysis even in the presence of oxygen, resulting in increased lactate production and reduced mitochondrial oxidation of pyruvate. Glucose metabolites are diverted to anabolic processes as a consequence, reducing pyruvate oxidation, hyperpolarizing the mitochondrial membrane potential, causing apoptotic resistance. Dichloroacetate (DCA) is a drug that can reverse the Warburg effect by inhibiting the pyruvate dehydrogenase kinases (PDKs), promoting oxidative metabolism of pyruvate. We are investigating in breast cancer cells (a) the effects of DCA on cell growth, (b) factors governing DCA sensitivity and (c) if DCA can enhance apoptosis induced by 4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid (PENAO), a novel anti-mitochondrial agent. At 5 mM DCA (48 hr treatment) there were 3-40% less viable cells present in MDA-MB-231, MCF7, MDA-MB-468, MCF10AT1, and T-47D breast cancer cell cultures. Growth of MCF10A non-cancerous cells was not affected, showing DCA selectively targets cancer cells. The PDKs have different sensitivities towards DCA inhibition (PDK2>PDK4>PDK1>PDK3). To determine if PDKs governed DCA sensitivity, PDK expression was examined by western blotting. In T-47D cells the expression of PDK2 (Ki 0.2 mM) and low levels of PDK1 (Ki 1 mM) and 3 (Ki 8 mM) correlated with their high sensitivity to DCA treatment. In MCF7 and MDA-MB-468 cells, high expression of PDK3 (Ki 8 mM) may explain their relative insensitivity to DCA. Extracellular lactate was also reduced by 50% at 1 mM and 5 mM DCA in T-47D and MCF7 cells respectively after 24 hr, indicating reversal of the Warburg effect, correlating with the PDK profiles. Induction of PDK1 in MCF7 cells under hypoxia increased sensitivity to DCA, showing the PDK profile still correlated with DCA sensitivity. The ability of DCA to enhance apoptosis induced by PENAO was also examined. The IC50 for PENAO (48 hr) was 3-13 µM for MDA-MB-468, MDA-MB-231, T-47D, MCF7 and MCF10AT1 cells, whereas 12 µM reduced cell viability by only 7% in the non-cancerous MCF10A cells. When combined with 5 mM DCA, the IC50 of PENAO for all cancer cell lines decreased by 15-70%, while toxicity to MCF10A cells was not increased. To measure apoptosis, cells were stained with annexin V and sorted by FACS. Treatment for 48 hr with 5 mM DCA and 5 µM PENAO doubled the proportion of apoptotic cells compared to PENAO alone on T-47D and MDA-MB-231 cells. DCA alone did not inhibit growth or induce apoptosis of MDA-MB-231 cells, thus showing potentiation of apoptosis. We have shown that DCA reverses the Warburg effect, inhibiting growth and enhancing apoptosis. PDKs may be a useful biomarker in determining whether DCA alone will be effective against different tumour types. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3228. doi:1538-7445.AM2012-3228

Optimization of the Antitumor Efficacy of a Synthetic Mitochondrial Toxin by Increasing the Residence Time in the Cytosol

Plasma membrane drug efflux pumps of the multidrug resistance associated protein (MRP) family blunt the effectiveness of anticancer drugs and are often associated with drug resistance. GSAO, a tripeptide trivalent arsenical that targets a key mitochondrial transporter in angiogenic endothelial cells, is an example of a compound whose efficacy is limited by tumor cell expression of MRP isoforms 1 and 2. A cysteine mimetic analogue of GSAO was made, PENAO, which accumulates in cells 85 times faster than GSAO due to increased rate of entry and decreased rate of export via MRP1/2. The faster rate of accumulation of PENAO corresponds to a 44-fold increase in antiproliferative activity in vitro and approximately 20-fold better antitumor efficacy in vivo. This information could be used to improve the efficacy of other small molecule cancer therapeutics.

Metabolism of the Tumor Angiogenesis Inhibitor 4-(N-(S-Glutathionylacetyl)amino)phenylarsonous Acid *

4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO) is a small, synthetic mitochondrial poison that targets angiogenic endothelial cells and is currently being tested in a Phase I/IIa clinical trial. The trivalent arsenical of GSAO reacts with and perturbs adenine nucleotide translocase of the inner mitochondrial membrane of endothelial cells, which leads to proliferation arrest. Three observations indicated that the γ-glutamyl residue of GSAO is cleaved at the endothelial cell surface by γ-glutamyl transpeptidase (γGT). GSAO was found to be an efficient substrate for γGT, endothelial cell accumulation and antiproliferative activity of GSAO was blunted by a competitive substrate and an active site inhibitor of γGT, and the level of cell surface γGT correlated strongly with the sensitivity of cells to GSAO. Using transport inhibitors, it was revealed that the resulting metabolite of GSAO cleavage by γGT, 4-(N-(S-cysteinylglycylacetyl)amino) phenylarsonous acid (GCAO), was transported across the plasma membrane by an organic anion transporter. Furthermore, GCAO is likely processed by dipeptidases in the cytosol to 4-(N-(S-cysteinylacetyl)amino) phenylarsonous acid (CAO), and it is this metabolite that reacts with mitochondrial adenine nucleotide translocase. Taken together, our findings indicate that γGT processing of GSAO at the cell surface is the rate-limiting step in its antiangiogenic activity. This information can explain the kidney toxicity at high doses of GSAO noted in preclinical studies and will aid in the anticipation of potential side effects in humans and in the design of better antimitochondrial cancer drugs.

The tumour metabolism inhibitors GSAO and PENAO react with cysteines 57 and 257 of mitochondrial adenine nucleotide translocase

BackgroundGSAO (4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid) and PENAO (4-(N-(S-penicillaminylacetyl)amino) phenylarsonous acid) are tumour metabolism inhibitors that target adenine nucleotide translocase (ANT) of the inner-mitochondrial membrane. Both compounds are currently being trialled in patients with solid tumours. The trivalent arsenical moiety of GSAO and PENAO reacts with two matrix facing cysteine residues of ANT, inactivating the transporter. This leads to proliferation arrest and death of tumour and tumour-supporting cells.ResultsThe two reactive ANT cysteine residues have been identified in this study by expressing cysteine mutants of human ANT1 in Saccharomyces cerevisiae and measuring interaction with the arsenical moiety of GSAO and PENAO. The arsenic atom of both compounds cross-links cysteine residues 57 and 257 of human ANT1.ConclusionsThe sulphur atoms of these two cysteines are 20 Å apart in the crystal structures of ANT and the optimal spacing of cysteine thiolates for reaction with As (III) is 3-4 Å. This implies that a significant conformational change in ANT is required for the organoarsenicals to react with cysteines 57 and 257. This conformational change may relate to the selectivity of the compounds for proliferating cells.

Mitochondria as targets in angiogenesis inhibition

Angiogenesis is integral to the growth and metastatic spread of tumours, and its targeting is an effective anti-tumour strategy. Currently hundreds of anti-angiogenic therapeutics exist in varying stages of development, a number of which have recently gained US Food and Drug Administration (FDA) approval for the treatment of various human cancers. One class of anti-angiogenic agents directly inhibit endothelial cell function and induce endothelial cell death so as to prevent their integration into new blood vessels. The mitochondria are the focal point for a variety of pro-apoptotic signals, and this review highlights those anti-angiogenic agents that involve the mitochondria in the execution of endothelial cell death. A brief overview of angiogenesis and the mitochondrial apoptotic pathway is also given.

Para to Ortho Repositioning of the Arsenical Moiety of the Angiogenesis Inhibitor 4-(N-(S-Glutathionylacetyl)Amino)Phenylarsenoxide Results in a Markedly Increased Cellular Accumulation and Antiproliferative Activity

The synthetic tripeptide arsenical 4-(N-(S-glutathionylacetyl)amino)p-phenylarsenoxide (p-GSAO) is an angiogenesis inhibitor that inactivates mitochondrial adenine nucleotide translocase (ANT) by cross-linking a pair of matrix-facing cysteine residues. This causes an increase in superoxide levels and proliferation arrest of endothelial cells followed by mitochondrial depolarization and apoptosis. p-GSAO induces proliferation arrest in endothelial cells and is a selective inhibitor of endothelial cells compared with tumor cells. An analogue of p-GSAO has been made in which the arsenical moiety is at the ortho instead of the para position on the phenyl ring. o-GSAO, like p-GSAO, bound to ANT in a dithiol-dependent manner but was approximately 8-fold more efficient than p-GSAO at triggering the mitochondria permeability transition in isolated mitochondria. o-GSAO was an approximately 50-fold more potent inhibitor of endothelial and tumor cell proliferation than p-GSAO. The mechanism of this effect was a consequence of approximately 300-fold faster rate of accumulation of o-GSAO in the cells, which is due, at least in part, to impaired export by the multidrug resistance-associated protein 1. Administration of o-GSAO to tumor-bearing mice delayed tumor growth by inhibiting tumor angiogenesis but there were side effects not observed with p-GSAO administration.