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Mitochondrial membrane permeabilization (MMP) is a rate-limiting step of apoptosis, including in anticancer chemotherapy. Adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP on the inner mitochondrial membrane in healthy cells. In addition, ANT can cooperate with Bax to form a lethal pore during apoptosis. Humans possess four distinct ANT isoforms, encoded by four genes, whose transcription depends on the cell type, developmental stage, cell proliferation, and hormone status. Here, we show that the ANT2 gene is up-regulated in several hormone-dependent cancers. Knockdown of ANT2 by RNA interference induced no major changes in the aspect of the mitochondrial network or cell cycle but provoked minor increase in mitochondrial transmembrane potential and reactive oxygen species level and reduced intracellular ATP concentration without affecting glycolysis. At expression and functional levels, ANT2 depletion was not compensated by other ANT isoforms. Most importantly, ANT2, but not ANT1, silencing facilitated MMP induction by lonidamine, a mitochondrion-targeted antitumor compound already used in clinical studies for breast, ovarian, glioma, and lung cancer as well as prostate adenoma. The combination of ANT2 knockdown with lonidamine induced apoptosis irrespective of the Bcl-2 status. These data identify ANT2 as an endogenous inhibitor of MMP and suggest that its selective inhibition could constitute a promising strategy of chemosensitization. (Cancer Res 2006; 66(18): 9143-52)

Supplementary Table S1 from Chemosensitization by Knockdown of Adenine Nucleotide Translocase-2

In the context of obesity, senescent cells accumulate in white adipose tissue (WAT). The cellular underpinnings of WAT senescence leading to insulin resistance are not fully elucidated. The objective of the current study was to evaluate the presence of WAT senescence early after initiation of high‐fat diet (HFD, 1–10 weeks) in 5‐month‐old male C57BL/6J mice and the potential role of energy metabolism. We first showed that WAT senescence occurred 2 weeks after HFD as evidenced in whole WAT by increased senescence‐associated ß‐galactosidase activity and cyclin‐dependent kinase inhibitor 1A and 2A expression. WAT senescence affected various WAT cell populations, including preadipocytes, adipose tissue progenitors, and immune cells, together with adipocytes. WAT senescence was associated with higher glycolytic and mitochondrial activity leading to enhanced ATP content in HFD‐derived preadipocytes, as compared with chow diet‐derived preadipocytes. One‐month daily exercise, introduced 5 weeks after HFD, was an effective senostatic strategy, since it reversed WAT cellular senescence, while reducing glycolysis and production of ATP. Interestingly, the beneficial effect of exercise was independent of body weight and fat mass loss. We demonstrated that WAT cellular senescence is one of the earliest events occurring after HFD initiation and is intimately linked to the metabolic state of the cells. Our data uncover a critical role for HFD‐induced elevated ATP as a local danger signal inducing WAT senescence. Exercise exerts beneficial effects on adipose tissue bioenergetics in obesity, reversing cellular senescence, and metabolic abnormalities., High‐fat diet (HFD) induced senescence of various white adipose tissue (WAT) cell types as one of the earliest events preceding mitochondrial dysfunction, inflammation, and fibrosis. In WAT of HFD‐fed mice and preadipocytes derived from HFD‐fed mice, ATP levels were increased. The anti‐senescence effects of exercise were partially dependent on purine catabolism as blocking xanthine oxidase activity increased ATP levels and inhibited exercise's rescue effects in subcutaneous WAT.

Pharmaceuticals are found in the environment but the impact of this contamination on human and animal health is poorly known. The liver could be particularly targeted since a significant number of these drugs are hepatotoxic, in particular via oxidative stress and mitochondrial dysfunction. Notably, the latter events can also be observed in liver diseases linked to obesity, so that the obese liver might be more sensitive to drug toxicity. In this study, we determined the effects of a chronic exposure to low doses of pharmaceuticals in wild-type and obese mice, with a particular focus on mitochondrial function. To this end, wild-type and ob/ob mice were exposed for 4 months to a cocktail of 11 pharmaceuticals provided in drinking water containing 0.01, 0.1, or 1 mg/L of each drug. At the end of the treatment, liver mitochondria were isolated and different parameters were measured. Chronic exposure to the pharmaceuticals reduced mitochondrial respiration driven by succinate and palmitoyl-l-carnitine in wild-type mice and increased antimycin-induced ROS production in ob/ob mice. Hyperglycemia and hepatic histological abnormalities were also observed in treated ob/ob mice. Investigations were also carried out in isolated liver mitochondria incubated with the mixture, or with each individual drug. The mitochondrial effects of the mixture were different from those observed in treated mice and could not be predicted from the results obtained with each drug. Because some of the 11 drugs included in our cocktail can be found in water at relatively high concentrations, our data could be relevant in environmental toxicology. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1375-1389, 2017.

International audience; Mitochondrial dysfunction can be a major mechanism whereby different drugs can induce adverse effects affecting different tissues such as liver, heart and skeletal muscle. In the most severe cases, drug-induced mitochondrial dysfunction can require a hospitalization, or lead to the death of the patient. Moreover, these adverse effects can lead to the withdrawal of drugs from the market, or earlier during clinical trials. Drugs can induce mitochondrial dysfunction by different mechanisms including inhibition of fatty acid oxidation, impairment of oxidative phosphorylation and respiratory chain activity as well as alteration of the integrity of the mitochondrial membranes. Some drugs also impair mitochondrial function via the production of reactive oxygen species and the generation of reactive metabolites, which can covalently bind to key mitochondrial proteins. The present chapter focuses on different drugs for which enough clinical and experimental evidence indicates the potential role of mitochondrial dysfunction in the pathogenesis of adverse effects such as liver injury, myopathy and cardiotoxicity acetaminophen, amiodarone, doxorubicin, nucleoside reverse transcriptase inhibitors (e.g. stavudine, zidovudine, didanosine), statins (e.g. atorvastatin, cerivastatin, simvastatin) and valproic acid. Notably, these drugs epitomize the diversity of the mechanisms whereby xenobiotics can induce mitochondrial dysfunction and also the variety of the targeted tissues. Other drugs affecting mitochondrial function by similar mechanisms are discussed more briefly in the present chapter. Because drug-induced mitochondrial dysfunction and related adverse events are major issues for public health and pharmaceutical companies, mitochondrial liability should be systematically investigated during preclinical safety studies. © Springer International Publishing AG, part of Springer Nature 2018.

The mitochondrial protein ANT2 is one of the four isoforms of the ADP/ATP translocase and is expressed in highly proliferating cells. ANT2 plays a crucial role in the maintenance of transmembrane potential and mitochondrial integrity in tumor cells by importing glycolytic ATP into the mitochondrial matrix. Thus, this protein is required for tumor cell survival and displays anti-apoptotic function. Recently, ANT2 upregulation was shown to be involved in drug resistance process in various cancer types. Considering ANT2 role in tumor cell metabolism, we searched for ANT2-ligand small molecules. Ligands were first identified by virtual screening of chemical library on 3D model of human ANT2 and validated as ADP/ATP translocase inhibitors using our screening platform on isolated mitochondria. Compound specificity for ANT2 isoform was validated by cellular knock-down and pull-down experiments. ANT2-ligands optimization lead to the selection of MTL105 compound that induce characteristic intrinsic apoptotic cell death, specifically in tumor cell lines at sub-µM concentrations with no effect on healthy cells, suggesting a strong safety margin. According to the NCI60 compare analysis, this compound constitutes a first-in-class product in cancer therapy with this MoA and would be particularly interesting to overcome multidrug-resistant cancers. Citation Format: Nelly Buron, Claire Pertuiset, Roxane Loyant, Cécile Martel, Mathieu Porceddu, Annie Borgne-Sanchez. Identification and optimization of pro-apoptotic molecules targeting adenine nucleotide translocator 2 (hANT2) in tumor mitochondria [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 788.

International audience; Numerous investigations have shown that mitochondrial dysfunction is a major mechanism of drug-induced liver injury, which involves the parent drug or a reactive metabolite generated through cytochromes P450. Depending of their nature and their severity, the mitochondrial alterations are able to induce mild to fulminant hepatic cytolysis and steatosis (lipid accumulation), which can have different clinical and pathological features. Microvesicular steatosis, a potentially severe liver lesion usually associated with liver failure and profound hypoglycemia, is due to a major inhibition of mitochondrial fatty acid oxidation (FAO). Macrovacuolar steatosis, a relatively benign liver lesion in the short term, can be induced not only by a moderate reduction of mitochondrial FAO but also by an increased hepatic de novo lipid synthesis and a decreased secretion of VLDL-associated triglycerides. Moreover, recent investigations suggest that some drugs could favor lipid deposition in the liver through primary alterations of white adipose tissue (WAT) homeostasis. If the treatment is not interrupted, steatosis can evolve toward steatohepatitis, which is characterized not only by lipid accumulation but also by necroinflammation and fibrosis. Although the mechanisms involved in this aggravation are not fully characterized, it appears that overproduction of reactive oxygen species by the damaged mitochondria could play a salient role. Numerous factors could favor drug-induced mitochondrial and metabolic toxicity, such as the structure of the parent molecule, genetic predispositions (in particular those involving mitochondrial enzymes), alcohol intoxication, hepatitis virus C infection, and obesity. In obese and diabetic patients, some drugs may induce acute liver injury more frequently while others may worsen the pre-existent steatosis (or steatohepatitis).

The adenine nucleotide translocator (ANT) is a mitochondrial bi-functional protein, which catalyzes the exchange of ADP and ATP between cytosol and mitochondria and participates in many models of mitochondrial apoptosis. The human adenine nucleotide translocator sub-family is composed of four isoforms, namely ANT1-4, encoded by four nuclear genes, whose expression is highly regulated. Previous studies have revealed that ANTI and 3 induce mitochondrial apoptosis, whereas ANT2 is anti-apoptotic. However, the role of the recently identified isoform ANT4 in the apoptotic pathway has not yet been elucidated. Here, we investigated the effects of stable heterologous expression of the ANT4 on proliferation, mitochondrial respiration and cell death in human cancer cells, using ANT3 as a control of pro-apoptotic isoform. As expected, ANT3 enhanced mitochondria-mediated apoptosis in response to lonidamine, a mitochondriotoxic chemotherapeutic drug, and staurosporine, a protein kinase inhibitor. Our results also indicate that the pro-apoptotic effect of ANT3 was accompanied by decreased rate of cell proliferation, alteration in the mitochondrial network topology, and decreased reactive oxygen species production. Of note, we demonstrate for the first time that ANT4 enhanced cell growth without impacting mitochondria] network or respiration. Moreover, ANT4 differentially regulated the intracellular levels of hydrogen peroxide without affecting superoxide anion levels. Finally, stable ANT4 overexpression protected cancer cells from lonidamine and staurosporine apoptosis in a manner independent of Bcl-2 expression. These data highlight a hitherto undefined cytoprotective activity of ANT4, and provide a novel dichotomy in the human ANT isoform sub-family with ANT1 and 3 isoforms functioning as pro-apoptotic while ANT2 and 4 isoforms render cells resistant to death inducing stimuli. (C) 2010 Elsevier Ltd. All rights reserved.

Increased production of amyloid-β (Aβ)42 peptide, derived from the amyloid-β protein precursor, and its subsequent aggregation into oligomers and plaques constitutes a hallmark of Alzheimer's disease (AD). We here report on a family of low molecular weight molecules, the Aftins (Amyloid-β Forty-Two Inducers), which, in cultured cells, dramatically affect the production of extracellular/secreted amyloid peptides. Aftins trigger β-secretase inhibitor and γ-secretase inhibitors (GSIs) sensitive, robust upregulation of Aβ42, and parallel down-regulation of Aβ38, while Aβ40 levels remain stable. In contrast, intracellular levels of these amyloids appear to remain stable. In terms of their effects on Aβ38/Aβ40/Aβ42 relative abundance, Aftins act opposite to γ-secretase modulators (GSMs). Aβ42 upregulation induced by Aftin-5 is unlikely to originate from reduced proteolytic degradation or diminished autophagy. Aftin-5 has little effects on mitochondrial functional parameters (swelling, transmembrane potential loss, cytochrome c release, oxygen consumption) but reversibly alters the ultrastructure of mitochondria. Aftins thus alter the Aβ levels in a fashion similar to that described in the brain of AD patients. Aftins therefore constitute new pharmacological tools to investigate this essential aspect of AD, in cell cultures, allowing (1) the detection of inhibitors of Aftin induced action (potential 'anti-AD compounds', including GSIs and GSMs) but also (2) the identification, in the human chemical exposome, of compounds that, like Aftins, might trigger sustained Aβ42 production and Aβ38 down-regulation (potential 'pro-AD compounds').

Mitochondrial membrane permeabilization (MMP) is a rate-limiting step of apoptosis, including in anticancer chemotherapy. Adenine nucleotide translocase (ANT) mediates the exchange of ADP and ATP on the inner mitochondrial membrane in healthy cells. In addition, ANT can cooperate with Bax to form a lethal pore during apoptosis. Humans possess four distinct ANT isoforms, encoded by four genes, whose transcription depends on the cell type, developmental stage, cell proliferation, and hormone status. Here, we show that the ANT2 gene is up-regulated in several hormone-dependent cancers. Knockdown of ANT2 by RNA interference induced no major changes in the aspect of the mitochondrial network or cell cycle but provoked minor increase in mitochondrial transmembrane potential and reactive oxygen species level and reduced intracellular ATP concentration without affecting glycolysis. At expression and functional levels, ANT2 depletion was not compensated by other ANT isoforms. Most importantly, ANT2, but not ANT1, silencing facilitated MMP induction by lonidamine, a mitochondrion-targeted antitumor compound already used in clinical studies for breast, ovarian, glioma, and lung cancer as well as prostate adenoma. The combination of ANT2 knockdown with lonidamine induced apoptosis irrespective of the Bcl-2 status. These data identify ANT2 as an endogenous inhibitor of MMP and suggest that its selective inhibition could constitute a promising strategy of chemosensitization. (Cancer Res 2006; 66(18): 9143-52)

orrigendum to “The fourth isoform of the adenine nucleotide ranslocator inhibits mitochondrial apoptosis in cancer cells” Int. J. Biochem. Cell Biol. 42 (2010) 623–629] indy Gallernea,2, Zahia Touata,1, Zhi Xiong Chenb, Cecile Martela,2, Eleonore Mayolaa,2, ssama Sharaf el deina,2, Nelly Buronc,3, Morgane Le Brasa, Etienne Jacototc,4, nnie Borgne-Sanchezc,3, Antoinette Lemoined, Christophe Lemairea,2, Shazib Pervaizb, atherine Brennera,∗,2

Since fifteen years ago, the mitochondrion has been progressively recognized as an integrator-coordinator of apoptosis. In this context recent anti-cancer drugs in development are targeting mitochondrial apoptotic machinery to induce tumor cell death. By this strategy, drugs can interact with apoptosis-regulating proteins of Bcl-2 family to trigger Bax/Bak oligomerization and mitochondrial membrane permeabilization. This event conducts to the release of pro-apoptotic factors such as cytochrome c into the cytosol that are required for caspases activation and apoptosis process. In this study, we selected another strategy and investigated the effect of ANT-targeting small molecules to induce cancer cells apoptosis. The adenine nucleotide translocator (ANT) is a mitochondrial protein of the inner membrane involved in the exchange of ADP and ATP between the mitochondrial matrix and the cytosol. Medicinal chemistry approach coupled with in silico studies yield to several small organic compounds, which proved to be specific for ANT and fulfill druggability criteria (good cell penetration and biodisponibility). We identified a family of compounds that inhibited ATP translocation and induced apoptosis by a pathway involving Bax-dependent cytochrome c release and caspases activation. One of the hit compound was optimized to obtain molecules able to induce apoptosis of HT-29 and BxPC3 cells at 100 nM. We are currently investigating which ANT isoform(s) is (are) targeted in cells. In vivo tumor regression experiments will be performed to achieve the proof of concept of ANT-ligands small molecules as an interesting anti-cancer strategy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4078. doi:10.1158/1538-7445.AM2011-4078

The phosphatidylinositol 3-kinase subunit PI3K is frequently mutated in human cancers and therefore represents an interesting therapeutic target. Cancer resistance to therapies is often related to expression of anti-apoptotic Bcl-2 family members which negatively regulate mitochondrial cell death. We investigated the influence of PI3K mutations on Bcl-2 family proteins both at the cellular and mitochondrial level. We used human breast epithelial cell lines with an endogenous “knock-in” PI3K mutation (HME-1 cells harbouring the H1047R activating mutation) from which we purified the mitochondria to homogeneity (95%). We first characterized these mitochondria for their sensitivity to reference compounds (Calcium, t-Bid) on 3 parameters: swelling, m loss and cytochrome c release. Mitochondrial preparations from wild-type and mutated HME-1 were analysed for their protein pattern in Bcl-2 family members. We observed accumulation of anti-apoptotic (Bcl-xl and Bcl-2) and pro-apoptotic (Bad) proteins in mitochondria isolated from PI3K mutated cells. We showed that the excess of anti-apoptotic proteins at the mitochondria does not change their sensitivity to the Bcl-2 family inhibitor, ABT-737. Investigations with PI3K-inhibitors are currently undergoing to determine the mitochondrial on-target biomarker for drug development. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-14.

Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance on malignant cells. A number of recent anti-cancer strategies are targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to isolate mitochondria from various origins with high purity, quality and functionality. We analyzed the effect of putative Bcl-2 inhibitors on both healthy and tumor isolated mitochondria. We measured mitochondrial membrane permeabilization (swelling), mitochondrial transmembrane potential (ΔΨm) and mitochondrial outer membrane permeabilization (MOMP) in both isolated healthy and tumoral cell mitochondria. Like t-Bid and some BH3 peptides, the molecule ABT-737 was found to present a tumor-specific mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We thus demonstrated that ABT-737 can induce a relatively large and targeted MOMP triggering the release of apoptotic proteins from tumoral cell mitochondria (Cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) however without any mitochondrial swelling. Furthermore, ABT-737 addition to isolated tumoral cell mitochondria induced specific Bax/Bak channel formation by oligomerizing monomeric Bax and/or Bak already inserted in the mitochondrial membrane. We also provided evidence that tumoral cell mitochondria can be either resistant or sensitive to ABT-737-induced MOMP, depending on their origin and interactions between pro- and anti-apoptotic proteins. Finally, this method based on MOMP is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against tumoral cell mitochondria but devoid of toxicity against healthy mitochondria. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1021.

International audience; Current limitations of chemotherapy include toxicity on healthy tissues and multidrug resistance of malignant cells. A number of recent anti-cancer strategies aim at targeting the mitochondrial apoptotic machinery to induce tumor cell death. In this study, we set up protocols to purify functional mitochondria from various human cell lines to analyze the effect of peptidic and xenobiotic compounds described to harbour either Bcl-2 inhibition properties or toxic effects related to mitochondria. Mitochondrial inner and outer membrane permeabilization were systematically investigated in cancer cell mitochondria versus non-cancerous mitochondria. The truncated (t-) Bid protein, synthetic BH3 peptides from Bim and Bak, and the small molecule ABT-737 induced a tumor-specific and OMP-restricted mitochondrio-toxicity, while compounds like HA-14.1, YC-137, Chelerythrine, Gossypol, TW-37 or EM20-25 did not. We found that ABT-737 can induce the Bax-dependent release of apoptotic proteins (cytochrome c, Smac/Diablo and Omi/HtrA2 but not AIF) from various but not all cancer cell mitochondria. Furthermore, ABT-737 addition to isolated cancer cell mitochondria induced oligomerization of Bax and/or Bak monomers already inserted in the mitochondrial membrane. Finally immunoprecipatations indicated that ABT-737 induces Bax, Bak and Bim desequestration from Bcl-2 and Bcl-xL but not from Mcl-1L. This study investigates for the first time the mechanism of action of ABT-737 as a single agent on isolated cancer cell mitochondria. Hence, this method based on MOMP (mitochondrial outer membrane permeabilization) is an interesting screening tool, tailored for identifying Bcl-2 antagonists with selective toxicity profile against cancer cell mitochondria but devoid of toxicity against healthy mitochondria.