Original ArticleAG311, a small molecule inhibitor of complex I and hypoxia-induced HIF-1α stabilization
Introduction
In addition to genetic and epigenetic heterogeneity, the concept of metabolic heterogeneity has increased recently. Considerable heterogeneity exists between different tumors, but also within the same tumor. This intratumoral heterogeneity is predominately mediated by genetic diversity, energy demand, and the proximity to vasculature, dictating glucose and oxygen availability. Until recently, cancer cells were thought to rely only on glycolysis for ATP production, even in the presence of sufficient oxygen supply [1], but metabolic heterogeneity demonstrates that there are multiple metabolic phenotypes, going beyond the upregulated glycolysis as observed by Warburg. One of these metabolic phenotypes is mitochondrial respiration, as cancer cells have been shown to have functional mitochondria capable of producing ATP [2], [3], [4].
Anticancer agents acting through mitochondrial inhibition have been described. For example, the anti-diabetic biguanides metformin and phenformin have been shown to exert their anticancer effect by inhibiting complex I, the first enzyme in the mitochondrial electron transport chain (ETC) [5], [6], [7], [8]. BAY-87-2243, another complex I inhibitor, has shown efficacy in a preclinical model of resistant melanoma [9]. The small molecule, IACS-10759, was specifically designed as a complex I inhibitor to target chemoresistant dormant tumors [9], [10]. Complex I, NADH-ubiquinone oxidoreductase, is a crucial player in mitochondrial respiration. It transfers electrons from NADH to reduce ubiquinone to ubiquinol resulting in proton translocation into the mitochondrial intermembrane space, which establishes an electrochemical gradient that is used for ATP synthesis. In order for this process to continue, molecular oxygen is required as the final electron acceptor. Regions of solid tumors often exhibit low oxygen tension (hypoxia), which has been shown to be involved in tumor development, chemo- and radioresistance, and metastasis in addition to tumor bioenergetics [11], [12]. These alterations in bioenergetic processes are mediated in part by increasing gene expression involved in glycolysis and by lowering the activity of the electron transport chain [13], [14], [15]. The major cellular adaptive response to hypoxia is tightly regulated by hypoxia-inducible transcription factor-1α, HIF-1α, which is stabilized in low oxygen tensions, thus an inhibition of complex I can prevent electron transfer and decrease oxygen consumption, which in turn could decrease HIF-1α stabilization [16], [17]. HIF-1α is capable of inducing a broad range of cellular responses including angiogenesis, resistance to apoptosis and tumor energetics/metabolism. Due to the importance of mitochondrial oxidative phosphorylation in hypoxia and in supporting tumor growth, progression and metastasis [18], [19], the development of mitochondrial inhibitors seems well justified.
Herein we investigate the molecular mechanism of a small molecule antitumor agent, AG311. We have previously shown that AG311 significantly reduced primary tumor growth and lung metastases in two breast cancer mouse models, by 81–85% [20]. Upon further investigation, a distinct metabolic mechanism for AG311 emerged. We reported that AG311 rapidly induced necrotic cell death, depolarized the mitochondrial membrane and severely reduced intracellular ATP levels [20]. In the current study, we further define the molecular and antitumor mechanisms and identify complex I of the electron transport chain (ETC) as a likely molecular target of AG311 in isolated cells and in tumors. We show that as a downstream consequence of complex I inhibition, AG311 reduced hypoxia-induced HIF-1α stabilization. Additionally, we show that AG311 synergizes with dichloroacetate (DCA) to increase cell death in cancer cells and increase tumor volume in a xenograft tumor mouse model.
Section snippets
Materials & methods
Additional information is described in the supplemental methods
Preferential cytotoxicity in glucose-deprived cancer cells and mitochondrial depolarization by AG311
Because cancer cells have an increased energy demand, induction of metabolic stress with glucose-depleted media can sensitize them to mitochondrial inhibitors. AG311 showed enhanced cytotoxicity in cancer cells (MDA-MB-435) treated in glucose-depleted medium, whereas docetaxel did not have an effect (Fig. 1A). AG311 has been shown to cause rapid mitochondrial membrane depolarization in cancer cells [20]. The mitochondrial depolarization was more profound in cancerous cells (MDA-MB-435),
Discussion
As a continuation of our previous findings showing that AG311 depolarizes mitochondrial membrane potential and induces necrotic cell death, here we explored the effect of AG311 on mitochondria function. We showed that the AG311-induced mitochondrial membrane depolarization was more pronounced in cancerous cells as compared to normal cells (Fig. 1B). In an endeavor to identify the molecular target of AG311, ETC complexes were tested. It was found that AG311 potently inhibited complex I of the
Acknowledgement
This work was supported, in part, by the National Institutes of Health, National Cancer Institute [Grant CA136944 (A.G.)], by College of Pharmacy startup funds (M.A.I.), and by a Grant-In-Aid of Research, Sigma Xi (A.B.). The authors acknowledge support from the Presbyterian Health Foundation Equipment grant (College of Pharmacy) and the Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health [Grant P20-GM103639]
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2022, Biochimica et Biophysica Acta - Reviews on CancerCitation Excerpt :Many mitochondria-targeting drugs with potential for cancer therapy target electron chain transport complexes. These drugs include complex I inhibitors, such as BAY 87–2243 [199], fenofibrate [200], AG311 [201], and drugs used for type 2 diabetes treatment, such as metformin [202] and canagliflozin [203]. Another complex I inhibitor, IACS-010759, is currently under investigation in phase I clinical trials [204,205].
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