6-Ketocholestanol is a recoupler for mitochondria, chromatophores and cytochrome oxidase proteoliposomes

Starkov, Anatoly A.; Bloch, Dmitry A.; Chernyak, Boris V.; Dedukhova, V.I.; Mansurova, S.E.; Severina, Inna I.; Simonyan, Ruben A.; Vygodina, Tatiana V.; Skulachev, Vladimir P.

doi:10.1016/s0005-2728(96)00134-x

Cited by 54 publications

(39 citation statements)

References 47 publications

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“…1, upper curve) that a very low concentration of uncoupler (1 X 10" 9 M SF6847) strongly inhibits state 4 H 2 0 2 production. This effect seems to be due to the SF6847 uncoupling activity rather than to other possible effects of this compound since 6-ketocholestanol, a recoupler specific for SF6847 and carbonylcyanide phenylhydrazone derivatives [15,16], caused a transient activation of the H2O2 generation inhibited by SF6847. Subsequent antimycin A addition strongly stimulated the H2O2 production, this fact confirming the original observation of Boveris and Chance [1].…”

Section: Resultsmentioning

confidence: 95%

High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria

1997

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Formation of Н 2 Ог has been studied in rat heart mitochondria, pretreated with H 2 0 2 and aminotriazole to lower their antioxidant capacity. It is shown that the rate of H 2 0 2 formation by mitochondria oxidizing 6 mM succinate is inhibited by a protonophorous uncoupler, ADP and phosphate, malonate, rotenone and myxothiazol, and is stimulated by antimycin A. The effect of ADP is abolished by carboxyatractylate and oligomycin. Addition of uncoupler after rotenone induces further inhibition of H 2 0 2 production. Inhibition of H 2 0 2 formation by uncoupler, malonate and ADP+Pi is shown to be proportional to the ΔΨ decrease by these compounds. A threshold ΔΨ value is found, above which a very strong increase in H2O2 production takes place. This threshold slightly exceeds the state 3 ΔΨ level. The data obtained are in line with the concept [Skulachev, V.P., Q. Rev. Biophys. 29 (1996), 169-202] that a high proton motive force in state 4 is potentially dangerous for the cell due to an increase in the probability of Superoxide formation.

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Section: Resultsmentioning

confidence: 95%

High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria

1997

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“…We did not observe restoration of mitochondrial coupling in the presence of CAT and GDP, inhibitors of the inner mitochondrial membrane proteins ANT and UCP, respectively. On the other hand, both SR4-and FCCP-mediated uncoupling were significantly inhibited by 6-KCH, a documented mitochondria recoupler (42). The mechanism of action of 6-KCH is not well understood but was suggested to incorporate into the mitochondrial membrane and induce a strong asymmetrical increase in the membrane dipole potential (52).…”

Section: Discussionmentioning

confidence: 99%

“…To further characterize putative SR4-induced uncoupling, HepG2 cells were incubated with the documented mitochondria recoupler 6-KCH, (42) and the MPT blocker CSA prior to exposure to SR4 or FCCP. 6-KCH completely prevented both SR4-and FCCP-stimulated OCR increase (Fig.…”

Section: Sr4 Increases Mitochondrial Respiration In Whole Cells and Imentioning

confidence: 99%

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

Figarola

Singhal

Tompkins

et al. 2015

Journal of Biological Chemistry

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Mitochondrial oxidative phosphorylation produces most of the energy in aerobic cells by coupling respiration to the production of ATP. Mitochondrial uncouplers, which reduce the proton gradient across the mitochondrial inner membrane, create a futile cycle of nutrient oxidation without generating ATP. Regulation of mitochondrial dysfunction and associated cellular bioenergetics has been recently identified as a promising target for anticancer therapy. Here, we show that SR4 is a novel mitochondrial uncoupler that causes dose-dependent increase in mitochondrial respiration and dissipation of mitochondrial membrane potential in HepG2 hepatocarcinoma cells. These effects were reversed by the recoupling agent 6-ketocholestanol but not cyclosporin A and were nonexistent in mitochondrial DNA-depleted HepG2 cells. In isolated mouse liver mitochondria, SR4 similarly increased oxygen consumption independent of adenine nucleotide translocase and uncoupling proteins, decreased mitochondrial membrane potential, and promoted swelling of valinomycin-treated mitochondria in potassium acetate medium. Mitochondrial uncoupling in HepG2 cells by SR4 results in the reduction of cellular ATP production, increased ROS production, activation of the energy-sensing enzyme AMPK, and inhibition of acetyl-CoA carboxylase and mammalian target of rapamycin signaling pathways, leading to cell cycle arrest and apoptosis. Global analysis of SR4-associated differential gene expression confirms these observations, including significant induction of apoptotic genes and down-regulation of cell cycle, mitochondrial, and oxidative phosphorylation pathway transcripts at 24 h post-treatment. Collectively, our studies demonstrate that the previously reported indirect activation of AMPK and in vitro anticancer properties of SR4 as well as its beneficial effects in both animal xenograft and obese mice models could be a direct consequence of its mitochondrial uncoupling activity. Hepatocellular carcinoma (HCC)2 is the most common and severe form of liver cancer, accounting for about 80 -90% of primary liver cancers and 5% of all human cancers. More than 600,000 deaths are attributed to HCC every year with 2:1 ratio for men versus women (1, 2). HCC is a primary cancer of hepatocytes that most typically occurs in the setting of known risk factors, including cirrhosis and chronic hepatitis B virus or hepatitis C virus infections (2, 3), although recently, several lines of evidence suggest that type 2 diabetes is also an independent risk factor for HCC development (4). HCC is an aggressive tumor and represents a major health problem as its incidence is increasing. Systemic chemotherapies have proven ineffective against advanced HCC, so it typically leads to death within 6 -20 months (5). Hepatocarcinogenesis is a multistep process involving inflammation, hyperplasia, and dysplasia that finally leads to malignant transformation. In recent years, mitochondria have been found to provide a novel targeting site for new anticancer drugs (known as "mitocans") that ca...

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“…After 3 min incubation oxygen consumption was measured as described below. In specified cases, mitochondria were treated for 3 min with 2 mg/ml oligomycin A prior to addition of regorafenib, and after 3 min incubation 200 mM 6-ketocholestanol (KC) was added to observe the recoupling effect (Starkov et al, 1997).…”

Section: Mitochondria Isolation and Drug Treatmentmentioning

confidence: 99%

Regorafenib impairs mitochondrial functions, activates AMP-activated protein kinase, induces autophagy, and causes rat hepatocyte necrosis

et al. 2015

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The tyrosine kinase inhibitor regorafenib was approved by regulatory agencies for cancer treatment, albeit with strong warnings of severe hepatotoxicity included in the product label. The basis of this toxicity is unknown; one possible mechanism, that of mitochondrial damage, was tested. In isolated rat liver mitochondria, regorafenib directly uncoupled oxidative phosphorylation (OXPHOS) and promoted calcium overload-induced swelling, which were respectively prevented by the recoupler 6-ketocholestanol (KC) and the mitochondrial permeability transition (MPT) pore blocker cyclosporine A (CsA). In primary hepatocytes, regorafenib uncoupled OXPHOS, disrupted mitochondrial inner membrane potential (MMP), and decreased cellular ATP at 1h, and triggered MPT at 3h, which was followed by necrosis but not apoptosis at 7h and 24h, all of which were abrogated by KC. The combination of the glycolysis enhancer fructose plus the mitochondrial ATPase synthase inhibitor oligomycin A abolished regorafenib induced necrosis at 7h. This effect was not seen at 24h nor with the fructose or oligomycin A separately. CsA in combination with trifluoperazine, both MPT blockers, showed similar effects. Two compensatory mechanisms, activation of AMP-activated protein kinase (AMPK) to ameliorate ATP shortage and induction of autophagy to remove dysfunctional mitochondria, were found to be mobilized. Hepatocyte necrosis was enhanced either by the AMPK inhibitor Compound C or the autophagy inhibitor chloroquine, while autophagy inducer rapamycin was strongly cytoprotective. Remarkably, all toxic effects were observed at clinically-relevant concentrations of 2.5-15μM. These data suggest that uncoupling of OXPHOS and the resulting ATP shortage and MPT induction are the key mechanisms for regorafenib induced hepatocyte injury, and AMPK activation and autophagy induction serve as pro-survival pathways against such toxicity.

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6-Ketocholestanol is a recoupler for mitochondria, chromatophores and cytochrome oxidase proteoliposomes

Cited by 54 publications

References 47 publications

High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria

High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria

SR4 Uncouples Mitochondrial Oxidative Phosphorylation, Modulates AMP-dependent Kinase (AMPK)-Mammalian Target of Rapamycin (mTOR) Signaling, and Inhibits Proliferation of HepG2 Hepatocarcinoma Cells

Regorafenib impairs mitochondrial functions, activates AMP-activated protein kinase, induces autophagy, and causes rat hepatocyte necrosis

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