SIRT3 Promotes BH3 Mimetic S1-induced Apoptosis Through Regulation Of Glucose Metabolism In Ovarian Cancer Cells

Aerobic glycolysis is a key hallmark of many cancers including ovarian cancer, which is characterized by enhanced glucose uptake and lactate secretion, and is abnormally dependent on glycolysis for ATP production even in the presence of oxygen. This metabolic phenotype with a high glucose dependence and increased glucose metabolism may facilitate the proliferation of cancer cells and enable them to evade apoptosis. While investigating cell death, mitochondria was shown to be under extensive metabolic control, and appear to determine whether cells respond to stress in an adaptive or suicidal way. Recently, the manipulation of metabolic derangement through inhibiting glycolysis and targeting mitochondrial apoptotic machinery was proposed as a new strategy to improve ovarian cancer therapy. However, the effect of Bcl-2 inhibitors on ovarian cancer cell metabolism remains unclear.SIRT3 is a member of the sirtuin family of NAD-dependent deacetylases, which preferentially localizes to mitochondria. Through the deacetylation of target enzymes, SIRT3 is involved in processes of energy metabolism, including the respiratory chain, tricarboxylic acid cycle, fatty acid oxidation, and ketogenesis. Moreover, SIRT3 was recently founded in mediating metabolic reprogramming by destabilizing hypoxia-inducible factor 1-alpha, a transcription factor that controls glycolytic gene expression. These findings imply that SIRT3 serves as an important therapeutic target involved in both cancer metabolism and apoptosis.We previously found that S1, a novel pan-BH3 mimetic targeting both Bcl-2 and Mcl-1, may inhibit cancer cell growth and induce apoptosis through mitochondrial pathways and endoplasmic reticulum stress-mediated pathways. The Bcl-2 antiapoptotic proteins are important cancer therapy targets; however, their role in cancer cell metabolism remains unclear. We found that the BH3-only protein mimetic S1, a novel pan Bcl-2 inhibitor, simultaneously interrupted glucose metabolism and induced apoptosis in human SKOV3 ovarian cancer cells, which was related to the activation of SIRT3, a stress-responsive deacetylase. S1 interrupted the cellular glucose metabolism mainly through causing damage to mitochondrial respiration and inhibiting glycolysis. Moreover, S1 upregulated the gene and protein expression of SIRT3, and induced the translocation of SIRT3 from the nucleus to mitochondria. SIRT3 silencing reversed the effects of S1 on glucose metabolism and apoptosis through increasing the level of HK-II localized to the mitochondria, while a combination of the glycolysis inhibitor 2-DG and S1 intensified the cytotoxicity through further upregulation of SIRT3 expression. This study underscores an essential role of SIRT3 in the antitumor effect of Bcl-2 inhibitors in human ovarian cancer through regulating both metabolism and apoptosis. The manipulation of Bcl-2 inhibitors combined with the use of classic glycolysis inhibitors may be rational strategies to improve ovarian cancer therapy. Method（1） The survival rate was examined using MTT assays in SKOV3 cells treated with S1. To initiate the treatment on palpable tumors, SKOV3 xenografts were inoculated subcutaneously into Balb/c nude mice.（2） The apoptotic chromatin condensation was observed by Hoechst 33342 staining using confocal microscopy. The apoptotic effects were further checked through detecting the expression of Bcl-2, Mcl-1, cytoplasmic cytochrome c and the activation of caspase-3 and PARP by western blot.（3） The effects of S1 on glucose metabolism and ATP production were examined through dectecting oxygen consumption rate（OCR） and extracellular acidification rates（ECAR） and the ATP level. The cellular metabolic profiles were also checked by measuring extracellular glucose and lactate levels.（4） The expression levels of the human glucose metabolism-related genes（84 genes） were examined in SKOV3 cells treated by S1（5μM, 6h exposure） using the RT2 Human Glucose Metabolism Profiler TM PCR Array technology.（5） LC/MS technology was used to detect the changes of intracellular metabolites, to observe the overall effect of S1 on cell metabolism, as well as the effect of SIRT3 activation on cell metabolism.（6） The gene and protein expression of SIRT3 were checked with q PCR and western blot. Immunofluorescence studies were carried out to check the colocalization of SIRT3 with TOM20, a mitochondrial marker. The nuclear and mitochondrial expression of SIRT3 were analyzed by western blot.（7） RNA Small interfering RNA（si RNA） sequences targeting human SIRT3（Gen Bank Accession NM₀12239） and a non-target sequence were constructed by Genechem（Shanghai, China）. The SIRT3 protein expression was checked by western blot. The survival rate was examined using MTT assays in transfected SKOV3 cells. The apoptotic effects were further checked through detecting the expression of caspase-3 and the activity of caspase-3/7. The expression of HK-II in the mitochondria was checked by western blot.（8） The effects of a combination of 2-DG with Bcl-2 inhibitors such as ABT737, ABT199, Oba on cell viability was checked by MTT assays. The expression of caspase-3 and SIRT3 were checked by western blot. Results（1） The viability of SKOV3 cells was shown to be decreased by S1 treatment. After S1 treatment, a significant reduction in tumor volume and weight was observed compared with the vehicle-treated control.（2） S1 decreased the expression of Bcl-2 and Mcl-1, and enhanced expression of cleaved caspase-3, cleaved PARP and cytoplasmic cytochrome c in SKOV3 cells. S1 efficiently induces apoptosis in SKOV3 cells.（3） S1 induced a significant decrease in OCR, whereas ECAR increased slightly. S1 was shown to decrease ATP levels in a dose-dependent manner.（4） Significant downregulation of gene expression indicative of glycolysis was induced by S1. The genes encoding enzymes involved in gluconeogenesis and tricarboxylic acid（TCA） cycle were upregulated by S1 treatment. S1 inhibits glycolysis and activates gluconeogenesis and TCA cycle at the m RNA level in SKOV3 cells.（5） Compared with control group, 146 different metabolites were screen out in S1-treated group by the metabonomics detection. Through metabolic pathway analysis using metabolites data combined with PCR array gene changes, we found that TCA, glycolysis/gluconeogenesis and the pentose phosphate pathway were the top three metabolic pathways that affected by S1.（6） By observing the gene and protein expression of SIRT3, we found that S1 upregulated SIRT3 in a time-dependent manner. A decrease in the nuclear expression of SIRT3 was matched by an increase in its colocalization with TOM20, a mitochondrial marker, suggesting that S1 induced the SIRT3 translocation from the nucleus to mitochondria.（7） SIRT3 knockdown alleviated S1-induced growth inhibition compared with control si RNA cells. A significant decrease in caspase-3/7 activity was shown in si SIRT3-SKOV3 cells. The consumption of glucose and lactate secretion after 12 h treatment with S1 were considerably higher in si SIRT3-SKOV3 than control-transfected cells. After S1 treatment, compared with NC group, 137 different metabolites were screen out in si-SIRT3 group by the metabonomics detection. Through metabolic pathway analysis using metabolites data, we found that Alanine-aspartate-glutamate metabolism pathway, Nitrogen metabolism pathway and Aminoacyl-t RNA metabolism pathway were the top three metabolic pathways that affected by S1.（8） 2-DG upregulated the activation of caspase 3 induced by S1, which correlated with the upregulation of SIRT3. Conclusion1. The BH3-only protein mimetic S1 interrupted glucose metabolism and induced apoptosis in human SKOV3 ovarian cancer cells, which was related to the activation of SIRT3.2. S1 interrupted the cellular glucose metabolism mainly through causing damage to mitochondrial respiration and inhibiting glycolysis.3. S1 up-regulated the gene and protein expression of SIRT3, and induced the translocation of SIRT3 from the nucleus to mitochondria.4. SIRT3 silencing reversed the effects of S1 on glucose metabolism and apoptosis, while a combination of the classic glycolysis inhibitor 2-DG and S1 intensified the cytotoxicity through further up-regulation of SIRT3 expression.Taken together, S1 appears to induce cell apoptosis and interrupt glucose metabolism in human ovarian cancer cells, partially through the activation of SIRT3. The manipulation of Bcl-2 inhibitors combined with the use of classic glycolysis inhibitors may be rational strategies to improve ovarian cancer therapy.