Bcl-2 Inhibitor ABT737 Enhances Apoptotic Sensitivity Via Regulating Glucose Metabolism Of Cisplatin-resistant Ovarian Cancer Cells

Cisplatin is one of the most widely used chemotherapeutic drugs and has been applied in the treatment of ovarian cancer.However,cisplatin resistance can develop,which results in poor prognosis and high patient mortality.Over the last few decades,many biological analyses of the mechanism of cisplatin resistance have been performed,and it is now recognized to be more complicated than originally thought.Recently,the main mechanism of cisplatin resistance in many cancers was thought to be related to evasion of apoptosis.Members of the Bcl-2 proteins family are the major regulators of the apoptotic process and were thought to inhibit apoptosis process.Mitochondria are the junction of cell regulation such as apoptosis,metabolism,redox and signal transduction.Recently,it has been suggested that antiapoptotic Bcl-2 protein located in the outer membrane of mitochondria inhibits mitochondrial complex I by reducing the inhibition of apoptotic protein Bax.Thus,apart from the role in the apoptotic process,the anti-apoptotic members of the Bcl-2 family may also play additional roles related to mitochondrial metabolism to regulate cell fate.Recently,ABT737and S1,powerful inhibitors of anti-apoptotic proteins Bcl-2 have been developed,and it has been found that these compounds can induce apoptosis of cell mitochondrial pathway,and provided a useful tool for studying mitochondrial function of drug-resistant tumor cells.Platinum resistance may be associated with metabolic changes in tumors.80years ago,it was thought that cancer cells are highly dependent on aerobic glycolysis to survive due to mitochondrial dysfunction,which is also called the“Warburg effect”.However,recent studies have demonstrated that the sources of ATP from mitochondria are also of great importance to cancer cells.Recently,evidence in support of the hypothesis that resistance to cytotoxic antineoplastic drugs induces a metabolic shift from aerobic glycolysis toward oxidative phosphorylation（OXPHOS）has been presented,demonstrating that metabolic plasticity plays an important role in tumor recurrence.Therefore,the metabolic phenotypes of drug-resistant cancer cells may differ for different drugs or cancer cell lines.Since the characteristics of glucose metabolism and the function of mitochondria may be more complex in tumor resistant cells,further exploration is needed.Sirtuins are highly conserved protein that regulate the physiological and energy needs of cells in response to metabolic signals.In the Sirtuins members of mitochondria,SIRT3 protein regulates the function of mitochondrial protein mainly through post-transcriptional deacetylation modification.The change of NAD⁺/NADH ratio affects SIRT3 activity.The decrease of SIRT3 activity can reduce the deacetylation modification of mitochondrial protein,inhibit the effective electron transport of electron transport chain,reduce the conversion of NADH to NAD⁺,and further reduce the activity of SIRT3.In combination with our previous experiments,ovarian cancer cisplatin-resistant cells showed high expression of Bcl-2,low expression of SIRT3.We speculate that SIRT3 may be a tumor suppressor gene for ovarian cancer resistant cells.In the process of apoptosis induced by chemotherapeutic drugs,there may be a close relationship between Bcl-2 and SIRT3.In this study,human ovarian cancer cell line SKOV3 and cisplatin-resistant SKOV3/DDP were used to investigate the regulatory effects of Bcl-2 and SIRT3 by using Bcl-2 protein inhibitor ABT737.To analyze the changes of glucose metabolism and mitochondrial function in SKOV3/DDP cells,and to elucidate the role of anti apoptotic Bcl-2 protein in the regulation of cisplatin resistance in ovarian cancer.Methods:1.The sensitivity to cisplatin of cisplatin-resistant SKOV3/DDP cells and cisplatin-sensitive SKOV3 cells was measured by MTT assay.Flow cytometric analysis of untreated SKOV3 or SKOV3/DDP cells to determine the percentage of cells in the G0/G1,S,or G2/M phases of the cell cycle.2.The glucose metabolism is measured by human glucose metabolism PCR array in SKOV3/DDP cells and SKOV3 cells.3.The need for glucose is determined by glucose analogue 2-NBDG and glucose consumption and lactate production were measured in the culture media using glucose and lactate kit,RT-PCR,Glycogen levels and MTT assay.4.The oxygen consumption rate（OCR）,intra oxygen concentration and extracellular acidification rates（ECAR）were determined using the fluorescent oxygen-sensitive and pH-sensitive probes Mito-Xpress and pH-Xtra.The ATP levels were quantified using ATP Kit.5.The mitochondrial produced reactive oxygen species is determined by MitoSox fluorescence and oxidant-sensitive dye DCFH-DA.The key enzyme G6PD of PPP measured by G6PD assay kit.Cellular NADPH content and NADPH/NADP⁺ratio,GSH and GSSG content enzymatic assay in SKOV3 and SKOV3/DDP cells.6.After exposure to ABT737,cell were subjected to MTT assay.The expression of glucose metabolism-related genes was determined using qRT-PCR.Cell apoptosis induction were determined by apoptosis detection kit.Spherical growth of 3D cells was observed by MCTS.7.After exposure to ABT737,the expression of SIRT3 and HIF-1αprotein was detected using immunoflurence and immunoprecipitation.The lactate production was determined using lactate kit.8.Cells were exposed to ABT737 and 2-DG and subjected to test expression of glucose metabolism-related genes was determined using qRT-PCR.The viability of monolayer cells or 3D spheroid by live/dead cell viability assay under fluorescence microscope.Results:1.Cisplatin-resistant SKOV3/DDP cells exhibited considerable resistance to cisplatin,while SKOV3 cells also exhibited sensitivity to cisplatin as determined by the MTT assay.SKOV3/DDP cells were preferentially enriched for G₀/G₁ quiescent cells and had a lower proliferation rate.2.Glucose metabolism is altered in SKOV3/DDP cells.The obtained results indicate the upregulation of glycolysis,the tricarboxylic acid cycle（TCA）cycle,and gluconeogenesis in SKOV3/DDP cells.3.SKOV3/DDP cells show a higher glucose demand,with increased glucose uptake and consumption,decreased lactate production,and overexpression of the glucose metabolism-related genes,elevated glycogen levels and less sensitivity to glucose deprivation.4.SKOV3/DDP cells exhibit an increase in oxygen consumption.The metabolic status of SKOV3/DDP cells might involve downregulation of glycolysis and a shift toward OXPHOS.5.SKOV3/DDP cells reset the redox balance by overexpressing the key enzyme G6PD（glucose-6-phosphate）of PPP（pentose phosphate pathway）to eliminate the cytotoxicity of highly elevated ROS.6.ABT737 sensitizes ovarian cancer cells to cisplatin treatment.ABT737decreased cell viability,glucose metabolism-related genes and oxygen consumption rate of SKOV3/DDP cells significantly compared with that of SKOV3 cells.And the growth of SKOV3/DDP spheroid was significantly inhibited.7.In comparison to SKOV3 cells,the expression of SIRT3 protein,the ratio of NAD⁺/NADH were lower and HIF-1αprotein was higher in SKOV3/DDP cells.After exposure to ABT737 or overexpression of SIRT3,the expression of SIRT3 is upregulated,HIF-1αprotein was downregulated,lactate production was reduced and in SKOV3/DDP cells.SIRT3-mediated deacetylation Of SOD2 and the subsequent increase of its antioxidant activity.8.When being combined with 2-deoxyglucose（2-DG）the anti-cancer effect of Bcl-2 inhibitor ABT737 was greatly potentiated.And the growth of SKOV3/DDP spheroid was significantly inhibited.Conclusions:1.In basal condition,the cell cycle distribution of cisplatin-resistant SKOV3/DDP cells is different from SKOV3 cells,suggesting a possible change in cell glucose metabolism.2.Compared with SKOV3 cells,the expression of glucose metabolism-related genes in SKOV3/DDP cells was significantly different,the dependence on glucose was increased,glycolysis level was relatively lower,and mitochondrial OXPHOS level was relatively higher.3.The stable overexpression of Bcl-2 was involved in the reprogramming of glucose metabolism in SKOV3/DDP cells shifting metabolism towards OXPHOS.Bcl-2 inhibitor ABT737 significantly inhibited the survival rate of 3D cells.the inhibition of Bcl-2 reduced the OXPHOS and sensitivity of SKOV3/DDP cells to cisplatin in a selective manner.The inhibition of glycolysis by 2-DG could increase the inhibitory effect of Bcl-2 inhibitor ABT737 on the growth of 3D cells and affect the level of GSH,ROS.4.ABT737 or overexpression SIRT3 significantly upregulates the expression of SIRT3 and downregulates HIF-1αprotein expression and lactate production in SKOV3/DDP cells.SIRT3-mediated deacetylation of SOD2 and the subsequent increase of its antioxidant activity.The downregulation of HIF-1αprotein further affects the transcription of glucose metabolism-related genes.Bcl-2 inhibitor ABT737 may interfere with glucose metabolism,mitochondrial oxidative phosphorylation,and balance of oxidation and antioxidation in cisplatin-resistant ovarian cancer cells.Mitochondrial dysfunction increases apoptotic sensitivity in cisplatin-resistant ovarian cancer cells.In this study,We focused on the unique metabolic pattern of human ovarian cancer cisplatin-resistant cell line SKOV3/DDP.Bcl-2 inhibitor ABT737 was used to explore the integration of apoptosis and metabolism of anti-apoptotic Bcl-2protein and to find the regulation mechanism of Bcl-2 protein in cisplatin resistant cells.This provides a basis for exploring the role of Bcl-2 in the metabolic reprogramming of cisplatin resistant ovarian cancer cells,and proposes a new therapeutic scheme for the unique metabolic pattern of cisplatin resistant ovarian cancer cells.