Nutrient Starvation Enhances Sensitivity Of Human Ovarian Cancer Cells To Apoptosis Induced By BH3 Mimetic

Ovarian cancer is the most common Gynecologic Cancers with high fatality.Due to decreased anti-tumor activity,Chemotherapy Resistance is the primary cause of patients` death resulted from tumor recurrence.Activation of oncogenes such as anti-apoptotic Bcl-2 proteins is considered to be a reason of tumor evasion of apoptosis in ovarian cancer cells.Thus,small-molecule inhibitors of Bcl-2 provide us a new direction for clinical treatment of ovarian cancer,and serve as a tool to investigate the role of Bcl-2 family proteins in apoptosis.ABT737 is one of the BH3 mimetic molecules,while tumors with high endogenous levels of Mcl-1 and side effects restrict its cytotoxic effects.Improving efficacy of ABT737 and side effects alleviation become a problem to be solved at present.Recently it has been found that tumor metabolism generally play an essential role in tumor therapy and prognosis.Tumor metabolism and cell death are closely related in every respect.Alteration of metabolic environment through nutrient limitation could enhance tumor cells sensitivity to chemotherapy drugs,and also cause Bcl-2 family proteins changes,implying that there are strong ties between Bcl-2 proteins and tumor metabolism.However,the link and regulation between nutrition and anti-apoptotic Bcl-2 proteins are not yet clear.Mitochondria serve as the main metabolic organelles play a link role in the regulation of cell death.And mitochondria are also highly dynamic organelles,which are in the balance of continuous fission and fusion.Mitochondria are very sensitive to metabolic stress,their morphologies usually changed under nutrient starvation.Furthermore,the balance of mitochondrial fusion and fission is tightly regulated by signal transduction and stress response pathways.Studies have confirmed that Bcl-2 family proteins can participate in the regulation of the balance of mitochondrial fusion and fission.Thus,we speculate that Bcl-2 proteins and tumor metabolism may determine cell survival and death via the regulation of mitochondrial dynamics.Therefore,we use Earle’s balanced salt solution（EBSS）as a nutrient starvation model and ABT737 as a small-molecule inhibitor of Bcl-2/Bcl-x L/Bcl-W.Human ovarian cancer cells SKOV3 were treated by EBSS combined with ABT737,and Glucose serves as nutrient supplement,to examine the role of tumor glycolysis in nutrient starvation resistance,and along with changes in mitochondrial dynamics,to explore the mechanism that nutrient starvation influences the sensitivity to apoptosis induced by BH3 mimetic.Methods:According to the design of the experiments,we set up the control group and the treatment group which included alone treatment group and combined treatment group,to conduct the following experiments respectively.（1）Cell viability was measured by MTT assay.Cellular morphology was determined by light microscopy.Hochest 33342 was used to stain and observe apoptotic nuclear morphology;apoptotic cells were observed by TUNEL staining and apoptotic rate was evaluated by flow cytometry.（2）The levels of ROS were measured by DCFH-DA staining.JC-1 staining and flow cytometry were used to determine mitochondrial potential.（3）Cell energy production was detected by ATP assay;production of lactic acid was measured by lactic acid production detection kit.（4）The m RNA levels of aerobic glycolysis-associated genes GLUT1,LDHA and HKII were evaluated by real-time PCR.（5）Nuclear and mitochondrial morphologies were observed by laser confocal microscope.Immunostainning and laser confocal microscope were applied to determine the co-location between Drp1 or Fis1 and mitochondria.（6）According to the sequence of Bcl-2（Gen Bank:NM₀00633）,p GPU6/ GFP/Neo/RNAi vector was used to construct RNAi recombinant plasmid named Si-Bcl-2.The transfected rate was observed by the cells percentage marked by green fluorescence.The protein expression of Bcl-2 was analyzed by Western blot.（7）Total protein were extracted,and the expression of aerobic glycolysis-associated proteins HKII,PDK1 and PDHB,mitochondrial fusion and fission protein OPA1,Mfn2,Drp1 and Fis1,Cleaved-Caspase3 and cytochrome C,and Bcl-2 family proteins were analyzed by Western blot.Both mitochondrial and cytoplasmic fraction proteins were extracted by cell mitochondria isolation kit.The mitochondrial fraction protein levels of Drp1,Fis1,OPA1 and Mfn2,and Bcl-2 family Bax/Bak were analyzed by Western blot.The cytoplasmic fraction protein levels of Omi/Htr A2,cytochrome C and Cleaved-Caspase3 were evaluated by Western blot.Results:1.ABT737 combined with EBSS can effectively reduce cell viability of SKOV3,but the same concentration of ABT737 or EBSS seldom reduced cell viability,Glucose supplement can alleviate the reduction of cell viability.Compared with EBSS alone group,ABT737 combined with EBSS decreased cell density and caused cell shrinkage.Both No-glucose DMEM alone and combined with ABT737 caused cell shrinkage with no obvious difference.2.ABT737 combined with EBSS caused nuclear condensation and fragmentation,Glucose supplement alleviated nuclear fragmentation significantly.And the numbers of apoptotic cells were increased significantly in the combination treatment,Glucose supplement reduced apoptotic cells obviously.3.ABT737 combined with EBSS enhanced ROS accumulation and decreased mitochondrial membrane potential,and promoted Omi/Htr A2 and cytochrome C release from mitochondria;while Glucose supplement decreased ROS production and maintained the mitochondrial membrane potential,and reduced Omi/Htr A2 and cytochrome C release.4.ABT737 combined with EBSS decreased ATP and lactic acid production,Glucose supplement increased both production significantly.EBSS promoted the expression of aerobic glycolysis-associated genes or proteins GLUT1,LDHA,HKII and PDK1,and inhibited PDHB expression.Combined with ABT737 decreased the expression of these genes and proteins,and increased PDHB expression.Glucose supplement restored the expression of aerobic glycolysis-associated genes and proteins.5.EBSS induced mitochondria fusion significantly.Combined with ABT737 promoted mitochondria fission,with nuclear indentations and deep grooves in 24 h.Glucose supplement reestablished the balance of mitochondrial fusion and fission,and decreased mitochondrial fission.6.ABT737 combined with EBSS decreased the protein levels of OPA1 and Mfn2,and increased the expression of Drp1 and Fis1.More Drp1 translocated to mitochondria,and more Fis1 expressed on mitochondria,but the co-location between Drp1（or Fis1）and mitochondria was decreased after Glucose supplement.7.ABT737 combined with EBSS influenced the expression of Bcl-2 family members,decreased the expression of Mcl-1 and increased the expression of BH3-only protein Noxa and pro-apoptotic Bax/Bak.The influence were alleviated after Glucose supplement.8.Silencing Bcl-2 combined with EBSS reduced cell viability,decreased the levels of Mcl-1 expression and increased the levels of Noxa and Bax/Bak expression,also reduced the levels of OPA1 and Mfn2 expression,and increased the levels of Drp1 and Fis1 expression.Conclusion:1.Nutrient starvation can enhance human ovarian cancer SKOV3 cells sensitivity to apoptosis induced by BH3 mimetic ABT737.2.ABT737 combined with nutrient starvation can significantly inhibit glycolytic function,and resulted in cell death due to insufficient energy supply.3.Nutrient starvation combined with ABT737 can promote mitochondrial fission,and mitochondrial fission may be an upstream event of apoptosis.The combination treatment may increase the mitochondrial damage,induce MOMP and release pro-apoptotic proteins.Nutrient starvation alone can induce mitochondrial fusion,indicating that mitochondrial fusion could confer tolerance to nutrient starvation in SKOV3 cells.4.ABT737 combined with nutrient starvation may influence the expression of Bcl-2 family proteins,inhibit expression of anti-apoptotic proteins,promote expression of pro-apoptotic proteins,and induce Bax/Bak mediated mitochondrial apoptosis.Silencing Bcl-2 combined with nutrient starvation can induce mitochondrial fission,suggesting that Bcl-2 family proteins may participate in regulating mitochondrial fusion and fission.5.Glucose supplement can recover glycolytic function impaired by ABT737 combined with nutrient starvation,and reestablish the balance of mitochondrial fusion and fission.It was further verified that the balance of mitochondrial fusion and fission can be co-regulated by glucose metabolism and Bcl-2 family.Above all,enhanced glycolysis and mitochondrial fusion may confer tolerance to nutrient starvation in human ovarian cancer SKOV3 cells.Combined with ABT737 can enhance SKOV3 cells sensitivity to apoptosis via impairing glycolysis,influencing Bcl-2 family and mitochondria dynamics.We try to examine the mechanism of mitochondrial dynamics,and clarify the interactive regulation between nutrient starvation and Bcl-2 family proteins via exploring nutrient starvation enhancing the sensitivity of the cells to apoptosis induced by BH3 mimetic.