PGC1? Participates In Cisplatin Resistance In Ovarian Cancer Through HSP70/HK2/VDAC1 Pathway

Background:Cisplatin is a traditional drug for treating tumor,which directly acts on killing tumor cells with DNA.It is recognized that chemotherapy drugs can not only interact with their own targets,but also kill tumor cells by inducing apoptosis.Therefore,inhibition of apoptosis can lead to increased cisplatin resistance in tumor cells.In addition,mitochondria are not only the core organelle of chemoresistance of cancer cells,but also the key step to control apoptosis of cancer cells.Therefore,induction of mitochondrial apoptosis may be one of the main ways for cancer to overcome chemotherapy resistance.In 1920s,Otto Warburg suggested that tumor cells were more inclined to use aerobic glycolysis to meet their energy needs.But more and more researches have shown that tumor cells can promote the survival of tumor cells through mitochondrial adaptation mechanisms such as mitochondrial biogenesis in order to cope with the stress of chemotherapy drugs.Therefore,the related molecules of mitochondrial biosynthesis in regulating mitochondrial pathway apoptosis needs to be explored urgently.As the main regulator of mitochondrial biogenesis,PGC1? is closely related to mitochondrial energy metabolism and apoptosis in tumor cells.Sancho P et al.Found that PGC1? can resist apoptosis of pancreatic cancer cells by increasing the expression of anti apoptotic protein BCL2 and reducing the expression of Pro-apoptotic protein BAX.Our early results showed that compared with SKOV3 cells,cisplatin resistant SKOV3/CDDP cells had a higher level of mitochondrial biogenesis.In addition,the expression of PGC1 ?was up-regulated in SKOV3/CDDP cells.The knockdown of PGC1 ? in SKOV3/CDDP cells showed that SKOV3/CDDP cells were more sensitive to cisplatin.After PGC1 ? was knocked down in SKOV3/CDDP cells,the expression of BCL2 decreased,the expression of BAX increased,and the apoptosis of SKOV3/CDDP cells increased.However,the mechanism of mitochondrial apoptosis is complex,and simple expression differences can not clearly explain the specific mechanism of PGC1? regulating SKOV3/CDDP cell apoptosis,which still needs to be further explored.The opening of mitochondrial permeability transition pore(MPTP)is the beginning of mitochondrial apoptosis cascade,and then apoptosis factor,cytochrome c and other factors are released into cytoplasm.Finally,caspase,a downstream factor of apoptosis,was activated to promote apoptosis.Therefore,the opening of MPTP is one of the key steps of mitochondrial apoptosis.In addition,hexokinase 2(HK2),as an important component of MPTP,can maintain the closure of MPTP and inhibit apoptosis by binding with voltage dependent anion channel 1(VDAC1)in mitochondria.Angelova et al.Found that mitochondrial mitotic protein MFF can promote the oligomerization of VDAC1 in cardiomyocytes.This weakened the binding between HK2 and VDAC1,and then promoted the opening of MPTP,leading to apoptosis.In addition,mitochondrial glycogen synthase kinase-3 ? can open MPTP by reducing the binding of HK2 and VDAC1,leading to apoptosis of melanoma cells.PGC1?,as the main regulatory molecule of mitochondrial biogenesis,is closely related to these mitochondrial related proteins.Therefore,the HK2/VDAC1 signaling pathway may provide a new idea for the mechanism of PGC1? regulating the apoptosis of chemotherapy-resistant tumor cells.Heat shock protein family/chaperones(HSPs)have the function of transporting proteins to mitochondria.Among them,HSP70 is one of the most important members of HSPs family,which can help J protein and other proteins transport to mitochondria.In addition,Henry Aceros et al.found that HSP70 can reduce the opening of MPTP during myocardial ischemia-reperfusion,and then apoptosis of cardiomyocytes decreased.In mice of HSP70 promoter HSF1 knockout,Liang Jun Yan et al found that the opening of MPTP in kidney cells induced the decrease of mitochondrial membrane potential.It is known that HK2/VDAC1 is the core mechanism of MPTP opening.Therefore,HSP70 may regulate the opening of MPTP through HK2/VDAC1 signaling pathway.Furthermore,some studies have found that PGC1? can reduce heat stress injury in mouse liver,kidney,and fibroblasts by promoting the expression of HSP70.However,the complex mechanisms of HSP70 and PGC1? under cisplatin stress are not clear.It is suggested that the mechanism of HSP70 and HK2 in mitochondrial apoptosis may provide a new strategy for PGC1? to regulate the apoptosis of chemotherapy-resistant tumor cells.In conclusion,we hypothesized that under cisplatin stress,PGC1? can promote the binding of HK2 and VDAC1,reduce the opening of MPTP and enhance cisplatin resistance of ovarian cancer by regulating the expression of HSP70.Knockdown of PGC1? can reduce the expression of HSP70 decreased,the combination of HK2 and PGC1? decreased,and the opening of MPTP increased,which reversed the resistance of ovarian cancer to cisplatin.Aim:In this study,we discussed the factors affecting the binding of HK2 and VDAC1 from the perspective of PGC1? regulating mitochondrial apoptosis and promoting cisplatin sensitivity,and clarified the regulatory mechanism of mitochondrial biogenesis and apoptosis,which provided a basis for targeting PGC1? to treat cisplatin resistance in ovarian cancer.Method:(1)In this study,human ovarian cancer SKOV3 and A2780 cells,cisplatin-resistant cells SKOV3/CDDP and A2780/CDDP cells were taken as the research objects,and the protein expression of PGC1? was detected by Western Blot.(2)After PGC1? was knockdown in SKOV3/CDDP and A2780/CDDP cells,MTT was used to detect the sensitivity of the cells to cisplatin.The expressions of anti-apoptotic protein BCL2,pro-apoptotic protein BAX and apoptosis downstream effector molecule c-caspase3 were detected by Western Blot.Mitochondrial membrane potential was detected by JC-1 staining flow cytometry in SKOV3/CDDP cells.(3)After PGC1? was knockdown in SKOV3/CDDP and A2780/CDDP cells,Western Blot was used to detect the changes of molecular proteins related to MPTP.Changes of mRNA level of MPTP related molecules by RT-qPCR.(4)After PGC1? was knockdown in SKOV3/CDDP and A2780/CDDP cells,then stimulated with cisplatin.Mitochondrial extraction experiment was used to detect the protein expression level of HK2 in mitochondria.Mitotracker stained mitochondria,and immunofluorescence was used to detect the co-localization of HK2 and mitochondria.(5)After PGC1? was knockdown in SKOV3/CDDP and A2780/CDDP cells,then stimulated with cisplatin.The binding changes between HK2 and VDAC1 were detected by immunoprecipitation.Co-localization of HK2 and VDAC1 was detected by immunofluorescence.(6)After PGC1? was knockdown in SKOV3/CDDP and A2780/CDDP cells,transcriptome sequencing was used to detect mRNA changes of transport protein family.The protein expression of HSPs family was detect by Western Blot.(7)We construct HSF1 luciferase reporter gene plasmid,and test whether PGC1? can regulate HSP70 at transcription level by luciferase reporter gene experiment.(8)constructing HSP70 overexpression plasmid,using PGC1? small molecule inhibitor SR-18292 in SKOV3/CDDP and A2780/CDDP cells,and overexpressing HSP70 plasmid at the same time.Mitochondrial extraction experiment was used to detect the changes of HK2 in mitochondria.Immunocoprecipitation was used to detect the binding changes between HK2 and VDAC1.The apoptosis was detected by AnnexinV/PI staining flow cytometry.(9)Overexpression plasmid of HSP70 SBD domain site mutation was constructed.The SR-18292 of PGC1? small molecule inhibitor was used in SKOV3/CDDP and A2780/CDDP cells,and HSP70 or HSP70S400K plasmid was overexpressed at the same time.Mitochondrial extraction experiment was used to detect the changes of HK2 in mitochondria.Immunocoprecipitation was used to detect the binding changes between HK2 and VDAC1.Results:(1)Compared with ovarian cancer SKOV3 and A2780,Western Blot showed that ovarian cancer cisplatin resistant cells SKOV3/CDDP and A2780/CDDP had higher expression level of PGC1?.The MTT showed that SKOV3/CDDP and A2780/CDDP cells were more sensitive to cisplatin after knocking down PGC1?.JC-1 staining flow cytometry showed that mitochondrial membrane potential of SKOV3/CDDP cells decreased.Western Blot showed that the apoptosis of SKOV3/CDDP and A2780/CDDP cells increased.(2)PGC1? was knocked down in SKOV3/CDDP and A2780/CDDP cells,and Western Blot showed that there was no significant change in MPTP-related molecular protein level.RT-qPCR showed that there was no significant change in the mRNA level of MPTP-related molecules.(3)After PGC1? was knocked down in SKOV3/CDDP and A2780/CDDP cells and stimulated by cisplatin,mitochondrial extraction experiment showed that the expression of HK2 in mitochondria was decreased.Immunocoprecipitation and immunofluorescence test showed that the binding of HK2 to VDAC1 decreased.(4)After PGC1? was knocked down in SKOV3/CDDP and A2780/CDDP cells,single cell sequencing showed that there were differences in the expression of heat shock related proteins.Western Blot showed that the expression of HSP70 protein decreased after PGC1?was knocked down in SKOV3/CDDP and A2780/CDDP cells.Luciferase reporter gene experiment showed that PGC1? could promote the transcription of HSP70 promoter HSF1.(5)After using PGC1? small molecule inhibitor SR-18292 and overexpressing HSP70 plasmid in SKOV3/CDDP and A2780/CDDP cells,the expression of HK2 in mitochondria increased slightly.Immuno-coprecipitation detection showed that the binding between HK2 and VDAC1 increased slightly.AnnexinV/PI staining and flow cytometry showed that apoptosis decreased.(6)After using PGC1? small molecule inhibitor SR-18292 and overexpressing HSP70 or HSP70S400K plasmid in SKOV3/CDDP and A2780/CDDP cells,the mitochondrial extraction experiment showed that the expression of HK2 in mitochondria decreased after overexpressing HSP70 S400K plasmid compared with HSP70 plasmid.Immuno-coprecipitation showed that the binding of HK2 to VDAC1 decreased after overexpression of HSP70S400K plasmid compared with that of HSP70 plasmid.Conclusion:(1)The inhibition of PGC1? may increase the sensitivity of ovarian cancer to cisplatin through mitochondrial apoptosis.(2)The inhibition of PGC1? may reduce the binding of mitochondrial HK2 and VDAC1 by regulating HSP70,and promote the opening of MPTP and apoptosis,thus reversing cisplatin resistance in ovarian cancer.(3)The 400-site amino acid of HSP70 promotes the expression of HK2 in mitochondria mediated by PGC1? and its binding to VDAC1.