Targeting Mitochondrial PHB2/OMA1/DELE1 Pathway Enhances Cisplatin-induced Apoptosis In Ovarian Cancer Cells

Ovarian cancer originates from the ovary or fallopian tube,preferentially metastasizes to the peritoneal peritoneum or omentum,and mainly spreads in the peritoneal cavity.So ovarian cancer is difficult to be diagnosed and has a high mortality rate.Cisplatin has been proved to induce apoptosis of ovarian cancer cells through mitochondrial or endoplasmic reticulum pathways.However,with the prolongation of treatment time and the variation of ovarian cancer,the therapeutic effect of cisplatin gradually deteriorates.Previous studies have proved that mitochondrial membrane permeability transition is a key step in tumor cell apoptosis induced by chemotherapeutic drugs.Our previous work found that targeting mitochondria and inducing endoplasmic reticulum stress increased the sensitivity of ovarian cancer to cisplatin.However,the regulatory mechanism affecting the mitochondrial membrane permeability of tumor cells is unclear,which hinders the development of drugs specifically targeting the mitochondrial membrane permeability transition of tumor cells.It has been reported that OMA1,the mitochondrial inner membrane(MIM)protease,is highly expressed in colorectal cancer and gastric cancer,but its expression is low in breast cancer,suggesting that OMA1 plays distinct functions in different cancers.Although the role of OMA1 in ovarian cancer is not clear,recent studies have found that activated OMA1 may promote the release of DELE1 to the cytoplasm and activate HRI and el F2? by cleaving DELE1,under the stimulus of mitochondrial uncoupler FCCP.In response to the stresses from different stimuli,the integrated stress response activates el F2? through the four kinases PERK,PKR,GCN2,and HRI.When multiple kinases are simultaneously activated,they will induce transcription factors such as CHOP,which disrupt the transcription of pro-survival proteins such as BCL2,and activate pro-death signal.PHB2 and SLP2,highly expressed in human ovarian cancer,have important roles in DNA transcription,nuclear signal transduction,mitochondrial structural integrity,cell division and cell membrane metabolism.Recent studies have found that PHB2 and SLP2 may be upstream molecules of OMA1.OMA1 is considered to be the key stress protease in tumor cell fate regulation.These data suggest that the PHB2/OMA1/DELE1 pathway can further elucidate the mechanism of tumor cell apoptosis induced by chemotherapeutic drugs.In this study,MIM stress protease OMA1 was used as point of penetration,combined with in vitro and in vivo experiments,to explore the mechanism that OMA1/DELE1/HRI may cooperate with PERK to amplify the interaction between mitochondria and endoplasmic reticulum,enhance cisplatin-induced apoptosis of ovarian cancer cells,and provide theoretical and experimental basis for targeting mitochondria to increase cisplatin sensitivity.Methods:1.To investigate the effect of mitochondrial stress inducer FCCP on the sensitivity of ovarian cancer cells to cisplatin.A2780 cells were treated with FCCP(2.5 ?M)and/or cisplatin(1 ?g/ml)for 24 h.ID8 cells were treated with FCCP(5 ?M)and/or cisplatin(4 ?g/ml)for 24 h.Drug sensitivity of ovarian cancer cells were detected by MTT assay.Apoptosis rates in ovarian cancer cells were analysed by flow cytometry.Apoptosis-related protein levels in ovarian cancer cells were measured by western blotting.2.To explore the effects of FCCP and cisplatin on mitochondrial proteins and mitochondrial inner and outer membranes of ovarian cancer cells.A2780 cells were treated with FCCP(2.5 ?M)and/or cisplatin(1 ?g/ml)for 12 h.ID8 cells were treated with FCCP(5 ?M)and/or cisplatin(4 ?g/ml)for 12 h.The expression of OMA1 and OPA1 in ovarian cancer cells was measured by western blotting.The structures of mitochondrial cristae and mitochondrial membranes in ovarian cancer cells were detected by transmission electron microscope.The mitochondrial membrane potential in ovarian cancer cells was detected by JC-1 staining.Mitochondria and cytoplasm were isolated,and the expression of cytochrome c in the cytoplasm of ovarian cancer cells was detected by Western blot.Immunofluorescence was used to detect the expression of cytochrome c in mitochondria and cytoplasm of ovarian cancer cells.3.To explore the effects of FCCP and cisplatin on endoplasmic reticulum stress and el F2?/ATF4 pathway in ovarian cancer cells.(1)A2780 cells were transfected with DELE1-HA for 24 h,then treated with FCCP and/or cisplatin for 6 h.The expression of DELE1 in ovarian cancer cells was measured by western blot.Mitochondria and cytoplasm were separated by Mitochondrial Isolation Kit,and the expression of DELE1 in cytoplasm of ovarian cancer cells was detected by Western blot.The binding of DELE1 to HRI in ovarian cancer cells was detected by immunocoprecipitation.(2)A2780 cells were treated with FCCP(2.5 ?M)and/or cisplatin(1 ?g/ml)for6 h.ID8 cells were treated with FCCP(5 ?M)and/or cisplatin(4 ?g/ml)for 6 h.The expression of p-PERK,p-el F2?,ATF4,ATF5,CHOP in ovarian cancer cells was detected by Western blot.The nucleus and cytoplasm were separated by Nucleus Isolation Kit,and the expression of ATF4,ATF5 and CHOP in the nucleus of ovarian cancer cells was detected by Western blot.The m RNA levels of ATF4,ATF5,CHOP and BCL2 family genes were detected by q RT-PCR.(3)A2780 cells were treated with FCCP(2.5 ?M)and/or cisplatin(1 ?g/ml)for12 h.ID8 cells were treated with FCCP(5 ?M)and/or cisplatin(4 ?g/ml)for 12 h.Mitochondria and cytoplasm were isolated by Mitochondrial Isolation Kit,and the expression of BAX in mitochondria of ovarian cancer cells was detected by Western blot.4.To explore the effect of knockdown OMA1 on the apoptosis of ovarian cancer cells and the corresponding mechanism.(1)A2780 cells were transfected with OMA1-sh RNA plasmids and Scr-sh RNA for 24 h and treated with FCCP and/or cisplatin for 24 h.Apoptosis rates,cell viabilities and expressions of proteins were detected by MTT assay,flow cytometry,and western blot,respectively.(2)The wild-type A2780 cells and the OMA1 stable knockdown A2780 cells were transfected with DELE1-HA for 24 h,then treated with FCCP and/or cisplatin for 6 h.The expression of DELE1 in ovarian cancer cells was evaluated by western blot.5.To explore the effects of FCCP and cisplatin on the interaction of PHB2 and SLP2 in ovarian cancer cells.A2780 cells were treated with FCCP(2.5 ?M)and/or cisplatin(1 ?g/ml)for 12 h.ID8 cells were treated with FCCP(5 ?M)and/or cisplatin(4 ?g/ml)for 12 h.The binding of SLP2 to PHB2 in ovarian cancer cells was detected by immunocoprecipitation.6.The effects of mitochondrial stress inducer FCCP and cisplatin in mice subcutaneous tumors were verified in vivo.The mouse subcutaneous tumor model of ovarian cancer cell ID8 was established.The mice were given intraperitoneal injections for two weeks,and the body weight and tumor volume were measured every day.Mice were sacrificed and the tumor tissues were stripped.Protein was extracted from tumor tissues and its expression was detected by Western blot.RNA was extracted from tumor tissues and the gene m RNA level was detected by q RT-PCR.The tumor tissue sections were prepared and the apoptosis rate was detected by TUNEL staining.Localization of SLP2 and PHB2 in tumour specimens was measured by immunofluorescence staining.Results:1.Both mitochondrial stress inducer FCCP and cisplatin inhibited cell viability,increased the rate of apoptosis,enhanced the expression of apoptotic protein cleaved caspase 3,and FCCP enhanced cisplatin-induced apoptosis,suggesting that FCCP increased the sensitivity of ovarian cancer cells to cisplatin.2.Compared to those in the control group and the group treated with cisplatin alone,OMA1 was activated,OPA1 was cleaved and mitochondrial cristae were decreased in the group treated with FCCP alone and the combined treatment group.In the group treated with FCCP alone,the mitochondrial membrane potential decreased,the mitochondrial outer membrane(MOM)was intact,and cytochrome c was not released into the cytoplasm.In the combined treatment group,the mitochondrial membrane potential decreased,the mitochondrial outer membrane was damaged,and cytochrome c was released into the cytoplasm.These data suggest that the apoptosis induced by the combined treatment of FCCP and cisplatin is related to the increase of mitochondrial outer membrane permeability.3.Compared to that in the control group and the group treated with cisplatin alone,DELE1 in the group treated with FCCP alone and the combined treatment group was cleaved and released into the cytoplasm to bind to HRI.Compared to that in the control group and the group treated with FCCP alone,the expression of p-PERK in the group treated with cisplatin alone and the combined treatment group was increased.Only in the combined treatment group,the phosphorylation level of el F2?,the m RNA level of ATF4,ATF5,CHOP,the expression of ATF5 and CHOP in the nucleus,the m RNA level of BIM,PUMA,NOXA and the expression of BAX in mitochondria increased,the m RNA level of MCL1 decreased.These data suggest that DELE1/HRI promotes PERK-induced el F2?/ ATF4 signal,triggering imbalance of BCL2 family members.4.Knockdown of OMA1 rescued the inhibitory effect on cell viability induced by the combined treatment with FCCP and cisplatin.The apoptosis rate,the cleavage of OPA1 and DELE1,the expression of ATF4,ATF5,CHOP,p-el F2? and the release of cytochrome c of the FCCP+cisplatin+OMA1-sh RNA group decreased compared to those of the FCCP+cisplatin+Src-sh RNA group.These data suggest that knockdown of OMA1 reverses the apoptosis through attenuating mitochondrial stress and el F2?/ATF4 pathway in ovarian cancer cells.5.Compared to those in the control group and the group treated with cisplatin alone,SLP2/PHB2 binding in the group treated with FCCP alone and the combined treatment group decreased,suggesting that disrupting PHB2/SLP2 complex may activite OMA1 in ovarian cancer cells.6.In mouse subcutaneous tumors,the combination of mitochondrial stress inducer FCCP and cisplatin activated OMA1,cleaved OPA1 and increased p-el F2?phosphorylation,upregulated ATF4,ATF5 and CHOP,which downregulated the m RNA level of Mcl1,upregulated the m RNA level of Bim,Puma and Noxa,and finally induced apoptosis and inhibited the growth of subcutaneous tumors.These data indicate that the combination of FCCP and cisplatin inhibits ovarian cancer growth in vivo through inducing apoptosis.Conclusions:1.FCCP-activated OMA1 induced mitochondrial inner membrane cristae remodeling in ovarian cancer cells by cleaving OPA1.Simultaneously,FCCP-activated OMA1 cleaved DELE1,activating HRI in cytoplasm.HRI and cisplatin-activated ER-associated protein kinase PERK,synergistically enhanced el F2?/ATF4 signal,increasing mitochondrial outer membrane permeability.2.Mitochondrial stress regulated OMA1/DELE1/HRI through PHB2/SLP2 complex,which was the main mechanism that nuclear-mitochondria interaction amplified the integrated stress response and increased cisplatin apoptosis sensitivity in ovarian cancer.3.Mitochondrial stress inducer FCCP increased the sensitivity of ovarian cancer to cisplatin by activating PHB2/OMA1/DELE1 pathway,in vivo and in vitro.