The Study Of Regulatory Effects Of Neddylation On Paclitaxel Resistance And The Related Mechanisms In Ovarian Cancer

Background and objective:Ovarian cancer is the most lethal malignancy of the gynecologic tract.Surgery and chemotherapy are the gold-standard treatments for ovarian cancer.Paclitaxel(PTX),combined with platinum,is the standard first-line chemotherapy for ovarian cancer.PTX also works in treating patients with platinum resistant or refractory ovarian cancer.PTX binds to tubulin in microtubules and stabilizes them against depolymerization,which interferes with the function of mitotic spindles,thereby resulting in mitotic arrest and apoptosis.However,the development of drug resistance becomes a major limitation for chemotherapy effectiveness.The mechanism of PTX resistance in ovarian cancer cells(OCCs)is complex and has not been fully elucidated.Neddylation is a post-translational protein modification in which the ubiquitin-like(Ubl)molecule NEDD8 is conjugated to substrate proteins.To the best of our knowledge,the neddylation pathway is executed by an enzymatic cascade consisting of NEDD8-activating enzyme E1(NAE),two NEDD8-conjugating enzyme E2s(UBE2M and UBE2F),and several substrate-specific NEDD8-E3 ligases.Neddylation mainly participates in regulating ubiquitination,stabilizing protein,regulating the activities of substrates and transcription factors.MLN4924(also named Pevonedistat),a small molecule inhibitor of NAE,can effectively block Neddylation.Several studies have reported that MLN4924 enhances cisplatin-induced DNA damage and apoptosis in ovarian cancer cells;however,it remains unknown whether and how neddylation affects PTX resistance.Here,we examined the effects of post-translational neddylation on the sensitivity of ovarian cancer cells HO8910 and OVA-W to PTX.This study helps to provide insights into the potential mechanism of PTX chemoresistance in ovarian cancer and provide new ideas for overcoming PTX resistance.Methods:1.By observing cellular morphology,clonogenic assays,cell viability assays(CCK)and wound healing assays,the effects of Neddylation specific inhibitor MLN4924 combined with paclitaxel on the proliferation and migration of HO8910 and OVA-W cells were determined.2.Apoptosis assays(flow cytometry),Western blot analyses were used to exam the effects of MLN4924 on PTX-induced apoptosis and measure expression levels of apoptosis-associated proteins.3.Immunofluorescence assays were used to examine the effects of PTX and MLN4924 on the organization and intracellular distribution of microtubule networks in HO8910 and OVA-W cells.4.We generated UBE2 M and/or UBE2 F stable knockdown HO8910 cells using sh RNA targeting UBE2 M and/or UBE2 F,and determined the roles these two Neddylation E2 s play in PTX-mediated cytotoxicity.Results:1.Compared with the PTX treatment group,the HO8910 and OVA-W cell vitality,proliferation and migration were significantly increased in PTX and MLN4924 co-treatment group.2.Compared with the PTX treatment group,the PTX-induced apoptosis of HO8910 cells was significantly attenuated by MLN4924 in PTX and MLN4924 co-treatment group.3.Compared with the PTX treatment group,the PTX-induced microtubule polymerization of HO8910 and OVA-W cells was significantly prevented by MLN4924 in PTX and MLN4924 co-treatment group.4.Compared with sh RNA-control cells,UBE2 M and/or UBE2 F stable knockdown HO8910 cells were less sensitive to PTX-mediated cytotoxicity and microtubule polymerization.Conclusions:1.Disruption of protein neddylation with the first-in-class inhibitor MLN4924 neutralized PTX-mediated antiproliferative,antimigration,and apoptotic effects,which contributed to PTX resistance in OCCs.2.Two Neddylation conjugating E2 enzymes,UBE2 M and UBE2 F,played essential roles in PTX-induced cytotoxicity and tubulin polymerization in OCCs.