Enhancement Of Cisplatin Sensitivity In Ovarian Cancer Cells By Roflumilast Via CAMP/PKA/CREB-FtMt Signaling Pathway

BackgroundThe incidence of ovarian cancer(OC)is ranked third in female genital malignancies.But the mortality rate of ovarian cancer is ranked first in female genital malignancies,which seriously threatens the life and health of women.Despite extensive cytoreductive surgery and cisplatin(DDP)-based post-operative chemotherapy in ovarian cancer patients,the annual survival rate of ovarian cancer patients is still hovering at 20%-30%,and the 10-year survival rate of ovarian cancer patients is only 4%-20%.There is lack of specific clinical symptoms and corresponding methods for early diagnosis of ovarian cancer,so that the majority of patients with ovarian cancer at the time of treatment has been in advanced stages,and it is difficult to achieve satisfactory tumor cell depletion goals.Therefore,postoperative chemotherapy is especially important to prolong the survival of patients.When ovarian cancer patients were treated with DDP-based chemotherapy,about 15%-25%patients were present primary drug resistance and among them about 80%patients were eventually developed to secondary DDP resistance.Therefore,drug resistance can directly affect the efficacy of chemotherapy and the prognosis of ovarian cancer patients.At present,exploring the mechanism of drug resistance in ovarian cancer and developing new drug-resistant reversing agent have become a hot spot in the field of oncology worldwide.Cyclic adenosine monophosphate(cAMP)could activate protein kinase A(PKA)and cyclic AMP response element-binding protein(CREB),playing important roles in gene regulation,cell migration,cell proliferation,cell apoptosis and mitochondrial homeostasis.Phosphodiesterases(PDEs)are responsible for hydrolyzation of cAMP to its inactive 5'-monophosphate.In recent years,several scholars have begun to explore whether inhibition of PDE4 could play an anti-tumor effect.Our previous study investigated the role of PDE4 inhibitor roflumilast in the treatment of ovarian cancer.After administration with roflumilast,the proliferations of ovarian cancer cell lines OVCAR3 and SKOV3 were significantly inhibited,and the cell apoptosis in those cells were significantly increased.Roflumilast activated the cAMP/PKA/CREB pathway and upregulated the mitochondrial ferritin(FtMt)level in OVCAR3 and SKOV3 cells.Overexpression of FtMt in OVCAR3 and SKOV3 cells induced cell apoptosis and inhibited tumour development.The PKA inhibitor H89 suppressed the expression of FtMt and restored the roflumilast-inhibited cell proliferation and-induced cell apoptosis,indicating the PKA pathway involved in the roflumilast-inhibited cell proliferation and-induced-cell apoptosis.In addition,downregulation of CREB significantly also reverses the effects of roflumilast on ovarian cancer cells and significantly inhibits the expression of FtMt.Those findings suggested that roflumilast promoted cell proliferation and inhibited cell apoptosis in ovarian cancer through upregulation of FtMt via cAMP/PKA/CREB pathway.DDP is a basic medicine for combined chemotherapy of ovarian cancer and plays an important role in the comprehensive treatment of ovarian cancer.However,the roles of roflumilast in DDP sensitivity of ovarian cancer cells are still unclear,the research may find a new breakthrough for cisplatin resistance in ovarian cancer.ObjectiveOur previous research proves that roflumilast up-regulated the expression of FtMt in ovarian cancer via cAMP/PKA/CREB signals,thereby inhibiting the cell proliferation and increased cell apoptosis in ovarian cancer.In this study,we aimed to investigate the roles of roflumilast in development of cisplatin sensitive ovarian cancer.Content and methodTwo ovarian cancer cell lines(OVCAR3 and SKOV3)were selected and divided into blank control group,DDP treatment group,DDP + roflumilast treatment group,and cells were transfected with CREB shRNA shRNA(short hairpin RNA)and then treated with DDP + roflumilast group.Cell viability,proliferation,apoptosis and other tests were performed,and the expression levels of cAMP,PKA,CREB,p-CREB and FtMt were examined,in order to confirm the role of roflumilast in the development of DDP-sensitive ovarian cancer.1.The effect of roflumilast on DDP sensitivity in ovarian cancer cells;2.The effect of roflumilast on CAMP/PKA/CREB-FtMt signaling pathway in ovarian cancer cells;3.Whether CREB shRNA can reverse the sensitivity of roflumilast to DDP sensitivity in ovarian cancer cells.Result1.Roflumilast significantly enhances the killing effect of DDP on ovarian cancer cells;2.Roflumilast induces expression of FtMt in ovarian cancer cells by activating cAMP/PKA/CREB signaling pathway;3.Down-regulation of CREB reverses roflumilast-mediated increases in DDP sensitivity in ovarian cancer cells.ConclusionRoflumilast enhances DDP sensitivity in ovarian cancer cells via activation of cAMP/PKA/CREB pathway to up-regulate the expression of downstream FtMt.These findings suggest that the PDE4 inhibitor-roflumilast may have potential as an anti-tumor drug,or may help overcome the clinical resistance of ovarian cancer.