The Mechanism Of Cancer-associated Fibroblasts Promoting Cisplatin-resisitance In Epithelial Ovarian Cancer Cells

Objective: The resistance to Cisplatin in ovarian cancer is one of the most important reasons for tumor recurrence and high mortality.Tumor microenvironment was closely related to tumor chemoresistance.Cancer-associated fibroblasts(CAFs)are the most abundant cel s in tumor microenvironment.In this study,we aimed to isolate CAFs from human ovarian cancer tissues and identify them,then explore the relationship between CAFs and Cisplatin-resisitance in ovarian cancer cells,finally il ustrate the possible mechanism.This investigation could provide a new insight into therapying the Cisplatin-resisitance in ovarian cancer.Methods: CAFs were isolated by tissue crawling method and enzymatic digestion method,and then detected the expression of vimentin(a mesenchymal marker),?-SMA(a specific marker of CAFs)and CK-8(an epithelial marker)by immunofluorescence and western blot.The conditoned medium of CAFs(CAFs-CM)was collected and mixed with fresh medium at a ratio of 1:1 and 2:1 to prepare coculture medium(Mix1:1 and Mix2:1).The half maximal inhibitory concentration(IC50)of Cisplatin for 24 h in A2780 and ES2 cells cultured in Normal,Mix1:1 and Mix2:1 was detected by CCK-8.Cell apoptotic rate in above culture conditions was detected by Flow cytometric.Meanwhile,cleaved caspase-3(an effector caspase),Bcl-2 and Survivin(two anti-apoptotic proteins)and the phosphorylation levels of STAT3(p-STAT3)in above groups were measured by Western blot.Then,Cryptotanshinone,a selective STAT3 inhibitor,was used to verify whether inhibition of STAT3 signaling could reverse Cisplatin-resisitance,apoptotic rates and the expression of Bcl-2,Survivin and p-STAT3 in A2780 and ES2 cells cultured in CAFs-CM.Results: The CAFs were isolated successfully and identified with a high expression of vimentin and ?-SMA but no CK-8 expression.The IC50 of Cisplatin in A2780 and ES2 cells cultured in CAFs-CM was increased in a CAFs-CM concentration-dependent manner,indicating that CAFs-CM could induce Cisplatinresisitance in ovarian cancer cells.Meanwhile,cell apoptotic rate was decreased in a CAFs-CM dose-dependent manner(Normal 20.02±1.69% vs Mix1:1 9.19±0.72% and Mix2:1 3.27±0.75% in A2780 cells,P<0.05)(Normal 21.73±1.62% vs Mix1:1 13±1.56% and Mix2:1 6.47±0.67% in ES2 cells,P<0.05).CAFs-CM attenuated the activation of casepase-3,and promoted the expression of anti-apoptotic proteins(Bcl-2,Survivin and p-STAT3).However,Cryptotanshinone,reverted the Cisplatinresisitance and apoptotic rate induced by cispaltin,as well as the expression of Bcl-2,Survivin and p-STAT3 in A2780 and ES2 cells.Conclusions: Here we provide the first evidence that CAFs-CM could attenuate the sensitivity to Cisplatin in ovarian cancer cel s.Our data suggested CAFs-CM could activate STAT3 signaling in ovarian cancer cells,thus promoting the expression of anti-apoptotic proteins Bcl-2 and Survivin,and then protect cel s from Cisplatininduced apoptosis.