Effects And Underlying Mechanisms Of OA On The Cisplatin-based Resistance Of Ovarian Cancer

Objective: To investigate the effects of oleic acid(OA)on cisplatin resistance in ovarian cancer(OVCA)and its mechanism,and to provide new targets and ideas for controlling OVCA metastasis and cisplatin resistance.Methods: 1.CCK-8 assay was used for detecting the effects of OA on the proliferation levels of human OVCA cell lines A2780 and OVCAR3 at different concentrations.2.CCK-8 assay was used to detect the IC50 of A2780 and OVCAR3 cells to PTX.3.The DDP-sensitive cell line A2780 and the DDP-resistant cell line OVCAR3 were selected as the research objects.CCK-8 assay was used to detect the the effects of OA on DDP and PTX resistance.Moreover,FABP4 inhibitor BMS309403 and PPARγ inhibitor GW9662 were used to do the blocking test respectively.4.Flow cytometry was used to detect the effects of OA on the apoptosis of A2780 and OVCAR3 cells induced by DDP using Annexin V-FITC/PI double staining.Moreover,FABP4 inhibitor BMS309403 and PPARγ inhibitor GW9662 were used to do the blocking test respectively.5.Western Blotting technology was used to detect the effects of different concentrations of OA at different times on the protein expression of FABP4,PPARγ and ABCG2 in A2780 and OVCAR3 cells.6.Western Blotting technology was used to detect the effects of OA or both OA and PA at different times on the protein expression of FABP4,PPARγ and ABCG2 in nuclear and plasma of A2780 and OVCAR3 cells.7.Western Blotting technology was used to detect the effects of OA on the protein expression of FABP4,PPARγ and ABCG2 in A2780 and OVCAR3 cells.Meanwhile,FABP4 inhibitor BMS309403 and PPARγ inhibitor GW9662 were used to do the blocking test respectively.8.Fatty acid transfer experiments combined with qualitative and quantitative oil red O-staining assay were used to detect the effects of OA on FA uptake of OVCA cells.Meanwhile,FABP4 inhibitor BMS309403 and PPARγ inhibitor GW9662 were used to do the blocking test respectively.9.Epithelial OVCA(EOC)clinical specimens with different FIGO stages were collected.Immunohistochemical assay was used to analyze the expression of FABP4,PPARγ and ABCG2,and the correlations between FABP4 and the expression of PPARγ and ABCG2 was analyzed,respectively.Results: 1.Results of CCK-8 assay showed that OA had no significant effect on the proliferation of OVCA cell lines A2780 and OVCAR3.2.Results of CCK-8 assay showed that the IC50 of PTX in A2780 and OVCAR3 cells was 0.698 μM and 0.064 μM,respectively.3.Results of CCK-8 assay showed that enhanced DDP tolerance of A2780 cells were remarkably improved with the treatment of 50 μM OA for 48 hours.Meanwhile,the enhanced DDP tolerance of OVCAR3 cells were remarkably improved with the treatment of 100 μM OA for 48 hours and 72 hours.FABP4 inhibitor BMS309403 and PPARγ inhibitor GW9662 could remarkably block the above effect respectively,which revealed that OA could promote the transport of fatty acid OA by up-regulating the expression of FABP4 and PPARγ,thus promoting DDP resistance in human OVCA cells.OA did not affect the tolerance of A2780 and OVCAR3 to PTX.4.Results of flow cytometry showed that there was no significant effect on the apoptosis rates of OVCA cells in the middle and late stages with the treatment of OA(A2780: 100 μM;OVCAR3: 200 μM)for 24 hours,but it could significantly decrease the apoptosis of A2780 and OVCAR3 cells induced by DDP.FABP4 inhibitor BMS309403 and PPARγ inhibitor GW9662 could partially block the above effect respectively,which indicated that OA may inhibit the apoptosis of OVCA cells by affecting the expression of FABP4 and PPARγ protein,thereby inhibiting the sensitivity of OVCA cells to DDP and enhancing the tolerance of OVCA cells to DDP.5.Results of Western Blotting assay showed that the protein expression levels of FABP4,PPARγ and ABCG2 in A2780 and OVCAR3 cells were remarkably improved with the treatment of 200 μM OA for 12 hours.6.Results of Western Blotting assay showed that OA alone or both OA with PA could significantly increase the protein expression levels of FABP4,PPARγ and ABCG2 in nuclear and cytoplasmic of A2780 and OVCAR3 cells.7.Results of Western Blotting assay showed that the protein expression levels of FABP4,PPARγ and ABCG2 in A2780 and OVCAR3 cells were remarkably improved with the treatment of 200 μM OA.Pretreated with FABP4 inhibitor BMS309403 and PPARγ inhibitor GW9662 for 2 hours,OA could inhibit the upregulation of ABCG2 in human OVCA cell lines,which indicated that OA could significantly increase the protein expression of ABCG2 through up-regulation of FABP4 and PPARγ signaling molecule,thus promoting the resistance of ovarian cancer cells to DDP.8.The results of oil red O-staining assay showed that the FA uptake of OVCA cells could be significantly enhanced after the treatment of 200 μM OA for 24 hours,while the uptake of fatty acid OA in OVCA cells could be significantly inhibited by both FABP4 inhibitor BMS309403 and PPARγ inhibitor GW9662.9.The results of Immunohistochemical(IHC)analysis of ovarian cancer pathological specimens showed that the expression of FABP4 was positively correlated with FIGO stage,ascites metastasis and lymph node metastasis of ovarian cancer.The protein expression of FABP4 was positively correlated with the protein expression levels of PPARγ and ABCG2.Conclusion: With intracorporal experiments of human OVCA cells and analysis of OVCA clinicopathological tissue samples,our present study revealed that OA could enhance the expression of ABCG2 via FABP4/PPARγ/ABCG2 signaling pathway,thus promoting the resistance of OVCA cells to DDP.This study indicated that FABP4 and PPARγ may be new targets to improve DDP chemoresistance of OVCA.