Study Of Relationship And Mechanism Between Grp78 And Drug Resistance In Ovarian Cancer

PART IStudy of relationship between GRP78 and drug resistance in ovarian cancer cellsObjective: To explore the relationship of GRP78 and chemoresistance in ovarian tumor.Methods: We made use of a pair of ovarian tumor cell lines, including C13K which was resistant to cisplatin and OV2008 which was sensitive to cisplatin. Both OV2008 cells and C13K cells were treated with different concentrations of cisplatin. And then chemosensitivity of cells to cisplatin were measured by methyl thiazolyl tetrazolium(MTT), while the change of cell morphology were detected by confocal microscope after staining with hoechest-33258. Meanwhile, the percentage of cell apoptosis was detected by flow cytometry after stained with AnnexinⅤ-PI. The expression of GRP78 protein were analyzed through Western blot.Results: The apopototis ratio of OV2008 cells was obviously higher than that of C13K cells after having been treated with the same consentration of cisplatin. The expression of GRP78 protein in C13K cells were significantly higher than those in OV2008 cells. At the same time, the expression of GRP78 protein in OV2008 cells after having been treated with cisplatin was lower than that in OV2008 control cells. While the expression of GRP78 protein in C13K cells did not indicate the difference.Conclusion: There was a negative correlation between the amount of GRP78 and sensitivity to cisplatin. GRP78 may play an significant role on the drug rsisitance of ovarian cancer. PART IIThe role of GRP78 gene in reversal of chemo-resistance in ovarian cancer cellsObjective: To investigate the effect of GRP78 on drug sensitivity by SiRNA-GRP78 and GRP78 induced agents such as 2-DG. At the same time, we design to demonstrate the mechanisms of drug resistance.Methods: We transfected SiRNA-GRP78 into C13K cells by lipofectamine 2000 accordingly. At the same time, OV2008 cells were treated with 2-DG. And then western blot was used to detect the change of GRP78 protein and P-Akt protein expression. After treated with cisplatin, the chemosensitivity of cells to cisplatin were measured by methyl thiazolyl tetrazolium(MTT), and then the cell apoptosis ratio and cell cycle were detected by flow cytometry.Results: After transiently transfection cells which were already transfected wit SiRNA-GRP78, the GRP78 protein expression was obviously decreased. and the P-Akt protein expression was changed correspondingly. It was observed that apoptosis rate of SiRNA-GRP78 transfected cells was much higher than those of SiRNA control. At the same time, it was observed that apoptosis rate of GRP78 overexpression cells was much higher than those of control. Futuremore, it was observed that P-Akt protein expression was changed correspondingly. And when TCN was used in the OV2008 cells, the apoptosis rate of GRP overexpression cells was much higher than those of control.Conclusions: Overexpresssion of GRP78 partially suppressed the apoptosis of humman ovarian cancer cells induced by cisplatin, and knockdown of GRP78 drastically enhanced apoptosis. The mechnism of drug resistance may be associated with PI3K/AKT pathway.