Expression Of GRP78 In Ovarian Cancer And It's Effection On Cisplatin Resistance In Ovarian Cancer Cells

Ovarian cancer is one of the most important causing death reasons in department of gynecology malignant tumor. In conventional clinical therapy, the first is surgical treatment in order to reduce the number of tumor cells, then combined chemotherapy. But the therapeutic efficacy of ovarian cancer was not good, and the recurrence rate was still high. The five year survival rate of ovarian cancer was extreme low, the mainly reason was the ovarian cancer cells were produced chemical resistance to Cisplatin.Endocytoplasmic reticulum was the most important organells in the cell, it was the place that regulated protein synthesis, folding after synthesis, collected, regulation of stress reaction and the concentration of calcium ion in the cells. The endocytoplasmic reticulum stress was the key traction when cells be responded to environmental agent. ERS could be induced by the changes of tumor microenvironment or anti-tumor medicine, it shows endocytoplasmic reticulum error folding, unfolded protein colleted and calcium ion balance confused in the cells. ERS activation unfolded protein reaction, UPR(unfolded protein reaction) include endocytoplasmic reticulum molecular chaperones GRP78/BIP (glucose-regulated protein 78/binding immunoglobulin protein), ERS reception protein PERK(PKR like ER kinase), IRE-1(inositol-requiring enzyme 1), ATF6 (activating transcription factor 6) ect. And the downstream signal conduction pathway. The moderate activity of UPR could be anti-apoptosis, promote tumor survival and the drug resistance, but the severe UPR could be induced cells to apoptosis.Clinical research show that the GRP78 protein was up-regulated in many malignant tumor, include lung cancer, liver cancer, breast cancer etc, and it can inhibit the role of inducing apoptosis by chemotherapeutics through many difference pathway.GRP78 played important role in endocytoplasmic reticulum, survival reaction, apoptosis regulation that induced by chemotherapeutics, the cell fate were influenced with reaction degree. Therefore presume that during the reaction by anti-tumor medicine, tumor drug-fast cells start endocytoplasmic reticulum, UPR reaction, produce the signal that promote tumor cells survival, and/or weaken cells apoptosis; GRP78 participate drug fast of tumor through regulated the signal pathway. In this study, we plan to research and approach tow question, the first is identification the difference of the endocytoplasmic stress and apoptosis reaction role that induced by cis-platinum between non-drug fast cells and drug fast cells; The second is approach the mechanism of GRP78 that participate endocytoplasmic reticulum stress mediated tumor drug fast, and approach the probably signal conduction pathway.Object:This study was designed to detect GRP78 in ovarian serous adenocarcinoma, and the relationship between the expression of GRP78 and development of ovarian cancer and explore inhibition of GRP78 expression on cisplatin-resistant human ovarian cancer cells and its mechanism, and to further explore GRP78 gene as a new target for therapy.,Methods:1. The expression of GRP78 in human ovarian carcinoma and its significance.(1) Detected 15 cases of normal ovarian tissue,18 cases of ovarian serous cystadenoma tissue,45 cases of ovarian serous adenocarcinoma of GRP78 expression by immunohistochemistry.(2) Using RT-PCR and Western-blot method for detection of normal ovarian tissue and ovarian serous adenocarcinoma of GRP78 mRNA and protein expression.(3) Statistical analysis of GRP78 expression in ovarian serous related clinical features.2. Inhibition of GRP78 expression reverses cisplatin-resistance in ovarian cancer cells and to explore its mechanism.(1) Human ovarian serous adenocarcinoma cell line SKOV3 cells and SKOV3/DDP cells in culture.(2) According to GRP78 gene sequences and RNAi design principles, building pSilencerTM3.1-H1 GRP78 siRNA plasmid and sequencing.(3) Cell transfection, and detection of GRP78 mRNA and protein expression levels by RT-PCR, Western-blot method.(4) Detection of cell survival by MTT assay. (5) With PI/AnnexinV double staining for quantitative detection of apoptosis, flow cytometry analysis on the calculation of cell apoptosis.(6) Detection of protein expression of CHOP in pSHlsi-GRP78 plasmid transfected cells by Western-blot method.(7) Utilizing Western-blot method to detect inhibition of GRP78 and cisplatin on GRP78, CHOP, pro-caspase 4, cleaved-caspase 4, pro-caspase 3, cleaved-caspase 3 protein expression.(8) With PI/AnnexinV double staining for quantitative detection of apoptosis, flow cytometry analysis and calculation on the rate of apoptosis, detection of inhibition of GRP78 expression on cisplatin induced apoptosis in ovarian cancer cells.(9) Detection of p-Akt, Akt, p-mTOR, mTOR protein expression in by Western-blot.(10) Detected XBP1 mRNA cut and CHOP mRNA expression by RT-PCR.Results:1. The expression of GRP78 in human ovarian carcinoma and its significance.Ovarian serous adenocarcinoma GRP78 positive cells was significantly higher than normal ovarian tissue and ovarian serous cystadenoma tissue; GRP78 in ovarian serous adenocarcinoma patients with high expression, but independent tumor grade, age and clinical Stage.The mRNA expression and protein expression in Ovarian serous adenocarcinoma was higher than normal ovarian tissue and ovarian serous cystadenoma tissue,but it was not relative with tumor grade.2. Inhibition of GRP78 expression reverses cisplatin-resistance in ovarian cancer cells and to explore its mechanism.(1) By restriction enzyme digestion and sequencing proved pSilencer TM3.1-H1 GRP78 siRNA recombinant plasmid was successfully constructed. Compared with the control group, GRP78 expression decreased and has statistically significant.(2) Inhibition of GRP78 significantly decreased the survival cells of SKOV3 cells and SKOV3/DDP cell, but a greater impact on the SKOV3/DDP cell survival.(3) Inhibit the expression of GRP78 induced apoptosis in SKOV3 cells and SKOV3/DDP.(4) CHOP protein expression in SKOV3/DDP cells was significantly lower than in SKOV3 cells; inhibition of GRP78 significantly increased the expression of CHOP protein in SKOV3/DDP cells.(5) Cisplatin effected the survival inhibition of SKOV3 cells and cells and SKOV3/DDP cells in a time-dependent and concentration-dependent form; the resistance of SKOV3/DDP cells to cisplatin is much higher than the parental SKOV3 cells, resistance index is 3.17.(6) Inhibition of GRP78 expression was significantly decreased of SKOV3 cells and SKOV3/DDP cells IC50 to cisplatin.(7) Cisplatin induced endoplasmic reticulum stress-mediated apoptosis in SKOV3 cells; inhibition of GRP78 expression could increase the SKOV3/DDP cells cleaved-caspase 4 and cleaved-caspase 3 expression and apoptosis rate.(8) Inhibition of GRP78 expression can reduce cisplatin-induced SKOV3/DDP cell p-Akt. p-mTOR expression, increased XBP1 mRNA cut clips and CHOP mRNA expression.Conclusion:1. GRP78 is highly expressed in human epithelial ovarian cancer, may be associated with ovarian cancer tumorigenisis and development.2. In this study, using RNA interference method to silence GRP78 expression in ovarian cancer cells, observing inhibition of GRP78 gene expression effected sensitivity of cisplatin-resistant human ovarian cancer cells to cisplatin, and exploring the role of GRP78 in mechanisms of chemo-resistance. In this study, inhibition of GRP78 expression reversed the cisplatin resistance in ovarian cancer cells. Suppression of GRP78 inhibited Akt/mTOR signaling pathway, increased endoplasmic reticulum stress-related transcription factor XBP1 mRNA. upregulated ER stress apoptosis-related protein CHOP expression and caspase 4 activation, and increased cisplatin induced apoptosis. It suggested that suppression of GRP78 has a positive role in solving the problem of cisplatin-resistant in ovarian cancer and gave a new revelation of targeted therapy of ovarian cancer.