| BackgroundOvarian cancer is one of the highest rates of gynecological malignant tumor, a serious threat to women’s lives and health. The trait of ovarian cancer is hidden onset, late diaognosis,early metastasis,quick progress and poor prognosis. When women see a doctor for abdominal distension, about 70 percent of them are in the later period of cancer(FIGO classification Ⅲ- Ⅳ period). The treatments of late oarian cancer are cytoreductive surgery and chenmotherapy based on platinum[1]. The aim of cytoreductive surgery is remove the tumor tissues at the most limits, but it is inevitable that possible remaining focus is exising, and become an important factor of ovarian cancer recurrence. So the application of the chemotherapy, thus greatly improved the treatment effect of ovarian cancer patients quality of life improved significantly, but taken together, 5 years survival rate remains near 30-40%, ovarian cancer patients with chemotherapy drug resistance is the main cause of treatment failure. At present the ovarian epithelial carcinoma mainly adopts chemotherapy regimens based on cisplatin, cisplatin by tumor cell DNA adduct formation, cause tumor cell DNA damage, which in turn make tumor cell DNA replication is inhibited, the regeneration of cell division stops, eventually kill tumor cells. However, the curative effect of cisplatin will recede because of the internal of cancer cells or acquired(drug induced) drug resistance. So far, the mechanism of cisplatin resistance is not entirely clear, but the nucleotide excision repair( NER) way to platinum drugs formation of DNA adduct cutting repair of platinum drugs in tumor drug resistance plays a very important role in the process of [2], and in NER pathway of excision repair crosscomp lementing p rotein1(ERCC1) is one of the most important repair proteins. ERCC1 in reducing tumor cells to a possibility in platinum chemotherapy drugs because it can timely DNA damage repair of platinum[3].Silence or interference off ERCC1 gene can increase the sensitivity of ovarian cancer cells to cisplatin [4].Mitogen activated protein kases(MAPK) signaling pathway in a variety of malignant tumors, including ovarian cancer cell proliferation, apoptosis and chemotherapy drugs and chemotherapy drug resistance play an important role [5-7], and in most tumors are not normal activated or abnormal expression. Studies have shown that excessive activation of p38 MAPK signaling pathways can lead to the occurrence of ovarian cancer and influence the prognosis of ovarian cancer[8]. MAPK signaling pathways play an important role in high ERCC1 gene expression in prostate cancer and non-small cell carcinoma of the tumor cells [9].Metformin is a routine oral medications for the treatment of type 2 diabetes [10, 11]. Recently two large epidemiological studies show that long-term use of metformin can reduce the incidence of ovarian cancer, and metformin can significantly extend ovarian cancer progression-free surial of patients with diabetes mellitus [12]. In the experiments in vivo and in vitro studies, metformin can inhibit the growth and development of a wide variety of tumor cells, including ovarian cancer [13, 14]. Especially metformin can increase non-small cell cancer, ovarian cancer cells sensitivity to chemotherapy drugs [15], it has been reported that metformin can improve the resistance of the breast cancer to chemotherapy drug, but the specific mechanism is unclear [16].This research through the CCK 8 method to detect human epithelial ovariancancer cells resistant strains C13 K and CP70 cell proliferation, cisplatin resistance and the change of cisplatin resistance from metformin. Application of si RNA transfection technique to inhibit the expression of ERCC1, p38 MAPK gene in drug-resistant cell respectively. Rt-pcr method was used to detect the the m RNA expression of ERCC1 gene before and after use of metformin in drug-resistant cells. And western blot method was used to detect ERCC1, AMPK, p-AMPK, p38 MAPK and p-p38 MAPK protein expression, discusses ERCC1 gene effects on ovarian cancer cells to cisplatin resistance. Discusses the cisplatin resistance change after the application of metformin to ovarian cancer cells, trying to learn more about whether metformin can improve the cisplatin resistance, as well as whether play a role by adjusting p38 MAPK signaling pathways and reduce ERCC1 gene expression, in order to provide new theoretical basis for the chemical treatment of ovarian cancer.ObjectiveWe applied is RNA to inhibit ERCC1 expression in C13 K and CP70 cells. CCK-8, real-time fluorescent quantitative-PCR and western blot were used to analyse the biological behavior that whether the metformin could reverse the cisplatin-resistance of ovarian cancer C13 K and CP70 cells to cisplatin and explore the molecular mechanism under it. The purpose was to provide new reflections on reverse the chemoresistnce clinically.Methods1. Si RNAtransfection inhibit ERCC1, p38 MAPK gene expression in C13 K and CP70 cells. 2. Cellproliferation method(CCK-8) detect the change of cisplatin resistance and improvementof metformin for resistant cells cisplatin resistance before and after the transfectionof ERCC1 si RNA. 3. Application the mothed of RT-PCR and Western blot to detectthe expression change of ERCC1, AMPK pathway, p38 MAPK signaling pathway beforeand after metformin act on the resistant cells.4. Statistical analysis: the SPSS Statistical packageprogram 17.0 was used for all analysis. All experiments are independent repeat3 times,One-way ANOVA was used to analyzed the multiple sets ofdata, LSD-t test was used to compare the different group. Significant level is=0.05.Results1.Metformin could reverse drug-resistance of C13 K and CP70 cells to cisplatin(P<0.05). 2.Metformin reduced the expression levels of ERCC1 m RNA and protein in C13 K and CP70 cells(P<0.05) and activated the AMPK signaling pathway(P<0.05), but this effect of metformin was blocked by AMPK blocking agent Compound C(P<0.01). 3.The p38 MAPK inhibitor SB203580 suppressed the expression of ERCC1 protein in C13 K and CP70 cells(P<0.05); while metformin could suppress the p38 MAPK signaling pathway(P<0.01), but not reduce the expression level of ERCC1 protein in C13 K and CP70 cells transfected with p38 MAPK si RNA(P<0.05).Conclusion1.The ERCC1 gene may probably regulate the resistance to cisplatin in ovarian cancer cells 2.Metformin may reverse the drug-resistance of cisplatin-resistant ovarian cancer cells to cisplatin. 3.Merformin may probably regulate the AMPK pathway and p38 MAPK pathway to reverse the cisplatin-resistance. |
PDF Full Text Request