The Study On The Relationship Between Insulin-like Growth Factor I Receptor And The Cisplatin Resistance In Epithelial Ovarian Cancer

Objective: Epithelial ovarian cancer is the most frequent form of ovarian cancer, when compared to other gynecological cancers, the fatality rate of ovarian cancer surpasses that of endometrial and cervical cancers put together. The majority of patients will experience a recurrence. In recent years, the scholars pay much attention to how to improve thesensitivity of the tumor cells to cisplatin.New treatment paradigms basedon our growing understanding of molecular pathways associated with cancer growth are under investigation. Over the last decade, accumulatingdata suggest that the Insulin-like growth factor1receptor (IGF-R) might be one such good therapeutic target in cancers, including ovarian cancer. However, the role of IGF-R in ovarian cancer warrants further description. In this study, we evaluated the potential of NVP-AEW-541thatis capable of inhibiting the phosphorylation and activation of the downstream pathway components of the IGF-IR to inhibit ovarian cancer cellproliferation.We establish the xenograft tumor model. Investigate the expression levels of IGF-1, IGF-IR protein after chemotherapy. Compare theinhabition rate and the sensitivity to cisplatin after the application of kinase inhibitor NVP-AEW541.Methods:1. The xenograft model in nude mouse with human ovarian cancer cellsSKOV3was established and tumor-derived cells were harvested. Forsubcutaneous tumor formation, SKOV3cells were injected s.c. into the thighof5-6-wk-old female nude mice. Drug treatment of mice was with20mg/kgof cisplatin（cDDP） once six days (total5times), whereas the other groupwas not treated. Harvest the tumor cells: SKOV3-P (not treated) and SKOV3-R (treated) after six weeks;2. IGF-I production was evaluated by immunocytochemistry and IGF-IRexpression and protein levels were evaluated by flow cytometry in theSKOV3-P and SKOV3-R cells;3. Investigate whether the SKOV3-P and SKOV3-R cells exposed to NVP-AEW541(0,1,5,10,20μM) for24h,48h,72h and96h underwentproliferation arrest by MTT assay;4. Investigate whether the SKOV3-P and SKOV3-R cells exposed to NVP-AEW541(0,1,5,10,20μM) and exogenous IGF-I protein (50ng/ml)for48h underwent proliferation arrest by MTT assay;5. Investigate whether the SKOV3-P and SKOV3-R cells exposed to cisplatin(0,0.25,2.5,25μg/ml) for48h underwent proliferation arrest by MTT assay，and analysed half maximal inhibitory concentration of cisplatin (IC50);6. Investigate whether the SKOV3-P and SKOV3-R cells exposed to NVP-AEW541(0,1,5,10,20μM) and cDDP(0,1,5,10,15,20μg/ml) for48hunderwent proliferation arrest by MTT assay;7. Compare the proportion of apoptotic cells by flow cytometry afterSKOV3-P and SKOV3-R cells were exposed to NVP-AEW541(10μM),NVP-AEW541(10μM)+cDDP (10μg/ml)for48h;8. Statistical methods: Data were evaluated using spss13.0statisticalsoftware, and data are expressed as mean±standard deviation. The significancedifference was determined using analysis of variance, LSD method and the ttest. P <0.05was considered significant.Results:1. The SKOV3-P and SKOV3-R cell lines produce IGF-I detectable byimmunocytochemistry，supporting the existence of an autocrine loop；Thelevel of the SKOV3-R cells was higher than that of SKOV3-P cells, and therewas significant difference (P<0.05);2. The SKOV3-P and SKOV3-R cell lines express IGF-IR detectable by FCM,and the expression level of the48h was higher than that of the24h, but therewas no significant difference (P>0.05);The expression level of the SKOV3-R cells was higher than that of SKOV3-P cells, and there was significantdifference (P<0.05);3. We investigated growth inhibitory effects by MTT assays，and NVP-AEW541induced apoptosis at the concentrations:3.1Dose-dependent:The SKOV3-P and SKOV3-R cells exposed to NVP-AEW541(0,1,5,10,20μM)for96h, the inhibitory rates were significant difference (P <0.05), andthere were also significant difference in each experimental group(P <0.05);3.2Time-dependent:The SKOV3-P and SKOV3-R cells exposed to NVP-AEW541for24h,48hand72h,and the inhibitory rates of the three time periods were significantdifference (P <0.05), and there were significant difference in each period (P<0.05);3.3The inhibition rates of the SKOV3-P cells were higher than those of the SKOV3-R cells and there were significant difference;4. Add back of50ng/ml of IGF-I for48h did not revert the NVP-AEW541induced inhibition of proliferation in the SKOV3-P and SKOV3-R cells(P>0.05);5.The inhibition effects of cisplatin on the SKOV3-P and SKOV3-R cells weredetermined by MTT assays, yielding IC50value of2.35±0.06μg/ml for theSKOV3-P cells whereas IC50value of15.87±0.13μg/ml for the SKOV3-Rcells；IC50value for the SKOV3-R cells was higher than that for theSKOV3-P cells, and there was significant difference（P <0.05）6. NVP-AEW541can improve the sensitivity of the SKOV3-P and SKOV3-Rcells to cDDP:The IC50value of2.35±0.06μg/ml for the SKOV3-P cells,and SKOV3-P cellsexposed to NVP-AEW54(1,5,10,20μM)，the IC50values of1.04±0.07μg/ml,0μg/ml,0μg/ml for the SKOV3-P cells, proving the sensitivity to cisplatinincreased（P <0.05）;The IC50value of15.87±0.13μg/ml for the SKOV3-R cells，and SKOV3-R cells exposed to NVP-AEW54(1,5,10,20μM), the IC50values of5.4 9±0.07μg/ml,1.14±0.11μg/ml,0μg/ml for the SKOV3-P cells, proving thesensitivity to cisplatin increased（P <0.05）Compared the sensitivity of the SKOV3-P cells to cisplatin with that of theSKOV3-R cells,there were significant difference（P <0.05）7. We analyzed the rate of apoptosis was a significant increase for theSKOV3-P and SKOV3-R cells exposed to drugs for48h by FCM:The SKOV3-P cells: compared NVP-AEW541with NVP-AEW541+cDDPthe rate of apoptosis was significant difference (P <0.05);The SKOV3-R cells: compared NVP-AEW541with NVP-AEW541+cDDPthe rate of apoptosis was significant difference (P <0.01).Compared the apoptosis rates of the SKOV3-P cells with that of the SKOV3-Rcells,there were significant difference（P <0.05）Conclusion:1. IGF-1and IGF-1R are present on the SKOV3-P, SKOV3-R cells, andthe levels of the SKOV3-R cells are higher than these of SKOV3-P cells, proving the overexpression of IGF-1and IGF-1R are definitely related to cisplatin resistance in ovarian cancer cells.2. IGF-1R kinase inhibitor NVP-AEW541can reduce the proliferation of theSKOV3-P, SKOV3-R cells. The inhibition rates of the SKOV3-P cells weremarkedly higher than those of the SKOV3-R cells.3. IGF-1R kinase inhibitor NVP-AEW541can increase the sensitivity tocisplatin and the rate of apoptosis in the SKOV3-P, SKOV3-R cells.The sensitivity to cisplatin and the apoptosis rates of the SKOV3-P cells were markedly higher than those of the SKOV3-R cells, proving inhibiting the insulin-like growth factor1receptor can revert the cisplatin resistance in ovarian cancer cells.