The Study On The Relationship Between Epidermal Growth Factor Receptor Signaling Pathway And The Cisplatin Resistance In Epithelial Ovarian Cancer SKOV3/DDP Cells

Epithelial ovarian cancer (EOC) is one of the most frequent maliganttumors in the female population. Because of a lack of early diagnosis andpreventive screening measures, approximately70%of all patients withovarian cancer are diagnosed at an advanced stage. The current managementof patients with advanced disease involves optimal surgical debulkingfollowed by chemotherapy. Although this treatment is highly effective,60-80%of women still die of this disease. The main reasons for poorprognosis are a high recurrence rate and resisitance to chemotherapeutics.Therefore, the development new therapy is critical for treatment of ovariancancer patients.Epidermal growth factor receptor (EGFR) signaling pathway causeswidespread concern of scholars. Accumulating evidence suggests that highexpression and constitutional activation of EGFR can be detected in mostcancer including ovarian cancer. EGFR signaling pathway is involved in manycellular process including cell proliferation, apoptosis, angiogenesis, adhesion,invasion and migration via the activation of PI3K/Akt and Ras/Raf/MEK/ERKdownstream signaling pathway. In addition, EGFR may mediatechemoresistance. Gefitinib (Iressa) is an EGFR-tyrosine kenase inhibitor(EGFR-TKI) that competitively inhibits binding of ATP at the ATP site onEGFR, and evidence suggests gefitinib can cause chemosensization in humantumors. Thus EGFR is a promising target for reversing the chemoresistance inovarian cancer.Objective:To investigate the relationship between activity of epidermal growthfactor receptor and the cisplatin resistance in ovarian cancer SKOV3/DDP cells.Methods:1The baseline expression of epidermal growth factor receptor (EGFR) andphosphorylated epidermal growth factor receptor (p-EGFR) protein in SKOV3and SKOV3/DDP cells were analyzed by Western blot.2The expression of EGFR, p-EGFR protein of SKOV3and SKOV3/DDPcells treated with cisplatin were analyzed by Western blot.3The cell proliferation of SKOV3and SKOV3/DDP cells treated withcisplatin at increasing concentrations (0.625,1.25,2.5,5,10,20μg/ml) weremeasured by MTT assay respectively, and the resistance index was calculated.4The cell proliferation of SKOV3and SKOV3/DDP cells treated withgefitinib at increasing concentrations (0.01,0.1,1,10,100μM) were measuredby MTT assay respectively.5The cell proliferation of SKOV3/DDP cells treated with gefitinib (1μM) andcisplatin (0.625,1.25,2.5,5,10,20μg/ml) combined were measured by MTTassay.6The apoptosis was evaluated by flow cytometry with gefitinib and cisplatinalone or combined in ovarian cancer cells.7The expression of EGFR, p-EGFR, Akt, p-Akt, ERK, p-ERK protein ofSKOV3/DDP cells with gefitinib and cisplatin alone or combined wereanalyzed by Western blot.8Statistical methods: Data were evaluated using SPSS13.0statistical software,and data were expressed as mean±standard deviation. The significancedifference was determined using analysis of variance, LSD method and the ttest. P <0.05was considered significant.Results:1Western blot analysis showed that the p-EGFR expression was significantlyenhanced in SKOV3/DDP compared with SKOV3cells (P＜0.05). However,there was no significant difference between the EGFR expression ofSKOV3/DDP and SKOV3cells (P>0.05).2Western blot analysis showed that compared with untreated cells, the p-EGFR expression was significantly enhanced with cisplatin treated inSKOV3and SKOV3/DDP cells (P＜0.05).3The inhibition effects of cisplatin on the SKOV3and SKOV3/DDP cellswere determined by MTT assays, yielding IC50value of3.74±0.03μg/ml forthe SKOV3cells and IC50value of17.66±0.58μg/ml for the SKOV3/DDPcells. The resistance index was4.72.4MTT assays showed that gefitinib could inhibit the growth of the SKOV3and SKOV3/DDP cells in dose-dependent manner. The inhibition rate ofSKOV3cells, induced by gefitinib, was significantly higher than that ofSKOV3/DDP cells and there was significant difference (P＜0.05).5MTT assays showed that the combined treatment of gefitinib and cisplatinsignificantly enhanced the antiproliferative effect compared with single-agenttreatment and there was significant difference (P＜0.05). The IC50value ofcombination of cispaltin with gefitinib to SKOV3/DDP cells was7.42±0.15,and the drug resistance reversal fold was2.38.6The Flow cytometry showed that the apoptosis rate of cisplatin+gefitinib,was significantly higher than that of cispaltin or gefitnib alone (P＜0.05), andthere was no significant difference between the apoptosis rate of cisplatin andgefitinib in SKOV3/DDP cells (P＞0.05). Compared cisplatin with cisplatin+gefitinib, the rate of apoptosis was significant difference in SKOV3/DDP cells(P <0.05) and compared the apoptosis rate of the SKOV3cells induced bycisplatin with that of the SKOV3/DDP cells, there was significant difference(P <0.05).7Western blot analysis showed gefitnib treatment could inhibit EGFR anddownstream signaling pathways Akt, ERK activation. The cisplatin treatmentupregulated the levels of p-EGFR, p-Akt, p-ERK protein expression. Whengefitinib was administered with cisplatin, their expressions weredownregulated.Conclusions:1p-EGFR is significantly enhanced in the SKOV3/DDP cells compared withthe SKOV3cells, proving the activation of EGFR signaling pathway may be involved in chemoresistance in SKOV3/DDP cells.2Cisplatin can activate EGFR signalling pathway, which decreases thechemotherapy sensitivity of ovarian cancer SKOV3/DDP cells. Gefitinib isable to block the activity of EGFR, Akt, ERK，and sensitise SKOV3/DDPcells to chemotherapy. Inhibiting EGFR activation may reverse the cisplatinresistance in ovarian cancer cells.