A Relevant Research Of The PI3K/AKT Signaling Pathway And Drug-resistance On Epithelial Ovarian Cancer In Vitro

Objective:This research investigated the PI3K/AKT signaling pathway and cisplatinresistance on ovarian epithelial cancer in vitro from the level of gene andprotein,explore its possible mechanism of drug resistance and may be an effectivereversion to find effective treatments to overcome the cisplatin resistance andprovide a certain amount of experiments in vitro.Methods:1. Epithelial ovarian cancer cell SKOV3cell were cultured in nutrient solutionof RPMI1640containing10%FBS and100u/ml of penicillin and streptomycinEpithelial ovarian cancer with the cisplatin-resistance SKOV3/DDP cell werecultured in nutrient solution of RPMI1640containing0.375ug/mlDDP,10%FBS and100u/ml of penicillin and streptomycin, two kinds of cells are cultured in incubatorin the situation of37℃,5%CO2.Draw the growth curve of the two kinds of cells.Though the growth curve can be concluded that SKOV3and SKOV3/DDP cells inlogarithmic phase for training after3to5days,in order to choose the right cells forthe experiment.2. The growing situation of both the SKOV3and SKOV3/DDP cells was assessedby MTT assay.3.The gene level of PI3K and Akt in the SKOV3and SKOV3/DDP cells wasmeasured by Quantative Real-time PCR,it could be shorted as Q-PCR.4. The expression of AKT,p-AKT and PI3K protein in the SKOV3andSKOV3/DDP cells was determined by Western blot.Results:1. Enzyme immune detector at490nm wavelength could determine the lightabsorbance value (OD), can indirectly mean the number of living cells. By MTTassay to detect the OD value of the SKOV3cell in the first day, the third day, the fifthday, the seventh day.OD was0.069±0.008,0.210±0.009,0.551±0.006,0.418±0.079, in the SKOV3/DDP cells the first day, the third day, the fifth day, the seventh day OD was0.030±0.004,0.147±0.010,0.329±0.005,0.252±0.058. The ODvalue of the SKOV3/DDP is lower than that of in the SKOV3cells,the difference wasstatistically significant.The SKOV3/DDP cells is growing slower than the SKOV3cells(P<0.05).2.By Q-PCR could find the2-△Ctof PI3K gene in the SKOV3cells is0.76±0.04and the2-△Ctof PI3K gene in the SKOV3/DDP cells is1.49±0.04,the level of PI3Kgene in the SKOV3/DDP cells is significantly higher than that of in the SKOV3cells,the difference was statistically significant(P<0.05).The2-△Ctof Akt gene in theSKOV3cells is1.37±0.03and the2-△Ctof Akt gene in the SKOV3/DDP cells is2.04±0.02,the Akt gene level in the SKOV3/DDP cells is significantly higher than that of inthe SKOV3cells, the difference was statistically significant(P<0.05).3. By Western-blotting could find the expression of PI3K protein in the SKOV3cells is0.80±0.03and the expression of PI3K protein in the SKOV3/DDP is1.43±0.01,the expression level of PI3K protein in the SKOV3/DDP cells is significantlyhigher than that in the SKOV3cells,the difference was statistically significant(P<0.05).The expression of Akt protein in the SKOV3is1.30±0.05and theexpression of Akt protein in the SKOV3/DDP cells is1.93±0.01, the expressionlevel of Akt protein in the SKOV3/DDP cells is significantly higher than that in theSKOV3cells, the difference was statistically significant(P<0.05).The expression ofp-Akt protein in the SKOV3cells is0.68±0.03and expression of p-Akt protein inthe SKOV3/DDP cells is1.06±0.06, the expression level of p-Akt protein in theSKOV3/DDP cells is significantly higher than that in the SKOV3cells, the differencewas statistically significant(P<0.05).Conclusion：1. The protein of Akt、p–Akt and PI3K is expressed in both the SKOV3andSKOV3/DDP cells. PI3K/Akt pathway is closely related to the ovarian epithelialcarcinoma.2. The expression of PI3K,Akt and p-Akt protein in the SKOV3/DDP cells ishigher than those in the SKOV3cells. The cisplatin resistance in the SKOV3/DDPcells is associated with the high expression of PI3K,Akt and p-Akt.3. The level of PI3K gene in the SKOV3/DDP cells is higher than that in the SKOV3cells. The level of Akt gene in the SKOV3/DDP cells is higher than that inthe SKOV3cells. SKOV3/DDP cells. It represents the mechanisms of the cisplatinresistance on the epithelial ovarian cancer is associated with high level of the PI3Kand Akt gene.