| Objective : To detect p-ERK protein expression in ovarian cancer specimens and cell lines,and to examine the effects of MEK inhibitor AZD6244 on cell proliferation,apoptosis as well as cell cycle of ovarian cancer cells.To explore the function and significance of M APK/ERK signaling pathway in the development and progression of ovarian cancer.Methods:(1)western blot was used to detect the p-ERK protein expression in different ovarian cancer cells,as well as the protein expressions of p-ERK,ERK,PARP,Cyclin D1,Caspase-3,Dyrk1 B in the cells after treated with MEK inhibitor AZD6244;(2)MTT assays was employed to determine the inhibitory effect of AZD6244 on the cell proliferation of OVCAR5,OVCAR8,SKOV3 and A2780S;(3)Flow cytometry was applied to determine the influence of AZD6244 on the cell apoptosis and cell cycle of OVCAR5,OVCAR8,SKOV3 and A2780S;(4)p-ERK protein has also been decte cted in ovarian cancer specim ens by immunohistochemistry.Results:(1)The protein p-ERK is widely expressed in various ovarian cancer cell lines such as SKOV3,OV2008,C13,A2780 S,A2780CP,OVCAR4,OVCAR5,OVCAR8 and CAOV3.After treatment of MEK inhibitor AZD6244(5,10?M),p-ERK in OVCAR5,OVCAR8,A2780 S and SKOV3 decreased significantly in dose-dependent manner while ERK stayed unchanged.Additionally,we found a reduction of the expression of Cyclin D1,Caspase-3 and Dyrk1 B in OVCAR5,OVCAR8 and A2780 S,compared with control groups.Specifically,cleaved PARP(89KDa)appeared in OVCAR5.However,it didn't make any difference to the expression of PARP,Caspase-3 and Cyclin D1 in SKOV3,except for the upregulation of Dyrk1 b.(2)Compared the cellular survival of different ovarian cancer cells after treated with MEK inhibitor AZD6244: MTT assays showed the inhibited growth of OVCAR5,OVCAR8 and A2780 S treated with different concentrations of MEK inhibitor AZD6244(0,2.5,5,10,25,50 and 100 ?M)for 48 hours,and the inhibitory effect was in the dose-dependent manner inhibition turns out to be significant as the concentration rises.The survival rates in all experimental groups are significantly lower than control groups(P<0.05).However,the inhibitory effect didn't seem to be obvious in SKOV3.The IC50 of OVCAR5,OVCAR8,A2780 S and SKOV3 were 3.88?M,12.99?M,39.06?M and >100?M,respectively.(3)We determined the influence of AZD6244(5,10?M)on apoptosis and cell cycle of different ovarian cancer cell lines by cytometry.The result showed that after 48 hours of AZD6244,the cell apoptosis rate of OVCAR5?OVCAR8 and A2780 S increased dose-dependently(P<0.05),but no significant was found with the cell apoptosis rate of SKOV3(P>0.05).In terms of cell cycle analysis,our study revealed that the experimetal groups(AZD6244,5,10?M)of OVCAR5 and OVCAR8 show a drastically increased proportion of cells which were arrested in G0/G1 phases and accompanied by a reduced proportion of cells in S phase,in a dose-dependent manner(P<0.05).In A2780 S,however,only in the group with higher concerntration of AZD6244(10?M)showed a significant difference comparing to the control group.Whereas,we didn't found any difference as to SKOV3 though(P>0.05).(4)We examined the protein p-ERK expression,collected in 104 patients of epithelial ovarian cancer,56 of which were patients in early stage and 48 in advanced stage.Among those cases,there are 8 diagnosed as low grade serous carcinoma,55 high grade serous carcinoma,10 clear cell carcinoma,15 ovarian endometrioid carcinoma and 16 mucinous carcinoma.Our study showed that p-ERK1/2 in low grade serous carcinoma was significantly higher expressed than that in high grade serous carcinoma(p < 0.05).Meanwhile,the amount of p-ERK1/2 expressed in clear cell carcinoma resembled that in low grade serous carcinoma(p > 0.05).However,a lower level of the p-ERK1/2 expression were both observed in mucinous carcinoma and low grade endometrial carcinoma(p < 0.05).And there seemed no significant correlationship between expression of p-ERK1/2 and the age,clinical stage as well as blood serum CA125 of the patients with ovarian cancer(p> 0.05).Conclusion:Our study showed that p-ERK1/2 was highly expressed in both the speciments and cells of epilethial ovarian cancer.MEK inhibitor AZD6244 downregulated the expression of p-ERK1/2 in ovarian cancer,accompanied by the decreased proliferation of ovarian cancer cells,and induction of cell apoptosis as well as G0/G1 phase arrest,coupled with the alteration of apoptosis-related protein PARP,caspase-3 as well as cell cycle regulation protein Cyclin D1.In conclusion,MAPK/ERK signaling pathway might play a key role in the development and progression of ovarian cancer,and will provide a novel option for molecular targeted therapies in ovarian cancer. |
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