Influence Of PI3K Inhibitor LY294002 To Expression And Phosphorylation Of ER-a In Human Breast Cancer Cell Line MCF-7

Objective using DMEM medium to cultivate breast cancer cell line MCF-7 ,and observing PI3K/Akt signal transduction pathway inhibitor LY294002 to expression of estrogen receptor alpha (ER-a) and Influence of phosphorylation of ER-a AF-1 domain in human breast cancer cell line MCF-7. discussing relation of PI3K/Akt signal transduction pathway and ER-a signal transduction pathway, and attempting to explain molacule biologic mechanism of hormone-resistant occurring in part of ER(+) breast cancer patients . prividing a reasonable clinical treatment project for them.Methods Using DMEM medium (including 10% FCS) to cultivate cell MCF-7 with monolayer adherence passage.After washed by phosphate buffer succus(PBS), the cultivated cell keep cultivating for four days in DMEM medium (including 5% FCS) without phenol red. the cultivated cell is grouped according to drug dosage: the first group(the blank control experiment group),there is no drug in the group. the second group(the low-dosage LY294002 experiment group),drug concentration is 5μmol/L in cell culture medium.the third group(the medium-dosage LY294002 experiment group),drug concentration is 10μmol/L in cell culture medium .and in the fourth group(the high-dosage LY294002 experiment group), drug concentration is 20μmol/L in cell culture medium . Then at 0 hour,24 hours,48 hours,72 hours collect all groups's cell to examine expression of ER-a and phosphorylation levels of ER-a AF-1 domain by Western blotting. Observing that how drug dosage and action time affects expressiong of ER-a and phosphorylation of ER-a AF-1 domain.finally use SPSS11.0 statistic software to analyse the result,and use SNK-q testing method to test difference of protein expression.Results 1,the expression of ER-a in human breast cancer cell line MCF-7: by SNK-q analyse, the expressions of protein ER-a in human breast cancer cell line MCF-7 at different action time in same dosage group does not exsit significance difference(P>0.05), the expressions of protein ER-a in human breast cancer cell line MCF-7 at different dosage at same action time does not exsit significance difference(P>0.05) 2, phosphorylation of ER-a AF-1 domain in human breast cancer cell line MCF-7: phosphorylation of ER-a AF-1 domain in human breast cancer cell line MCF-7 at different action time in same dosage group by SNK-q testing and analyzing shows: phosphorylation of ER-a AF-1 domain in MCF-7 at drug different action time does not exsit significance difference in the low-dosage group(P>0.05).phosphorylation of ER-a AF-1 domain in MCF-7 at drug different action time exsit significance difference between medium -dosage group and high-dosage group(P<0.05),it decreases along with time continues,and has no obviously change after acted by drug for 48 hours. phosphorylation of ER-a AF-1 domain in human breast cancer cell line MCF-7 in different dosage at same action time by SNK-q testing and analyzing shows: At different time ,phosphorylation of ER-a AF-1 domain in MCF-7 does not exsit significance difference between the blank control group and the low-dosage group(P>0.05),it exsits significance difference between medium -dosage,high-dosage group and the blank control group(P<0.05), and it drops clearly with dosage increases..Conclusion 1.PI3K/Akt inhibitor LY294002 has no obviously Influence to expression of protein ER-a in human breast cancer cell line MCF-7. 2.PI3K inhibitor LY294002 can inhibite phosphorylation of ER-a AF-1 domain in human breast cancer cell line MCF-7. 3.inhibitory action of PI3K inhibitor LY294002 to phosphorylation of ER-a AF-1 domain in human breast cancer cell line MCF-7 is drug dosage- and time- dependent. When drug has acted for 48 hours, It is the most strong that drug effect depends on drug dosage.