Regulation Of Chemokines CXCL12, CXCL8 And CX₃CL1 By Estrogen In ER Positive Human Breast Cancer Cells

Objective:Our study is to investigate effect of estrogen on chemokines expression profile in estrogen receptor(ER) positive human breast cancer cells,and to explore the mechanism.Methods:Oligo GEArray chemokines and chemokine receptors microarray was used to screen targeting chemokines and chemokine receptors in ER positive human breast cancer cells treated with estradiol.RT-PCR was performed to verify the impact of estradiol on corresponding chemokines(CXCL12,CXCL8,CXCL1 and CCL2) mRNA expression in MCF-7(ER⁺),T47D(ER⁺) and MDA-MB-231(ER) cells. RT-qPCR(real-time PCR) and ELISA was utilized to detect the change of chemokines (CXCL12,CXCL8,CX3CL1) mRNA expression and extracellular protein level at different time point treated with different dose of estradiol in absence or presence of ER antagonist fulvestrant(ICI182,780) in MCF-7 cells(ER⁺).CCK-8 kit and FCM was employed to analyze effect of estrodiol in breast cancer MCF-7 cell's growth and cell cycle.Results:Estradiol could stimulate cell growth and increase S-phrase distribution in ER positive human breast cancer cell MCF-7;while decrease of G2-M phrase, increase of S phrase and apoptosis augment were observed in MCF-7 cell when treated with estradiol combination with ICI182,780.Microarray analysis showed that estradiol could up-regulate CXCL12,CXCL8,CXCL1 and CCL2 mRNA expression while down-regulate CX₃CL1 rnRNA expression.CXCL12 mRNA was up-regulated 4.4 folds treated with estradiol compared with vehicle control.Same changes were observed by RT-PCR.But estradiol did not influence these chemokines mRNA expression in ER negative breast cancer cell MDA-MB-231.Further study revealed that the most impact of estradiol on chemokine expression was observed when MCF-7 was treated with 10 nmol/L estradiol.These impacts appeared from 1 hour to 24 hours after estradiol treatment.In addition,estradiol had parallel impact on CXCL12, CXCL8 chemokine extracellular protein secretion.However,estradiol down-regulated CX₃CL1 mRNA expression while up-regulated CX₃CL1 extracellular protein secretion.These regulations could be inverted or diminished by ICI182,780 in ER positive breast cancer cell MCF-7.For example,CXCL12 mRNA expression and extracellular protein level was much lower in estradiol+ICI182,780 group than in estradiol group.Conclusions:Our study demonstrates that CXCL12,CXCL8 and CX₃CL1 may be target of estrogen in breast cancer cells and that stradiol could broadly and diversely regulate chemokine mRNA expression profile and extracellular protein level in ER positive human breast cancer cell MCF-7.This effect at least,in part,is mediated through the estrogen receptor signaling.