The Effect Of Neuregulin On Proliferation Invasion And Metastasis Of Non-Overexpression ErbB2 Breast Cancer Cells

Objective:To explore whether there was activation of NRG/ErbB2 ligand signal transduction pathway in non-overexpression ErbB2 breast cancer cells, MDA-MB-231 and MCF-7 cells were treated with ErbB2 kinase inhibitor AG825. Then to investigate the proliferation,invasion and metastasis effect of NRG/ErbB2 signal transduction pathway on non-overexpression ErbB2 breast cancer cells MDA-MB-231, and its molecular mechanisms were analyzed.Methods : Non-overexpression ErbB2 breast cancer cells MDA-MB-231 and MCF-7 were investigated in the study. Growth curves of the two cell lines were obtained by the trypan blue exclusion method and cell count .The expressions of NRG were detected by immunocytochemistry and Western-blot. MDA-MB-231 cells and MCF-7 cells were treated with ErbB2 kinase inhibitor AG825. The effect of AG825 on the proliferation was investigated by MTT assay to compare the different effects between the two kinds of cells. The intermediate concentration IC50 in 48 hours in MDA-MB-231 cells was determined. MDA-MB-231 cells were treated with 40μmol/L AG825 for 48h hours, and invasion and metastasis of MDA-MB-231 cells were evaluated with transwell chamber; the expression of MMP-2 and MMP-9 enzymatic activity was detected by gelatin zymography. The protein expressions of MMP-2, HIF-1αand mtp53 proteins were detected by Western-blot. The cell cycle and apoptosis were carried out by flow cytometry; the HIF-1αmRNA was detected by RT-PCR.Results:Compared with MCF-7 cells, MDA-MB-231 cells present stronger ability for growth. MDA-MB-231 cells expressed a relatively higher level of neuregulin by immunocytochemistry method, and MCF-7 cells did not express NRG. In the Western-blot, the positive reaction band has been found in 44KD which coincides with the molecular weight of NRG in MDA-MB-231 cells. When MDA-MB-231 cells and MCF-7 cells were treated with AG825, MDA-MB-231 cells were inhibited more obviously. The intermediate concentration IC50 of AG825 in MDA-MB-231 cells was 56.59μmol/L. MDA-MB-231 cells were treated with 40μmol/L AG825 for 48h hours, and invasion and metastasis were also depressed(P <0.05). The expressions of MMP-2 and MMP-9 enzymatic activity were lower(P <0.05). The expression levels of MMP-2,HIF-1αand mtp53 were decreased(P <0.05). The expression level of HIF-1αmRNA was decreased(P <0.05). The cell cycle was arrested in G0/G1 phase(P <0.05), and the apoptosis rate was increased(P <0.05). Conclusion:Our study indicates that neuregulin were synthesized in MDA-MB-231 cells as transmembrane proteins, and neuregulin could activate ErbB2 receptor signal transduction pathway by autocrine or paracrine action. NRG/ErbB2 signal transduction pathway can affect MMP-2 expression and induce invasion and metastasis. Cell proliferation, inhibition of apoptosis and invasion can be induced by up-regulation of mtp53 and HIF-1αlevel.