Role Of Endogenous H₂S Synthetase-CSE In Human Breast Cancer Metastasis And The Effect And Mechanism Of The Novel CSE Inhibitor I157172 Against Breast Cancer Metastasis

OBJECTIVE:To investigate the role and mechanism of cystathionine-γ-lyase（CSE）and the novel CSE inhibitor I157172 in breast cancer metastasis.METHODS:Immunohistochemistry,Western blot and quantitative real-time PCR（qRT-PCR） assays were used to detect the expression level of CSE in human breast cancer tissues and cells.CSE siRNA and pCMV-EGFP-hCSE were transfected into MDA-MB-231 cells respectively and MTS,scratch and transwell chamber assays were performed to analyze the effects of different CSE expression levels on proliferation,migration and invasion of breast cancer cells in vitro.The xenograft tumor model of human breast cancer cells MDA-MB-231 and stably CSE knockdown MDA-MB-231 were established in nude mice,and the effects of different CSE expression levels on lung metastasis of breast cancer were analyzed by H&E.Western blot was used to detect the effects of different CSE expression levels on the expression of MMPs and VEGF signal pathway-related proteins（PI3K,Akt,pAkt,FAK,Paxillin,Ras,Raf,ERK1/2 and pERK1/2）in human breast cancer MDA-MB-231 cells,in order to explore the mechanism of CSE expression regulating breast cancer metastasis.A novel CSE inhibitor I157172 was obtained by computer virtual screening method,and methylene blue method was used to detect its effect on H₂S production in MDA-MB-231 cells,in order to evaluate its inhibitory activity on CSE.MTS,EdU and transwell chamber assays were used to detect the inhibitory effects of I157172 on proliferation,migration and invasion of breast cancer cells.Western blot was used to detect the effects of I157172 on the expression of MMPs and VEGF signal pathway-related proteins（PI3K,Akt,pAkt,FAK,Paxillin,Ras,Raf,ERK1/2 and pERK1/2）in human breast cancer MDA-MB-231 cells,in order to evaluate the role and mechanism of I157172 in inhibiting breast cancer metastasis.Results:1.Immunohistochemistry,Western Blot and qRT-PCR results showed that the levels of CSE mRNA and protein in breast cancer patient tissues with lymph node metastasis was higher than ones with non-lymph node metastasis.The levels of CSE mRNA and protein in high metastatic breast cancer MDA-MB-231 cells was also significantly higher than that in non-metastatic breast cancer MCF-7 cells.2.MTS,scratch and transwell results showed that down-regulation of CSE expression inhibited the growth,migration and invasion of human breast cancer MDA-MB-231 cells,while up-regulation of CSE expression promoted the growth,migration and invasion of human breast cancer MDA-MB-231 cells.3.H&E staining showed that down-regulation of CSE expression inhibited lung metastasis of human breast cancer MDA-MB-231 cells in BALB/c-nu mice.4.Western blot results showed that down-regulation of CSE expression reduced the levels of MMP2 and MMP9 in human breast cancer MDA-MB-231 cells,while up-regulation of CSE expression increased the levels of MMP2 and MMP9 in stably CSE knockdown MDA-MB-231 cells.5.Western blot results showed that down-regulation of CSE expression reduced the levels of VEGF and its downstream pathway-related proteins（PI3K,Akt,pAkt,FAK,Paxillin,Ras,Raf,ERK1/2and pERK1/2）in human breast cancer MDA-MB-231 cells,while up-regulation of CSE expression increased the levels of VEGF and its downstream pathway-related proteins（PI3K,Akt,pAkt,FAK,Paxillin,Ras,Raf,ERK1/2 and pERK1/2）in stably CSE knockdown MDA-MB-231 cells.6.Western blot and methylene blue results showed that the novel CSE inhibitor I157172,which was screened by computer virtual screening,reduced the levels of CSE protein and H₂S in MDA-MB-231cells,which indicated I157172 possessed the inhibitory activity of CSE protein.7.MTS,EdU and transwell results showed that the novel CSE inhibitor I157172 inhibited the proliferation,migration and invasion of human breast cancer MDA-MB-231 cells.8.Western blot results showed that the novel CSE inhibitor I157172 reduced the levels of MMP2 and MMP9 in human breast cancer MDA-MB-231 cells.9.Western blot results showed that the novel CSE inhibitor I157172 reduced the levels of VEGF and its downstream pathway-related proteins（PI3K,Akt,pAkt,FAK,Paxillin,Ras,Raf,ERK1/2 and pERK1/2）in human breast cancer MDA-MB-231 cells.Conclusion:1.High expression of CSE promotes the metastasis of breast cancer.2.CSE promotes the metastasis of breast cancer via VEGF signaling pathway.3.The novel CSE inhibitor I157172 significantly inhibits the proliferation,migration and invasion of breast cancer cells by inhibiting VEGF signaling pathway.