ABSTRACT
The aim of this study was to investigate the effects of hirsutine on apoptosis of breast cancer cells and its possible mechanism. The MCF-10A, MCF-7 and MDA-MB-231 cells were treated with hirsutine at different concentrations for 48 h or incubated with 160 μmol/L hirsutine for 24, 48, and 72 h. The MCF-10A cell line is a non-tumorigenic epithelial cell line, and the MCF-7 and MDA-MB-231 are human breast adenocarcinoma cell lines. CCK-8 assay was employed to detect the cell viability. Flow cytometry was used to assay the apoptosis and mitochondrial membrane potential (MMP). The protein expressions of Bcl-2, Bax, cleaved-caspase 9, cleaved-caspase 3 and cytochrome C (Cyt C) in the MDA-MB-231 cells were detected by Western blotting. The results showed that hirsutine remarkably reduced the viability of MCF-7 and MDA-MB-231 cells in a time- and dose-dependent manner (P < 0.05) with IC50 values of 447.79 and 179.06 μmol/L, respectively. In the MDA-MB-231 cells, hirsutine induced apoptosis and depolarization of MMP (P < 0.05), released Cyt C from mitochondria (P < 0.05), and activated caspase 9 and caspase 3 (P < 0.05). However, these effects induced by hirsutine were all inhibited by cyclosporin A (CsA) (P < 0.05), a specific inhibitor of mitochondrial permeability transition pore (MPTP). In addition, hirsutine down-regulated the protein level of Bcl-2 and up-regulated the protein level of Bax (P < 0.05). These results suggest that hirsutine may induce apoptosis of human breast cancer MDA-MB-231 cells through decreasing the ratio of Bcl-2 to Bax, opening MPTP, releasing Cyt C from mitochondria, and activating caspase 9 and caspase 3.
ABSTRACT
AIM:To investigate the effect of hirsutine on hypoxia-induced migration and invasion abilities of human breast cancer MCF-7 cells and its possible mechanism. METHODS:CCK-8 assay was employed to detect the cyto-toxic effect of hirsutine on the MCF-7 cells. Cell migration was observed by wound healing assay,and cell invasion ability was measured by Transwell invasion assay. Western blot was used to analyze the protein levels of hypoxia-inducible factor-1α(HIF-1α),Snail,E-cadherin and matrix metalloproteinase-9 (MMP-9). The mRNA levels of HIF-1α was detected by RT-PCR. RESULTS:Hirsutine remarkably reduced the cell viability from 32 μmol/L(P<0.05),and the IC50value was 62.82 μmol/L. In hypoxia state,MCF-7 cells showed more powerful capabilities of migration and invasion (P<0.05), higher protein levels of HIF-1α,Snail and MMP-9 (P<0.05),lower protein level of E-cadherin(P<0.05),and higher mRNA level of HIF-1α (P<0.05). These hypoxia-induced effects were all inhibited by hirsutine at 16 μmol/L (P<0.05),apart from the mRNA level of HIF-1α. CONCLUSION:Hirsutine inhibits hypoxia-induced migration and inva-sion in human breast cancer MCF-7 cells most likely via down-regulation of the protein levels of HIF-1α,Snail and MMP-9,and up-regulation of the protein level of E-cadherin.