| Purpose:This article aims to explore the effects of San Huang decoction on the moderation of Aurora kinase A(AURKA) and the biological behavior of breast cancer, including proliferation, apoptosis, adhesion, migration, invasion, Epithelial-to-Mesenchymal Transition (EMT) markers and angiogenesis, and elucidate the underlying relationship between them. In addition, we also primarily explore the effect of San Huang decoction on the chemosensitivity of breast cancer cells to tamoxifen and epirubicin.Methods:1.The inhibition of MCF-7 and MDA-MB-231 cells proliferation was determined by CCK-8 assay. The apoptosis of MCF-7 and MDA-MB-231 cells was detected by AnnexinV-FITC/PI Staining. The expression of apoptosis-related proteins were determined by Western Blot analysis.2.Besides, The expression of mRNA and protein of AURKA and p53 were examined by qRT-PCR analysis and Western Blot analysis respectively. The knockdown of AURKA in MCF-7 and MDA-MB-231 cells was made by Aurora A small interfering RNA (siRNA). CCK-8 assay was employed to determine the effect of San Huang decoction on the growth of AURKA-downregulated MCF-7 and MDA-MB-231 cells.3.The cell adhesion assay was used to examine the change of MCF-7 and MDA-MB-231 cells adhesion caused by San Huang decoction. The effects of San Huang decoction on the migration and invasion of MCF-7 and MDA-MB-231 cells was determined by cell transwell assay, and the expression of EMT markers was detected by Western Blot analysis.4.Moreover, cell transwell assays and Matrigel assay were respectively used to examine the effect of San Huang decoction on migration and tubule formation of human umbilical vein endothelial cells (HUVEC)s grown in the supernatant from MCF-7 and MDA-MB-231 cells with or without AURKA-knockdown. The secretion of VEGF and its transcription were investigated by ELISA assay and qRT-PCR analysis respectively.5.At last, CCK-8 assay and AnnexinV-FITC/PI staining were employed to examine the effect of San Huang decoction combined with tamoxifen/epirubicin on the proliferation and apoptosis of MCF-7 cells, and the effect of San Huang decoction with epirubicin on the proliferation and apoptosis of MDA-MB-231 cells, the effects of which on apoptosis related proteins were determined by Western Blot analysis.Results:1.San Huang decoction inhibited the proliferation of MCF-7 and MDA-MB-231 cells in a dose-dependent manner (P<0.05). The effect of inhibition caused by San Huang decoction 48 hours after delivering to breast cancer cells was better than 24 hours(P<0.05), although similar as 72 hours(P>0.05). San Huang decoction was also found to induce apoptosis in both MCF-7 and MDA-MB-231 cell lines. Consistent with cellular results, San Huang decoction treatment significantly increased the apoptosis-related protein level of cleaved-PARP(c-PARP) and Bax, and down-regulated Bcl-2.2.San Huang decoction decreased the expression of mRNA and protein of AURKA and increased that of p53. With the knockdown of AURKA by small interfering RNA (siRNA), the inhibition of MCF-7 cells proliferation caused by San Huang decoction was decreased by 50.0%(from 49.2% to 24.8%), and the anti-growth effect of San Huang decoction on MDA-MB-231 cells reduced from 51.5% to 33.7%.3.San Huang decoction remarkably downregulated the adhesion, migration and invasion of MCF-7 and MDA-MB-231 cells (P<0.01). In addition, similar with the effect of knockdown of AURKA, San Huang decoction improved the protein level of the epithelial marker of E-cadherin and decreased the mesenchymal markers of N-cadherin, vimentin, snail and slug with notable statistical significance (P<0.01)4.Parallel with silencing AURKA, SHD reduced the migration and tubule formation of HUVECs cultured in the supernatant from MCF-7 and MDA-MB-231 cells (P<0.01).Moreover, San Huang decoction regulated VEGF secretion and transcription level with important statistical significance (P<0.01)5.Coadministration of San Huang decoction and tamoxifen enhanced the effect of tamoxifen on inhibiting the proliferation, inducing the apoptosis and regulating apoptosis-related proteins of MCF-7 cells. Combined with San Huang decoction, the effect of epirubicin treatment of MCF-7 and MDA-MB-231 cells was enhanced in proliferation, apoptosis and apoptosis-related proteins with significant statistical difference (P<0.01).Conclusion:In conclusion, our findings indicated that San Huang decoction was able to regulated the mRNA and protein level of AURKA in MCF-7 and MDA-MB-231 cells. Based on this point, San Huang decoction preformed to induce apoptosis of breast cancer cells to inhibit their growth, moderate EMT markers to inhibit the migration and invasion and downregulate breast tumor neovascularization through downregulating VEGF transcription level. Moreover, San Huang decoction improved the chemosensitivity of breast cancer cells to tamoxifen and epirubicin. |
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