花青素单一成分矢车菊-3-O-葡萄糖苷抑制B16-F10细胞增殖和迁移的机理

Mechanism of the anthocyanin single component cyanidin-3-O-glucoside inhibiting proliferation and migration of B16-F10 cells

本研究旨在探讨花青素单一成分矢车菊-3-O-葡萄糖苷(cyanidin-3-O-glucoside, Cy-3-glu)对小鼠黑色素瘤细胞B16-F10增殖和迁移的影响,并阐明相关机理。用不同浓度Cy-3-glu处理B16-F10细胞,采用CCK-8法检测细胞存活率,划痕法检测细胞迁移情况,流式细胞仪检测细胞周期,real-time PCR检测细胞周期相关基因的m RNA表达水平,Western blot检测p-AKT、E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)和波形蛋白(vimentin)的相对表达水平,并用小动物活体成像仪检测B16-F10细胞在C57BL/6J小鼠体内的生长和迁移情况。结果显示,Cy-3-glu能明显抑制体外B16-F10细胞的生长(P <0.001)和迁移(P <0.01),使细胞周期阻滞在S期。Cy-3-glu作用后p-AKT (P <0.05)、N-cadherin和vimentin (P <0.001)表达水平显著下降,E-cadherin表达水平升高(P <0.05)。饲喂Cy-3-glu饲料的肿瘤模型C57BL/6J小鼠肿瘤大小和重量显著减小(P <0.05),肿瘤的转移水平也明显降低。以上结果提示,Cy-3-glu通过抑制PI3K/AKT信号、调节细胞黏附和迁移信号使B16-F10细胞周期停滞在S期,最终抑制其在体内和体外的增殖和迁移。

To study the effects of the anthocyanin single component cyanidin-3-O-glucoside(Cy-3-glu) on the proliferation and migration of mouse melanoma cells and to elucidate the underlying mechanisms, B16-F10 cells were treated with different concentrations of Cy-3-glu. Cell viability was analyzed by a CCK-8 method. Cell migration was determined by the callus scratching technique. Cell cycle was measured by the flow cytometry. The expression levels of genes involved in cell cycle regulation were detected by real-time PCR. Protein expression levels of p-AKT, E-cadherin, N-cadherin and vimentin were analyzed by Western blot. The growth and migration of B16-F10 cells in C57 BL/6 J mice were monitored by the cryogenically cooled IVIS-imaging system. The results showed that Cy-3-glu significantly inhibited the growth(P < 0.001) and migration(P < 0.01) of B16-F10 cells, and arrested the cell cycle in the S phase. After Cy-3-glu treatment, the expression levels of p-AKT(P < 0.05), N-cadherin and vimentin(P < 0.001) were decreased significantly, and the expression level of E-cadherin was dramatically increased(P < 0.05). The size and weight of tumors and tumor metastasis in mice fed with a diet containing Cy-3-glu were significantly reduced(P < 0.05). In conclusion, Cy-3-glu inhibits proliferation and migration of B16-F10 cells by inhibiting the PI3 K/AKT signaling pathway, cell adhesion and migration signals.