白屈菜红碱对人卵巢癌SKOV3细胞迁移、侵袭和上皮-间质转化的影响

Effect of chelerythrine on migration, invasion, and epithelial-mesenchymal transition of human ovarian cancer SKOV3 cells

摘要

目的 探讨白屈菜红碱（CHE）对人卵巢癌SKOV3细胞迁移、侵袭和上皮-间质转化（EMT）的抑制作用，阐明其相关作用机制。 方法 体外培养SKOV3细胞，分为对照组和2.5、5.0、10.0、20.0及40.0 μmol·L^－1 CHE组，采用噻唑蓝（MTT）法检测各组细胞增殖抑制率。体外培养SKOV3细胞，分为对照组、转移生长因子β1（TGF-β1）组、TGF-β1+5 μmol·L^－1 CHE组和TGF-β1+10 μmol·L^－1 CHE组，采用细胞划痕实验检测各组细胞迁移率，Transwell小室实验检测各组细胞中迁移细胞数和侵袭细胞数，Westren blotting法检测各组细胞中E-钙黏蛋白（E-cadherin）、N-钙黏蛋白（N-cadherin）和波形蛋白（Vimentin）蛋白表达水平，免疫荧光染色法检测各组细胞中E-cadherin和N-cadherin荧光强度。 结果 MTT法，与对照组比较，5.0、10.0、20.0和40.0 μmol·L^－1 CHE组细胞增殖抑制率明显升高（P<0.05或P<0.01）。细胞划痕实验，与对照组比较，TGF-β1组细胞迁移率明显升高（P<0.01）；与TGF-β1组比较，TGF-β1+5 μmol·L^－1 CHE组和TGF-β1+10 μmol·L^－1 CHE组细胞迁移率明显降低（P<0.01）。Transwell小室实验，与对照组比较，TGF-β1组细胞中迁移细胞数和侵袭细胞数明显增加（P<0.05）；与TGF-β1组比较，TGF-β1+5 μmol·L^－1 CHE组和TGF-β1+10 μmol·L^－1 CHE组细胞中迁移细胞数和侵袭细胞数明显减少（P<0.01）。Westren blotting法，与对照组比较，TGF-β1组细胞中E-cadherin蛋白表达水平明显降低（P<0.01），N-cadherin和Vimentin蛋白表达水平明显升高（P<0.05或P<0.01）；与TGF-β1组比较，TGF-β1+5 μmol·L^－1 CHE组和TGF-β1+10 μmol·L^－1 CHE组细胞中E-cadherin蛋白表达水平明显升高（P<0.01），N-cadherin和Vimentin蛋白表达水平明显降低（P<0.01）。免疫荧光染色，与对照组比较，TGF-β1组细胞中E-cadherin荧光强度明显降低，N-cadherin荧光强度明显升高；与TGF-β1组比较，TGF-β1+5 μmol·L^－1 CHE组和TGF-β1+10 μmol·L^－1 CHE组细胞中E-cadherin荧光强度明显升高，N-cadherin荧光强度明显降低。 结论 CHE能够抑制人卵巢癌SKOV3细胞增殖、迁移、侵袭和EMT。

Abstract

Objective To discuss the inhibitory effect of chelerythrine （CHE） on the migration， invasion， and epithelial-mesenchymal transition （EMT） of the human ovarian cancer SKOV3 cells，and to clarify the associated mechanism. Methods The SKOV3 cells were cultured in vitro and divided into control group and 2.5， 5.0， 10.0， 20.0， and 40.0 μmol·L^－1 CHE groups.Methylthiazolydiphenyl-tetrazolium（MTT） assay was used to detect the inhibitory rates of proliferation of the cells in various groups. The SKOV3 cells were cultured in vitro and divided into control group， transforming growth factor-β1 （TGF-β1） group， TGF-β1+5 μmol·L^－1 CHE group， and TGF-β1+10 μmol·L^－1 CHE group.Cell scratch assay was used to detect the migration rates of the cells in various groups； Transwell chamber assay was used to detect the numbers of migration and invasion cells in various groups； Western blotting method was used to detect the expression levels of E-cadherin， N-cadherin， and Vimentin proteins in the cells in various groups； immunofluorescence staining method was used to detect the fluorescence intensities of E-cadherin and N-cadherin in the cells in various groups. Results The MTT assay results showed that compared with control group， the inhibitory rates of proliferation of the cells in 5.0， 10.0， 20.0， and 40.0 μmol·L^－1 CHE groups were significantly increased （P<0.05 or P<0.01）. The cell scratch assay results showed that compared with control group， the migration rate of the cells in TGF-β1 group was increased （P<0.01）； compared with TGF-β1 group， the migration rates of the cells in TGF-β1+5 μmol·L^－1 CHE group and TGF-β1+10 μmol·L^－1 CHE group were significantly decreased （P<0.01）. The Transwell chamber assay results showed that compared with control group， the numbers of migration and invasion cells in TGF-β1 group were significantly increased （P<0.05）； compared with TGF-β1 group， the numbers of migration and invasion cells in TGF-β1+5 μmol·L^－1 CHE group and TGF-β1+10 μmol·L^－1 CHE group were significantly decreased （P<0.01）. The Western blotting results showed that compared with control group， the expression level of E-cadherin protein in the cells in TGF-β1 group was significantly decreased （P<0.01）， while the expression levels of N-cadherin and Vimentin proteins were increased （P<0.05 or P<0.01）； compared with TGF-β1 group， the expression levels of E-cadherin protein in the cells in TGF-β1+5 μmol·L^－1 CHE group and TGF-β1+10 μmol·L^－1 CHE group were significantly increased （P<0.01）， and the expression levels of N-cadherin and Vimentin proteins were significantly decreased （P<0.01）. The immunofluorescence staining results showed that compared with control group， the fluorescence intensity of E-cadherin in the cells in TGF-β1 group was decreased， and the fluorescence intensity of N-cadherin was increased； compared with TGF-β1 group， the fluorescence intensities of E-cadherin in the cells in TGF-β1+5 μmol·L^－1 CHE group and TGF-β1+10 μmol·L^－1 CHE group were significantly increased， and the fluorescence intensities of N-cadherin were decreased. Conclusion CHE can inhibit the proliferation， migration， invasion， and EMT of the human ovarian cancer SKOV3 cells.

关键词

白屈菜红碱 / 卵巢肿瘤 / 上皮-间质转化 / 转化生长因子β1 / 细胞迁移 / 细胞侵袭

Key words

Chelerythrine / Ovarian neoplasm / Epithelial-mesenchymal transition / Transforming growth factor-β1 / Cell migration / Cell invasion

Author summay

周 佳（1995－），女，吉林省长春市人，在读博士研究生，主要从事中药药剂和新药开发方面的研究。