天津医药

天津医药 ›› 2017, Vol. 45 ›› Issue (7): 691-695.doi: 10.11958/20170329

• 细胞与分子生物学 • 上一篇    下一篇

肉叶芸香碱对黑色素瘤 A375 细胞上皮间充质转化的影响

赵庆芳，霍焱， 姜战胜，刘东颖   

  1. 1 天津医科大学眼科医院药剂科 （邮编 300384）； 2 天津市肿瘤医院中医肿瘤科
  • 收稿日期:2017-03-14 修回日期:2017-06-05 出版日期:2017-07-15 发布日期:2017-08-08

Effects of harmine on the epithelial mesenchymal transition of melanoma A375 cells

ZHAO Qing-fang,HUO Yan,JIANG Zhan-sheng,LIU Dong-ying   

  1. 1 Department of Pharmacy, Tianjin Medical University Eye Hospital, Tianjin 300384, China;2 Department of Chinese Medicine Oncology, Tianjin Medical University Cancer Institute and Hospital
  • Received:2017-03-14 Revised:2017-06-05 Published:2017-07-15 Online:2017-08-08

PDF (PC)

3780

摘要： 目的 探讨肉叶芸香碱对黑色素瘤 A375 细胞的抑制作用及影响机制。方法 （1）体外培养的黑色素瘤A375 细胞， 分别给予 0 （对照）、 0.5、 1、 2、 5、 10、 20、 50 和 100 mg/L 浓度的肉叶芸香碱处理 48 h， CCK-8 法检测各浓度组细胞存活率， 确定实验浓度。（2）细胞经 0、 1、 2 mg/L 肉叶芸香碱处理后， 分别采用细胞划痕实验和 Transwell 实验检测各组细胞迁移和侵袭能力； Western blot 实验检测上皮间充质转化（EMT）相关蛋白 E-钙黏蛋白（E-cadherin）、N-钙黏蛋白 （N-cadherin）， Snail 和 p53 的表达水平。（3） 细胞设对照组 （仅转染空载体）、 空载体组 （经 2 mg/L 肉叶芸香碱处理 24 h 后， 转染空载体）、 Snail 转染组 （经 2 mg/L 肉叶芸香碱处理 24 h 后， 转染 Snail cDNA）， 转染后检测各组细胞迁移和侵袭能力。结果 （1）肉叶芸香碱浓度≥2 mg/L 时， 随着处理浓度的升高， A375 细胞存活率较对照组明显降低（P < 0.05）， 后续实验选取 0、 1、 2 mg/L 肉叶芸香碱进行处理。（2）随着肉叶芸香碱处理浓度的升高， 各组划痕区细胞数和穿膜细胞数均明显下降， 细胞的迁移和侵袭能力逐渐减弱； 同时 EMT 相关蛋白 E-cadherin、 p53 表达升高， N-cadherin、 Snail 表达降低。（3） 细胞转染实验结果显示， 转染 Snail 后， 细胞的迁移和侵袭能力较空载体组有所提高。结论 肉叶芸香碱可抑制 A375 细胞的增殖， 降低其转移和侵袭能力。这可能是通过激活 p53 后降低 Snail 表达， 进而升高 E-cadherin， 下调 N-cadherin 来抑制 EMT 过程而实现的。

关键词: 肉叶芸香, 黑素瘤, 上皮间充质转化, Snail

Abstract: Objective To explore the inhibitory effect of harmine on melanoma A375 cells and its mechanism thereof.Methods (1) Melanoma A375 cells were treated with harmine at 0, 0.5, 1, 2, 5, 10, 20, 50 and 100 mg/L for 48 h in vitro.CCK-8 method was used to detect the cell viability and confirm the experimental concentrations. (2) After the cells were treated with 0, 1, 2 mg/L harmine, the scratch and transwell assays were used to detect the cell migration and invasion ability. Western blot assay was used to detect the expression levels of epithelial mesenchymal transition (EMT) - related protein E-cadherin, N-cadherin, Snail and p53. (3) Three groups of cells were set up. The control group was transfected with empty vector only. The empty vector group was transfected with empty vector after treated with 2 mg/L harmine for 24 h. The Snail transfection group was transfected with Snail cDNA after treated with 2 mg/L harmine for 24 h. The cell migration and invasion ability were detected after the transfection. Results (1) When the concentration of harmine was above 2 mg/L, the survival rate of A375 cells was significantly lower than that of the control group with the increase of harmine concentrations(P < 0.05). Then, the concentrations of 0, 1 and 2 mg/L of harmine were used in the following experiments. (2) With the increase of the harmine concentrations, the number of cells in the scratched area and the number of trans-membrane cells in each group were significantly decreased. The migration and invasion ability of the cells were decreased gradually. The expression levels of E- cadherin and p53 were increased, while the expression levels of N- cadherin and Snail were decreased. (3) Cell transfection experiments showed that the migration and invasion ability of the cells were increased compared with those of empty vector group after transfection with Snail. Conclusion Harmine can inhibit the proliferation of A375 cells and decrease the abilities of metastasis and invasion, which may be achieved by decreasing the expression of Snail after activating the p53, thereby increasing E-cadherin and down-regulating N-cadherin to inhibit the EMT process.

Key words: harmine, melanoma, epithelial mesenchymal transition, Snail

中图分类号: