内皮素3／内皮素受体B对A375细胞NF—κB／Bcl-2相关蛋白A1抗凋亡通路的调节

Endothelin-3/endothelin receptor B （ET-3/ETRB） regulates the NF-KB/Bfl-1 anti-apoptotic pathway in a malignant melanoma cell line A375

目的研究内皮素3（ET-3）对人恶性黑素瘤（MM）A375细胞核因子（NF）-κB／Bfl-1抗凋亡通路的调节。方法用ET一3（100nmol／L）刺激A375细胞24h后，流式细胞仪检测细胞凋亡率。RT—PCR及Western印迹测定不同浓度ET-3（0、1、10、100nmol／L）及其与内皮素受体B（ETRB）阻断剂BQ788联合干预A375细胞后，Bfl-1在mRNA和蛋白质水平的表达。Western印迹检测相同条件干预下磷酸化NF—KB（pNF—KB）的蛋白表达。结果ET-3可以降低A375细胞的凋亡率（F=10．68，P〈0．05）。ET-3对A375细胞Bn-1mRNA和蛋白表达水平的影响呈浓度依赖性上调，BQ788明显阻断ET-3上调Bn-1的效应（P〈0．01）。Western印迹检测结果显示，ET-3显著上调pNF—KB的表达（P〈0．05），BQ788干预下其表达水平降低，接近对照组，ET-3（100nmol／L）＋BQ788组与100nmol／LET-3组比较，差异有统计学意义（P〈0．01）。结论ET-3／ETRB通过激活NF—κB／Bfl-1抗凋亡通路抑制A375细胞凋亡。

Objective To investigate the modulation of ET-3 on the nuclear factor （NF）-KB/Bfl-1 anti- apoptotic pathway in a malignant melanoma cell line A375. Methods Flow cytometry was performed to detect the apoptosis in cultured A375 cells after treatment with ET-3 of I00 nmol/L for 24 hours. ET-3 of various concentrations （0, 1, 10, 100 nmol/L） was used to treat some A375 ceils with or without the pretreatment with the ETRB antagonist BQ788; after another 2d-hour culture, RT-PCR and Western blot were conducted to examine the mRNA expression of Bfl-I and protein expressions of Bfl-1 and ETRB, respectively. Results The 24-hour treatment with ET-3 of 100 nmol/L significantly reduced the apoptosis rate of A375 cells （F = 10.68, P 〈 0.05）. The mRNA and protein expressions of Bfl-1 were up-regulated by ET-3 in a concentration dependent manner （both P 〈 0.01）, while BQ788 significantly blocked the ET-3-induced up-regulation （F = 420.38, 229.49, both P 〈 0.01 ）. The protein expression of pNF-κB in A375 cells was also enhanced by ET-3 of different concentrations （all P 〈 0.05）, but the enhancement was suppressed by BQ788, and there was a significant difference in the protein expression of pNF-κB between cells treated with ET-3 of 100 nmol/L and those treated with the combination of ET-3 of 100 nmol/L and BQ788 （F = 255.46, P 〈 0.01）. Conclusion ET-3/ETRB inhibits the apoptosis in A375 cells likely by activating the NF-κB/Bfl-1 anti-apoptotic pathway.