ABSTRACT
[Objective] To explore the role of endothelin-3 (ET-3) on epithelial-to-mesenchymal transition in a malignant melanoma cell line A375.[Methods] A375 cells were cultured in vitro and classified into 3 groups to be treated with ET-3 at 100 nmol/L (ET-3 group),co-cultured with ET-3 at 100 nmol/L and endothelin receptor B (ETRB) antagonist BQ788 at 100 μmo1/L (ETRB antagonist group),or to remain untreated (blank control group).After additional 24-hour culture,Transwell chamber assay was used to detect the invasive capability of A375 cells,real time-PCR to measure the mRNA expressions of Twist and Slug,and Western blot to determine the protein expression of E-cadherin,vimentin,Twist and Slug.The changes in the morphology of A375 cells were observed.Data were assessed by analysis of variance and Scheffe's method.[Results] In the Transwell assay,the number of A431 cells permeating through the basement membrane was 4.200 ± 0.837,9.400 ± 0.548 and 3.400 ± 0.894 respectively in the blank control group,ET-3 group and ETRB antagonist group (F =88.44,P ＜ 0.01 ),suggesting that ET-3 could promote the metastasis of A375 ceils,while BQ788 could block the promotive effect of ET-3.The epithelial-to-mesenehymal transition was obvious in cells treated with ET-3 alone,but was inapparent in cells treated with ET-3 and BQ788.The ET-3 at 100 nmol/Lsignificantly decreased the protein expression of E-cadhefin from 0.33 ± 0.002 (blank control group) to 0.28 ±0.007,but increased that of vimentin from 0.83 ± 0.014 (blank control group) to 1.16 ± 0.003,while BQ788upregulated the E-cadherin expression to 0.42 ± 0.008 and downregulated the vimentin expression to 0.75 ±0.030,and significant differences were observed in the E-cadherin expression and vimentin expression among the ET-3 group,ETRB antagonist group and blank control group (F =329.98,262.94,respectively,both P ＜ 0.01 ).A significant increase was observed in the mRNA and protein expression of Slug (F=376.94,288.87,both P＜ 0.01 )and Twist (F=215.62,156.96,P＜ 0.01 and 0.05) in A375 cells after treatment with ET-3.[Conclusion] ET3/ETRB axis may promote the epithelial-to-mesenchymal transition in A375 cells likely by regulating the expression of E-cadherin,vimentin and two important transcription factors Twist and Slug.
ABSTRACT
Objective To investigate the modulation of ET-3 on the nuclear factor (NF)-κB/Bfl-1 antiapoptotic 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 100 nmol/L for 24 hours. ET-3 of various concentrations (0, 1, 10, 100 nmol/L) was used to treat some A375 cells with or without the pretreatment with the ETRB antagonist BQ788; after another 24-hour culture, RT-PCR and Western blot were conducted to examine the mRNA expression of Bfl-1 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.