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 μmol/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 cells, while BQ788 could block the promotive effect of ET-3. The epithelial-to-mesenchymal 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/L significantly decreased the protein expression of E-cadherin 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 BQ788 upregulated 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 ET-3/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.

Key words: Epithelial-to-Mesenchymal Transition