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.
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Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60 percent of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.
Subject(s)
Humans , Cell Culture Techniques/methods , /pharmacology , Epithelial Cells/cytology , Intestinal Mucosa/cytology , Intestine, Small/cytology , Thermolysin/pharmacology , Cell Differentiation , Cell Line , Cell Movement , Cell Proliferation , Epithelial Cells/drug effects , Fetus , Intestinal Mucosa/embryology , Intestine, Small/embryologyABSTRACT
In order to investigate the expression of endothelin receptor B (ETR-B) in human ma-lignant melanoma (MM) cells A375 and SK-mel-1 and the proliferative effects of endothelin 3 (ET3) on A375 cells, RT-PCR was applied to detect the expression of ETR-B gene in human MM cells A375 and SK-mel-1. MTT method was used to evaluate the growth enhancing effects of ET3 on A375 cell line in vitro. The results showed that ETR-B gene was expressed in both MM A375 and SK-mel-1 cells. ET3 had stronger ability to enhance the proliferation of A375 cells in vitro in a con- centration-dependent manner. It was suggested that ET3/ETR-B might play an important proliferative role in MM.
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Objective To investigate the relationship between gene expression of endothelin-3(ET-3) and inflammation of acute pancreatitis(AP) in rats.Methods Fifty-four rats were divided randomly into 4 groups:sham operation group,AP group,arterial injection group and vein injection group.AP was induced by reverse intra-bile duct infusion 4.5% sodium taurocholate,treated with low dose dopamine 〔5 ?g/(kg?min)〕 by injecting arterial or tail vein.Rats were sacrificed at 1,6 and 24 h after the induction of AP.The mRNA expression of ET-3 was evaluated by semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR) and pathological changes was observed in rats.Results Expression of ET-3 mRNA could be detected from 1 up to 24 h after the induction of pancreatitis.Expression of ET-3 mRNA of sham operation group was decreased significantly compared with other three groups.Expression of ET-3 mRNA showed a significant decrease by arterial injection dopamine than that by tail vein(P
ABSTRACT
AIM To study mechanism of endothelin (ET) on canine pulmonary veins. METHODS The isometric tension of pulmonary venous strips was recorded. RESULTS ET 3 and IRL1640 produced contraction in pulmonary venous strips. ET 3 induced contraction was markedly suppressed by BQ123 (P