RESUMO
UNLABELLED: The study was aimed on analysis of human beta-defensin-1-4 (hBDs) mRNA expression in cultured thyroid cancer cells and evaluation of effects of recombinant hBD-2 (rec-hBD-2) on growth patterns, migration properties and expression of E-cadherin and vimentin in these cells. METHODS: The study was performed on cultured follicular thyroid cancer WRO cells, papillary thyroid cancer TPC1 cells, and anaplastic thyroid cancer KTC-2 cells. For analysis of hBD-1-4 mRNA expression in thyroid cancer cells, semiquantitative RT-PCR was used. Effects of rec-hBD-2 on cell proliferation, viability, and migration were analyzed using direct cell counting, MTT test, and scratch assay respectively. Expression of vimentin and E-cadherin was evaluated by quantitative PCR (qPCR). RESULTS: By the data of RT-PCR, all three studied thyroid cancer cell lines express hBD-1 and -4 mRNA, but not hBD-2 mRNA, while hBD-3 expression was detected in WRO and KTC-2 cells. The treatment of TPC-1, WRO, and KTC-2 cells with 100-1000 nM rec-hBD-2 resulted in significant concentration-dependent suppression of cell proliferation, viability, and migratory property. By the data of qPCR, significant up-regulation of vimentin expression was registered in KTC-2 and WRO cells treated with 500 nM rec-hBD-2. Significant down-regulation of E-cadherin expression (p < 0.05) was detected only in KTC-2 cells treated with the defensin. Also, it has been shown that TPC-1 cells treated with 500 nM rec-hBD-2 acquired more elongated morphology. CONCLUSION: The data demonstrate that hBD-2 in concentrations higher than 100 nM exerts significant concentration-dependent suppression of thyroid cancer cell growth and migration, and affects vimentin and E-cadherin expression dependent on histologic type of thyroid cancer cells.
Assuntos
Adenocarcinoma Folicular/genética , Carcinoma Papilar/genética , Neoplasias da Glândula Tireoide/genética , beta-Defensinas/genética , Adenocarcinoma Folicular/metabolismo , Adenocarcinoma Folicular/patologia , Apoptose , Caderinas/genética , Caderinas/metabolismo , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Movimento Celular , Proliferação de Células , Humanos , Técnicas In Vitro , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias da Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/patologia , Células Tumorais Cultivadas , Vimentina/genética , Vimentina/metabolismo , Cicatrização , beta-Defensinas/metabolismoRESUMO
BACKGROUND AND AIM: Human beta-defensin-2 (hBD-2) is an antimicrobial cationic peptide capable to control human carcinoma cell growth via cell cycle regulation. The present study was aimed on determination of hBD-2 influence on the growth patterns and malignant potential of cultured human melanoma cells. METHODS: The study was performed on cultured human melanoma cells of mel Z and mel Is lines treated with recombinant hBD-2 (rec-hBD-2); cell viability, proliferation, cell cycle distribution, and anchorage-independent growth were analyzed using MTT test, direct cell counting, flow cytometry, and colony forming assay respectively. Expression and/or phosphorylation levels of proteins involved in cell cycle control were evaluated by Western blotting. RESULTS: The treatment of mel Z and mel Is cells with rec-hBD-2 in a concentration range of 100-1000 nM resulted in a concentration-dependent suppression of cell proliferation, viability, and colony forming activity. It has been shown that rec-hBD-2 exerts its growth suppression effects via significant downregulation of B-Raf expression, activation of pRB and upregulation of p21(WAF1) expression, downregulation of cyclin D1 and cyclin E resulting in cell cycle arrest at G1/S checkpoint. CONCLUSION: According to obtained results, hBD-2 exerts its growth suppression effect toward human melanoma cells via downregulation of B-Raf, cyclin D1 and cyclin E expression, upregulation of p21(WAF1) expression and activation of pRB.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Pontos de Checagem do Ciclo Celular/genética , Melanoma/genética , beta-Defensinas/genética , Apoptose/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Ciclina D1/biossíntese , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/patologia , Proteínas Proto-Oncogênicas B-raf/biossínteseRESUMO
AIM: The aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4). METHODS: hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells. To purify soluble fusion GST-hBD-4 protein, affinity chromatography was applied. Rec-hBD-4 was cleaved from the fusion protein with thrombin, and purified by reverse phase chromatography on Sep-Pack C18. Effects of rec-hBD-4 on proliferation, viability, cell cycle distribution, substrate-independent growth, and mobility of cultured human cancer cells of A431, A549, and TPC-1 lines were analyzed by direct cell counting technique, MTT assay, flow cytofluorometry, colony forming assay in semi-soft medium, and wound healing assay. RESULTS: Rec-hBD-4 was expressed in bacterial cells as GST-hBD-4 fusion protein, and purified by routine 3-step procedure (affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography). Analysis of in vitro activity of rec-hBD-4 toward three human cancer cell lines has demonstrated that the defensin is capable to affect cell behaviour in concentration-dependent manner. In 1-100 nM concentrations rec-hBD-4 significantly stimulates cancer cell proliferation and viability, and promotes cell cycle progression through G2/M checkpoint, greatly enhances colony-forming activity and mobility of the cells. Treatment of the cells with 500 nM of rec-hBD-4 resulted in opposite effects: significant suppression of cell proliferation and viability, blockage of cell cycle in G1/S checkpoint, significant inhibition of cell migration and colony forming activity. CONCLUSION: Recombinant human beta-defensin-4 is biologically active peptide capable to cause oppositely directed effects toward biologic features of cancer cells in vitro dependent on its concentration.
