1. Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells
O Gerashchenko, E Zhuravel, O Skachkova, N Khranovska, V Pushkarev, P Pogrebnoy, M Soldatkina Exp Oncol. 2014 Mar;36(1):17-23.
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.
2. Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells
O L Gerashchenko, E V Zhuravel, O V Skachkova, N N Khranovska, V V Filonenko, P V Pogrebnoy, M A Soldatkina Exp Oncol. 2013 Jun;35(2):76-82.
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.
3. Human beta-defensin-2 controls cell cycle in malignant epithelial cells: in vitro study
E Zhuravel, T Shestakova, O Efanova, Yu Yusefovich, D Lytvin, M Soldatkina, P Pogrebnoy Exp Oncol. 2011 Sep;33(3):114-20.
Aim: In the present research we analyze the mechanism of human beta-defensin-2 (hBD-2) influence on cultured malignant epithelial cell growth. Materials and methods: The analysis of a concentration-dependent effect of recombinant hBD-2 (rec-hBD-2) on cell growth patterns and cell cycle distribution has been performed in vitro with 2 cell lines (human lung adenocarcinoma A549 cells and human epidermoid carcinoma A431 cells) using MTT test, flow cytometry and direct cell counting. To study intracellular localization of hBD-2 immunocytofluorescent and immunocytochemical analyses were applied, and effect of hBD-2 on signal cascades involved in cell cycle regulation has been studied by Western blotting. Results: According to our data, rec-hBD-2 exerts a concentration-dependent effect on the viability of cultured A549 and A431 cells. It causes proproliferative effect at concentrations below 1 nM, significant suppression of cell proliferation at concentration range from 10 nM to 1 μM (p<0.05), and cell death at higher concentrations. Using flow cytometry we have demonstrated that hBD-2 dependent growth suppression is realized via cell cycle arrest at G1/S phase (p<0.05). Also, we have registered significant activation of pRB and decreased expression of Cyclin D1 in cells treated with the defensin compared to untreated control cells, while the expression of p53 remains unaffected. The study of intracellular localization of hBD-2 in these cells has revealed that exogeneously added defensin molecules enter the cells, are distributed throughout the cytoplasm and could be detected in cell nuclei. The model study using A549 cells treated with 1,25-(OH)(2)D(3) has shown similar cell growth suppression effect of native endogenously produced hBD-2. Conclusion: The results of our study suggest that in malignant epithelial cells hBD-2 may control cell growth via arrest of G1/S transition and activation of pRB.
