This work is devoted to analyze antitumor activity of recombinant hBD-2-4 (rec-hBD-2-4) against cultured human cancer cells. Analysis of biologic activity of rec-hBD-2 influence on the growth patterns and malignant potential of cultured cancer cells has been carried out using eight different human tumor cell lines of different histogenesis. The effect of rec-hBD-2 on viability and proliferation of cultured cancer cells was analyzed using MTT assay and direct counting of cells. It has been shown that rec-hBD-2 caused significant concentration-dependent suppression of proliferation of tumor cells of different histogenesis. Moreover, treatment of the cells with nanomolar concentrations of rec-hBD-2 (10 nM - 1 mkM) resulted in significant dose-dependent decrease of cell viability. Flow cytometry analysis of cell cycle distribution of the cells treated with 10 nM-1 mkM rec-hBD-2 for 48 h has demonstrated a concentration-dependent cell growth arrest at G1/S checkpoint. The results of Western blotting analysis have shown that treatment with rec-hBD-2 resulted in slight downregulation of pRB expression and its complete dephosphorylation, significant downregulation of cyclin D1, cyclin E, CDK4 and B-Raf expression levels, and significant upregulation of p21WAF1 in hBD-2-treated cells. At the same time p53 expression level remained unaffected. It was shown that treatment with 100 nM of rec-hBD-2 resulted in insignificant decrease of colony numbers compared to control cells, while addition of 500 nM of rec-hBD-2 into cell incubation medium significantly inhibited ability of cells to form colonies in semi-soft medium. Also, hBD-2 dependent suppression of cancer cell migration and alterations in cell morphology toward more elongated mesenchymal phenotype have been observed. The study of expression of two common EMT markers - vimentin and E-cadherin - has been carried out using qPCR analysis in cells treated with 500 nM rec-hBD-2 for 24 h. It has been found that effects of hBD-2 on vimentin and E-cadherin expression levels strongly depended on cancer cell histological type. Analysis of biologic activity of rec-hBD-4 was carried out in three human cancer cell lines - A431, A549 and TPC-1. As it has been shown by direct cell counting technique rec-hBD-4 exerted a concentration-dependent effect on the cell proliferation and stimulated cell proliferation at concentrations from 1 to 100 nM and significantly suppressed at concentrations ? 500 nM. Effects of hBD-4 were similar in A431 and A549 cells but TPC-1 cells seem to be less sensitive to low nanomolar concentrations of rec-hBD4. Data have shown that in low concentrations (0.1-100 nM) rec-hBD-2 caused insignificant increase of viability of A431, TPC-1 and A549 cells. In all three cell lines treated with 500 nM of rec-hBD-4, a decrease of cell viability has been recorded while treatment of the cells with rec-hBD-4 at concentrations higher than 1 µM caused significant suppression of cell viability. So, biologic effects of nanomolar concentrations of rec-hBD-4 are of bimodal character, unlike to these of recombinant hBD-2. Such multimodal effects of rec-hBD-4 on cell viability were found to be reflected in its effects on cell cycle distribution. When the cells treated with 1 nm - 1 µM rec-hBD-4 were subjected to flow cytofluorometry analysis, it has been revealed that treatment of the cells with 100 nM rec-hBD-4 resulted in significant stimulation of cell cycle in G2/M checkpoint, with 500 nM - in significant blockage of cell cycle in G1/S checkpoint; higher concentrations of hBD-4 insignificantly suppressed cell cycle progression. The results of Western blotting analysis have shown that incubation of the cells with 100 nM rec-hBD-4 caused upregulation of B-Raf and cyclin B1 expression levels, and their downregulation in the case of higher concentrations of rec-hBD-4. At the same time treatment with rec-hBD-4 in concentration 100 nM-1 mkM resulted in dephosphorylation of pRB. Colony-forming activity of the cells treated with 100 nM rec-hBD-4 significantly increased compared to control untreated cells, with 500 nM - significantly decreased, while in the presence of 1 mkM of the defensin no visible colonies were developed. Unlike hBD-2 and hBD-4, at concentration range from 1 nM to 5 mkM rec-hBD-3 has been shown to cause no significant effects on proliferation and viability of cultured cancer cell lines. At the same time, as it has been demonstrated in MTT assay, while being noncytotoxic, rec-hBD-3 has been found to potentiate antiproliferative effects of clinically well established anti-mitotic chemotherapy medication - docetaxel. The studies of combined effects of rec-hBDs on viability of A431 cells have demonstrated significant potentiation of cytoxicity in the case of use of rec-hBD-2 and rec-hBD-3 or rec-hBD-2 and rec-hBD-4 combinations compared to that of rec-hBD-2 only. In contrary, if rec-hBD-3 was used in combination with rec-hBD-4, its antagonistic effects toward cell cytotoxicity were observed. So, this study has shown that inducible human beta-defensins can enhance or attenuate the effects of each other toward cultured tumor cell viability.
