*2.3. The E*ff*ect of rHct-S3 on EGF-Induced Neoplastic Transformation of Normal Cells and Colony Formation of Cancer Cells*

The effects of rHct-S3 on neoplastic transformation of JB6 Cl41 cells induced by EGF, colony formation and growth of cancer cells were studied by soft agar assay, which is considered to be the most accurate type of in vitro test for detecting malignant transformation of cells [29]. The actinoporin was found to inhibit the EGF-induced neoplastic transformation of JB6 Cl41 cells by 10% ± 5.0, 23% ± 2.5, and 34% ± 0.2 at subtoxic concentrations of 1, 2, and 4 μM, respectively (Figure 3a,b). Moreover, rHct-S3 decreased the number of colonies of HT-29 cells by 25% ± 1.8, 33% ± 0.1, and 47% ± 0.9, at concentrations of 1, 2, and 4 μM, respectively, compared to non-treated cells (control) (Figure 3c). At the same doses, rHct-S3 inhibited the colony formation of MDA-MB-231 cells by 17% ± 2.4, 20% ± 2.5, 37% ± 1.2 and SK-MEL-28 cells by 18% ± 1.5, 24% ± 0.5, 34% ± 3.6, respectively (Figure 3d,e). Because activity of rHct-S3 was most pronounced in colorectal carcinoma HT-29 cells, further experiments were carried out with this cell line. It should be noted that chemotherapeutic drug, cisplatin, used as a positive control in this study, inhibited colony formation of HT-29, MDA-MB-231, and SK-MEL-28 cells by 46%, 75%, and 39% at a non-cytotoxic dose of 3 μM, respectively (Figure 3). These results indicate that the actinoporin has a promissing anticancer potential.
