Assessment of Cytotoxicity and Induction of Apoptosis by Cytolysin-A in MCF-7 Human Breast Cancer Cell Line

A protein called cytolysin A or ClyA, encoded by certain bacteria species, can cause cytotoxicity. Although the ClyA protein is not typically expressed at detectable levels in most E. coli strains, here it was successfully overproduced and purified by cloning the structural gene into a hns mutant strain. The cytotoxicity of the purified cytolysin was assessed on two MCF-7 cancer cell lines and HDF normal cell line using the MTT assay. Flow cytometry was employed to examine the cytolysin's ability to induce apoptosis in cancer cells. In addition, a Western blot analysis was carried out to evaluate the expression levels of P53, Bcl2, and Bax proteins. The results revealed that cytolysin exhibited dose-dependent and time-dependent toxicity towards cancer cells, while showing minimal toxicity against normal cells, indicating its selective action against cancer cells. Cytolysin had an IC50 value of 3.29 µg/ml against MCF-7 cancer cells and 12.6 µg/ml against HDF normal cells. Flow cytometry results further demonstrated that cytolysin induced apoptosis in cancer cells, evidenced by increased expression of p53 and BCL2, as well as decreased in Bax, in gene and protein levels. These findings underscore the potential of cytolysin as a targeted therapy for cancer, highlighting its selective cytotoxic effect on cancer cells. Keywords: Cytolysin-A, Breast cancer, Cytotoxicity, Flowcytometry, Western blotting