Due to the adverse side effects of current cancer chemotherapeutics development of targeted anti-cancer medications is under intensive focus. In the present study a fusion protein consisting of the catalytic and translocation domains of diphtheria toxin fused to BR2, a cancer specific cell penetrating peptide, was produced as a new targeted anti-cancer agent and its cytotoxic effects on various cell lines including MCF-7 and HeLa (as cancerous), and HEK293 and HUVEC (as normal) cell lines was evaluated.
DT386-BR2 protein was expressed in E. coli cells followed by protein purification using affinity chromatography and authenticated by SDS-PAGE and Western blotting. Next, the purified protein was subjected to evaluation of its cyto-lethal effects on the mentioned cell lines at various concentrations. DT386 and BR2 alone were also produced and used as negative controls. In this regard, MTT assay followed by flow cytometry were used for cytotoxicity assessment and prediction of induced cell death mechanism.
SDS-PAGE and Western blotting confirmed expression of DT386-BR2 fusion protein by revealing a band of about 47kDa. In case of the cancerous cell lines, i.e. HeLa and MCF-7 cells, significant reduction in survival percent was shown when compared to the controls. However, no significant cytotoxicity was observed in case of the normal cell lines, HUVEC and HEK293 cells. In addition, DT386 and BR2 alone did not show any significant cytolethal effects. The IC50 of DT386-BR2 for HeLa and MCF-7 was about 0.55 and 2.08 μg/ml, respectively. Furthermore, flow cytometry revealed dose dependent apoptosis induction by DT386-BR2 after 12 hrs.
This chimeric protein showed promising cancer cell specific cytotoxicity and might be considered as a new drug candidate for treatment of some types of cancer. Further studies regarding non-specific cytotoxicity and in vivo animal and preclinical studies of this protein are undergoing.
Isfahan University of Medical Sciences
Research Deputy of Isfahan University of Medical Sciences
All authors have declared no conflicts of interest.