Evaluating cytotoxic effects of recombinant fragaceatoxin C pore forming toxin against AML cell lines

Objective(s): Current therapeutic strategies for cancer are associated with side effects and lack of specificity in treatments. Biological therapies including monoclonal antibodies and immune effectors have been the subject of multiple research projects. Pore-forming proteins may become the other biological strategy to overcome the problems associated with current treatments. But detailed mechanisms of their action on target membranes remained to be elucidated. We aimed to study the cytotoxic effects of recombinant form of fragaceatoxin C on AML cell lines HL-۶۰ and KG-۱. Materials and Methods: We cloned the FraC gene in pET-۲۸a (+) bacterial expression vector and the expressed recombinant FraC protein was purified by affinity chromatography. Then, cytotoxic effects of the recombinant protein were examined on two AML cell lines, HL-۶۰ and KG-۱. Effects of serum and calcium ion were explored by hemolysis assay in more details. Results: Our results showed that the recombinant C-terminal polyhistidine-tagged FraC protein has potent cytotoxic effects on both AML cell lines, with IC۵۰=۵.۶, and ۴.۶ µg.ml-۱ for HL-۶۰ and KG-۱ cells, respectively. Serum showed dose-dependent and also time-dependent inhibitory effects on the hemolytic and cytotoxic activities of the FraC protein. Pre-incubation of the toxin with different concentrations of calcium ion also inhibited hemolytic activity of FraC toxin.Conclusion: Results of the present study showed that FraC has potential anti-tumor effects. By detailed investigation of the inhibition mechanism of serum and calcium effects in the future, it can be possible to design target sites for clinical applications of the toxin.

Keywords:

Acute myeloid leukemia , Fragaceatoxin C , HL-۶۰ cell , KG-۱ cell , Pore-forming toxin , Recombinant expression