The study of cytotoxicity and membrane interaction of D-LAK antimicrobial peptides

Abstract

Tuberculosis (TB) remains a major global heath problem. Mismanagement of TB leads to the development of multidrug-resistant tuberculosis (MDR-TB). There is an urgent need to develop new and effective therapeutics against MDR-TB. Antimicrobial peptides (AMPs) have emerged as a new class of anti-TB agent against drug-resistant TB. AMPs are an essential component of the innate immune system that defend against invading pathogens. AMPs exhibit broad-spectrum antimicrobial activity against a wide range of microorganisms. It is interesting to determine the therapeutic potential of AMPs against MDR-TB for clinical use. This study aimed to study the cytotoxicity of D-LAK AMPs and their combinations with anti-TB drugs on THP-1 cells and HEP G2 cells and to investigate the cell membrane interaction of D-LAK peptides. MTT assay was performed to determine the cytotoxicity of D-LAK120-A, D-LAK120-HP13, isoniazid (INH) and rifampicin (RIF) on THP-1 cells and HEP G2 cells. The cytotoxicity of the combinations of D-AMPs/anti-TB drugs (D-LAK120-A/INH, D-LAK120-A/RIF, D-LAK120-HP13/INH andD-LAK120-HP13/RIF) on THP-1 cells and HEP G2 cells were also studied using MTT assay to determine the combination effect of D-AMPs with anti-TB drugs. IC50 values were obtained from MTT assay to determine the concentrations at which these agents were safe for use. A fluorescent dye, calcein-AM, was used in cell-membrane interaction studies. Flow cytometry and confocal microscope experiments were performed to elucidate the mechanism of membrane activity of D-LAK120-A and D-LAK120-HP13. From the two cell membrane interaction studies, it was found that D-LAK120-A and D-LAK120-HP13 had interactions on the cell surface of THP-1 cells. There was an obvious cytotoxic effect at concentration of 16 μM and above for both peptides on THP-1 cells in the cell membrane interaction study. Future studies will be focused on the anti-TB effects of these peptides in combination with anti-TB drugs on MDR-TB at their non-toxic concentrations.