Ac-93253 Inhibits Intracellular Growth of Mycobacteria in Human Macrophages by Inducing Apoptosis in Mitochondrial-Dependent Manner

Abstract

Introduction: Ac-93253 iodide induces apoptosis in NSCLC cancer cells. Our study found that it is bactericidal against Mycobacterium tuberculosis (M.tb), and it induces apoptosis upon treatment of non-cytotoxic concentration. Due to the primary finding, we want to decipher the mechanism of apoptosis and its effect on the survival of intracellular mycobacteria human macrophages. Methods: We have checked the expression of apoptosis-inducing genes and proteins through qRT-PCR and western blotting in Ac-93253 treated and mycobacterial-infected dTHP-1 cells. To confirm the apoptosis, We isolate DNA fragments and analyze them through agarose gel electrophoresis. To validate the above findings, we have used Annexin V and propidium iodide staining to check the accumulation of phosphatidylserine in the plasma membrane's outer leaflet. To quantify the loss of ∆ m we used Rhodamine 123 staining, and stained cells were analyzed through flow cytometry. To validate the role of ∆ m, we used cyclosporine A1 and checked its effect on apoptosis and anti-mycobacterial activity. To understand the canonical pathway of apoptosis, we checked the secretion of TNFα through sandwich ELISA. Result: Through MTT and trypan blue exclusion assay, 0.5 μM of Ac-93253 was found to be non-cytotoxic even after 72 hours of treatment in phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 (dTHP-1) cells. Significant up-regulation in the expression of various pro-apoptotic genes like Bcl-2, Bax, and Bad and the cleaved caspase 3 was observed upon treatment with a non-cytotoxic dose of Ac93253. Ac-93253 treatment also leads to DNA fragmentation and increased phosphatidylserine accumulation in the outer leaflet of the plasma membrane. Ac-93253 treatment also effectively reduced the intracellular growth of mycobacteria in infected dTHP-1 cells, and ZVAD-FMK broad-range apoptosis inhibitor significantly brought back the intracellular mycobacterial growth in Ac-93253 treated macrophages. Conclusion: These findings suggest apoptosis may be the probable effector response through which Ac-93253 manifests its antimycobacterial property