Coompo Research Chemicals

In the present study, we investigated the effect of mahanine on the activation of the apoptotic pathway in human leukemia U937 cells. Various end points were used to screen for apoptosis: Morphological changes in cells, the relative numbers of viable and apoptotic cells; translocation of membrane bound phosphatidylserine and DNA analysis. We found that mahanine-induced apoptosis in U937 cells involved activation of caspases, including caspase-3, release of cytochrome c into cytosol, loss of mitochondrial membrane permeability, and decreased levels of cellular ATP. Pretreatment of cells with cyclosporine A, prior to/concomitant with exposure to mahanine, effectively prevented the deleterious effects of the alkaloid on cellular integrity and viability. As mitochondrial permeability is known to be important in the regulation of cytochrome c release, our observations indicate that mitochondria are the principal target of mahanine. More specifically, we propose that mahanine causes the mitochondrial membranes to lose their permeability, resulting in caspase-3 activation and apoptosis.

Mahanine inhibits growth and induces apoptosis in both androgen-responsive, LNCaP and androgen-independent, PC3 cells by targeting cell survival pathway.

Mahanine can reverse an epigenetically silenced gene, RASSF1A in prostate cancer cells by inhibiting DNMT activity that in turn down-regulates a key cell cycle regulator, cyclin D1. Mahanine therefore, promises an encouraging therapeutic choice for advanced prostatic cancer.

Mahanine has a dose- and time-dependent anti-proliferative activity in acute lymphoid (MOLT-3) and chronic myeloid (K562) leukemic cell lines and in the primary cells of leukemic and myeloid patients, with minimal effect on normal immune cells including CD34(+) cells. Leukemic cells underwent phosphatidylserine externalization and DNA fragmentation, indicating mahanine-induced apoptosis. An increase in reactive oxygen species suggests that the mahanine-induced apoptosis was mediated by oxidative stress. A significant drop in the Bcl2/Bax ratio, the loss of mitochondrial transmembrane potential as well as cytochrome c release from the mitochondria to the cytosol suggested involvement of the mitochondrial pathway of apoptosis. Cytochrome c release was followed by the activation of caspase-9, caspase-3 and caspase-7, and cleavage of PARP in both MOLT-3 and K562 cells. In MOLT-3 cells, formation of the Fas-FasL-FADD-caspase-8 heterotetramer occurred, leading to the cleavage of Bid to its truncated form, which consequently resulted in formation of the mitochondrial transmembrane pore. The incubation of MOLT-3 cells with mahanine in the presence of caspase-8 inhibitor or FasL-neutralizing NOK-2 antibody resulted in the decrease of mahanine-induced cell death. Mahanine was also a potent inhibitor of K562 xenograft growth, which was evident in an athymic nude mice model. In summary, these results provide evidence for involvement of the death receptor-mediated extrinsic pathway of apoptosis in the mahanine-induced anticancer activity in MOLT-3 cells, but not in K562 cells, which are deficient in Fas/FasL.