Combination of Nimbolide and TNF--Increases Human Colon Adenocarcinoma Cell Death through JNK-mediated DR5 Up- regulation

Tumor necrosis factor (TNF-), an inflammatory cytokine that plays an important role in the control of cell proliferation, differentiation, and apoptosis, has previously been used in anti-cancer therapy. However, the therapeutic applications of TNF- are largely limited due to its general toxicity and anti-apoptotic influence. To overcome this problem, the present study focused on the effect of active constituents isolated from a medicinal plant on TNF--induced apoptosis in human colon adenocarcinoma (HT-29) cells. Nimbolide from Azadirachta indica was evaluated for cytotoxicity by methyl tetrazolium 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay and phase contrast microscopy. Effects on apoptotic signaling proteins were investigated using Western blot analysis. Nimbolide showed cytotoxicity against HT-29 cells that was significantly different from the control group (<0.01), a concentration of 10 M significantly inducing cell death (<0.01). In combination with TNF-, nimbolide significantly enhanced-induced cell death. In apoptotic pathway, nimbolide activated c-Jun N-terminal kinase (JNK) phosphorylation, BH3 interacting-domain death agonist (Bid) and up-regulated the death receptor 5 (DR5) level. In the combination group, nimbolide markedly sensitized TNF--induced JNK, Bid, caspase-3 activation and the up-regulation of DR5. Our findings overall indicate that nimbolide may enhance TNF--mediated cellular proliferation inhibition through increasing cell apoptosis of HT-29 cells by up-reglation of DR5 expression via the JNK pathway.