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

  • Boonyarat, Chantana (Faculty of Pharmaceutical Sciences, Khon Kaen University) ;
  • Yenjai, Chavi (Natural Products Research Unit, Department of Chemistry and Center of Excellence for Innovation in Chemistry, Faculty of Science, Khon Kaen University) ;
  • Reubroycharoen, Prasert (Department of Chemical Technology, Faculty of Science, Chulalongkorn University) ;
  • Waiwut, Pornthip (Faculty of Pharmaceutical Sciences, Ubon Ratchathani University)
  • Published : 2016.05.01

Abstract

Tumor necrosis factor ($TNF-{\alpha}$), 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 $TNF-{\alpha}$ 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 $TNF-{\alpha}$-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 (p<0.01), a concentration of $10{\mu}M$ significantly inducing cell death (p<0.01). In combination with $TNF-{\alpha}$, 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 $TNF-{\alpha}$-induced JNK, Bid, caspase-3 activation and the up-regulation of DR5. Our findings overall indicate that nimbolide may enhance $TNF-{\alpha}$-mediated cellular proliferation inhibition through increasing cell apoptosis of HT-29 cells by up-reglation of DR5 expression via the JNK pathway.

Keywords

$TNF-{\alpha}$;apoptosis;HT-29 human colon adenocarcinoma cells;nimbolide;DR5;JNK pathway

Acknowledgement

Supported by : Chulalongkorn University

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