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Activation of Apoptosis by 1-Hydroxy-5,7-Dimethoxy-2-Naphthalene-Carboxaldehyde, a Novel Compound from Aegle marmelos

¹ Department of Medicine, ² OU Cancer Institute, and ³ Department of Cell Biology, University of Oklahoma Health Sciences Center; ⁴ Swaasth, Inc., Oklahoma City, Oklahoma; ⁵ Centre for Biotechnology, Anna University, Chennai, India; ⁶ Chittaranjan National Cancer Institute, Kolkata, India; ⁷ VHS Leprosy Research Centre, Erode, India; and ⁸ National Institute of Immunology, JNU Complex, New Delhi, India

Requests for reprints: Shrikant Anant, University of Oklahoma Health Sciences Center, 920 Stanton L. Young Boulevard, WP1360, Oklahoma City, OK 73104. Fax: 405-271-5450; E-mail: shrikant-anant{at}ouhsc.edu.

Key Words: TNF- • chemotherapy • proliferation • apoptosis • marmelin

We have identified a natural compound that activates apoptosis of epithelial cancer cells through activation of tumor necrosis factor- (TNF-), TNF receptor (TNFR)-associated death domain (TRADD), and caspases. The molecule 1-hydroxy-5,7-dimethoxy-2-naphthalene-carboxaldehyde (HDNC, marmelin) was isolated and characterized from ethyl acetate fraction of extracts of Aegle marmelos. HDNC treatment inhibited the growth of HCT-116 colon cancer tumor xenografts in vivo. Immunostaining for CD31 showed that there was a significant reduction in microvessels in the HDNC-treated animals, coupled with decreased cyclooxygenase-2, interleukin-8, and vascular endothelial growth factor mRNA. Using hexoseaminidase assay, we determined that HDNC inhibits proliferation of HCT-116 colon and HEp-2 alveolar epithelial carcinoma cells. Furthermore, the cancer cells showed increased levels of activated caspase-3 and induced G₁ cell cycle arrest, which was suppressed by caspase-3 inhibitors. HDNC induced TNF-, TNFR1, and TRADD mRNA and protein expression. Moreover, caspase-8 and Bid activation, and cytochrome c release, were observed, suggesting the existence of a cross-talk between death receptor and the mitochondrial pathways. HDNC inhibited AKT and extracellular signal-regulated kinase phosphorylation both in cells in culture and in tumor xenografts. In addition, electrophoretic mobility shift assay and luciferase reporter assays showed that HDNC significantly suppressed TNF-–mediated activation and translocation of nuclear factor-B (NF-B). This was further confirmed by Western blot analysis of nuclear extracts wherein levels of RelA, the p65 component of NF-B, were significantly less in cells treated with HDNC. Together, the data suggest that the novel compound HDNC (marmelin) is a potent anticancer agent that induces apoptosis during G₁ phase of the cell cycle and could be a potential chemotherapeutic candidate. [Cancer Res 2008;68(20):8573–81]