Subramaniam Dharmalingam, Giridharan Periyasamy, Murmu Nabendu, Shankaranarayanan Nallakandy P, May Randal, Houchen Courtney W, Ramanujam Rama P, Balakrishnan Arun, Vishwakarma Ram A, Anant Shrikant
Department of Medicine, OU Cancer Institute, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Cancer Res. 2008 Oct 15;68(20):8573-81. doi: 10.1158/0008-5472.CAN-08-2372.
We have identified a natural compound that activates apoptosis of epithelial cancer cells through activation of tumor necrosis factor-alpha (TNF-alpha), 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 G(1) cell cycle arrest, which was suppressed by caspase-3 inhibitors. HDNC induced TNF-alpha, 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-alpha-mediated activation and translocation of nuclear factor-kappaB (NF-kappaB). This was further confirmed by Western blot analysis of nuclear extracts wherein levels of RelA, the p65 component of NF-kappaB, 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 G(1) phase of the cell cycle and could be a potential chemotherapeutic candidate.
我们已经鉴定出一种天然化合物，它通过激活肿瘤坏死因子-α（TNF-α）、TNF受体（TNFR）相关死亡结构域（TRADD）和半胱天冬酶来激活上皮癌细胞的凋亡。从印度枳椇提取物的乙酸乙酯馏分中分离并鉴定出分子1-羟基-5,7-二甲氧基-2-萘甲醛（HDNC，木蝴蝶素）。HDNC处理在体内抑制了HCT-116结肠癌肿瘤异种移植的生长。CD31免疫染色显示，HDNC处理的动物体内微血管显著减少，同时环氧化酶-2、白细胞介素-8和血管内皮生长因子mRNA水平降低。使用己糖胺酶测定法，我们确定HDNC抑制HCT-116结肠癌细胞和HEp-2肺泡上皮癌细胞的增殖。此外，癌细胞显示活化的半胱天冬酶-3水平升高，并诱导G(1)期细胞周期停滞，这被半胱天冬酶-3抑制剂所抑制。HDNC诱导TNF-α、TNFR1和TRADD mRNA及蛋白表达。此外，还观察到半胱天冬酶-8和Bid活化以及细胞色素c释放，表明死亡受体和线粒体途径之间存在相互作用。HDNC在培养细胞和肿瘤异种移植中均抑制AKT和细胞外信号调节激酶磷酸化。此外，电泳迁移率变动分析和荧光素酶报告基因分析表明，HDNC显著抑制TNF-α介导的核因子-κB（NF-κB）活化和易位。核提取物的蛋白质印迹分析进一步证实了这一点，其中在HDNC处理的细胞中，NF-κB的p65成分RelA的水平显著降低。总之，数据表明新型化合物HDNC（木蝴蝶素）是一种有效的抗癌剂，可在细胞周期的G(1)期诱导凋亡，可能是一种潜在的化疗候选药物。
