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Purpose: To study the effect of moscatilin (purified from the stem of orchid Dendrobrium loddigesii) on the proliferation of human colorectal cancer HCT-116 cells in vitro and in vivo. Experimental Design: The growth inhibition of moscatilin was screened on several human cancer cell lines. The effect of moscatilin on tubulin was detected in vitro. Following moscatilin treatment on HCT-116 cells, c-Jun NH 2 -terminal protein kinase (JNK) and caspase activation was studied by Western blot analysis, and DNA damage was done by Comet assay. Specific JNK inhibitor SP600125 was cotreated to reverse moscatilin-induced apoptosis. Tumor growth inhibition of moscatilin was done on HCT-116 xenograft models. Results: Moscatilin induced a time-dependent arrest of the cell cycle at G 2 -M, with an increase of cells at sub-G 1 . Moscatilininhibited tubulin polymerization, suggesting that it might bind to tubulins. Moscatilin also induced the phosphorylation ofJNK1/2. SP600125 significantly inhibited the activation of caspase-9 and caspase-3 and the subsequent moscatilin-induced apoptosis. The data suggest that JNK activation may contribute to moscatilin-mediated apoptosis signaling. A parallel experiment showed that SP600125 significantly inhibitsTaxol-and vincristine-induced HCT-116 cell apoptosis. This suggests that the JNK activation may be a common mechanism for tubulinbinding agents. Moreover, moscatilin induces DNA damage, phosphorylation of H2AX and p53, and up-regulation of p21. Our HCT-116 xenograft models show the in vivo efficacy of moscatilin. Conclusions: In summary, our results suggest that moscatilin induces apoptosis of colorectal HCT-116 cells via tubulin depolymerization and DNA damage stress and that this leads to the activation of JNK and mitochondria-involved intrinsic apoptosis pathway.

Inflammation and low oxygen diffusion are recognized characteristics of cardiovascular diseases such as atherosclerosis. Evodiamine, extracted from the traditional Chinese herb, Evodia rutaecarpa, is a bioactive anti-inflammatory alkaloid. The objective of this study was to investigate whether evodiamine could repress hypoxia-induced inflammatory response. We showed that evodiamine repressed not only COX-2 and iNOS expression but also prostaglandin E2 release in a concentration-dependent manner under hypoxic conditions. Furthermore, our studies indicated that COX-2 mRNA was inhibited by evodiamine, implying that transcriptional activity is involved in the mechanistic pathway. It is striking that hypoxia-inducible factor 1alpha (HIF-1alpha) inhibitor, camptothecin, suppressed hypoxia-induced COX-2 expression rather than pyrrolidine dithiocarbamate, a nuclear factor kappaB inhibitor. In addition, our studies have confirmed that evodiamine inhibited HIF-1alpha, which accounted for the transcriptional activity of COX-2, rather than nuclear factor kappaB in RAW264.7 cells. Finally, evodiamine did not affect either the level of HIF-1alpha mRNA or the degradation rate of HIF-1alpha protein, but it regulated the translational process of HIF-1alpha. We found that hypoxia-evoked phosphorylation of Akt and p70S6K was blocked after evodiamine treatment, in addition to the inhibition of phosphorylation of 4E-BP. These results suggest that the mechanism of repression of hypoxia-induced COX-2 expression by evodiamine is through the inhibition of HIF-1alpha at the translational level and is primarily mediated via dephosphorylation of Akt and p70S6K. Therefore, evodiamine could be an effective therapeutic agent against inflammatory diseases involving hypoxia.

Hepatocellular carcinoma is highly chemoresistant to currently available chemotherapeutic agents. In this study, 2 ¶-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone (CHM-1), a synthetic 6,7-substituted 2-phenyl-4-quinolone, was identified as a potent and selective antitumor agent in human hepatocellular carcinoma. CHM-1 induced growth inhibition of HA22T, Hep3B, and HepG2 cells in a concentration-dependent manner but did not obviously impair the viability of normal cells at the IC 50 for liver cancer cells. CHM-1-induced apoptosis was also characterized by immunofluorescence microscopy. CHM-1 interacted with tubulin at the colchicine-binding site, markedly inhibited tubulin polymerization both in vitro and in vivo, and disrupted microtubule organization. CHM-1 caused cell cycle arrest at G 2 -M phase by activating Cdc2/cyclin B1 complex activity. CHM-1-induced cell death, activation of Cdc2 kinase activity, and elevation of MPM2 phosphoepitopes were profoundly attenuated by roscovitine, a specific cyclin-dependent kinase inhibitor. CHM-1 did not modulate the caspase cascade, and the pan-caspaseinhibitor z-VAD-fmk did not abolish CHM-1-induced cell death. However, CHM-1 induced the translocation of apoptosis-inducing factor (AIF) from mitochondria to the nucleus. Small interfering RNA targeting of AIF substantially attenuated CHM-1-induced AIF translocation. Importantly, CHM-1 inhibited tumor growth and prolonged the lifespan in mice inoculated with HA22T cells. In conclusion, we show that CHM-1 exhibits a novel antimitotic antitumor activity against human hepatocellular carcinoma both in vitro and in vivo via a caspase-independent pathway. CHM-1 is a promising chemotherapeutic agent worthy of further development into a clinical trial candidate for treating cancer, especially hepatocellular carcinoma. [Mol Cancer Ther 2008;7(2):350 -60]