apicularen-a and Colonic-Neoplasms

Apicularen A is a novel antitumor agent and strongly induces death in tumor cells. In this study, we synthesized apicularen A acetate, an acetyl derivative of apicularen A, and investigated its antitumor effect and mechanism in HM7 colon cancer cells. Apicularen A acetate induced apoptotic cell death and caspase-3 activation; however, the pan-caspase inhibitor Z-VAD-fmk could not prevent this cell death. Apicularen A acetate induced the loss of mitochondrial membrane potential and the translocation of apoptosis-inducing factor (AIF) from mitochondria. In addition, apicularen A acetate significantly decreased tubulin mRNA and protein levels and induced disruption of microtubule networks. Taken together, these results indicate that the mechanism of apicularen A acetate involves caspase-independent apoptotic cell death and disruption of microtubule architecture.

Topics: Apoptosis; Apoptosis Inducing Factor; Blotting, Western; Bridged Bicyclo Compounds, Heterocyclic; Cell Line, Tumor; Colonic Neoplasms; Down-Regulation; Flow Cytometry; Fluorescent Antibody Technique; Humans; Membrane Potential, Mitochondrial; Microtubules; Protein Transport; Reverse Transcriptase Polymerase Chain Reaction; Tubulin

Apicularen A has been shown to cause growth inhibition and apoptosis in several cancer cell lines. However, the mechanisms of apicularen A-induced cell death and in vivo effects remain unclear. In this study, we investigated the molecular mechanisms of apicularen A-induced cell death in HM7 human colon cancer cells in vitro and anticancer activity in vivo.. We tested cytotoxicity with a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, apoptosis with DNA fragmentation assay, mitochondrial membrane potential, and cell cycle with fluorescence-activated cell sorting. Caspase activation was done by fluorometry. Alterations of microtubule structure, tubulin protein, and mRNA level were assessed by immunofluorescence, Western blot, and reverse transcription-PCR. In vivo studies were assessed using nude mice tumor cell growth in xenograft model and liver colonization assay.. Apicularen A treatment of HM7 cells inhibited cell growth and this inhibition was partially rescued by z-VAD-fmk. Apicularen A caused accumulation of sub-G(1)-G(0), DNA fragmentation, Fas ligand induction, and activation of caspase-8 and caspase-3, but mitochondrial membrane potential was not changed. Furthermore, beta-tubulin protein and mRNA were decreased by apicularen A, but in vitro polymerization of tubulin was not affected. Concurrently, apicularen A-treated cell showed disruption of microtubule architecture. In in vivo studies, apicularen A reduced tumor volume by approximately 72% at the end of a 15-day treatment. Moreover, apicularen A reduced liver colonization as much as 95.6% (50 microg/kg/d).. Apicularen A induces cell death of HM7 cells through up-regulating Fas ligand and disruption of microtubule architecture with down-regulation of tubulin level. These findings indicate that apicularen A is a promising new microtubule-targeting compound.

Topics: Animals; Bridged Bicyclo Compounds, Heterocyclic; Caspase 3; Caspase 8; Cell Death; Cell Line, Tumor; Colonic Neoplasms; DNA Fragmentation; Enzyme Activation; Fas Ligand Protein; Gene Expression Regulation; Gene Expression Regulation, Neoplastic; Humans; Liver; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Microtubules; Tubulin