Apicularen A induces cell death through Fas ligand up-regulation and microtubule disruption by tubulin down-regulation in HM7 human colon cancer cells

Abstract

Purpose: 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.

Experimental design: 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.

Results: 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).

Conclusion: 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.