Molecular signaling cascade in DNA bisintercalator, echinomycin-induced apoptosis of HT-29 cells - Evidence of the apoptotic process via activation of the cytochrome c-ERK-caspase-3 pathway

Abstract

The DNA-interactive drug, echinomycin, is a potent antitumor agent, which is able to induce apoptosis in a multitude of cancer cell lines. Previously, we showed that echinomycin strongly inhibited the growth of a variety of cancer cell lines, and the activation of mitogen-activated protein kinases (MAPK) in human colon cancer cells (HT-29). However, little information currently exists regarding the details of intracellular signaling pathways such as the MAPK, mitochondrial, and caspase pathways. In order to clarify this issue, we verified the plausible molecular signaling cascade by performing an immunobiochemical apoptosis experiment involving the mitochondrial and caspase pathways. The apoptotic process of HT-29 cells was accompanied by the activation of procaspase-9, -3 and cytochrome c release. Both caspase and MAPK inhibitors were used in the determination of the specific roles of MAPK and caspase in echinomycin-induced apoptosis. ERK (PD98059) or caspase-3-specific (Z-DEVD-FMK) inhibitors were discovered to significantly attenuate echinomycin-induced apoptosis. PD98059 treatment or overexpression of kinase-inactive ERK did not alter the echinomycin-induced cytochrome c release into the cytosol, but did diminish the activation of procaspase-3. Also, Z-DEVD-FMK was found to have no effect on either cytochrome c release or ERK activation. Taken together, these results indicate that cytochrome c release, and the activation of ERK and caspase-3 in the final apoptosis pathway are all relevant factors in echinomycin-induced apoptosis. To our knowledge, this study is the first to delineate the echinomycin's direct detrimental effects on colon cancer cells. (c) 2005 Elsevier Ltd. All rights reserved.