Apoptosis-inducing activity of the antimicrobial peptide cecropin of <italic>Musca domestica</italic> in human hepatocellular carcinoma cell line BEL-7402 and the possible mechanism

Abstract

We studied the apoptosis-inducing properties of the antimicrobial peptide cecropin of Musca domestica in human hepatocellular carcinoma cell line BEL-7402 and its underlying mechanism. Proliferation inhibition of the human hepatocellular carcinoma BEL-7402 cells and the human normal liver cells were determined by the MTT assay, and the cell viability was determined by trypan blue dye exclusion assay. The apoptotic tumor cells treated with cecropin were examined by transmission electron microscopy and terminal-deoxynucleotidyl transferase mediated nick end labeling. The apoptosis rate was measured by flow cytometry (FCM) with PI/Annexin-V double staining. Western blot analysis and RT-PCR were used to determine the expression levels of proteins involved in apoptosis, such as Fas, Fas-L, caspase-8, and caspase-3. The experimental results showed that Musca domestica cecropin inhibited the proliferation of human hepatocellular carcinoma BEL-7402 cells in dosedependent and time-dependent manners, without affecting the proliferation of normal liver cells. FCM showed that the cell apoptosis rates were 5.1+/-0.11%, 8.1+/-0.04%, and 10.9+/-0.15% after the treating with 100 mM cecropin for 24, 48, and 72 h, respectively. The rates of apoptosis were 5.4+/-0.14% and 8.0+/-0.13% after the treating with 25 and 50 microM cecropin for 72 h, respectively. Western blot analysis and RT-PCR showed that the apoptosisrelated molecules including Fas, Fas-L, caspase-8 and caspase-3 were activated. This study showed that the antimicrobial peptide cecropin-inducing apoptosis in human hepatocellular carcinoma BEL-7402 cells, which might be associated with upregulation of Fas, Fas-L, and caspase-8 and caspase-3 and triggering extrinsic apoptotic pathway.