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Antimicrobial peptide m2163 or m2386 identified from Lactobacillus casei ATCC 334 can trigger apoptosis in the human colorectal cancer cell line SW480.

Research paper by Tsung-Lin TL Tsai, An-Chieh AC Li, Yi-Chieh YC Chen, Yi-Shun YS Liao, Thy-Hou TH Lin

Indexed on: 06 Jan '15Published on: 06 Jan '15Published in: Tumor Biology



Abstract

Ribosomal synthesized antimicrobial peptides (AMPs) are widely distributed in nature and are toxic to certain microorganisms. Some of these AMPs are found to exhibit cytotoxic activity against the growth of cancer cells and thus have obvious anticancer potential. Here, we have studied the antiproliferation on the human colorectal cancer cell line SW480 of two AMPs, namely m2163 and m2386, identified by us from a lactic acid bacterium Lactobacillus casei ATCC 334 previously. A half maximal inhibitory concentration (IC50) of 40 μg/ml is determined first using the MTT (3-(4, 5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay for either peptide m2163 or m2386. The apoptosis in treated SW480 cells by either peptide m2163 or m2386 is analyzed using flow cytometry with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide double staining. These analyses show that a substantial population of treated SW480 cells can undergo apoptosis by either peptide m2163 or m2386. The real-time quantitative polymerase chain reaction (qPCR) and Western blot analyses are subsequently used to study how the apoptosis is induced in the treated SW480 cells by either peptide m2163 or m2386. While m2163 is found to induce the expression of Fas and TRAILR1, the expression of Fas, TNFR1, and TRAILR1 death receptors on the cell surface of treated SW480 cells is found to be induced by m2386. Further, the expression of some mitochondria-related apoptosis proteins such as Smac is found to be also induced, suggesting that either peptide m2163 or m2386 can trigger both the extrinsic and intrinsic apoptosis pathways. The cell membrane permeability is greatly enhanced upon treatment with either peptide m2163 or m2386 as analyzed by the flow cytometry using both FITC-labeled peptides. The flow cytometry is also used to analyze the fluorescence intensity given by FITC-m2163 in either the mitochondria or cytoplasm fraction of the treated and fractionated SW480 cells. It is found that the detected fluorescence intensity of the mitochondria fraction is much weaker than that of the cytoplasm one, suggesting that most of the FITC-m2163 peptides are located in the cytoplasm rather than the mitochondria. This is further confirmed by a confocal microscopy study that either peptide m2163 or m2386 can localize on the cell membrane for a substantial length of time and then penetrate into the cell cytoplasm to induce the apoptosis.