Research Of Amphibian Antimicrobiol Peptides Temporin-1CEa On The Anticancer Mechanisms Of Breast Cancer Cells

Amphibian antimicrobial peptides as important mediators of innate immunity of livingorganisms, is a class of peptides with antimicrobial and anticancer activity in the process ofdefence response against pathogenic micro-organisms. Temporin-1CEa, one17amino acidsamphibian antimicrobial peptide precursor isolated from the Chinese brown frog Rana skin, ispositively charged with amphiphilic α-helix structure and exhibit good anti-cancer effect.The main purpose of the present study is to explore the anti-cancer effects and thepossible mechanisms of temporin-1CEa on breast cancer cells. We firstly determined thegrowth inhibition activity of temporin-1CEa against variety of tumor cell lines including threekinds of breast cancer lines (MCF-7, MDA-MB231, Bcap37cell line) using MTT method.Then the manner of peptide-induced cell death and the possible mechanisms of the anticancereffects of temporin-1CEa were investigated through lactate dehydrogenase (LDH) assay andAnnexin V/PI double staining assay. The tumor cell membrane lytic effect of temporin-1CEa,membrane potential and membrane permeability were also detected using fluorescence probeDiBAC4(3) and Calcein-Am/EthD-1. Finally, using the fluorescent probes Rhodamine123and Fluo3-AM, the peptide-induced tumor cell mitochondrial membrane potential andintracellular calcium release were analyzed.The results showed that the amphibian antimicrobial peptide temporin-1CEa couldrapidly (after1hour peptide exposure) induce cell death for all12kinds of tested tumor cellslines in a dose-dependent manner, with no significant cytotoxicity and growth inhibition tonormal cell. Moreover, among12tested cancer cell lines, the melanoma and breast cancerwere particularly sensitive to temporin-1CEa-induced cytotoxicity. Therefore, in the preasentstudy, three breast cancer cells were selected for further exploring the mechanism ofanticancer action of the antimicrobial peptides temporin-1CEa.Further research results suggested that the main target of temporin-1CEa was thebiomembrane. Temporin-1CEa could firstly increase the membrane potential and themembrane permeability of breast cancer cells, After influx into the cells, the peptide couldfurther disrupt the mitochondrial membrane, induce mitochondrial membrane potentialcollapse, impaire normal supply of cellular energy, and finaly trigger tumor cell death. Moreover, the enhanced concentration of intracellular calcium may also contribute to thecytotoxic effects of temporin-1CEa.