Used anticancer 374913-63-0 therapeutics, such as selective cytotoxicity for cancer cells, bypass of the multidrug-resistance mechanism, and synergism effects in combination therapy [6]. Many AMPs damage the cellular membrane as part of their killing mechanism. Although the interactions that take place between AMPs and the outer membrane leaflet of neoplasticeukaryotic cells are not completely understood, the mechanism by which AMPs interacts with microbial cytoplasmic membranes may provide important clues to this process. The net negative charge that is conferred upon many cancer cells as a result of differential branching and sialic acid content of N-linked glycans associated with transmembrane glycoproteins [7], as well as the elevated cell surface anionic molecules such as Fruquintinib site phosphatidylserine [8,9] and Oglycosylated mucins [10,11], is believed to promote electrostatic interactions with AMPs at the cancer cell surface. Then the membrane-bound AMPs disrupted cell membrane through pore formation or membrane destabilization [12]. Besides the direct membrane-destructing effect, some researchers have suggested that AMPs might exert cytolytic activity against cancer cells through ion-permeable channel formation in the cell membrane [13] or other non-membranolytic intracellular actions [14?6]. Temporin-1CEa is a cationic amphiphilic antimicrobial peptide isolated from the skin secretions of the Chinese brown frog (Rana chensinensis). We have recently reported that temporin-1CEa exhibits rapid cytotoxic activity against both microorganisms and human cancer cells [17,18]. In the present study, 
we have therefore continued our investigations for elucidation of anticancerMechanisms of Temporin-1CEa Induced Cytotoxicitymechanisms of temporin-1CEa. The study presents the anticancer activity of temporin-1CEa against two human breast cancer cell lines MDA-MB-231 and MCF-7 utilizing MTT assay and LDH leakage assay for cell death, together with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for providing insights into morphological changes. In addition, cell-membrane permeability is determined using flow cytometry and an annexin-V-FITC/propidium-iodide protocol. The intracellular calcium ion and reactive oxygen species (ROS) concentrations and mitochondrial membrane potential were also evaluated. The results presented here may provide new insights helping to understand the direct membrane-destruction effect and intracellular mechanisms of temporin-1CEa in breast 
cancer cells.Results Temporin-1CEa Induces Breast Cancer Cell DeathAs shown in Fig. 1, both MTT assay and LDH leakage assay indicated that treatment of cancer cells with temporin-1CEa induced cell death in a concentration-dependent manner. The formazan production (optical density) measured in MTT assay (Fig.1A) was reduced after one hour of incubation with peptides; meanwhile, the extracellular LDH activity value (optical density) was enhanced in LDH leakage assay (Fig.1B). Moreover, the in vitro cytotoxicity assay also indicated that MCF-7 cancer cells were more vulnerable to the temporin-1CEa-induced cytotoxicity than MDA-MB-231 cells. For instance, after one hour exposure to 40 mM temporin-1CEa, the percentages of cells viability and cytotoxicity as assessed by MTT assay and LDH leakage assay were 22 64 and 61 67 for MCF-7 cell line, and were 61 62 and 32 68 for MDA-MB-231 cell line.quadrant), cells with membrane lipid asymmetry and PS exposure (FITC-annexin V pos.Used anticancer therapeutics, such as selective cytotoxicity for cancer cells, bypass of the multidrug-resistance mechanism, and synergism effects in combination therapy [6]. Many AMPs damage the cellular membrane as part of their killing mechanism. Although the interactions that take place between AMPs and the outer membrane leaflet of neoplasticeukaryotic cells are not completely understood, the mechanism by which AMPs interacts with microbial cytoplasmic membranes may provide important clues to this process. The net negative charge that is conferred upon many cancer cells as a result of differential branching and sialic acid content of N-linked glycans associated with transmembrane glycoproteins [7], as well as the elevated cell surface anionic molecules such as phosphatidylserine [8,9] and Oglycosylated mucins [10,11], is believed to promote electrostatic interactions with AMPs at the cancer cell surface. Then the membrane-bound AMPs disrupted cell membrane through pore formation or membrane destabilization [12]. Besides the direct membrane-destructing effect, some researchers have suggested that AMPs might exert cytolytic activity against cancer cells through ion-permeable channel formation in the cell membrane [13] or other non-membranolytic intracellular actions [14?6]. Temporin-1CEa is a cationic amphiphilic antimicrobial peptide isolated from the skin secretions of the Chinese brown frog (Rana chensinensis). We have recently reported that temporin-1CEa exhibits rapid cytotoxic activity against both microorganisms and human cancer cells [17,18]. In the present study, we have therefore continued our investigations for elucidation of anticancerMechanisms of Temporin-1CEa Induced Cytotoxicitymechanisms of temporin-1CEa. The study presents the anticancer activity of temporin-1CEa against two human breast cancer cell lines MDA-MB-231 and MCF-7 utilizing MTT assay and LDH leakage assay for cell death, together with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) for providing insights into morphological changes. In addition, cell-membrane permeability is determined using flow cytometry and an annexin-V-FITC/propidium-iodide protocol. The intracellular calcium ion and reactive oxygen species (ROS) concentrations and mitochondrial membrane potential were also evaluated. The results presented here may provide new insights helping to understand the direct membrane-destruction effect and intracellular mechanisms of temporin-1CEa in breast cancer cells.Results Temporin-1CEa Induces Breast Cancer Cell DeathAs shown in Fig. 1, both MTT assay and LDH leakage assay indicated that treatment of cancer cells with temporin-1CEa induced cell death in a concentration-dependent manner. The formazan production (optical density) measured in MTT assay (Fig.1A) was reduced after one hour of incubation with peptides; meanwhile, the extracellular LDH activity value (optical density) was enhanced in LDH leakage assay (Fig.1B). Moreover, the in vitro cytotoxicity assay also indicated that MCF-7 cancer cells were more vulnerable to the temporin-1CEa-induced cytotoxicity than MDA-MB-231 cells. For instance, after one hour exposure to 40 mM temporin-1CEa, the percentages of cells viability and cytotoxicity as assessed by MTT assay and LDH leakage assay were 22 64 and 61 67 for MCF-7 cell line, and were 61 62 and 32 68 for MDA-MB-231 cell line.quadrant), cells with membrane lipid asymmetry and PS exposure (FITC-annexin V pos.