探討SALF、epinecidin-1及pardaxin抗菌肽之抗菌、抗發炎及抗癌活性

Abstract

Anti-microbial peptides (AMPs) have many functions, such as anti-bacteria, anti-inflammation, and anti-cancer. Considering the anti-bacterial function of AMPs, we initially synthesized a series of truncated AMP derivatives of shrimp anti-lipopolysaccharide factor (SALF), epinecidin-1, and pardaxin. We then determined the minimum inhibitory concentrations (MICs) of these derivatives against eight bacterial species. We also examined the synergistic effect between peptide and non-peptide antibiotics. We found that epinecidin-0217 and pardaxin-0127 exhibited a broad range of activity against Gram-positive and Gram-negative bacteria. Moreover, a combination of these AMPs with antibiotics resulted in a synergistic improvement in the anti-bacterial activity against methicillin-resistant Staphylococcus aureus. Considering the anti-inflammatory function of AMPs, we performed ELISA to show that SALF inhibits the production of inflammatory cytokines, tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-6, and IL-1β, as well as the secretion of monocyte chemoattractant protein (MCP)-1 in cervical epithelial cells treated with lipopolysaccharide (LPS). We also conducted RT-PCR to demonstrate that SALF suppresses il-6, il-8, il-1α, and mcp-1 expressions. Moreover, we provided evidence that SALF may regulate vaginal epithelial cell immune responses via mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB pathways. We then conducted ELISA to confirm that SALF downregulates the secretion of pro-inflammatory cytokines (TNF-α, IL-1α, IL-6, IL-8, and MCP-1) in cervical epithelial cells infected by Trichomonas vaginalis. We futher employed real-time PCR to show that SALF affects the expression of several pro-inflammatory genes. Conducting Western blot analysis, we demonstrated that SALF treatment inhibits T. vaginalis infection of cervical epithelial cells via p38 and NF-κB pathways. These pathways were observed by using appropriate inhibitors and by performing ELISA/Western blot analysis. Considering the anti-cancer function of AMPs, we investigated the cytotoxic activity of SALF on four cervical cancer cell lines. We performed fluorescence-activated cell sorting analysis to show that SALF induces cervical cancer cell death and G2/M phase arrest in vitro. Furthermore, we observed the localization of SALF to the cytosol and nuclei of human epithelial carcinoma (HeLa) cells at 24 h after treatment by confocal imaging. AO/EtBr and DNA fragmentation results indicated that SALF induces apoptosis in HeLa cells. Moreover, we used various caspase inhibitors in vitro and immunohistochemistry in vivo to show that caspase-3 and caspase-9 are involved in SALF-induced apoptosis. We also showed that SALF induces mitochondrial membrane depolarization associated with caspase-dependent pathway in HeLa cells. Our findings indicated that SALF exhibits promising anti-cancer activity by triggering apoptosis. SALF may be applied in single or combinatorial therapy against cervical cancer. We also reported that epinecidin-0217 and pardaxin-0127 inhibit the growth of HeLa and fibrosarcoma (HT-1080) cells.