Epinecidin-1, an antimicrobial peptide from fish (Epinephelus coioides) which has an antitumor effect like lytic peptides in human fibrosarcoma cells.

Epinecidin-1, a synthetic 21-mer antimicrobial peptide originally identified from grouper (Epinephelus coioides), specifically exhibited high antimicrobial activities against both Gram-negative and Gram-positive bacteria. In the current study we report on the in vitro cytotoxicity of the peptide, an important factor before it can be considered for further applications in cancer therapy. The cytotoxicity of epinecidin-1 was investigated against several cancer cells (A549, HA59T/VGH, HeLa, HepG2, HT1080, RAW264.7, and U937) and normal cells (AML-12, NIH3T3, and WS-1) with the MTT assay, and the inhibition of cancer cell growth was confirmed by a soft agar assay and scanning electron microscopy. However, cell variations were detected with AO/EtBr staining, while apoptosis and necrosis gene expressions in HT1080 cells after treatment with the epinecidin-1 peptide and Nec-1 showed that epinecidin-1 had an anti-necrosis function in HT1080 cells. The data presented here indicate that epinecidin-1 has in vitro antitumor activity against the HT1080 cell line, and functions like lytic peptides. In addition, our results suggest that epinecidin-1 may prove to be an effective chemotherapeutic agent for human fibrosarcoma cells in the future.

Stable expression in a Chinese hamster ovary (CHO) cell line of bioactive recombinant chelonianin, which plays an important role in protecting fish against pathogenic infection.

Chelonianin, originally isolated from the shrimp (Penaeus monodon), exhibits antimicrobial effects in vitro and in vivo and is used to treat infectious fish diseases. Herein, we report that the recombinant chelonianin protein fused to a fluorescent protein (rcf protein) was expressed from a stably transfected Chinese hamster ovary (CHO) cells. The in vitro experiments showed that the rcf protein exhibited antimicrobial activity against several bacteria, while the recombinant fluorescent protein alone did not. In addition, pretreatment and post-treatment with the rcf protein were both effective in promoting a significant decrease in fish mortality and decreasing the number of infectious bacteria. We utilized the quantitative reverse-transcriptase polymerase chain reaction technique to survey the levels of gene expressions of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide synthase 1 induced in response to bacterial infection in experiments with tilapia (Oreochromis mossambicus). Our results indicated that the rescue of fish treated with the rcf protein may involve regulation of TNF-alpha expression. Collectively, chelonianin inhibited the production of an inflammatory mediator and reduced mortality in fish during bacterial challenge, suggesting that it has potential as a therapeutic or prophylactic drug for use against bacterial infectious diseases.

Organization and promoter analysis of the grouper (Epinephelus coioides) epinecidin-1 gene.

Epinecidin-1, an antimicrobial peptide documented in some fish, is an essential component of the innate immune response in fish, but little is known about its gene regulation. To better understand the molecular mechanism controlling transcription of the epinecidin-1 gene, we cloned and sequenced the genes and promoter regions of three epinecidin-1 peptides from the grouper (Epinephelus coioides). These genes have the potential to encode three putative epinecidin-1 peptides with either a short or a long 5'-untranslated region (UTR). These epinecidin-1 genes, numbered 124-1 (gene structure: 5 exons), 124-2 (gene structure: 5 exons), and 961 (gene structure: 4 exons), have 3' UTR sequences that dramatically differ by being located on different exons in clones 124 and 961. To address the roles of lipopolysaccharide (LPS) and poly(I):poly(C) in regulating epinecidin-1 expression, serial deletions were prepared in the promoter region of two clones that contained three genes. Different fragments of the epinecidin-1 5'-flanking region were transfected into U937 (human histiocytic lymphoma) and ZFL (zebrafish liver) cells and then treated with 0, 1, 10, and 100 mug/mL LPS or poly(I):poly(C). The results showed that after treatment with 10 mug/mL LPS, high promoter activity was observed in the 0.6-kb promoter fragment (of clone 961). Promoter deletions showed that hepatocyte nuclear factor (HNF)-1 was required for a maximal response of epinecidin-1 961 promoter activity after LPS treatment in ZFL cells. Morphological studies of transgenic zebrafish indicated that the 2-kb epinecidin-1 124-1 promoter-driven GFP transcripts appeared in the eye and skin as confirmed by immunohistochemical staining. These results indicate that the 2-kb epinecidin-1 124-1 promoter is active in a tissue-specific manner.

Gene expression and localization of the epinecidin-1 antimicrobial peptide in the grouper (Epinephelus coioides), and its role in protecting fish against pathogenic infection.

Epinecidin-1 is an antimicrobial peptide and plays a vital role in protecting fish against pathogenic infection. As a mimic of a grouper epinecidin-1 peptide, it has tertiary structures that closely resemble those of pleurocidin found in the winter flounder (Pleuronectes americanus). The tissue-specific, lipopolysaccharide (LPS)-stimulation-specific, and poly(I):poly(C)-stimulation-specific expressions of the grouper (Epinephelus coioides) epinecidin-1 antimicrobial peptide were determined using a comparative reverse-transcription polymerase chain reaction. Results of the tissue distribution analysis revealed high levels of epinecidin-1 messenger RNA (mRNA) in the head kidneys, intestines, and skin. Expression of epinecidin-1 mRNA was dose-dependently stimulated by both LPS and poly(I):poly(C). Immunohistochemical analysis with the polyclonal antiserum of a grouper epinecidin-1 peptide (rabbit polyclonal antibody) showed that the peptide was localized with the epinecidin-1 antibody in the gills and intestines. Two synthetic peptides of the grouper epinecidin-1 peptide (g-ple 22-51 and g-ple 22-42) and one winter flounder pleurocidin as a control exhibited high antimicrobial activities against gram-negative or gram-positive bacteria. In addition, peptide treatment was effective in promoting a significant increase in fish survival after the injection of Vibrio vulnificus in tilapia (Oreochromis mossambicus) and grouper. These results are relevant to the design of prophylactic and therapeutic strategies to counter bacterial infections, especially for preventing or ameliorating immune defects in fish during bacterial infections.

Shrimp (Penaeus monodon) anti-lipopolysaccharide factor reduces the lethality of Pseudomonas aeruginosa sepsis in mice.

We investigated the efficacy of amino acids 55-76 of the synthetic shrimp anti-lipopolysaccharide factor peptide (SALF(55-76) cyclic peptide), the C-terminal part of the shrimp anti-lipopolysaccharide factor. This study was conducted to elucidate the effects of the antiseptic action of this peptide. The SALF(55-76) cyclic peptide was tested against bacterial clinical isolates and showed broad-spectrum antimicrobial activity. Transmission electron microscopic (TEM) examination of SALF(55-76) cyclic peptide-treated Pseudomonas aeruginosa showed that severe swelling preceded cell death and breakage of the outer membrane; the intracellular inclusion was found to have effluxed extracellularly. When mice were treated with the SALF(55-76) cyclic peptide before bacterial challenge with P. aeruginosa, the peptide highly protected mice against death by sepsis. The P. aeruginosa recovered from SALF(55-76) cyclic peptide-treated mice after 4 h exhibited reduced bacterial growth similar to that recovered from vancomycin-treated mice. In addition, the syntheses of inflammatory cytokines, such as interleukin (IL)-2, IL-4, IL-10, IL-12, IL-13, interferon-gamma, and tumor necrosis factor [TNF]-alpha, were significantly upregulated 4 h after SALF(55-76) cyclic peptide treatment except for IL-4 in the liver. The expressions of Toll-like receptor 4 (Tlr4), Irf3, myd88, and Tram, were considerably elevated, but only Tlr4 existed in the spleen 4 h after SALF(55-76) cyclic peptide treatment. The prophylactic administration of SALF(55-76) cyclic peptide was begun the TNF-alpha response in comparison to untreated mice by an ELISA analysis. Due to its multifunctional properties, the SALF(55-76) cyclic peptide may become an important prophylaxis against and therapy for bacterial infectious diseases, as well as for septic shock.