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Susan E. Douglas - One of the best experts on this subject based on the ideXlab platform.

  • Pleurocidin-family cationic antimicrobial peptides are cytolytic for breast carcinoma cells and prevent growth of tumor xenografts.
    Breast cancer research : BCR, 2011
    Co-Authors: Ashley L. Hilchie, Susan E. Douglas, Aleksander Patrzykat, Carolyn D. Doucette, Devanand M. Pinto, David W. Hoskin
    Abstract:

    Introduction Cationic antimicrobial peptides (CAPs) defend against microbial pathogens; however, certain CAPs also exhibit anticancer activity. The purpose of this investigation was to determine the effect of the Pleurocidin-family CAPs, NRC-03 and NRC-07, on breast cancer cells.

  • A Novel Target-Specific, Salt-Resistant Antimicrobial Peptide against the Cariogenic Pathogen Streptococcus mutans
    Antimicrobial agents and chemotherapy, 2011
    Co-Authors: Junni Mai, Susan E. Douglas, Raymond T. Syvitski, Jeffrey W. Gallant, Xiao-lin Tian, Nadine Merkley, Zakia Biswas, Junqi Ling
    Abstract:

    In this study, we constructed and evaluated a target-specific, salt-resistant antimicrobial peptide (AMP) that selectively targeted Streptococcus mutans, a leading cariogenic pathogen. The rationale for creating such a peptide was based on the addition of a targeting domain of S. mutans ComC signaling peptide pheromone (CSP) to a killing domain consisting of a portion of the marine-derived, broad-spectrum AMP Pleurocidin to generate a target-specific AMP. Here, we report the results of our assessment of such fusion peptides against S. mutans and two closely related species. The results showed that nearly 95% of S. mutans cells lost viability following exposure to fusion peptide IMB-2 (5.65 μM) for 15 min. In contrast, only 20% of S. sanguinis or S. gordonii cells were killed following the same exposure. Similar results were also observed in dual-species mixed cultures of S. mutans with S. sanguinis or S. gordonii. The peptide-guided killing was further confirmed in S. mutans biofilms and was shown to be dose dependent. An S. mutans mutant defective in the CSP receptor retained 60% survival following exposure to IMB-2, suggesting that the targeted peptide predominantly bound to the CSP receptor to mediate killing in the wild-type strain. Our work confirmed that IMB-2 retained its activity in the presence of physiological or higher salt concentrations. In particular, the fusion peptide showed a synergistic killing effect on S. mutans with a preventive dose of NaF. In addition, IMB-2 was relatively stable in the presence of saliva containing 1 mM EDTA and did not cause any hemolysis. We also found that replacement of serine-14 by histidine improved its activity at lower pH. Because of its effectiveness, salt resistance, and minimal toxicity to host cells, this novel target-specific peptide shows promise for future development as an anticaries agent.

  • The zebrafish embryo as a tool for screening and characterizing Pleurocidin host-defense peptides as anti-cancer agents.
    Disease models & mechanisms, 2011
    Co-Authors: Michael G. Morash, Susan E. Douglas, Jeffrey W. Gallant, Anna Robotham, Christina M. Ridley, Kelly H. Soanes
    Abstract:

    The emergence of multidrug-resistant cancers and the lack of targeted therapies for many cancers underscore an unmet need for new therapeutics with novel modes of action towards cancer cells. Host-defense peptides often exhibit selective cytotoxicity towards cancer cells and show potential as anti-cancer therapeutics. Here, we screen 26 naturally occurring variants of the peptide Pleurocidin for cytotoxic and anti-cancer activities, and investigate the underlying mechanism of action. Cytotoxicities were assessed in vitro using cell-based assays and in vivo using zebrafish embryos. Morphological changes were assessed by both transmission and scanning electron microscopy, and functional assays were performed on zebrafish embryos to investigate the mechanism of cell death. A total of 14 peptides were virtually inactive against HL60 human leukemia cells, whereas 12 caused >50% death at ≤32 μg/ml. Morphological changes characteristic of oncosis were evident by electron microscopy after only 1 minute of treatment with 32 μg/ml of variant NRC-03. Only two peptides were hemolytic. Four peptides showed no toxicity towards zebrafish embryos at the highest concentration tested (25 μM; ∼64 μg/ml) and one peptide was highly toxic, killing 4-hour-post-fertilization (hpf) embryos immediately after exposure to 1 μM peptide. Four other peptides killed embryos after 24 hours of exposure at 1 μM. Most peptides caused mortality at one or more developmental stages only after continuous exposure (24 hours) with higher lethal doses (≥5 μM). Pleurocidin NRC-03 bound to embryos and induced the release of superoxide, caused an increase in the number of TUNEL-positive nuclei, and caused membrane damage and the loss of embryonic epithelial integrity, marked by the exclusion of cells from the outer epithelium and the appearance of F-actin within the circumferential cells of the repair site. Our results indicate that specific Pleurocidin variants are attractive cancer-selective agents that selectively induce cell death in target cells but leave non-target cells such as erythrocytes and non-transformed cells unaffected.

  • Abstract #850: Cytotoxic activity of two Pleurocidin-like cationic antimicrobial peptides against human breast cancer cells
    Cancer Research, 2009
    Co-Authors: Ashley L. Hilchie, Susan E. Douglas, Aleksander Patrzykat, David W. Hoskin
    Abstract:

    Cationic antimicrobial peptides are small peptides that constitute an important defense against microbial pathogens and, in some cases, also exhibit potent cytotoxic activity against cancer cells. Atlantic pleuronectid flatfish are a particularly rich source of cationic antimicrobial peptides, including NRC-03 and NRC-07, which are Pleurocidin-like peptides derived from winter and yellowtail flounder, respectively. The purpose of this investigation was to evaluate the potential of NRC-03 and NRC-07 as novel cytotoxic agents for the treatment of breast cancer. Colorimetric MTT and LDH-release assays showed that NRC-03 and NRC-07 rapidly killed MDA-MB-231, MDA-MB-468, MCF-7 and T47D human breast cancer cells in a time- and dose-dependent manner. Additionally, NRC-03 and NRC-07 were cytotoxic towards cisplatin-resistant human ovarian cancer cell lines. Colorimetric hemolytic assays revealed that neither NRC-03 nor NRC-07 possessed hemolytic activity. Human fibroblasts and umbilical vein endothelial cells were not harmed by NRC-03 or NRC-07; however, high concentrations of NRC-03 and NRC-07 were lytic for human T cells and mammary epithelial cells, suggesting that both peptides require modification to enhance breast cancer cell selectivity. Fluorescent microscopy revealed that fluorochrome-labeled NRC-03 and NRC-07 rapidly bound to the surface of breast cancer cells, but not human dermal fibroblasts, suggesting that peptide binding was required for cell death. Propidium iodide staining, as well as scanning electron microscopy, showed that NRC-03 and NRC-07 rapidly caused pore formation in the cellular membrane of breast cancer cells, suggesting that cell death was mediated by membrane lysis. However, flow cytometric analysis of dihydroethidium- or DiOC 6 -stained breast cancer cells revealed that NRC-03- and NRC-07-mediated cytotoxicity was also associated with reactive oxygen species production and mitochondrial membrane destabilization, respectively. Nevertheless, the pan-caspase inhibitor BOC-D-FMK did not prevent NRC-03- and NRC-07-induced cytotoxicity, supporting the hypothesis that NRC-03 and NRC-07 act primarily through a membrane-lytic mechanism. We conclude that NRC-03 and NRC-07 warrant further investigation as novel therapeutic agents for the treatment of breast cancer. Supported by Natural Science and Engineering Research Council of Canada, the Cancer Research Training Program, and the Canadian Breast Cancer Foundation - Atlantic Region Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 850.

  • abstract 850 cytotoxic activity of two Pleurocidin like cationic antimicrobial peptides against human breast cancer cells
    Cancer Research, 2009
    Co-Authors: Ashley L. Hilchie, Susan E. Douglas, Aleksander Patrzykat, David W. Hoskin
    Abstract:

    Cationic antimicrobial peptides are small peptides that constitute an important defense against microbial pathogens and, in some cases, also exhibit potent cytotoxic activity against cancer cells. Atlantic pleuronectid flatfish are a particularly rich source of cationic antimicrobial peptides, including NRC-03 and NRC-07, which are Pleurocidin-like peptides derived from winter and yellowtail flounder, respectively. The purpose of this investigation was to evaluate the potential of NRC-03 and NRC-07 as novel cytotoxic agents for the treatment of breast cancer. Colorimetric MTT and LDH-release assays showed that NRC-03 and NRC-07 rapidly killed MDA-MB-231, MDA-MB-468, MCF-7 and T47D human breast cancer cells in a time- and dose-dependent manner. Additionally, NRC-03 and NRC-07 were cytotoxic towards cisplatin-resistant human ovarian cancer cell lines. Colorimetric hemolytic assays revealed that neither NRC-03 nor NRC-07 possessed hemolytic activity. Human fibroblasts and umbilical vein endothelial cells were not harmed by NRC-03 or NRC-07; however, high concentrations of NRC-03 and NRC-07 were lytic for human T cells and mammary epithelial cells, suggesting that both peptides require modification to enhance breast cancer cell selectivity. Fluorescent microscopy revealed that fluorochrome-labeled NRC-03 and NRC-07 rapidly bound to the surface of breast cancer cells, but not human dermal fibroblasts, suggesting that peptide binding was required for cell death. Propidium iodide staining, as well as scanning electron microscopy, showed that NRC-03 and NRC-07 rapidly caused pore formation in the cellular membrane of breast cancer cells, suggesting that cell death was mediated by membrane lysis. However, flow cytometric analysis of dihydroethidium- or DiOC 6 -stained breast cancer cells revealed that NRC-03- and NRC-07-mediated cytotoxicity was also associated with reactive oxygen species production and mitochondrial membrane destabilization, respectively. Nevertheless, the pan-caspase inhibitor BOC-D-FMK did not prevent NRC-03- and NRC-07-induced cytotoxicity, supporting the hypothesis that NRC-03 and NRC-07 act primarily through a membrane-lytic mechanism. We conclude that NRC-03 and NRC-07 warrant further investigation as novel therapeutic agents for the treatment of breast cancer. Supported by Natural Science and Engineering Research Council of Canada, the Cancer Research Training Program, and the Canadian Breast Cancer Foundation - Atlantic Region Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 850.

Thomas T. Chen - One of the best experts on this subject based on the ideXlab platform.

  • RNA-Seq analysis of differentially expressed genes relevant to innate and adaptive immunity in cecropin P1 transgenic rainbow trout (Oncorhynchus mykiss).
    BMC genomics, 2018
    Co-Authors: Yueh-chiang Han, Chun-mean Lin, Thomas T. Chen
    Abstract:

    In the past years, our laboratory successfully generated transgenic rainbow trout bearing cecropin P1 transgene. These fish exhibited resistant characteristic to infection by Aeromonas salmonicida, Infectious Hematopoietic Necrosis Virus (IHNV) and Ceratomyxa shasta (a parasitic pathogen). Previously, treating rainbow trout macrophage cells (RTS-11) with cecropin B, Pleurocidin and CF17, respectively, resulted in elevated expression of two pro-inflammatory genes, e.g. cyclooxygenase-2 (cox-2) and interleukin-1β (il-1β). In addition, a profiling of global gene expression by 44 k salmonid microarray analysis was conducted, and the results showed that immune relevant processes have been perturbed in cecopin P1 transgenic rainbow trout. Therefore, we hypothesized that cecropin P1 may not only eliminate pathogens directly, but also modulate the host immune systems, leading to increased resistance against pathogen infections. To confirm this hypothesis, we performed de novo mRNA deep sequencing (RNA-Seq) to analyze the transcriptomic expression profiles in three immune competent tissues of cecropin P1 transgenic rainbow trout. De novo sequencing of mRNA of the rainbow trout spleen, liver and kidney tissues were conducted by second-generation Illumina system, followed by Trinity assembly. Tissue specific unigenes were obtained, and annotated according to the Gene Ontology (GO) and the Nucleotide Basic Local Alignment Search Tool (BLAST). Over 2000 differentially expressed genes (DEGs) were determined by normalized ratio of Reads Per Kilobase of transcript per million mapped reads (RPKM) among the transgenic and non-transgenic fish in a tissue specific manner, and there were 82 DEGs in common among the three tissues. In addition, the enrichment analysis according to Gene Ontology Biological Process (GO:BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) based pathway analysis associated with innate/adaptive immunity of fish were also performed to illustrate the altered immune-related functions in each tissue. According to the RNA-Seq data, the correlations between alteration of gene expression profiles and the functional perturbations of the host immune processes were revealed. In comparison with the results of cDNA microarray analysis conducted by Lo et al., the overall results supported our hypothesis that the gene product of cecropin P1 transgene may not only directly eliminate pathogens, but also modulate the host immune system. Results of this study present valuable genetic information for Oncorhynchus mykiss, and will benefit future studies on the immunology of this fish species.

  • RNA-Seq analysis of differentially expressed genes relevant to innate and adaptive immunity in cecropin P1 transgenic rainbow trout (Oncorhynchus mykiss)
    BMC, 2018
    Co-Authors: Yueh-chiang Han, Chun-mean Lin, Thomas T. Chen
    Abstract:

    Abstract Background In the past years, our laboratory successfully generated transgenic rainbow trout bearing cecropin P1 transgene. These fish exhibited resistant characteristic to infection by Aeromonas salmonicida, Infectious Hematopoietic Necrosis Virus (IHNV) and Ceratomyxa shasta (a parasitic pathogen). Previously, treating rainbow trout macrophage cells (RTS-11) with cecropin B, Pleurocidin and CF17, respectively, resulted in elevated expression of two pro-inflammatory genes, e.g. cyclooxygenase-2 (cox-2) and interleukin-1β (il-1β). In addition, a profiling of global gene expression by 44 k salmonid microarray analysis was conducted, and the results showed that immune relevant processes have been perturbed in cecopin P1 transgenic rainbow trout. Therefore, we hypothesized that cecropin P1 may not only eliminate pathogens directly, but also modulate the host immune systems, leading to increased resistance against pathogen infections. To confirm this hypothesis, we performed de novo mRNA deep sequencing (RNA-Seq) to analyze the transcriptomic expression profiles in three immune competent tissues of cecropin P1 transgenic rainbow trout. Results De novo sequencing of mRNA of the rainbow trout spleen, liver and kidney tissues were conducted by second-generation Illumina system, followed by Trinity assembly. Tissue specific unigenes were obtained, and annotated according to the Gene Ontology (GO) and the Nucleotide Basic Local Alignment Search Tool (BLAST). Over 2000 differentially expressed genes (DEGs) were determined by normalized ratio of Reads Per Kilobase of transcript per million mapped reads (RPKM) among the transgenic and non-transgenic fish in a tissue specific manner, and there were 82 DEGs in common among the three tissues. In addition, the enrichment analysis according to Gene Ontology Biological Process (GO:BP), and Kyoto Encyclopedia of Genes and Genomes (KEGG) based pathway analysis associated with innate/adaptive immunity of fish were also performed to illustrate the altered immune-related functions in each tissue. Conclusions According to the RNA-Seq data, the correlations between alteration of gene expression profiles and the functional perturbations of the host immune processes were revealed. In comparison with the results of cDNA microarray analysis conducted by Lo et al., the overall results supported our hypothesis that the gene product of cecropin P1 transgene may not only directly eliminate pathogens, but also modulate the host immune system. Results of this study present valuable genetic information for Oncorhynchus mykiss, and will benefit future studies on the immunology of this fish species

  • Effects of linear cationic α-helical antimicrobial peptides on immune-relevant genes in trout macrophages
    Developmental and comparative immunology, 2005
    Co-Authors: P. Peter Chiou, Jenny Khoo, Niels C. Bols, Sue Douglas, Thomas T. Chen
    Abstract:

    There are increasing evidence of the potential role of antimicrobial peptides in the regulation of immune responses in mammalian species. However, the effects of these peptides in fish have yet to be investigated. In this study, we examined the transcriptional expression profile of representative immune-relevant genes in a trout macrophage cell line, RTS11, in response to three linear cationic alpha-helical antimicrobial peptides (insect cecropin B, fish Pleurocidin and a cecropin analogue CF17). The expression levels of two pro-inflammatory genes, interleukin-1 beta (IL-1 beta) and cyclo-oxygenase-2 (COX-2), increased in the peptide-treated RTS11 cells. The peptides did not appear to affect the expression levels of representative genes associated with antigen presentation, interferon response or JAK/STAT signal transduction. Furthermore, the induction of IL-1 beta and COX-2 in RTS11 by lipopolysaccharide was not adversely affected by these three antimicrobial peptides. Overall, the data indicate a pro-inflammatory effect of the three cationic antimicrobial peptides in the inflammatory response of salmonid species, suggesting a potential application of these peptides as immune adjuvant for fish vaccination.

Dong Gun Lee - One of the best experts on this subject based on the ideXlab platform.

  • Concentration-Dependent Mechanism Alteration of Pleurocidin Peptide in Escherichia coli
    Current Microbiology, 2016
    Co-Authors: June Young Lee, Dong Gun Lee
    Abstract:

    Cationic antimicrobial peptides (CAPs) are essential components of the innate immune system. Most CAPs exert antimicrobial effects via membrane-active mechanisms, while high concentrations of CAPs are associated with non-selective cytotoxicity. We originally hypothesized that a sub-lethal concentration of CAPs was able to exert antibacterial activity, by interacting with negatively charged nucleic acids, and not by damaging bacterial membranes. We selected Pleurocidin (Ple) and Escherichia coli as experimental models of CAPs and bacteria, respectively. Whereas Ple distinctly acted on bacterial membranes in a concentration-dependent manner, the cell viability was almost similar regardless the peptide concentration. To address how Ple retained its antibacterial activity in a low concentration, we particularly focused on the induction of intracellular apoptosis-like death (ALD). Finally, it was suggested that a sub-lethal concentration of Ple led to ALD in E. coli , mediated by caspase-like protein and RecA. To the best of our knowledge, this is the first study showing that alterations of CAP mechanisms are concentration dependent in bacteria.

  • The influence of the N-terminal region of antimicrobial peptide Pleurocidin on fungal apoptosis.
    Journal of microbiology and biotechnology, 2013
    Co-Authors: Hyemin Choi, Dong Gun Lee
    Abstract:

    In our previous study, the 25-mer antimicrobial peptide Pleurocidin (Ple) had been thought to induce apoptosis in Candida albicans. This study demonstrated that reactive oxygen species (ROS) production was a major cause of Ple-induced apoptosis. Four truncated analogs were synthesized to understand the functional roles in the N- and C-terminal regions of Ple on the apoptosis. Ple, Ple (4-25), Ple (1-22), and Ple (1-19) produced ROS, including hydroxyl radicals, on the order of [Ple > Ple (1-22) > Ple (4-25) > Ple (1-19)], whereas Ple (7-25) did not induce any ROS production. The results suggested that the N-terminal deletion affected the ROS-inducing activities much more than that of the C-terminal deletion, and net hydrophobicity [Ple > Ple (1-22) > Ple (4-25) > Ple (1-19) > Ple (7-25)] was related to ROS generation rather than other primary factors like net charge. Hence, we focused on the N-terminal-truncated peptides, Ple (4-25) and Ple (7-25), and examined other apoptotic features, including mitochondrial membrane depolarization, caspase activation, phosphatidylserine externalization, and DNA and nuclear fragmentation. The results also confirmed the disappearance of apoptotic activity of Ple (7-25) by the truncation of the N-terminal region (1-6) and the specific activity patterns between Ple and analogs. In conclusion, the N-terminal region of Ple played an important role in apoptosis.

  • antimicrobial peptide Pleurocidin synergizes with antibiotics through hydroxyl radical formation and membrane damage and exerts antibiofilm activity
    Biochimica et Biophysica Acta, 2012
    Co-Authors: Hyemin Choi, Dong Gun Lee
    Abstract:

    Abstract Background Pleurocidin, a 25-mer antimicrobial peptide (AMP), is known to exert bactericidal activity. However, the synergistic activity and mechanism(s) of Pleurocidin in combination with conventional antibiotics, and the antibiofilm effect of the peptide are poorly understood. Methods The interaction between Pleurocidin and antibiotics was evaluated using checkerboard assay. To study the mechanism(s) involved in their synergism, we detected hydroxyl radical formation using 3′-(p-hydroxyphenyl) fluorescein, measured the NAD + /NADH ratio by NAD + cycling assay, observed change in bacterial viability with the hydroxyl radical scavenger thiourea, and investigated cytoplasmic membrane damage using propidium iodide. Also, the antibiofilm effect of Pleurocidin was examined with the tissue culture plate method. Results All combinations of Pleurocidin and antibiotics showed synergistic interaction against bacterial strains (fractional inhibitory concentration index (FICI) ≤ 0.5) except for Enterococcus faecium treated with a combination of the peptide and ampicillin (FICI = 0.75). We identified that Pleurocidin alone and in combinations with antibiotics induced formation of hydroxyl radicals. The oxidative stress was caused by a transient NADH depletion and the addition of thiourea prevented bacterial death, especially in the case of the combined treatment of Pleurocidin and ampicillin showing synergisms. The combination of Pleurocidin and erythromycin increased permeability of bacterial cytoplasmic membrane. Additionally, Pleurocidin exhibited a potent inhibitory effect on preformed biofilm of bacterial organisms. In conclusion, Pleurocidin synergized with antibiotics through hydroxyl radical formation and membrane-active mechanism, and exerted antibiofilm activity. General significance The synergistic effect between Pleurocidin and antibiotics suggests the AMP is a potential therapeutic agent and adjuvant for antimicrobial chemotherapy.

  • Influence of the N- and C-terminal regions of antimicrobial peptide Pleurocidin on antibacterial activity.
    Journal of microbiology and biotechnology, 2012
    Co-Authors: Jaeyong Cho, Hyemin Choi, Dong Gun Lee
    Abstract:

    Pleurocidin, a 25-mer antimicrobial peptide, has been known to exhibit potent antibacterial activity. To investigate the functional roles in N- and C-terminal regions of Pleurocidin on the antibacterial activity, we designed four truncated analogs. The antibacterial susceptibility testing showed that Pleurocidin and its analogs exerted antibacterial effect against various bacterial strains and further possessed specific activity patterns corresponding with their hydrophobic scale [Pleurocidin > Anal 3 (1-22) > Anal 1 (4-25) > Anal 4 (1-19) > Anal 2 (7-25)]. Fluorescence experiments using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 3,3'-dipropylthiadicarbocyanine iodide [diSC3(5)] indicated that the differences in antibacterial activity of the peptides were caused by its membrane-active mechanisms including membrane disruption and depolarization. Blue shift in tryptophan fluorescence demonstrated that the decrease in net hydrophobicity attenuates the binding affinity of Pleurocidin to interact with plasma membrane. Therefore, the present study suggests that hydrophobicity in the N- and C-terminal regions of Pleurocidin plays a key role in its antibacterial activity.

  • Antimicrobial peptide Pleurocidin synergizes with antibiotics through hydroxyl radical formation and membrane damage, and exerts antibiofilm activity
    Biochimica et biophysica acta, 2012
    Co-Authors: Hyemin Choi, Dong Gun Lee
    Abstract:

    Pleurocidin, a 25-mer antimicrobial peptide (AMP), is known to exert bactericidal activity. However, the synergistic activity and mechanism(s) of Pleurocidin in combination with conventional antibiotics, and the antibiofilm effect of the peptide are poorly understood. The interaction between Pleurocidin and antibiotics was evaluated using checkerboard assay. To study the mechanism(s) involved in their synergism, we detected hydroxyl radical formation using 3'-(p-hydroxyphenyl) fluorescein, measured the NAD(+)/NADH ratio by NAD(+) cycling assay, observed change in bacterial viability with the hydroxyl radical scavenger thiourea, and investigated cytoplasmic membrane damage using propidium iodide. Also, the antibiofilm effect of Pleurocidin was examined with the tissue culture plate method. All combinations of Pleurocidin and antibiotics showed synergistic interaction against bacterial strains (fractional inhibitory concentration index (FICI)≤0.5) except for Enterococcus faecium treated with a combination of the peptide and ampicillin (FICI=0.75). We identified that Pleurocidin alone and in combinations with antibiotics induced formation of hydroxyl radicals. The oxidative stress was caused by a transient NADH depletion and the addition of thiourea prevented bacterial death, especially in the case of the combined treatment of Pleurocidin and ampicillin showing synergisms. The combination of Pleurocidin and erythromycin increased permeability of bacterial cytoplasmic membrane. Additionally, Pleurocidin exhibited a potent inhibitory effect on preformed biofilm of bacterial organisms. In conclusion, Pleurocidin synergized with antibiotics through hydroxyl radical formation and membrane-active mechanism, and exerted antibiofilm activity. The synergistic effect between Pleurocidin and antibiotics suggests the AMP is a potential therapeutic agent and adjuvant for antimicrobial chemotherapy. Copyright © 2012 Elsevier B.V. All rights reserved.

Aleksander Patrzykat - One of the best experts on this subject based on the ideXlab platform.

  • Pleurocidin-family cationic antimicrobial peptides are cytolytic for breast carcinoma cells and prevent growth of tumor xenografts
    Breast Cancer Research, 2011
    Co-Authors: Ashley L. Hilchie, Aleksander Patrzykat, Carolyn D. Doucette, Devanand M. Pinto, Susan Douglas, David W. Hoskin
    Abstract:

    Introduction Cationic antimicrobial peptides (CAPs) defend against microbial pathogens; however, certain CAPs also exhibit anticancer activity. The purpose of this investigation was to determine the effect of the Pleurocidin-family CAPs, NRC-03 and NRC-07, on breast cancer cells. Methods MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) and acid phosphatase cell-viability assays were used to assess NRC-03- and NRC-07-mediated killing of breast carcinoma cells. Erythrocyte lysis was determined with hemolysis assay. NRC-03 and NRC-07 binding to breast cancer cells and normal fibroblasts was assessed with fluorescence microscopy by using biotinylated-NRC-03 and -NRC-07. Lactate dehydrogenase-release assays and scanning electron microscopy were used to evaluate the effect of NRC-03 and NRC-07 on the cell membrane. Flow-cytometric analysis of 3,3'-dihexyloxacarbocyanine iodide- and dihydroethidium-stained breast cancer cells was used to evaluate the effects of NRC-03 and NRC-07 on mitochondrial membrane integrity and reactive oxygen species (ROS) production, respectively. Tumoricidal activity of NRC-03 and NRC-07 was evaluated in NOD SCID mice bearing breast cancer xenografts. Results NRC-03 and NRC-07 killed breast cancer cells, including drug-resistant variants, and human mammary epithelial cells but showed little or no lysis of human dermal fibroblasts, umbilical vein endothelial cells, or erythrocytes. Sublethal doses of NRC-03 and, to a lesser extent, NRC-07 significantly reduced the median effective concentration (EC_50) of cisplatin for breast cancer cells. NRC-03 and NRC-07 bound to breast cancer cells but not fibroblasts, suggesting that killing required peptide binding to target cells. NRC-03- and NRC-07-mediated killing of breast cancer cells correlated with expression of several different anionic cell-surface molecules, suggesting that NRC-03 and NRC-07 bind to a variety of negatively-charged cell-surface molecules. NRC-03 and NRC-07 also caused significant and irreversible cell-membrane damage in breast cancer cells but not in fibroblasts. NRC-03- and NRC-07-mediated cell death involved, but did not require, mitochondrial membrane damage and ROS production. Importantly, intratumoral administration of NRC-03 and NRC-07 killed breast cancer cells grown as xenografts in NOD SCID mice. Conclusions These findings warrant the development of stable and targeted forms of NRC-03 and/or NRC-07 that might be used alone or in combination with conventional chemotherapeutic drugs for the treatment of breast cancer.

  • Pleurocidin-family cationic antimicrobial peptides are cytolytic for breast carcinoma cells and prevent growth of tumor xenografts.
    Breast cancer research : BCR, 2011
    Co-Authors: Ashley L. Hilchie, Susan E. Douglas, Aleksander Patrzykat, Carolyn D. Doucette, Devanand M. Pinto, David W. Hoskin
    Abstract:

    Introduction Cationic antimicrobial peptides (CAPs) defend against microbial pathogens; however, certain CAPs also exhibit anticancer activity. The purpose of this investigation was to determine the effect of the Pleurocidin-family CAPs, NRC-03 and NRC-07, on breast cancer cells.

  • Abstract #850: Cytotoxic activity of two Pleurocidin-like cationic antimicrobial peptides against human breast cancer cells
    Cancer Research, 2009
    Co-Authors: Ashley L. Hilchie, Susan E. Douglas, Aleksander Patrzykat, David W. Hoskin
    Abstract:

    Cationic antimicrobial peptides are small peptides that constitute an important defense against microbial pathogens and, in some cases, also exhibit potent cytotoxic activity against cancer cells. Atlantic pleuronectid flatfish are a particularly rich source of cationic antimicrobial peptides, including NRC-03 and NRC-07, which are Pleurocidin-like peptides derived from winter and yellowtail flounder, respectively. The purpose of this investigation was to evaluate the potential of NRC-03 and NRC-07 as novel cytotoxic agents for the treatment of breast cancer. Colorimetric MTT and LDH-release assays showed that NRC-03 and NRC-07 rapidly killed MDA-MB-231, MDA-MB-468, MCF-7 and T47D human breast cancer cells in a time- and dose-dependent manner. Additionally, NRC-03 and NRC-07 were cytotoxic towards cisplatin-resistant human ovarian cancer cell lines. Colorimetric hemolytic assays revealed that neither NRC-03 nor NRC-07 possessed hemolytic activity. Human fibroblasts and umbilical vein endothelial cells were not harmed by NRC-03 or NRC-07; however, high concentrations of NRC-03 and NRC-07 were lytic for human T cells and mammary epithelial cells, suggesting that both peptides require modification to enhance breast cancer cell selectivity. Fluorescent microscopy revealed that fluorochrome-labeled NRC-03 and NRC-07 rapidly bound to the surface of breast cancer cells, but not human dermal fibroblasts, suggesting that peptide binding was required for cell death. Propidium iodide staining, as well as scanning electron microscopy, showed that NRC-03 and NRC-07 rapidly caused pore formation in the cellular membrane of breast cancer cells, suggesting that cell death was mediated by membrane lysis. However, flow cytometric analysis of dihydroethidium- or DiOC 6 -stained breast cancer cells revealed that NRC-03- and NRC-07-mediated cytotoxicity was also associated with reactive oxygen species production and mitochondrial membrane destabilization, respectively. Nevertheless, the pan-caspase inhibitor BOC-D-FMK did not prevent NRC-03- and NRC-07-induced cytotoxicity, supporting the hypothesis that NRC-03 and NRC-07 act primarily through a membrane-lytic mechanism. We conclude that NRC-03 and NRC-07 warrant further investigation as novel therapeutic agents for the treatment of breast cancer. Supported by Natural Science and Engineering Research Council of Canada, the Cancer Research Training Program, and the Canadian Breast Cancer Foundation - Atlantic Region Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 850.

  • abstract 850 cytotoxic activity of two Pleurocidin like cationic antimicrobial peptides against human breast cancer cells
    Cancer Research, 2009
    Co-Authors: Ashley L. Hilchie, Susan E. Douglas, Aleksander Patrzykat, David W. Hoskin
    Abstract:

    Cationic antimicrobial peptides are small peptides that constitute an important defense against microbial pathogens and, in some cases, also exhibit potent cytotoxic activity against cancer cells. Atlantic pleuronectid flatfish are a particularly rich source of cationic antimicrobial peptides, including NRC-03 and NRC-07, which are Pleurocidin-like peptides derived from winter and yellowtail flounder, respectively. The purpose of this investigation was to evaluate the potential of NRC-03 and NRC-07 as novel cytotoxic agents for the treatment of breast cancer. Colorimetric MTT and LDH-release assays showed that NRC-03 and NRC-07 rapidly killed MDA-MB-231, MDA-MB-468, MCF-7 and T47D human breast cancer cells in a time- and dose-dependent manner. Additionally, NRC-03 and NRC-07 were cytotoxic towards cisplatin-resistant human ovarian cancer cell lines. Colorimetric hemolytic assays revealed that neither NRC-03 nor NRC-07 possessed hemolytic activity. Human fibroblasts and umbilical vein endothelial cells were not harmed by NRC-03 or NRC-07; however, high concentrations of NRC-03 and NRC-07 were lytic for human T cells and mammary epithelial cells, suggesting that both peptides require modification to enhance breast cancer cell selectivity. Fluorescent microscopy revealed that fluorochrome-labeled NRC-03 and NRC-07 rapidly bound to the surface of breast cancer cells, but not human dermal fibroblasts, suggesting that peptide binding was required for cell death. Propidium iodide staining, as well as scanning electron microscopy, showed that NRC-03 and NRC-07 rapidly caused pore formation in the cellular membrane of breast cancer cells, suggesting that cell death was mediated by membrane lysis. However, flow cytometric analysis of dihydroethidium- or DiOC 6 -stained breast cancer cells revealed that NRC-03- and NRC-07-mediated cytotoxicity was also associated with reactive oxygen species production and mitochondrial membrane destabilization, respectively. Nevertheless, the pan-caspase inhibitor BOC-D-FMK did not prevent NRC-03- and NRC-07-induced cytotoxicity, supporting the hypothesis that NRC-03 and NRC-07 act primarily through a membrane-lytic mechanism. We conclude that NRC-03 and NRC-07 warrant further investigation as novel therapeutic agents for the treatment of breast cancer. Supported by Natural Science and Engineering Research Council of Canada, the Cancer Research Training Program, and the Canadian Breast Cancer Foundation - Atlantic Region Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 850.

  • Novel Antimicrobial Peptides Derived from Flatfish Genes
    Antimicrobial agents and chemotherapy, 2003
    Co-Authors: Aleksander Patrzykat, Jeffrey W. Gallant, Jung-kil Seo, Jennifer Pytyck, Susan E. Douglas
    Abstract:

    We report on the identification of active novel antimicrobials determined by screening both the genomic information and the mRNA transcripts from a number of different flatfish for sequences encoding antimicrobial peptides, predicting the sequences of active peptides from the genetic information, producing the predicted peptides chemically, and testing them for their activities. We amplified 35 sequences from various species of flatfish using primers whose sequences are based on conserved flanking regions of a known antimicrobial peptide from winter flounder, Pleurocidin. We analyzed the sequences of the amplified products and predicted which sequences were likely to encode functional antimicrobial peptides on the basis of charge, hydrophobicity, relation to flanking sequences, and similarity to known active peptides. Twenty peptides were then produced synthetically and tested for their activities against gram-positive and gram-negative bacteria and the yeast Candida albicans. The most active peptide (with the carboxy-terminus amidated sequence GWRTLLKKAEVKTVGKLALKHYL, derived from American plaice) showed inhibitory activity over a concentration range of 1 to 8 micro g/ml against a test panel of pathogens, including the intrinsically antibiotic-resistant organism Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and C. albicans. The methods described here will be useful for the identification of novel peptides with good antimicrobial activities.

Jeffrey W. Gallant - One of the best experts on this subject based on the ideXlab platform.

  • A Novel Target-Specific, Salt-Resistant Antimicrobial Peptide against the Cariogenic Pathogen Streptococcus mutans
    Antimicrobial agents and chemotherapy, 2011
    Co-Authors: Junni Mai, Susan E. Douglas, Raymond T. Syvitski, Jeffrey W. Gallant, Xiao-lin Tian, Nadine Merkley, Zakia Biswas, Junqi Ling
    Abstract:

    In this study, we constructed and evaluated a target-specific, salt-resistant antimicrobial peptide (AMP) that selectively targeted Streptococcus mutans, a leading cariogenic pathogen. The rationale for creating such a peptide was based on the addition of a targeting domain of S. mutans ComC signaling peptide pheromone (CSP) to a killing domain consisting of a portion of the marine-derived, broad-spectrum AMP Pleurocidin to generate a target-specific AMP. Here, we report the results of our assessment of such fusion peptides against S. mutans and two closely related species. The results showed that nearly 95% of S. mutans cells lost viability following exposure to fusion peptide IMB-2 (5.65 μM) for 15 min. In contrast, only 20% of S. sanguinis or S. gordonii cells were killed following the same exposure. Similar results were also observed in dual-species mixed cultures of S. mutans with S. sanguinis or S. gordonii. The peptide-guided killing was further confirmed in S. mutans biofilms and was shown to be dose dependent. An S. mutans mutant defective in the CSP receptor retained 60% survival following exposure to IMB-2, suggesting that the targeted peptide predominantly bound to the CSP receptor to mediate killing in the wild-type strain. Our work confirmed that IMB-2 retained its activity in the presence of physiological or higher salt concentrations. In particular, the fusion peptide showed a synergistic killing effect on S. mutans with a preventive dose of NaF. In addition, IMB-2 was relatively stable in the presence of saliva containing 1 mM EDTA and did not cause any hemolysis. We also found that replacement of serine-14 by histidine improved its activity at lower pH. Because of its effectiveness, salt resistance, and minimal toxicity to host cells, this novel target-specific peptide shows promise for future development as an anticaries agent.

  • The zebrafish embryo as a tool for screening and characterizing Pleurocidin host-defense peptides as anti-cancer agents.
    Disease models & mechanisms, 2011
    Co-Authors: Michael G. Morash, Susan E. Douglas, Jeffrey W. Gallant, Anna Robotham, Christina M. Ridley, Kelly H. Soanes
    Abstract:

    The emergence of multidrug-resistant cancers and the lack of targeted therapies for many cancers underscore an unmet need for new therapeutics with novel modes of action towards cancer cells. Host-defense peptides often exhibit selective cytotoxicity towards cancer cells and show potential as anti-cancer therapeutics. Here, we screen 26 naturally occurring variants of the peptide Pleurocidin for cytotoxic and anti-cancer activities, and investigate the underlying mechanism of action. Cytotoxicities were assessed in vitro using cell-based assays and in vivo using zebrafish embryos. Morphological changes were assessed by both transmission and scanning electron microscopy, and functional assays were performed on zebrafish embryos to investigate the mechanism of cell death. A total of 14 peptides were virtually inactive against HL60 human leukemia cells, whereas 12 caused >50% death at ≤32 μg/ml. Morphological changes characteristic of oncosis were evident by electron microscopy after only 1 minute of treatment with 32 μg/ml of variant NRC-03. Only two peptides were hemolytic. Four peptides showed no toxicity towards zebrafish embryos at the highest concentration tested (25 μM; ∼64 μg/ml) and one peptide was highly toxic, killing 4-hour-post-fertilization (hpf) embryos immediately after exposure to 1 μM peptide. Four other peptides killed embryos after 24 hours of exposure at 1 μM. Most peptides caused mortality at one or more developmental stages only after continuous exposure (24 hours) with higher lethal doses (≥5 μM). Pleurocidin NRC-03 bound to embryos and induced the release of superoxide, caused an increase in the number of TUNEL-positive nuclei, and caused membrane damage and the loss of embryonic epithelial integrity, marked by the exclusion of cells from the outer epithelium and the appearance of F-actin within the circumferential cells of the repair site. Our results indicate that specific Pleurocidin variants are attractive cancer-selective agents that selectively induce cell death in target cells but leave non-target cells such as erythrocytes and non-transformed cells unaffected.

  • Novel Antimicrobial Peptides Derived from Flatfish Genes
    Antimicrobial agents and chemotherapy, 2003
    Co-Authors: Aleksander Patrzykat, Jeffrey W. Gallant, Jung-kil Seo, Jennifer Pytyck, Susan E. Douglas
    Abstract:

    We report on the identification of active novel antimicrobials determined by screening both the genomic information and the mRNA transcripts from a number of different flatfish for sequences encoding antimicrobial peptides, predicting the sequences of active peptides from the genetic information, producing the predicted peptides chemically, and testing them for their activities. We amplified 35 sequences from various species of flatfish using primers whose sequences are based on conserved flanking regions of a known antimicrobial peptide from winter flounder, Pleurocidin. We analyzed the sequences of the amplified products and predicted which sequences were likely to encode functional antimicrobial peptides on the basis of charge, hydrophobicity, relation to flanking sequences, and similarity to known active peptides. Twenty peptides were then produced synthetically and tested for their activities against gram-positive and gram-negative bacteria and the yeast Candida albicans. The most active peptide (with the carboxy-terminus amidated sequence GWRTLLKKAEVKTVGKLALKHYL, derived from American plaice) showed inhibitory activity over a concentration range of 1 to 8 micro g/ml against a test panel of pathogens, including the intrinsically antibiotic-resistant organism Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, and C. albicans. The methods described here will be useful for the identification of novel peptides with good antimicrobial activities.

  • Identification, structure and differential expression of novel Pleurocidins clustered on the genome of the winter flounder, Pseudopleuronectes americanus (Walbaum).
    European journal of biochemistry, 2003
    Co-Authors: Susan E. Douglas, Aleksander Patrzykat, Jennifer Pytyck, Jeffrey W. Gallant
    Abstract:

    Antimicrobial peptides form one of the first lines of defense against invading pathogens by killing the microorganisms and/or mobilizing the host innate immune system. Although over 800 antimicrobial peptides have been isolated from many different species, especially insects, few have been reported from marine fish. Sequence analysis of two genomic clones (15.6 and 12.5 kb) from the winter flounder, Pseudopleuronectes americanus (Walbaum) resulted in the identification of multiple clustered genes for novel Pleurocidin-like antimicrobial peptides. Four genes and three pseudogenes (Psi) are encoded in these clusters, all of which have similar intron/exon boundaries but specify putative antimicrobial peptides differing in sequence. Pseudogenes are easily detectable but have incorrect initiator codons (ACG) and often contain a frameshift(s). Potential promoters and binding sites for transcription factors implicated in regulation of expression of immune-related genes have been identified in upstream regions by comparative genomics. Using reverse transcription-PCR assays, we have shown for the first time that each gene is expressed in a tissue-specific and developmental stage-specific manner. In addition, synthetic peptides based on the sequences of both genes and pseudogenes have been produced and tested for antimicrobial activity. These data can be used as a basis for prediction of antimicrobial peptide candidates for both human and nonhuman therapeutants from genomic sequences and will aid in understanding the evolution and transcriptional regulation of expression of these peptides.

  • Cloning and developmental expression of a family of Pleurocidin-like antimicrobial peptides from winter flounder, Pleuronectes americanus (Walbaum).
    Developmental and comparative immunology, 2001
    Co-Authors: Susan E. Douglas, Jeffrey W. Gallant, Zhiyuan Gong, Choy L. Hew
    Abstract:

    Abstract Low molecular weight antimicrobial peptides are an important component of the innate immune system in animals, yet they have not been examined widely in fish. Of particular interest is their expression during development and in response to environmental conditions and disease. Here, we report the isolation of four genomic sequences encoding putative antimicrobial peptides from the winter flounder, Pleuronectes americanus (Walbaum), as well as reverse transcription-PCR products from two tissues that form the first defensive barrier to microbes — skin and intestine. Alignment of the predicted polypeptide sequences shows a conserved hydrophobic signal peptide of 22 amino acids followed by 25 amino acids that are identical (WF2) or homologous to the amino acid sequence of Pleurocidin, followed by a conserved acidic portion. Southern hybridisation analysis indicates that related peptides are encoded in the genomes of other flatfish species. Northern and RT-PCR analyses of RNA from multiple tissues show that two of the Pleurocidin genes are expressed predominantly in the skin whereas two other genes are expressed mainly in the intestine. RT-PCR assays of total RNA from larvae of different ages provide the first evidence of developmental expression of antimicrobial peptides in fish and indicate that the Pleurocidin gene is first expressed at 13 days post-hatch in winter flounder.