RESUMEN
BACKGROUND: Pancreatic cancer is one of the leading causes of cancer death in Western societies. Its late diagnosis and resistance to chemotherapies result in a high mortality rate; thus, the development of more effective therapies for the treatment of pancreatic cancer is strongly warranted. Usnic acid (UA) is a secondary metabolite of lichens that shows modest antiproliferative activity toward cancer cells. Recently, we reported the synthesis of a UA pyrazole derivative, named 5, which was more active than the parent compound toward cervical cancer cells. Here, its anticancer potential has been evaluated in detail in other cancer cells, particularly pancreatic cancer cells. METHODS: The impact of UA and derivative 5 on cell viability, morphology, cell cycle, and death was assessed using the MTT test, electron microscopy, flow cytometry, and immunoblotting, respectively. The calcium ions level was detected fluorometrically. In vivo, the anticancer activity of 5 was evaluated in a murine xenograft model. RESULTS: Derivative 5 inhibited the viability of different cancer cells. Noncancerous cells were less sensitive. It induced the release of calcium ions from the endoplasmic reticulum (ER) and ER stress, which was manifested by cell vacuolization. It was accompanied by G0/G1 cell cycle arrest and cell death of pancreatic cancer cells. When applied to nude mice with xenografted pancreatic cancer cells, 5 inhibited tumor growth, with no signs of kidney or liver toxicity. CONCLUSIONS: UA derivative 5 is superior to UA inhibiting the growth and proliferation of pancreatic cancer cells. ER stress exaggeration is a mechanism underlying the activity of derivative 5.
RESUMEN
Derivatives of usnic acid (UA), a secondary metabolite from lichens, were synthesized to improve its anticancer activity and selectivity. Recently we reported the synthesis and activity of an UA isoxazole derivative, named 2b, against cancer cells of different origins. Herein, the molecular mechanisms underlying its activity and efficacy in vivo were tested. The viability of breast cancer or normal cells has been tested using an MTT assay. Cell and organelle morphology was analyzed using light, electron and fluorescence microscopy. Gene expression was evaluated by RNAseq and protein levels were evaluated by Western blotting. In vivo anticancer activity was evaluated in a mice xenograft model. We found that 2b induced massive vacuolization which originated from the endoplasmic reticulum (ER). ER stress markers were upregulated both at the mRNA and protein levels. ER stress was caused by the release of Ca2+ ions from the ER by IP3R channels which was mediated, at least partly, by phospholipase C (PLC)-synthetized 1,4,5-inositol triphosphate (IP3). ER stress led to cell death with features of apoptosis and paraptosis. When applied to nude mice with xenografted breast cancer cells, 2b stopped tumour growth. In mice treated with 2b, vacuolization was observed in tumour cells, but not in other organs. This study shows that the antiproliferative activity of 2b relates to the induction of ER stress in cancer, not in healthy, cells and it leads to breast cancer cell death in vitro and in vivo.
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Neoplasias de la Mama , Animales , Apoptosis , Benzofuranos , Neoplasias de la Mama/tratamiento farmacológico , Muerte Celular , Línea Celular Tumoral , Estrés del Retículo Endoplásmico , Femenino , Humanos , Isoxazoles , Ratones , Ratones DesnudosRESUMEN
Vibrio cholerae represents a constant threat to public health, causing widespread infections, especially in developing countries with a significant number of fatalities and serious complications every year. The standard treatment by oral rehydration does not eliminate the source of infection, while increasing antibiotic resistance among pathogenic V. cholerae strains makes the therapy difficult. Thus, we assessed the antibacterial potential of plant-derived phytoncides, isothiocyanates (ITC), against V. cholerae O365 strain. Sulforaphane (SFN) and 2-phenethyl isothiocyanate (PEITC) ability to inhibit bacterial growth was assessed. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values indicate that these compounds possess antibacterial activity and are also effective against cells growing in a biofilm. Tested ITC caused accumulation of stringent response alarmone, ppGpp, which indicates induction of the global stress response. It was accompanied by bacterial cytoplasm shrinkage, the inhibition of the DNA, and RNA synthesis as well as downregulation of the expression of virulence factors. Most importantly, ITC reduced the toxicity of V. cholerae in the in vitro assays (against Vero and HeLa cells) and in vivo, using Galleria mellonella larvae as an infection model. In conclusion, our data indicate that ITCs might be considered promising antibacterial agents in V. cholerae infections.
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Antibacterianos/farmacología , Cólera/dietoterapia , Isotiocianatos/farmacología , Mariposas Nocturnas/microbiología , Sulfóxidos/farmacología , Vibrio cholerae/efectos de los fármacos , Animales , Biopelículas/efectos de los fármacos , Línea Celular , Chlorocebus aethiops , ADN/biosíntesis , Modelos Animales de Enfermedad , Guanosina Tetrafosfato/biosíntesis , Células HeLa , Humanos , Pruebas de Sensibilidad Microbiana , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , ARN/biosíntesis , Células Vero , Vibrio cholerae/patogenicidad , Virulencia/efectos de los fármacos , Factores de Virulencia/biosíntesisRESUMEN
Usnic acid is a secondary metabolite abundantly found in lichens, for which promising cytotoxic and antitumor potential has been shown. However, knowledge concerning activities of its derivatives is limited. Herein, a series of usnic acid derivatives were synthesized and their antiproliferative potency against cancer cells of different origin was assessed. Some of the synthesized compounds were more active than usnic acid. Compounds 2a and 2b inhibited survival of all tested cancer cell lines in a dose- and time-dependent manner. Their IC50 values after 48 h of treatment were ca. 3 µM for MCF-7 and PC-3 cells and 1 µM for HeLa cells, while 3a and 3b revealed antiproliferative activity only against HeLa cells. All active usnic acid derivatives induced G0/G1 arrest and a drop in the fraction of HeLa cells in the S and G2/M phases. Compounds 2a and 2b decreased the clonogenic potential of the cancer cells evaluated and induced cell cycle arrest at the G0/G1 phase and apoptosis in MCF-7 cells. Moreover, they induced massive cytoplasmic vacuolization, which was associated with elevated dynein-dependent endocytosis, a process that has not been reported for usnic acid and indicates a novel mechanism of action of its synthetic derivatives. This work also shows that naturally occurring usnic acids are promising lead compounds for the synthesis of derivatives with more favorable properties against cancer cells.
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Antineoplásicos/farmacología , Benzofuranos/síntesis química , Benzofuranos/farmacología , Proliferación Celular/efectos de los fármacos , Antineoplásicos/química , Benzofuranos/química , Células HeLa , Humanos , Células MCF-7RESUMEN
BACKGROUND: Lichens that were used in traditional medicine for ages produce numerous secondary metabolites, however our knowledge about biological activities of substances secreted by separated bionts is scarce. The main objectives of this study were to isolate and find optimal conditions for the growth of mycelia from three common lichen-forming fungi, i.e. Caloplaca pusilla, Protoparmeliopsis muralis and Xanthoria parietina and to evaluate antibacterial and antiproliferative activities of their acetone extracts. METHODS: Agar disc diffusion and broth microdilution methods were used to test antimicrobial activity against six species of bacteria. MTT method, flow cytometry assay and DAPI staining were applied to test antiproliferative activity of selected extracts against MCF-7 (human breast adenocarcinoma), PC-3 (human prostate cancer) and HeLa (human cervix adenocarcinoma) cancer cells. RESULTS: P. muralis strongly inhibited the growth of Gram-positive bacteria, i.e. Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus and Staphylococcus epidermidis (MICs from 6.67 to 100.00 µg mL-1). X. parietina grown on PDA and G-LBM media decreased HeLa or MCF-7 cancer cells viability with IC50 values of about 8 µg mL-1, while C. pusilla grown on G-LBM medium showed the highest potency in decreasing MCF-7 (7.29 µg mL-1), PC-3 (7.96 µg mL-1) and HeLa (6.57 µg mL-1) cancer cells viability. We also showed induction of apoptosis in HeLa, PC-3 and MCF-7 cell lines treated with increasing concentrations of C. pusilla extract. CONCLUSION: We showed that selected acetone extracts demonstrated a strong antimicrobial and anticancer effects that suggests that aposymbiotically cultured lichen-forming fungi can be a source of antibacterial and antiproliferative compounds.
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Antibacterianos/farmacología , Antineoplásicos/farmacología , Hongos/química , Líquenes/microbiología , Antibacterianos/aislamiento & purificación , Antineoplásicos/aislamiento & purificación , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Hongos/crecimiento & desarrollo , Bacterias Grampositivas/efectos de los fármacos , Humanos , Micelio/química , Micelio/crecimiento & desarrolloRESUMEN
Until recently, only two small regulatory RNAs encoded by lambdoid bacteriophages were known. These transcripts are derived from paQ and pO promoters. The former one is supposed to act as an antisense RNA for expression of the Q gene, encoding a transcription antitermination protein. The latter transcript, called oop RNA, was initially proposed to have a double role, in establishing expression of the cI gene and in providing a primer for DNA replication. Although the initially proposed mechanisms by which oop RNA could influence the choice between two alternative developmental pathways of the phage and the initiation of phage DNA replication were found not true, the pO promoter has been demonstrated to be important for both regulation of phage development and control of DNA replication. Namely, the pO-derived transcript is an antisense RNA for expression of the cII gene, and pO is a part of a dual promoter system responsible for regulation of initiation of DNA synthesis from the oriλ region. Very recent studies identified a battery of small RNAs encoded by lambdoid bacteriophages existing as prophages in chromosomes of enterohemorrhagic Escherichia coli strains. Some of them have very interesting functions, like anti-small RNAs.
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Bacteriófago lambda/genética , Escherichia coli Enterohemorrágica/genética , Plásmidos/genética , ARN Viral/genética , Bacteriófagos/genética , Regulación Viral de la Expresión Génica , Genoma Bacteriano , Regiones Promotoras Genéticas , ARN sin Sentido , ARN Pequeño no Traducido/genéticaRESUMEN
BACKGROUND: It is generally believed that there are many natural sources of as yet unknown bioactive compounds with a high biotechnological potential. However, the common method based on the use of cell extracts in the preliminary screening for particular molecules or activities is problematic as amounts of obtained compounds may be low, and such experiments are hardly reproducible. Therefore, the aim of this work was to test whether a novel strategy to search for previously unknown biological activities can be efficient. This strategy is based on construction of metagenomic libraries and employment of Escherichia coli strains as cell factories producing compounds of properties potentially useful in biotechnology. RESULTS: Three cyanobacterial metagenomic libraries were constructed in the fosmid system. The libraries were screened for various biological activities. Extracts from selected E. coli clones bearing constructs with fragments of cyanobacterial genomes revealed antimicrobial or anticancer activities. Interestingly, stimulation of growth of host bacteria bearing particular plasmids with certain cyanobacterial genes was detected, suggesting a potential possibility for improvement of E. coli cultivation during biotechnological production. The most interesting plasmids were sequenced, and putative mechanisms of biological effects caused by cyanobacterial gene products are discussed. CONCLUSIONS: The strategy of exploring cyanobacteria as sources of bioactive compounds, based on E. coli cell factories producing compounds due to expression of genes from metagenomic libraries, appears to be effective.
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Cianobacterias/genética , Escherichia coli/genética , Biblioteca de Genes , Vectores Genéticos , Metagenoma , MetagenómicaRESUMEN
The exo-xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. In this report, using bacteriophage λ and Shiga toxin-converting bacteriophage Ï24Β, we demonstrate that the presence of this region on a multicopy plasmid results in impaired lysogenization of Escherichia coli and delayed, while more effective, induction of prophages following stimulation by various agents (mitomycin C, hydrogen peroxide, UV irradiation). Spontaneous induction of λ and Ï24Β prophages was also more efficient in bacteria carrying additional copies of the corresponding exo-xis region on plasmids. No significant effects of an increased copy number of genes located between exo and xis on both efficiency of adsorption on the host cells and lytic development inside the host cell of these bacteriophages were found. We conclude that genes from the exo-xis region of lambdoid bacteriophages participate in the regulation of lysogenization and prophage maintenance.
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Bacteriófago lambda/genética , Toxina Shiga/metabolismo , Escherichia coli Shiga-Toxigénica/patogenicidad , Escherichia coli Shiga-Toxigénica/virología , Activación Viral , Secuencia de Aminoácidos , Bacteriófago lambda/fisiología , Datos de Secuencia Molecular , Plásmidos , Profagos/genética , Profagos/fisiología , Alineación de Secuencia , Toxina Shiga/genética , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/fisiologíaRESUMEN
BACKGROUND: Isothiocyanates derived from the Brassicaceae plants possess chemopreventive and anticancer activities. One of them is sulforaphene (SF), which is abundant in Rhapanus sativus seeds. The underlying mechanism of its anticancer activity is still underexplored. PURPOSE: SF properties make it an interesting candidate for cancer prevention and therapy. Thus, it is crucial to characterize the mechanism of its activity. STUDY DESIGN: We investigated the mechanism of antiproliferative activity of SF in breast cancer cells differing in growth factor receptors status and lacking functional p53. METHODS: Viability of SKBR-3 and MDA-MB-231 breast cancer cells treated with SF was determined by SRB and clonogenic assays. Cell cycle, cell death and oxidative stress were analyzed by flow cytometry or microscopy. The levels of apoptosis and autophagy markers were assessed by immunoblotting. RESULTS: SF efficiently decreased the viability of breast cancer cells, while normal cells (MCF10A) were less sensitive to the analyzed isothiocyanate. SF induced G2/M cell cycle arrest, as well as disturbed cytoskeletal organization and reduced clonogenic potential of the cancer cells. SF induced apoptosis in a concentration-dependent manner which was associated with the oxidative stress, mitochondria dysfunction, increased Bax:Bcl2 ratio and ADRP levels. SF also potentiated autophagy which played a cytoprotective role. CONCLUSIONS: SF exhibits cytotoxic activity against breast cancer cells even at relatively low concentrations (5-10µM). This is associated with induction of the cell cycle arrest and apoptosis. SF might be considered as a potent anticancer agent.
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Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Isotiocianatos/farmacología , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Neoplasias de la Mama/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Estrés Oxidativo/efectos de los fármacos , Raphanus/químicaRESUMEN
Metagenomic studies have become increasingly popular. They allow for the estimation of biodiversity in complex populations. This diversity presents an enormous but largely unexpected genetic and biological pool and can be exploited for the recovery of novel genes, entire metabolic pathways and their products. Generally metagenomic study is a genomic analysis of organisms by direct extraction and cloning of DNA from their natural environment. The most common problems of modern metagenomics are as follows: majority of the microorganisms present in the environment cannot be cultivated by standard techniques, DNA extraction methods are not very effective, isolated DNA is contaminated with various compounds, a choice for a screening method is not obvious.
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ADN/aislamiento & purificación , Metagenómica , Ecosistema , Aguas del AlcantarilladoRESUMEN
Metagenomics is a powerful tool to better understand the microbial niches, especially these from extreme habitats like oceans and seas, hot springs or deserts. However, one who is going to face the metagenomic studies should realize the challenges which might occur in the course of experiments. This manuscript indicates common problems in function-driven metagenomics, especially factors that influence gene expression are taken into account. Codon usage bias, internal cell accumulation, correct protein folding or presence of proper initiation factors are discussed and possible ways to overcome these problems are proposed. Finally, the annotation process is described, including possible limitations that one should take under consideration. What is more, the most popular databases for metagenomic data are mentioned and discussed.
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Metagenómica , Microbiota , CodónRESUMEN
Cyanobacteria are a rich source of biologically active compounds used in pharmacology and biotechnology. Due to their high capacity of adaptation, which is reflected in the production of diverse metabolites, including toxins, these microorganisms are able to inhabit very different environments. In this work, water and ethanol extracts from 11 cyanobacterial strains derived from the Baltic Sea (Microcystis, Synechocystis, Leptolyngbya, Pseudanabaena, Lyngbya, Phormidium, Nodularia and Anabaena genera) were screened for anticancer activity. MCF-7 human breast cancer and HeLa cervical cancer cell lines, as well as HDFa normal human fibroblasts, were used. Three extracts derived from Pseudanabaena sp., Pseudanabaena cf. galeata and Microcystis aeruginosa revealed potent and selective antiproliferative activities against cancer cells. The mechanism of the anticancer activity was explored in MCF-7 cells, and was found to rely on the inhibition of the pro-survival Akt kinase and induction of cell death. The peptide profiles of selected cyanobacterial extracts were determined using LC-MS/MS, and classes of bioactive compounds that might be potentially responsible for the observed anticancer activities are presented.
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Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Cianobacterias/química , Toxinas Marinas/farmacología , Línea Celular , Cromatografía Liquida , Células HeLa , Humanos , Células MCF-7 , Toxinas Marinas/química , Océanos y Mares , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Espectrometría de Masas en TándemRESUMEN
A microRNA-size (20-nt long) molecule has been identified in Escherichia coli after induction of Shiga toxin-converting bacteriophage Φ24B. This small RNA, named 24B_1, is encoded in the lom-vb_24B_43 region of the phage genome, and apparently it is produced by cleavage of a larger transcript. A phage devoid of 24B_1 revealed decreased efficiency of lysogenization, quicker prophage induction after provoking the SOS response, higher efficiency of progeny phage production during the lytic cycle and less efficient adsorption on the host cells. Expression of most of phage genes was drastically increased after infection of E. coli by the Φ24BΔ24B_1 phage. Since 24B_1 may impair expression of the d_ant gene, coding for an anti-repressor, these results may explain the mechanism of regulations of the physiological processes by this small RNA due to impaired activity of the cI repressor and changed expression of vast majority of phage genes. To our knowledge, this is the first example of functional microRNA-size molecule in bacterial cells.
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Colifagos/metabolismo , Escherichia coli/virología , Regulación Viral de la Expresión Génica/fisiología , MicroARNs/biosíntesis , ARN Viral/biosíntesis , Toxina Shiga , Colifagos/genética , Escherichia coli/genética , Escherichia coli/metabolismo , MicroARNs/genética , ARN Viral/genética , Respuesta SOS en Genética/genéticaRESUMEN
Lambdoid bacteriophages serve as useful models in microbiological and molecular studies on basic biological process. Moreover, this family of viruses plays an important role in pathogenesis of enterohemorrhagic Escherichia coli (EHEC) strains, as they are carriers of genes coding for Shiga toxins. Efficient expression of these genes requires lambdoid prophage induction and multiplication of the phage genome. Therefore, understanding the mechanisms regulating these processes appears essential for both basic knowledge and potential anti-EHEC applications. The exo-xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. Recent report indicated that the Ea8.5 protein, encoded in this region, contains a newly discovered fused homeodomain/zinc-finger fold, suggesting its plausible regulatory role. Moreover, subsequent studies demonstrated that overexpression of the exo-xis region from a multicopy plasmid resulted in impaired lysogenization of E. coli and more effective induction of λ and Ф24B prophages. In this report, we demonstrate that after prophage induction, the increase in phage DNA content in the host cells is more efficient in E. coli bearing additional copies of the exo-xis region, while survival rate of such bacteria is lower, which corroborated previous observations. Importantly, by using quantitative real-time reverse transcription PCR, we have determined patterns of expressions of particular genes from this region. Unexpectedly, in both phages λ and Ф24B, these patterns were significantly different not only between conditions of the host cells infection by bacteriophages and prophage induction, but also between induction of prophages with various agents (mitomycin C and hydrogen peroxide). This may shed a new light on our understanding of regulation of lambdoid phage development, depending on the mode of lytic cycle initiation.
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Bacteriófago lambda/genética , Bacteriófagos/genética , Regulación Viral de la Expresión Génica , Profagos/genética , Activación Viral/genética , Escherichia coli Enterohemorrágica , Toxina Shiga/metabolismoRESUMEN
Shiga toxin-producing Escherichia coli (STEC) strains belong to the group of pathogens that cause bloody diarrhea and hemorrhagic colitis with often severe complications. The main problem with human pathogenic E. coli strains, including STEC, is a wide spectrum of phenotypes and clinical manifestations. It is related to a variety of exchangeable genetic elements, like plasmids, bacteriophages, transposons and pathogenicity islands, that take part in horizontal gene transfer which influences creation of new dangerous bacterial strains. A good example of this phenomenon is a novel Shiga toxin-producing E. coli O104:H4 serotype that was associated with a widespread and severe foodborne disease outbreak in Germany in 2011. The O104:H4 strain was created by a number of horizontal gene transfer events between two distinct pathogens, resulting in the emergence of the new, atypical strain. That outbreak proved that also rare and unusual serotypes of STEC may be a significant risk factor and that the procedures recommended for STEC detection were not suitable to deal with this kind of pathogens. With respect to new combinations of chromosomal and extrachromosomal elements in susceptible bacterial hosts, epidemics and frequent human infections caused by STEC strains, we suggest that more attention should be paid to the development and improvement of diagnostic methods. It is difficult to determine STEC bacteria by general microbiological, biochemical and immunological assays, because strains can vary dramatically in their phenotypic and serotypic properties. It is postulated that standardized genetic tests, based on detection of features most frequently presented by STEC, particularly those located on easily exchangeable elements (such as Shiga toxin-encoding phages), can be more adequate for STEC detection.
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Cromosomas Bacterianos , Infecciones por Escherichia coli , Escherichia coli Shiga-Toxigénica , Cromosomas Bacterianos/genética , Cromosomas Bacterianos/metabolismo , Brotes de Enfermedades/prevención & control , Infecciones por Escherichia coli/diagnóstico , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/patología , Enfermedades Transmitidas por los Alimentos/diagnóstico , Enfermedades Transmitidas por los Alimentos/epidemiología , Enfermedades Transmitidas por los Alimentos/genética , Enfermedades Transmitidas por los Alimentos/patología , Alemania , Humanos , Escherichia coli Shiga-Toxigénica/genética , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Escherichia coli Shiga-Toxigénica/patogenicidadRESUMEN
The marine environment is estimated to be one of the most significant sources of biological activity in the world. In the last few decades an increase in the research intensity conducted on marine microorganisms has been observed, which confirms the great potential of these organisms in the field of bioactive compounds' production. In order to efficiently use the natural resources of the marine environment, metagenomics can be applied. This powerful technique allows for efficient screening of microbial biodiversity for bioactive compounds. The primary aim of this review is to present some aspects of the construction of metagenomic libraries, and strategies of screening for novel bioactives in the marine surrounding. This paper also illustrates several examples of the application of metagenomic methods in the discovery of novel enzymes and drugs in various marine environments.