RGD peptide inhibits tumor growth by affecting angiogenesis related signaling pathway of laryngeal cancer stem cells / El péptido RGD inhibe el crecimiento tumoral al afectar la vía de señalización relacionada con la angiogénesis de las células madre del cáncer de laringe

SUMMARY: This study is to investigate the role and mechanism of RGD peptide in laryngeal cancer stem cells (CSCs). Laryngeal cancer CD133+Hep-2 CSCs were sorted by flow cytometry. RGD peptide was co-cultured with sorted laryngeal CSCs. Cell proliferation was detected with CCK-8 assay. The mRNA levels of VEGF/VEGFR2/STAT 3/HIF-1α were detected with RT-PCR. The proteins of VEGF/ VEGFR2/STAT 3/HIF-1α were detected with Western blot. The sorted CSCs were inoculated into nude mice. Tumor volume was measured. Integrin αvβ3 expression in tumor tissues was analyzed with immunohistochemistry. The results showed that the ratio of CD133+ CSCs to the total number of cells was 1.34±0.87 %, while CD133-non-tumor stem cells accounted for 95.0±5.76 %. The sorted cancer stem cells grew well. The RGD peptide significantly inhibited the proliferation of CD133+Hep-2 laryngeal CSCs in a dose-dependent manner. The RGD peptide significantly inhibited the mRNA of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a concentration-dependent manner. Consistently, the RGD peptide significantly inhibited the protein expression of VEGFR2, STAT3 and HIF-1α in laryngeal CSCs in a dose-dependent manner. At the same time, in vivo tumor experiments showed that the RGD peptide significantly inhibited tumor volume but not the body weight. Furthermore, RGD peptide significantly inhibited the expression of tumor angiogenesis-related protein integrin αvβ3. Our findings demonstrate that RGD peptide inhibits tumor cell proliferation and tumor growth. The underlying mechanism may that RGD inhibits tumor angiogenesis-related signaling pathways, thus affecting the tumor angiogenesis, and decreasing the progression of human laryngeal CSCs.

Este estudio se realizó para investigar el papel y el mecanismo del péptido RGD en las células madre del cáncer de laringe (CSC). Las CSC CD133+Hep-2 de cáncer de laringe se clasificaron mediante citometría de flujo. El péptido RGD se cocultivó con CSC laríngeas clasificadas. La proliferación celular se detectó con el ensayo CCK-8. Los niveles de ARNm de VEGF/VEGFR2/ STAT 3/HIF-1α se detectaron con RT-PCR. Las proteínas de VEGF/ VEGFR2/STAT 3/HIF-1α se detectaron con Western blot. Las CSC clasificadas se inocularon en ratones nudos. Se midió el volumen del tumor. La expresión de integrina αvβ3 en tejidos tumorales se analizó con inmunohistoquímica. Los resultados mostraron que la proporción de CSC CD133+ con respecto al número total de células fue de 1,34 ± 0,87 %, mientras que las células madre no tumorales CD133 representaron el 95,0 ± 5,76 %. Las células madre cancerosas clasificadas crecieron bien. El péptido RGD inhibió significativamente la proliferación de CSC laríngeas CD133+Hep-2 de una manera dependiente de la dosis. El péptido RGD inhibió significativamente el ARNm de VEGFR2, STAT3 y HIF-1α en CSC laríngeas de manera dependiente de la concentración. De manera consistente, el péptido RGD inhibió significativamente la expresión proteica de VEGFR2, STAT3 y HIF-1α en CSC laríngeas, de manera dependiente de la dosis. Al mismo tiempo, los experimentos con tumores in vivo mostraron que el péptido RGD inhibía significativamente el volumen del tumor pero no el peso corporal. Además, el péptido RGD inhibió significativamente la expresión de la proteína integrina αvβ3 relacionada con la angiogénesis tumoral. Nuestros hallazgos demuestran que el péptido RGD inhibe la proliferación de células tumorales y el crecimiento tumoral. El mecanismo subyacente puede ser que RGD inhiba las vías de señalización relacionadas con la angiogénesis tumoral, afectando así la angiogénesis tumoral y disminuyendo la progresión de las CSC laríngeas humanas.

Ghrelin attenuates the growth of HO-8910 ovarian cancer cells through the ERK pathway

Ovarian cancer is one of the most common causes of death from gynecologic tumors and is an important public health issue. Ghrelin is a recently discovered bioactive peptide that acts as a natural endogenous ligand of the growth hormone secretagogue receptor (GHSR). Several studies have identified the protective effects of ghrelin on the mammalian reproductive system. However, little research has been done on the effects of ghrelin on ovarian cancer cells, and the underlying mechanisms of these effects. We sought to understand the potential involvement of mitogen-activated protein kinases (MAPKs) in ghrelin-mediated inhibition of growth of the ovarian line HO-8910. We applied different concentrations of ghrelin and an inhibitor of the ghrelin receptor (D-Lys3-GHRP-6) to HO-8910 cells and observed the growth rate of cells and changes in phosphorylation of the MAPKs ERK1/2, JNK and p38. We discovered that ghrelin-induced apoptosis of HO-8910 cells was though phosphorylated ERK1/2, and that this phosphorylation (as well as p90rsk phosphorylation) was mediated by the GHSR. The ERK1/2 pathway is known to play an essential part in the ghrelin-mediated apoptosis of HO-8910 cells. Hence, our study suggests that ghrelin inhibits the growth of HO-8910 cells primarily through the GHSR/ERK pathway.

Comparison of antibiotic resistance and virulence factors in pigmented and Non-pigmented Pseudomonas aeruginosa / Comparación de la resistencia antibiótica y los factores de virulencia en las Pseudomonas aeruginosa pigmentadas y no pigmentadas

OBJECTIVES: Pseudomonas aeruginosa produces multiple virulence factors that have been implicated in pathogenesis and quorum sensing. The aim of this study was to determine differences in the virulence factors of pigmented and non-pigmented P aeruginosa isolates. METHODS: Associations were assessed between pigment production (pyocyanin and pyoverdin) and production of DNase, elastase, lipase, protease, siderophore, twitching motility, antibiotic resistance patterns and virulence-associated genes in 57 non-duplicate P aeruginosa isolates from wounds, sputum, urine, high vaginal swab (HVS), ear, eye and respiratory tract swabs and aspirates of peritoneum and ulcers. RESULTS: Most (82.5%) of the isolates produced either pigment. Pigmented isolates produced more frequently and significant more (p < 0.05) DNase, elastase, lipase protease, and siderophore. Imipenem was the only antibiotic to which all isolates were susceptible (p < 0.05), while 93% and 32% were resistant to tetracycline and norfloxacin, respectively. There was however no significant difference between pigmented and non-pigmented isolates when antibiotic resistance was compared. While isolates had multiple virulence-associated genes, exoS (51%), rhlA (37%) and rhlB (46%) were the predominant genes detected. Except for exoY, genes were present in pigmented isolates more frequently than in non-pigmented isolates. CONCLUSION: The results of this study suggest that antibiotic resistance per se might not be associated with the pigment production in P aeruginosa. However, pigment production appeared to be more significantly associated with multi-drug resistance, presence ofvirulence-associated genes, and expression ofcertain virulence factors, most notably elastase, protease, siderophore and DNase activity. Since pigment production is easy to determine, this might to be a good starting point to identify the virulence status ofan isolate.

OBJETIVO: Pseudomonas aeruginosa produce múltiples factores de virulencia que han estado implicados en patogénesis y detección de quórum (quorum sensing). El objetivo de este estudio fue determinar las diferencias en los factores de virulencia de aislados de P aeruginosa pigmentada y no pigmentada. MÉTODO: Se evaluaron las asociaciones entre la producción de pigmentos (piocianina y pioverdina) y la producción de Dnasa, elastasa, lipasa, proteasa, sideróforos, motilidad asociada a superficies (twitching), patrones de resistencia antibiótica, y genes asociados con virulencia en 57 aislados de P aeruginosa no duplicados, de heridas, esputo, orina, exudado vaginal, exudados de oídos, ojos, y vías respiratorias, y aspirados de peritoneo y úlceras. RESULTADOS: La mayor parte (82.5%) de los aislados produjeron uno de los pigmentos. Los aislados pigmentados produjeron con mayor frecuencia y más significativamente (p < 0.05). Dnasa, elastasa, lipasa, proteasa, y siderósforos. Imipenem fue el único antibiótico al que todos los aislados eran susceptibles (p < 0.05), mientras que el 93% y el 32% fueron resistentes a la tetraciclina y a la norfloxacina, respectivamente. Sin embargo, no hubo diferencia significativa entre los aislados pigmentados y los no pigmentados cuando se comparaba la resistencia antibiótica. Si bien los aislados tenían múltiples genes asociados con la virulencia, exoS (51%), rhlA (37%) y rhlB (46%) fueron los genes predominantes detectados. Con excepción de exoY, los genes estuvieron presentes en aislados pigmentados con mayor frecuencia que en los aislados no pigmentados. CONCLUSIÓN: Los resultados de este estudio sugieren que la resistencia antibiótica per se podría no estar asociada con la producción de pigmentos en P aeruginosa. Sin embargo, la producción de pigmentos parecía estar asociada más significativamente con la resistencia a las multidrogas, la presencia de genes asociados con la virulencia, y la expresión de ciertos factores de virulencia, en particular la actividad de la elastasa, la proteasa, los sideróforos, y la Dnasa. Puesto que la producción de pigmentos es fácil de determinar, esto podría ser un buen punto de partida para identificar el estado de virulencia de un aislado.

Ghrelina e secretagogos do hormônio de crescimento (GHS): modulação da secreção do hormônio de crescimento e perspectivas terapêuticas: [revisão] / Ghrelin and growth hormone secretagogues (GHS): modulation of growth hormone secretion and therapeutic applications: [review]

A secreção do hormônio de crescimento (GH) é modulada pelo hormônio liberador de hormônio de crescimento (GHRH) e pela somatostatina. Na última década foi descoberto um terceiro mecanismo de controle, envolvendo os secretagogos de GH (GHS). A ghrelina, o ligante endógeno do receptor dos GHS, é um peptídeo acilado produzido no estômago, que também é sintetizado no hipotálamo. Este peptídeo é capaz de liberar GH, além de aumentar a ingesta alimentar. A ghrelina endógena parece amplificar o padrão básico de secreção de GH, ampliando a resposta do somatotrofo ao GHRH, estimulando múltiplas vias intracelulares interdependentes. Entretanto, seu local de atuação predominante é o hipotálamo. Neste trabalho, será apresentada revisão sobre a descoberta da ghrelina, os mecanismos de ação e o possível papel fisiológico dos GHS e da ghrelina na secreção de GH e, finalmente, as possíveis aplicações terapêuticas destes compostos.

Growth hormone-releasing hormone (GHRH) and somatostatin modulate growth hormone (GH) secretion. A third mechanism was discovered in the last decade, involving the action of growth hormone secretagogues (GHS). Ghrelin, the endogenous ligand of the GHS-receptor, is an acylated peptide mainly produced by the stomach, but also synthesized in the hypothalamus. This compound increases both GH release and food intake. Endogenous ghrelin might amplify the basic pattern of GH secretion, optimizing somatotroph responsiveness to GHRH, activating multiple interdependent intracellular pathways. However, its main site of action is the hypothalamus. In the current paper it is reviewed the available data on the discovery of this peptide, the mechanisms of action and possible physiological roles of the GHS and ghrelin on GH secretion, and finally, the possible therapeutic applications of these compounds.

Structural model and ligand interactions of the Xanthomonas axonopodis pv. citri oligopeptide-binding protein

The oligopeptide-binding protein, OppA, ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides by several bacterial species. In the present study, we report a structural model and an oligopeptide docking analysis of the OppA protein expressed by Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The X. citri OppA structural model showed a conserved three-dimensional structure, irrespective of the low amino acid identities with previously defined structures of Bacillus subtilis and Salmonella typhimurium orthologs. Oligopeptide docking analysis carried out with the proposed model indicated that the X. citri OppA preferentially binds tri- and tetrapeptides. The present study represents the first structural analysis of an OppA ortholog expressed by a phytopathogen and contributes to the understanding of the physiology and nutritional strategies of X. citri.

Improvement of bovine ß-lactoglobulin production and secretion by Lactococcus lactis

The stabilizing effects of staphylococcal nuclease (Nuc) and of a synthetic propeptide (LEISSTCDA, hereafter called LEISS) on the production of a model food allergen, bovine ß-lactoglobulin (BLG), in Lactococcus lactis were investigated. The fusion of Nuc to BLG (Nuc-BLG) results in higher production and secretion of the hybrid protein. When LEISS was fused to BLG, the production of the resulting protein LEISS-BLG was only slightly improved compared to the one obtained with Nuc-BLG. However, the secretion of LEISS-BLG was dramatically enhanced (~10- and 4-fold higher than BLG and Nuc-BLG, respectively). Finally, the fusion of LEISS to Nuc-BLG resulting in the protein LEISS-Nuc-BLG led to the highest production of the hybrid protein, estimated at ~8 æg/ml (~2-fold higher than Nuc-BLG). In conclusion, the fusions described here led to the improvement of the production and secretion of BLG. These tools will be used to modulate the immune response against BLG via delivery of recombinant lactococci at the mucosal level, in a mouse model of cow's milk allergy.

Linear competitive inhibition of human tissue kallikrein by 4-aminobenzamidine and benzamidine and linear mixed inhibition by 4-nitroaniline and aniline

Hydrolysis of D-valyl-L-leucyl-L-arginine p-nitroanilide (7.5-90.0 `M) by human tissue kallikrein (hK1) (4.58-5.27 nM) at pH 9.0 and 37ºC was studied in the absence and in the presence of increasing concentrations of 4-aminobenzamidine (96-576 `M), benzamidine (1.27-7.62 mM), 4-nitroaniline (16.5-66 `M) and aniline (20-50 mM). The kinetic parameters determined in the absence of inhibitors were: Km = 12.0 + or - 0.8 `M and k cat = 48.4 + or - 1.0 min-1. The data indicate that the inhibition of hK1 by 4-aminobenzamidine and benzamidine is linear competitive, while the inhibition by 4-nitroaniline and aniline is linear mixed, with the inhibitor being able to bind both to the free enzyme with a dissociation constant Ki yielding an EI complex, and to the ES complex with a dissociation constant Ki', yielding an ESI complex. The calculated Ki values for 4-aminobenzamidine, benzamidine, 4-nitroaniline and aniline were 146 + or - 10, 1,098 + or - 91, 38.6 + or - 5.2 and 37,340 + or - 5,400 `M, respectively. The calculated Ki' values for 4-nitroaniline and aniline were 289.3 + or - 92.8 and 310,500 + or - 38,600 `M, respectively. The fact that Ki'>Ki indicates that 4-nitroaniline and aniline bind to a second binding site in the enzyme with lower affinity than they bind to the active site. The data about the inhibition of hK1 by 4-aminobenzamidine and benzamidine help to explain previous observations that esters, anilides or chloromethyl ketone derivatives of Nalpha-substituted arginine are more sensitive substrates or inhibitors of hK1 than the corresponding lysine compounds

Mecanismos de absorçäo de aminoácidos e oligopeptídios: controle e implicaçöes na dietoterapia humana / Mechanisms of amino acids and oligopeptides absorption in humans

Os mecanismos envolvidos na absorção de aminoácidos e di-tripeptídeos são revistos direcionadamente às implicações na nutrição humana. As membranas borda em escova e basolateral são atravessadas por aminoácidos e ditripeptídeos mediante mecanismos passivos (difusão simples ou facilitada) ou ativos (c0-transportadores de Na+ ou H+). O sistema ativo dependente de Na+ ocorre principalmente na borda em escova e a difusão simples na basolateral. Ambas as membranas apresentam o transporte passivo facilitado. Os aminoácidos livres usam tanto o sistema passivo, como o ativo, enquanto os di-tripeptídeos utilizam mais o sistema ativo (H+). A borda em escova possui sistemas distintos para aminoácidos livres e di-tripeptídios, com os primeiros utilizando preferentemente o Na+ dependente e os demais o H+ - dependente. Os aminoácidos livres são transportados em diferentes velocidades por carreadores, em mecanismos saturáveis e competitivos, com especificidade para aminoácidos básicos, ácidos e neutros. Os di e tripeptídeos têm pelo menos dois carreadores, ambos H+ dependentes. Na membrana basolateral os aminoácidos livres são transportados por difusão simples enquanto os di-tripeptídios o são ativamente por processo de troca aniônica. A principal regulação do transporte transmembrana de aminoácidos e di-tripepídios é a concentração desses substratos ao nível da membrana. Afora isso, di e tripeptídios são mais eficientemente absorvidos que os aminoácidos livres os quais por sua vez o são melhor que os tetra ou mais peptídios. Desta forma, o di-tripeptídios resultam em maior retenção nitrogenada que os demais e são particularmente úteis nos casos de redução da capacidade absortiva intestinal. Os peptídios não absorvidos são fermentados pelas bactérias colônicas resultando ácidos graxos de cadeia curta, ácidos dicarboxílicos, compostos fenólicos e amônia. Os ácidos graxos e a amônia são fixados pelas bactérias para produção de energia e aminoácidos, respectivamente.

Spontaneous Kanamycin-resistant Escherichia coli mutant with altered periplasmic oligopeptide permease protein (OPPA) and impermeability to aminoglycosides

A spontaneous kanamycin-resistant Escherichia coli mutant, showing cross resitance to five other aminoglycosides and absence of the OppA protein was isolated. [3H]- dihydrostreptomycin uptake is reduced in this mutant, implying that the oligopeptide transport system in involved in accumulation of aminoglucosides, although apparently not related with aminoglycoside permeability alteration due to bacterial adaptation to osmotic changes.

Copper(II) as an efficient scavenger of singlet molecular oxygen.

Reactive oxygen species (ROS) are considered to play an important role in tissue injury that damages DNA, proteins, carbohydrates and lipids. Increased production of ROS and/or decreased efficiency of antioxidant defense system has been shown to contribute to a number of degenerative processes including cancer and AIDS. Among the various forms of ROS, singlet oxygen (1O2), which is generated predominantly in photosensitization reactions, is of particular physiologic significance because of its selectively long life in aqueous solution, its ability to cross the cell membrane barrier and high reactivity towards biomolecules. In the present study, the 1O2 scavenging potential of Cu(II) has been evaluated by (i) generating 1O2 by photosensitization of rose bengal (RB), (ii) establishing 1O2 quenching with recognized 1O2 scavengers like sodium azide, DABCO and (iii) examining the effect of Cu(II) in scavenging of 1O2. The results revealed that Cu(II) inhibited the rate of 1O2 production by 88%, 68%, 40%, 21% and 10% at a concentration of 10(-2) M, 5 x 10(-3) M, 10(-3) M; 5 x 10(-4) M, and 10(-4) M, respectively. Under similar experimental condition, sodium azide or DABCO at 10(-2) M inhibited the 1O2 production by 86% and 88%, respectively. Other 1O2 generating photosensitizer like hematoporphyrin, riboflavin and methylene blue also produced identical results with Cu(II) but Fe(II), Fe(III), Zn(II) or As(III) did not produce any quenching of 1O2. Presence of a copper binding peptide (Gly-Gly-His) in the reaction system reduced the 1O2 scavenging capacity of Cu(II) by 52-66% depending upon the UV dose. The 1O2 scavenging property of metal ion appears to have an advantage to reduce the oxidative damage of photodynamic reactions in order to prevent ROS-induced toxicity reactions.

Subsite study of human pepsin in disease.

The synthetic peptides AC-Glu-Phe-Phe (NO2)-Arg-amide (peptide VP) and AC-Ile-Glu-Phe-Phe (NO2)-Arg-amide (peptide VIP) are more readily hydrolyzed by human pepsin in gastric juice of patients of gastritis than those of duodenal ulcer and normal subjects. The kinetic parameters suggest that S3 subsite of the enzyme plays a role in the elevation of enzyme activity in gastric disease.

Thermolysin: a peptide forming enzyme.

Thermolysin, a thermostable endopeptidase, is recognised as a potential peptide bond forming enzyme. The importance of structural properties and its stereospecific nature towards peptide bond formation is described. Thermolysin's use in the keystep of the preparation of an artificial sweetener 'aspartame' is highlighted.