RESUMO
Mass spectrometry has become the most prominent yet evolving technology in quantitative proteomics. Today, a number of label-free and label-based approaches are available for the relative and absolute quantification of proteins and peptides. However, the label-based methods rely solely on the employment of stable isotopes, which are expensive and often limited in availability. Here we propose a label-based quantification strategy, where the mass difference is identified by the differential alkylation of cysteines using iodoacetamide and acrylamide. The alkylation reactions were performed under identical experimental conditions; therefore, the method can be easily integrated into standard proteomic workflows. Using high-resolution mass spectrometry, the feasibility of this approach was assessed with a set of tryptic peptides of human serum albumin. Several critical questions, such as the efficiency of labeling and the effect of the differential alkylation on the peptide retention and fragmentation, were addressed. The concentration of the quality control samples calculated against the calibration curves were within the ±20% acceptance range. It was also demonstrated that heavy labeled peptides exhibit a similar extraction recovery and matrix effect to light ones. Consequently, the approach presented here may be a viable and cost-effective alternative of stable isotope labeling strategies for the quantification of cysteine-containing proteins.
Assuntos
Acrilamida , Cisteína , Iodoacetamida , Proteômica , Iodoacetamida/química , Alquilação , Cisteína/química , Cisteína/análise , Acrilamida/química , Acrilamida/análise , Humanos , Proteômica/métodos , Espectrometria de Massas/métodos , Marcação por Isótopo/métodos , Peptídeos/química , Peptídeos/análise , Espectrometria de Massas em Tandem/métodosRESUMO
A high-resolution HILIC-MS/MS method was developed to analyze anthranilic acid derivatives of N-glycans released from human serum alpha-1-acid glycoprotein (AGP). The method was applied to samples obtained from 18 patients suffering from high-risk malignant melanoma as well as 19 healthy individuals. It enabled the identification of 102 glycan isomers separating isomers that differ only in sialic acid linkage (α-2,3, α-2,6) or in fucose positions (core, antenna). Comparative assessment of the samples revealed that upregulation of certain fucosylated glycans and downregulation of their nonfucosylated counterparts occurred in cancer patients. An increased ratio of isomers with more α-2,6-linked sialic acids was also observed. Linear discriminant analysis (LDA) combining 10 variables with the highest discriminatory power was employed to categorize the samples based on their glycosylation pattern. The performance of the method was tested by cross-validation, resulting in an overall classification success rate of 96.7%. The approach presented here is significantly superior to serological marker S100B protein in terms of sensitivity and negative predictive power in the population studied. Therefore, it may effectively support the diagnosis of malignant melanoma as a biomarker.
Assuntos
Melanoma/sangue , Orosomucoide/metabolismo , Biomarcadores Tumorais/sangue , Cromatografia/métodos , Glicosilação , Humanos , Polissacarídeos/sangue , Espectrometria de Massas em Tandem/métodos , ortoaminobenzoatos/químicaRESUMO
Poly(ADP-ribose) polymerase-1 (PARP1) is a pro-inflammatory protein, whose pro-inflammatory properties were demonstrated in human. The pro-inflammatory properties of PARP1 were shown in Th1- and Th2-mediated inflammatory pathologies, but not Th17-mediated inflammation. Thus, we studied the role of PARP1 in the imiquimod-induced model of psoriasis. To our surprise, in imiquimod-induced psoriasis, PARP1 acted as an anti-inflammatory factor and its genetic deletion exacerbated symptoms. We showed that in the absence of PARP1, the epidermis thickened and the number of TUNEL-positive cells decreased in the epidermis. These data indicate programmed cell death is decreased in keratinocytes. Changes in involucrin expression suggest that keratinocyte differentiation is hampered. Furthermore, epidermal expression of IL6 increased in the psoriasiform lesions of PARP1 knockout mice, suggesting that the inflammatory response is also derailed in the absence of PARP1. Finally, we showed that PARP1 expression is reduced in human psoriatic lesions compared with control skin samples. In imiquimod-treated HPV-KER keratinocytes, PARP inhibition recapitulated the in vivo findings, namely keratinocyte hyperproliferation; furthermore, the mRNA expression of psoriasis-associated cytokines (IL6, IL1ß, IL8, IL17 and IL23A) was also induced. The inhibition of TRPV1 abrogated the effects of the combined imiquimod + PARP inhibitor treatment.
Assuntos
Citocinas/genética , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Psoríase/fisiopatologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Humanos , Imiquimode/farmacologia , Inflamação/genética , Interleucina-6/metabolismo , Queratinócitos , Masculino , Camundongos , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Psoríase/induzido quimicamente , Psoríase/patologia , RNA Mensageiro/metabolismo , Índice de Gravidade de Doença , Canais de Cátion TRPV/antagonistas & inibidores , Células Th17RESUMO
Our study aimed at finding a mechanistic relationship between the gut microbiome and breast cancer. Breast cancer cells are not in direct contact with these microbes, but disease could be influenced by bacterial metabolites including secondary bile acids that are exclusively synthesized by the microbiome and known to enter the human circulation. In murine and bench experiments, a secondary bile acid, lithocholic acid (LCA) in concentrations corresponding to its tissue reference concentrations (< 1 µM), reduced cancer cell proliferation (by 10-20%) and VEGF production (by 37%), aggressiveness and metastatic potential of primary tumors through inducing mesenchymal-to-epithelial transition, increased antitumor immune response, OXPHOS and the TCA cycle. Part of these effects was due to activation of TGR5 by LCA. Early stage breast cancer patients, versus control women, had reduced serum LCA levels, reduced chenodeoxycholic acid to LCA ratio, and reduced abundance of the baiH (7α/ß-hydroxysteroid dehydroxylase, the key enzyme in LCA generation) gene in fecal DNA, all suggesting reduced microbial generation of LCA in early breast cancer.
Assuntos
Apoptose/efeitos dos fármacos , Bactérias/metabolismo , Neoplasias da Mama/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Detergentes/farmacologia , Ácido Litocólico/farmacologia , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Prognóstico , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Mass spectrometry is a highly sensitive high-throughput instrumental analytical technique. It is used to determine the molecular mass, but also gives information on molecular structure amd is used for quantitation as well. Although it was developed over 100 years ago, it continues to evolve, both with respect to figures of merit (like sensitivity) and with respect to applications in various novel fields of science and technology. Mass spectrometry is capable of studying macromolecules (like proteins and protein complexes), and has very high sensitivity, now compounds at the atto- or zeptomol level can also be studied. Mass spectrometry can be coupled to separation techniques, and can be used to analyze complex mixtures, trace level compounds in biological matrices like active pharmaceutical ingredients or metabolites. In recent years in proteomics research has become a major new direction. In the present review we briefly introduce basic mass spectrometry techniques (ion surces, analyzers), combinations with chromatography (GC/MS, HPLC/MS), CEI MS) and tandem mass spectrometry. We also introduce two novel methods, mass spectrometry "imaging" and "lab-on-a-chip" technology.
Assuntos
Arabidopsis/química , Cromatografia Gasosa-Espectrometria de Massas/métodos , Cromatografia Gasosa-Espectrometria de Massas/tendências , Humanos , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Espectrometria de Massas/tendências , Estrutura Molecular , Proteínas/química , Proteômica/métodos , Proteômica/tendências , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas em Tandem/tendênciasRESUMO
Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme. However, recent investigations have attributed unique roles to PARP-2 in metabolic regulation in the liver. We assessed changes in hepatic lipid homeostasis upon the deletion of PARP-2 and found that cholesterol levels were higher in PARP-2(-/-) mice as compared to wild-type littermates. To uncover the molecular background, we analyzed changes in steady-state mRNA levels upon the knockdown of PARP-2 in HepG2 cells and in murine liver that revealed higher expression of sterol-regulatory element binding protein (SREBP)-1 dependent genes. We demonstrated that PARP-2 is a suppressor of the SREBP1 promoter, and the suppression of the SREBP1 gene depends on the enzymatic activation of PARP-2. Consequently, the knockdown of PARP-2 enhances SREBP1 expression that in turn induces the genes driven by SREBP1 culminating in higher hepatic cholesterol content. We did not detect hypercholesterolemia, higher fecal cholesterol content or increase in serum LDL, although serum HDL levels decreased in the PARP-2(-/-) mice. In cells and mice where PARP-2 was deleted we observed decreased ABCA1 mRNA and protein expression that is probably linked to lower HDL levels. In our current study we show that PARP-2 impacts on hepatic and systemic cholesterol homeostasis. Furthermore, the depletion of PARP-2 leads to lower HDL levels which represent a risk factor to cardiovascular diseases.
Assuntos
Colesterol/metabolismo , Lipoproteínas HDL/sangue , Fígado/metabolismo , Poli(ADP-Ribose) Polimerases/fisiologia , Animais , Células Hep G2 , Humanos , Masculino , Camundongos , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/fisiologiaAssuntos
Antineoplásicos/administração & dosagem , Carcinoma Hepatocelular/tratamento farmacológico , Cateterismo Periférico/instrumentação , Cisplatino/administração & dosagem , Artéria Hepática , Neoplasias Hepáticas/tratamento farmacológico , Dispositivos de Acesso Vascular , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/efeitos adversos , Carcinoma Hepatocelular/patologia , Cateterismo Periférico/efeitos adversos , Cateterismo Periférico/métodos , Cisplatino/efeitos adversos , Esquema de Medicação , Desenho de Equipamento , Feminino , Artéria Hepática/diagnóstico por imagem , Humanos , Infusões Intra-Arteriais , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Miniaturização , Agulhas , Dados Preliminares , Radiografia Intervencionista , Estudos Retrospectivos , Resultado do TratamentoRESUMO
A stable visible-light-driven photocatalyst (λ≥450â nm) for water oxidation is reported. Rhodium substitution into the pyrochlore Y2 Ti2 O7 is demonstrated by monitoring Vegard's law evolution of the unit-cell parameters with changing rhodium content, to a maximum content of 3 % dopant. Substitution renders the solid solutions visible-light active. The overall rate of oxygen evolution is comparable to WO3 but with superior light-harvesting and surface-area-normalized turnover rates, making Y2 Ti1.94 Rh0.06 O7 an excellent candidate for use in a Z-scheme water-splitting system.
RESUMO
Poly(ADP-ribose) polymerase (PARP)-2 is a nuclear enzyme that belongs to the PARP family and PARP-2 is responsible for 5-15 % of total cellular PARP activity. PARP-2 was originally described in connection to DNA repair and in physiological and pathophysiological processes associated with genome maintenance (e.g., centromere and telomere protection, spermiogenesis, thymopoiesis, azoospermia, and tumorigenesis). Recent reports have identified important rearrangements in gene expression upon the knockout of PARP-2. Such rearrangements heavily impact inflammation and metabolism. Metabolic effects are mediated through modifying PPARγ and SIRT1 function. Altered gene expression gives rise to a complex phenotype characterized primarily by enhanced mitochondrial activity that results both in beneficial (loss of fat, enhanced insulin sensitivity) and in disadvantageous (pancreatic beta cell hypofunction upon high fat feeding) consequences. Enhanced mitochondrial biogenesis provides protection in oxidative stress-related diseases. Hereby, we review the recent developments in PARP-2 research with special attention to the involvement of PARP-2 in transcriptional and metabolic regulation.
Assuntos
Reparo do DNA , Poli(ADP-Ribose) Polimerases/fisiologia , Transcrição Gênica , Animais , Montagem e Desmontagem da Cromatina , Regulação da Expressão Gênica , Instabilidade Genômica , Humanos , Camundongos , Modelos Genéticos , Estresse Oxidativo/genética , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Estrutura Terciária de Proteína , Sirtuína 1/metabolismo , Sirtuína 1/fisiologia , Espermatogênese/genéticaRESUMO
Poly(ADP-ribose) polymerase 2 (PARP2) alongside PARP1 are responsible for the bulk of cellular PARP activity, and they were first described as DNA repair factors. However, research in past decades implicated PARPs in biological functions as diverse as the regulation of cellular energetics, lipid homeostasis, cell death, and inflammation. PARP activation was described in Th2-mediated inflammatory processes, but studies focused on the role of PARP1, while we have little information on PARP2 in inflammatory regulation. In this study, we assessed the role of PARP2 in a Th17-mediated inflammatory skin condition, psoriasis. We found that PARP2 mRNA expression is increased in human psoriatic lesions. Therefore, we studied the functional consequence of decreased PARP2 expression in murine and cellular human models of psoriasis. We observed that the deletion of PARP2 attenuated the imiquimod-induced psoriasis-like dermatitis in mice. Silencing of PARP2 in human keratinocytes prevented their hyperproliferation, maintained their terminal differentiation, and reduced their production of inflammatory mediators after treatment with psoriasis-mimicking cytokines IL17A and TNFα. Underlying these observations, we found that aromatase was induced in the epidermis of PARP2 knock-out mice and in PARP2-deficient human keratinocytes, and the resulting higher estradiol production suppressed NF-κB activation, and hence, inflammation in keratinocytes. Steroidogenic alterations have previously been described in psoriasis, and we extend these observations by showing that aromatase expression is reduced in psoriatic lesions. Collectively, our data identify PARP2 as a modulator of estrogen biosynthesis by epidermal keratinocytes that may be relevant in Th17 type inflammation. KEY MESSAGES : PARP2 mRNA expression is increased in lesional skin of psoriasis patients. PARP2 deletion in mice attenuated IMQ-induced psoriasis-like dermatitis. NF-κB activation is suppressed in PARP2-deficient human keratinocytes. Higher estradiol in PARP2-deficient keratinocytes conveys anti-inflammatory effect.
Assuntos
Dermatite , Psoríase , Animais , Humanos , Camundongos , Aromatase/metabolismo , Dermatite/metabolismo , Dermatite/patologia , Modelos Animais de Doenças , Imiquimode/efeitos adversos , Inflamação/metabolismo , Queratinócitos/metabolismo , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Psoríase/genética , Psoríase/metabolismo , RNA Mensageiro/metabolismo , Pele/metabolismoRESUMO
Four fused nitrogen-containing ring systems were investigated by electrospray ionization-tandem mass spectrometry: Pyridazino-indoles, pyridazino-quinolines, a pyrimido-quinoline derivative and pyrimido-cinnolines. Fragmentation patterns of these compounds are discussed and compared. Several characteristic cross-ring fragments were formed mainly on the pyridazine and pyrimidine rings of the ring systems. The connected Cl, NO2 , Me, Ph and more extended heterocyclic substituents influenced the fragmentation.
Assuntos
Nitrogênio , Espectrometria de Massas por Ionização por Electrospray , Nitrogênio/química , Espectrometria de Massas por Ionização por Electrospray/métodosRESUMO
Altered tryptophan (TRP) metabolism may have an important role in migraine susceptibility through its main metabolites, serotonin and kynurenine (KYN). Both affect pain processing and stress response by interfering with neural and brain hypersensitivity and by interacting with chemokines and cytokines that control vascular and inflammatory processes. The involvement of these pathways in migraine has been widely studied, but acute citalopram neuroendocrine challenge on TRP metabolism and cytokine profile has not been investigated yet. In our study, females with episodic migraine without aura and healthy controls were studied before and after acute citalopram or placebo in a double-blind setting. At baseline, increased TRP/large neutral amino acid (LNAA) ratio and decreased RANTES chemokine concentration were detected in migraine patients compared to controls. The challenge induced a significant increase in TRP, KYN, and TRP/LNAA in healthy controls, but not in migraine patients. Furthermore, migraine attack frequency negatively correlated with KYN/TRP ratio and positively correlated with the neuroendocrine-challenge-induced KYN concentration increase. Our results support a decreased breakdown of TRP via KYN pathway and a failure to modulate TRP-KYN pathway during citalopram-induced acute stress together with an increased vascular sensitivity in migraine. These mechanisms may provide useful drug targets for future drug development.
Assuntos
Transtornos de Enxaqueca , Triptofano , Citalopram/farmacologia , Citalopram/uso terapêutico , Método Duplo-Cego , Feminino , Humanos , Cinurenina/metabolismo , Transtornos de Enxaqueca/tratamento farmacológico , Serotonina , Triptofano/metabolismoRESUMO
Thermal stability of lactase (ß-galactosidase) enzyme has been studied by a variety of physico-chemical methods. ß-galactosidase is the main active ingredient of medications for lactose intolerance. It is typically produced industrially by the Aspergillus oryzae filamentous fungus. Lactase was used as a model to help understand thermal stability of enzyme-type biopharmaceuticals. Enzyme activity (hydrolyzation of lactose) of ß-galactosidase was determined after storing the solid enzyme substance at various temperatures. For a better understanding of the relationship between structure and activity changes we determined the mass and size of the molecules with gel electrophoresis and dynamic light scattering and detected aggregation processes. A bottom-up proteomic procedure was used to determine the primary amino acid sequence and to investigate changes in the N-glycosylation pattern of the protein. NMR and CD spectroscopic methods were used to observe changes in higher order structures and to reveal relationships between structural and functional changes.
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The purpose of this report was to describe the reorientation of the chimney graft technique to downsize brachial artery access during thoracic endovascular aortic repair and thus preserve left subclavian artery flow. In the case described herein, the chimney graft was advanced not from the brachial or axillary artery, but from the common femoral artery, over a brachiofemoral pull-through wire. The chimney graft was then turned out into the ascending aorta by balloon dilatation via percutaneous brachial access ("reorientation"). Despite the use of a large-diameter chimney graft, the chimney technique with percutaneous brachial access was successfully performed using the reorientation technique.
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Changes to bacterial metabolite-elicited signaling, in oncobiosis associated with breast cancer, plays a role in facilitating the progression of the disease. We show that indoxyl-sulfate (IS), a tryptophan metabolite, has cytostatic properties in models of breast cancer. IS supplementation, in concentrations corresponding to the human serum reference range, suppressed tumor infiltration to the surrounding tissues and metastasis formation in a murine model of breast cancer. In cellular models, IS suppressed NRF2 and induced iNOS, leading to induction of oxidative and nitrosative stress, and, consequently, reduction of cell proliferation; enhanced oxidative and nitrosative stress are crucial in the subsequent cytostasis. IS also suppressed epithelial-to-mesenchymal transition vital for suppressing cellular movement and diapedesis. Furthermore, IS rendered cells hypometabolic, leading to a reduction in aldehyde-dehydrogenase positive cells. Pharmacological inhibition of the pregnane-X receptor using CH223191 and the aryl-hydrocarbon receptor using ketoconazole diminished the IS-elicited effects, suggesting that these receptors were the major receptors of IS in these models. Finally, we showed that increased expression of the human enzymes that form IS (Cyp2E1, Sult1A1, and Sult1A2) is associated with better survival in breast cancer, an effect that is lost in triple negative cases. Taken together, IS, similar to indolepropionic acid (another tryptophan metabolite), has cytostatic properties and higher expression of the metabolic machinery responsible for the formation of IS supports survival in breast cancer.
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Pancreatic adenocarcinoma is one of the most lethal cancers in both men and women, with a median five-year survival of around 5%. Therefore, pancreatic adenocarcinoma represents an unmet medical need. Neoplastic diseases, such as pancreatic adenocarcinoma, often are associated with microbiome dysbiosis, termed oncobiosis. In pancreatic adenocarcinoma, the oral, duodenal, ductal, and fecal microbiome become dysbiotic. Furthermore, the pancreas frequently becomes colonized (by Helicobacter pylori and Malassezia, among others). The oncobiomes from long- and short-term survivors of pancreatic adenocarcinoma are different and transplantation of the microbiome from long-term survivors into animal models of pancreatic adenocarcinoma prolongs survival. The oncobiome in pancreatic adenocarcinoma modulates the inflammatory processes that drive carcinogenesis. In this review, we point out that bacterial metabolites (short chain fatty acids, secondary bile acids, polyamines, indole-derivatives, etc.) also have a role in the microbiome-driven pathogenesis of pancreatic adenocarcinoma. Finally, we show that bacterial metabolism and the bacterial metabolome is largely dysregulated in pancreatic adenocarcinoma. The pathogenic role of additional metabolites and metabolic pathways will be identified in the near future, widening the scope of this therapeutically and diagnostically exploitable pathogenic pathway in pancreatic adenocarcinoma.
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Altered serotonergic neurotransmission is a key factor in several neurologic and psychiatric disorders such as migraine. Human and animal studies suggest that chronically low interictal serotonin levels of plasma and brain may facilitate increased activity of the trigeminovascular pathway, and may contribute to development of repeated migraine attacks. However, brain serotonin synthesis is affected by the concentration of tryptophan, its metabolites and a number of amino acids. In this work a simple and robust LC-MS/MS method for the quantitative determination of valine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, serotonin and kynurenine in human plasma has been developed and validated. Sample preparation was achieved by protein precipitation, using trifluoroacetic acid. Chromatographic separation was carried out on a Supelco Ascentis® Express C18 column (3.0 mm i.d.â¯×â¯150â¯mm, 2.7⯵m) equipped with an Agilent Zorbax Eclipse XDB C8 guard-column under isocratic conditions at a flow rate of 0.4â¯mL/min, over a 6.5â¯min run time. Mobile phase was 0.2% trifluoroacetic acid - acetonitrile (85:15, v/v). The eight analytes and two internal standards were ionized by positive electrospray ionization and detected in multiple reaction monitoring mode. A "fit-for-purpose" validation approach was adopted using surrogate matrix for the preparation of calibration samples. The calibration curves of all analytes showed excellent linearities with a correlation coefficient (r2) of 0.998 or better. Spiked surrogate matrix samples and pooled human plasma were used as quality control samples. Intra-day and inter-day precisions were less than 11.8% and 14.3%, and accuracies were within the ranges of 87.4-114.3% and 87.7-113.3%, respectively. Stability of the components in standard solutions, surrogate matrix, pooled plasma and processed samples were found to be acceptable under all relevant conditions. No significant carryover effect was observed. The surrogate matrix behaved parallel to human plasma when assessed by standard addition method and diluting the authentic matrix with surrogate matrix. The method was successfully applied for analysis of 800 human plasma samples to support a clinical study.
Assuntos
Aminoácidos/sangue , Serotonina/sangue , Espectrometria de Massas em Tandem/métodos , Aminoácidos/metabolismo , Técnicas Biossensoriais , Calibragem , Cromatografia Líquida de Alta Pressão , Humanos , Cinurenina/análise , Limite de Detecção , Modelos Lineares , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Solventes/química , Espectrometria de Massas por Ionização por Electrospray , Triptofano/metabolismoRESUMO
Oncobiotic transformation of the gut microbiome may contribute to the risk of breast cancer. Recent studies have provided evidence that the microbiome secretes cytostatic metabolites that inhibit the proliferation, movement, and metastasis formation of cancer cells. In this study, we show that indolepropionic acid (IPA), a bacterial tryptophan metabolite, has cytostatic properties. IPA selectively targeted breast cancer cells, but it had no effects on non-transformed, primary fibroblasts. In cell-based and animal experiments, we showed that IPA supplementation reduced the proportions of cancer stem cells and the proliferation, movement, and metastasis formation of cancer cells. These were achieved through inhibiting epithelial-to-mesenchymal transition, inducing oxidative and nitrosative stress, and boosting antitumor immune response. Increased oxidative/nitrosative stress was due to the IPA-mediated downregulation of nuclear factor erythroid 2-related factor 2 (NRF2), upregulation of inducible nitric oxide synthase (iNOS), and enhanced mitochondrial reactive species production. Increased oxidative/nitrosative stress led to cytostasis and reductions in cancer cell stem-ness. IPA exerted its effects through aryl hydrocarbon receptor (AHR) and pregnane X receptor (PXR) receptors. A higher expression of PXR and AHR supported better survival in human breast cancer patients, highlighting the importance of IPA-elicited pathways in cytostasis in breast cancer. Furthermore, AHR activation and PXR expression related inversely to cancer cell proliferation level and to the stage and grade of the tumor. The fecal microbiome's capacity for IPA biosynthesis was suppressed in women newly diagnosed with breast cancer, especially with stage 0. Bacterial indole biosynthesis showed correlation with lymphocyte infiltration to tumors in humans. Taken together, we found that IPA is a cytostatic bacterial metabolite, the production of which is suppressed in human breast cancer. Bacterial metabolites, among them, IPA, have a pivotal role in regulating the progression but not the initiation of the disease.
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There is a great need for efficient and cost-effective melanoma screening, but this is not yet solved. Epidemiological studies on trends in melanoma incidence by tumour thickness, anatomical site and demographical data can help to improve public health efforts regarding earlier melanoma diagnosis. We aimed to study the trends in the incidence and characteristics of patients and their melanoma in North-East Hungary from 2000 to 2014. Data were obtained from a university hospital-based registry. A total of 1509 cutaneous invasive melanomas of 1464 patients were included in the study. A moderate but significant increase in incidence was observed in the region [average annual percentage change: 3.04 (0.07; 6.11); P = 0.045], with a breakpoint in 2007. From 2001 to 2007, the trend was increasing [APC: 9.84 (3.52; 16.55); P=0.006], but it stalled from 2007 [APC: -2.45 (-5.99; 1.23); P = 0.164]. However, in the age groups over the age of 60 years, where the standardised incidence was the highest, the incidence continued to rise. Furthermore, older age, male sex and trunk or lower extremity localization were found to be associated with thicker melanomas. Our results support that regular screening examination for melanoma would be desirable for people over the age of 60 years.
Assuntos
Melanoma/epidemiologia , Sistema de Registros/estatística & dados numéricos , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Hungria/epidemiologia , Incidência , Masculino , Melanoma/classificação , Melanoma/patologia , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Fatores de RiscoRESUMO
In breast cancer patients, the diversity of the microbiome decreases, coinciding with decreased production of cytostatic bacterial metabolites like lithocholic acid (LCA). We hypothesized that LCA can modulate oxidative stress to exert cytostatic effects in breast cancer cells. Treatment of breast cancer cells with LCA decreased nuclear factor-2 (NRF2) expression and increased Kelch-like ECH associating protein 1 (KEAP1) expression via activation of Takeda G-protein coupled receptor (TGR5) and constitutive androstane receptor (CAR). Altered NRF2 and KEAP1 expression subsequently led to decreased expression of glutathione peroxidase 3 (GPX3), an antioxidant enzyme, and increased expression of inducible nitric oxide synthase (iNOS). The imbalance between the pro- and antioxidant enzymes increased cytostatic effects via increased levels of lipid and protein oxidation. These effects were reversed by the pharmacological induction of NRF2 with RA839, tBHQ, or by thiol antioxidants. The expression of key components of the LCA-elicited cytostatic pathway (iNOS and 4HNE) gradually decreased as the breast cancer stage advanced. The level of lipid peroxidation in tumors negatively correlated with the mitotic index. The overexpression of iNOS, nNOS, CAR, KEAP1, NOX4, and TGR5 or the downregulation of NRF2 correlated with better survival in breast cancer patients, except for triple negative cases. Taken together, LCA, a metabolite of the gut microbiome, elicits oxidative stress that slows down the proliferation of breast cancer cells. The LCA-oxidative stress protective pathway is lost as breast cancer progresses, and the loss correlates with poor prognosis.