RESUMO
Carboxylesterases, historically referred as non-specific esterases, are ubiquitous hydrolases with high catalytic efficiency. Without exceptions, all mammalian species studied contain multiple forms of carboxylesterases. While having been widely studied in humans and experimental animals, these enzymes remain to be characterized in farm animals. In this study, we showed that pig liver esterase 1 (PLE1) and pig liver esterase 6 (PLE6) were highly active toward amoxicillin (AMO) and ampicillin (AMP), two major antibiotics that are widely used in food-supplements. Mass-spectrometric analysis established that the hydrolysis occurred at the ß-lactam amide bond and the hydrolysis drastically decreased or completely eliminated the antibacterial activity. Furthermore, hydrolytic activity and proteomic analysis suggested that trace PLEs existed in pig plasma and contributed little to the hydrolysis of AMO and AMP. These results suggested that carboxylesterases-based hydrolysis determines the therapeutic intensity of these and related antibiotics and the magnitude of the determination occurs in a species-dependent manner.
Assuntos
Carboxilesterase/genética , Fígado/enzimologia , Proteômica , Amoxicilina/química , Amoxicilina/farmacologia , Ampicilina/química , Ampicilina/farmacologia , Animais , Antibacterianos/farmacologia , Catálise , Humanos , Hidrólise/efeitos dos fármacos , Fígado/efeitos dos fármacos , Suínos , beta-LactamasRESUMO
Xuezhikang capsule (XZK) is a traditional Chinese medicine that contains lovastatin (Lv) for hyperlipidemia treatment, although it has fewer side effects than Lv. However, the pharmacokinetic mechanisms contributing to its distinct efficacy and low side effects are unclear. Mice were fed a high-fat diet (HFD) for 6 weeks to induce hyperlipidemia. We first conducted the pharmacokinetic studies in HFD mice following oral administration of Lv (10 mg/kg, i.g.) and found that HFD remarkably decreased the active form of Lv (the lovastatin acid, LvA) exposure in the circulation system, especially in the targeting organ liver, with a declined conversion from Lv to LvA, whereas the Lv (responsible for myotoxicity) exposure in muscle markedly increased. Then we compared the pharmacokinetic profiles of Lv in HFD mice after the oral administration of XZK (1200 mg/kg, i.g.) or an equivalent dose of Lv (10 mg/kg, i.g.). A higher exposure of LvA and lower exposure of Lv were observed after XZK administration, suggesting a pharmacokinetic interaction of some ingredients in XZK. Further studies revealed that HFD promoted the inflammation and inhibited carboxylesterase (CES) activities in the intestine and the liver, thus contributing to the lower transformation of Lv into LvA. In contrast, XZK inhibited the inflammation and upregulated CES in the intestine and the liver. Finally, we evaluated the effects of monacolins and phytosterols, the fractional extracts of isoflavones, on inflammatory LS174T or HepG2 cells, which showed that isoflavones inhibited inflammation, upregulated CES, and markedly enhanced the conversion of Lv into LvA. For the first time, we provide evidence that isoflavones and Lv in XZK act in concert to enhance the efficacy and reduce the side effects of Lv.
Assuntos
Medicamentos de Ervas Chinesas/uso terapêutico , Hiperlipidemias/tratamento farmacológico , Isoflavonas/farmacologia , Lovastatina/análogos & derivados , Lovastatina/uso terapêutico , Administração Oral , Animais , Carboxilesterase/genética , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Medicamentos de Ervas Chinesas/administração & dosagem , Medicamentos de Ervas Chinesas/metabolismo , Medicamentos de Ervas Chinesas/farmacocinética , Humanos , Inflamação/tratamento farmacológico , Lovastatina/administração & dosagem , Lovastatina/metabolismo , Lovastatina/farmacocinética , Masculino , Camundongos Endogâmicos C57BL , Receptor de Pregnano X/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Rapid wound healing and subsequent formation of the apical epithelial cap (AEC) are believed to be required for successful appendage regeneration in amphibians. Despite the significant role of AEC in limb regeneration, its role in tail regeneration and the mechanisms that regulate the wound healing and AEC formation are not well understood. We previously identified Xenopus laevis es1, which is preferentially expressed in wounded regions, including the AEC after tail regeneration. In this study we established and characterized transgenic Xenopus laevis lines harboring the enhanced green fluorescent protein (EGFP) gene under control of an es1 gene regulatory sequence (es1:egfp). The EGFP reporter expression was clearly seen in several regions of the embryo and then declined to an undetectable level in larvae, recapitulating the endogenous es1 expression. After amputation of the tadpole tail, EGFP expression was re-activated at the edge of the stump epidermis and then increased in the wound epidermis (WE) covering the amputation surface. As the stump started to regenerate, the EGFP expression became restricted to the most distal epidermal region, including the AEC. EGFP was preferentially expressed in the basal or deep cells but not in the superficial cells of the WE and AEC. We performed a small-scale pharmacological screening for chemicals that affected the expression of EGFP in the stump epidermis after tail amputation. The EGFP expression was attenuated by treatment with an inhibitor for ERK, TGF-ß or reactive oxygen species (ROS) signaling. These treatments also impaired wound closure of the amputation surface, suggesting that the three signaling activities are required for es1 expression in the WE and successful wound healing after tail amputation. These findings showed that es1:egfp Xenopus laevis should be a useful tool to analyze molecular mechanisms regulating wound healing and appendage regeneration.
Assuntos
Carboxilesterase/genética , Elementos Facilitadores Genéticos/genética , Epiderme/fisiologia , Genes Reporter , Proteínas de Fluorescência Verde/genética , Regiões Promotoras Genéticas/genética , Regeneração/fisiologia , Cauda/fisiologia , Transgenes , Proteínas de Xenopus/fisiologia , Xenopus laevis/fisiologia , Amputação Cirúrgica , Animais , Animais Geneticamente Modificados , Avaliação Pré-Clínica de Medicamentos , Células Epidérmicas , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/análise , Larva , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Cauda/lesões , Cicatrização/efeitos dos fármacos , Cicatrização/fisiologia , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/crescimento & desenvolvimentoRESUMO
Salvia miltiorrhiza root (Danshen) is widely used in Asia for its cardiovascular benefits and contains both hydrophilic phenolic acids and lipophilic tanshinones, which are believed to be responsible for its therapeutic efficacy. This review summarized the effects of these bioactive components from S. miltiorrhiza roots on pharmacokinetics of comedicated drugs with mechanic insights regarding alterations of protein binding, enzyme activity, and transporter activity based on the published data stemming from both in vitro and in vivo human studies. In vitro studies indicated that cytochrome P450 (CYP450), carboxylesterase enzyme, catechol-O-methyltransferase, organic anion transporter 1 (OAT1) and OAT3, and P-glycoprotein were the major targets involved in S. miltiorrhiza-drug interactions. Lipophilic tanshinones had much more potent inhibitory effects towards CYPs activities compared to hydrophilic phenolic acids, evidenced by much lower Ki values of the former. Clinical S. miltiorrhiza-drug interaction studies were mainly conducted using CYP1A2 and CYP3A4 probe substrates. In addition, the effects of coexisting components on the pharmacokinetic behaviors of those noted bioactive compounds were also included herein.
Assuntos
Doenças Cardiovasculares/tratamento farmacológico , Medicamentos de Ervas Chinesas/uso terapêutico , Interações Ervas-Drogas , Salvia miltiorrhiza/química , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Carboxilesterase/genética , Doenças Cardiovasculares/patologia , Catecol O-Metiltransferase/genética , Sistema Enzimático do Citocromo P-450/genética , Medicamentos de Ervas Chinesas/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteína 1 Transportadora de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/genéticaRESUMO
Glandular trichomes of cultivated tomato (Solanum lycopersicum) and many other species throughout the Solanaceae produce and secrete mixtures of sugar esters (acylsugars) on the plant aerial surfaces. In wild and cultivated tomato, these metabolites consist of a sugar backbone, typically glucose or sucrose, and two to five acyl chains esterified to various positions on the sugar core. The aliphatic acyl chains vary in length and branching and are transferred to the sugar by a series of reactions catalyzed by acylsugar acyltransferases. A phenotypic screen of a set of S. lycopersicum M82 × Solanum pennellii LA0716 introgression lines identified a dominant genetic locus on chromosome 5 from the wild relative that affected total acylsugar levels. Genetic mapping revealed that the reduction in acylsugar levels was consistent with the presence and increased expression of two S. pennellii genes (Sopen05g030120 and Sopen05g030130) encoding putative carboxylesterase enzymes of the α/ß-hydrolase superfamily. These two enzymes, named ACYLSUGAR ACYLHYDROLASE1 (ASH1) and ASH2, were shown to remove acyl chains from specific positions of certain types of acylsugars in vitro. A survey of related genes in M82 and LA0716 identified another trichome-expressed ASH gene on chromosome 9 (M82, Solyc09g075710; LA0716, Sopen09g030520) encoding a protein with similar activity. Characterization of the in vitro activities of the SpASH enzymes showed reduced activities with acylsugars produced by LA0716, presumably contributing to the high-level production of acylsugars in the presence of highly expressed SpASH genes.
Assuntos
Carboxilesterase/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Metabolismo dos Carboidratos , Carboxilesterase/genética , Mapeamento Cromossômico , Genes de Plantas , Hidrólise , Solanum lycopersicum/genética , Proteínas de Plantas/genética , Solanum/genética , Solanum/metabolismo , Sacarose/análogos & derivados , Sacarose/química , Sacarose/metabolismo , Tricomas/metabolismoRESUMO
BACKGROUND: Serine hydrolases (SHs) are among the largest classes of enzymes in humans and play crucial role in many pathophysiological processes of cancer. We have undertaken a comprehensive proteomic analysis to assess the differential expression and cellular localization of SHs, which uncovered distinctive expression of Carboxylesterase 2 (CES2), the most efficient carboxyl esterase in activating the prodrug irinotecan into SN-38, in pancreatic ductal adenocarcinoma (PDAC). We therefore assessed the extent of heterogeneity in CES2 expression in PDAC and its potential relevance to irinotecan based therapy. METHODS: CES2 expression in PDAC and paired nontumor tissues was evaluated by immunohistochemistry. CES2 activity was assessed by monitoring the hydrolysis of the substrate p-NPA and correlated with irinotecan IC50 values by means of Pearson's correlation. Kaplan-Meier and Cox regression analyses were applied to assess the association between overall survival and CES2 expression in patients who underwent neoadjuvant FOLFIRINOX treatment. All statistical tests were two-sided. RESULTS: Statistically significant overexpression of CES2, both at the mRNA and protein levels, was observed in PDAC compared with paired nontumor tissue (P < .001), with 48 of 118 (40.7%) tumors exhibiting high CES2 expression. CES2 activity in 11 PDAC cell lines was inversely correlated with irinotecan IC50 values (R = -0.68, P = .02). High CES2 expression in tumor tissue was associated with longer overall survival in resectable and borderline resectable patients who underwent neoadjuvant FOLFIRINOX treatment (hazard ratio = 0.14, 95% confidence interval = 0.04 to 0.51, P = .02). CONCLUSION: Our findings suggest that CES2 expression and activity, by mediating the intratumoral activation of irinotecan, is a contributor to FOLFIRINOX sensitivity in pancreatic cancer and CES2 assessment may define a subset of patients likely to respond to irinotecan based therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Camptotecina/análogos & derivados , Carboxilesterase/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/enzimologia , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/enzimologia , Adulto , Idoso , Camptotecina/administração & dosagem , Carboxilesterase/genética , Linhagem Celular Tumoral , Quimioterapia Adjuvante , Feminino , Fluoruracila/administração & dosagem , Humanos , Imuno-Histoquímica , Irinotecano , Estimativa de Kaplan-Meier , Masculino , Espectrometria de Massas , Pessoa de Meia-Idade , Terapia Neoadjuvante/métodos , Compostos Organoplatínicos/administração & dosagem , Oxaliplatina , RNA Mensageiro/metabolismo , Radioterapia Adjuvante , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de RNA , Análise Serial de Tecidos , Resultado do Tratamento , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In this study, neural stem cells (NSCs)-derived enzyme/prodrug therapy (NDEPT) was used to treat primary lung cancer or metastatic lung cancer in the brain. To confirm the anti-tumor effect of NSCs expressing carboxyl esterase (CE), A549 lung cancer cells were treated with HB1.F3.CE cells and CPT-11. A significant decrease in the viability/proliferation of lung cancer cells was observed compared to negative controls or cells treated with CPT-11 alone. To produce a mouse model of primary lung cancer or lung cancer metastasis to the brain, A549 cells were implanted in the dorsal area of the mouse or right hemisphere. CM-DiI pre-stained stem cells were implanted near the primary lung cancer tumor mass or in the contralateral brain. Two days after stem cells injection, mice were inoculated with CPT-11 (13.5 kg/mouse/day) via intraperitoneal injection. In the primary lung cancer mouse models, tumor mass was 80% lower in response to HB1.F3.CE in conjunction with CPT-11, while it was only reduced by 40% in the group treated with CPT-11 alone. Additionally, therapeutic efficacy of co-treatment with stem cells and CPT-11 was confirmed by detection of apoptosis and necrosis in primary and metastatic lung cancer tissues. By secreting VEGF, tumor cells modulate Erk1/2 and Akt signaling and migration of stem cells. This further increased tumor-selectivity of stem cell/prodrug co-therapy. Overall, these results indicate that NSCs expressing the therapeutic gene may be a powerful tool for treatment of primary lung cancer or metastasis of lung cancer to the brain.
Assuntos
Camptotecina/análogos & derivados , Carboxilesterase/biossíntese , Neoplasias Pulmonares/terapia , Células-Tronco Neurais/transplante , Animais , Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , Carboxilesterase/genética , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Irinotecano , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Células-Tronco Neurais/enzimologia , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The bioavailability of therapeutic agents can be improved by using prodrugs which have better passive diffusion than the active agents. Intestinal hydrolysis is an important reaction in the bioconversion of prodrugs, and may be the rate-limiting factor in their absorption. Carboxylesterase (CES) is ubiquitous in most organs and is located in the endoplasmic reticulum. Single-pass perfusion experiments in rat intestine have shown that CES is the main enzyme involved in intestinal first-pass hydrolysis. In man, intestinal CESs belong to the CES2 gene family and their activity is nearly constant along the jejunum and ileum. The predominant human intestinal CES, hCE2, preferentially hydrolyzes prodrugs in which the alcohol group of a pharmacologically active molecule has been modified by the addition of a small acyl group. In preclinical animal models, CES2 isozymes are also the major intestinal enzymes although they have different substrate specificities to human CES2, while CES1 isozymes and other unidentified enzymes are also present. It is therefore difficult to predict human intestinal absorption from animal experiments. Caco-2 cells mainly express the human CES1 isozyme, hCE1, which shows quite different substrate specificity from hCE2, making Caco-2 cells unsuitable for prediction of human intestinal absorption of prodrugs. However, we have developed a novel experimental method for predicting the human intestinal absorption of prodrugs using Caco-2 cells in which CES-mediated hydrolysis has been inhibited. The expression of hCE2 shows inter-individual variation and is regulated by several mechanisms, such as gene polymorphism and epigenetic processes. There are no reports suggesting that severe toxicity is associated with prodrugs due to genetic polymorphism of the CES2 gene.
Assuntos
Carboxilesterase/metabolismo , Intestino Delgado/enzimologia , Pró-Fármacos/administração & dosagem , Pró-Fármacos/farmacocinética , Administração Oral , Animais , Disponibilidade Biológica , Biotransformação , Carboxilesterase/genética , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Hidrólise , Absorção Intestinal , Mucosa Intestinal/enzimologia , Isoenzimas/metabolismo , Especificidade de Órgãos , Filogenia , Especificidade da EspécieAssuntos
Avaliação Pré-Clínica de Medicamentos , Isoenzimas , Modelos Animais , Preparações Farmacêuticas/metabolismo , Primatas , Animais , Carboxilesterase/genética , Carboxilesterase/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Humanos , Preparações Farmacêuticas/administração & dosagem , Primatas/genética , Primatas/metabolismoRESUMO
Doxazolidine (Doxaz) is a functionally distinct formaldehyde conjugate of doxorubicin (Dox) that induces cancer cell death in Dox-sensitive and resistant cells. Pentyl PABC-Doxaz (PPD) is a prodrug of Doxaz that is activated by carboxylesterase 2 (CES2), which is expressed by liver, non-small-cell lung, colon, pancreatic, renal, and thyroid cancer cells. Here, we demonstrate that in two murine models, PPD was effective at slowing tumor growth and demonstrated markedly reduced cardiotoxic and nephrotoxic effects, as well as better tolerance, relative to Dox. Hepatotoxicity, consistent with liver expression of the murine CES2 homologue, was induced by PPD. Unlike irinotecan, a clinical CES2-activated prodrug, PPD produced no visible gastrointestinal damage. Finally, we demonstrate that cellular response to PPD may be predicted with good accuracy using CES2 expression and Doxaz sensitivity, suggesting that these metrics may be useful as clinical biomarkers for sensitivity of a specific tumor to PPD treatment.
Assuntos
Carbamatos/metabolismo , Carbamatos/farmacologia , Carboxilesterase/metabolismo , Doxorrubicina/análogos & derivados , Oxazóis/metabolismo , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacologia , Animais , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Western Blotting , Carbamatos/química , Carbamatos/toxicidade , Carboxilesterase/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Doxorrubicina/química , Doxorrubicina/metabolismo , Doxorrubicina/farmacologia , Doxorrubicina/toxicidade , Avaliação Pré-Clínica de Medicamentos , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Neoplasias/enzimologia , Neoplasias/patologia , Oxazóis/farmacologia , Pró-Fármacos/química , Pró-Fármacos/toxicidade , Ratos , Análise de RegressãoRESUMO
Levels of insecticide resistance, carboxylesterase activity, carboxylesterase expression, and the cDNA sequence of carboxylesterase gene were investigated in malathion resistant and susceptible strains of cotton aphids, Aphis gossypii (Glover). The resistant strain (MRR) exhibited 80.6-fold resistance to malathion compared to the susceptible strain (MSS) in cotton aphids. Five substrates, alpha-naphthyl acetate (alpha-NA), beta-naphthyl acetate (beta-NA), alpha-naphthyl propionate (alpha-NPr), alpha-naphthyl butyrate (alpha-NB), alpha-naphthyl caprylate (alpha-NC) and S-methyl thiobutyrate (S-MTB) were used to determine carboxylesterase activity in MRR and MSS strains of cotton aphids. Carboxylesterase activity was significantly higher in MRR strain than in MSS strain, 3.7-fold for alpha-NA, 3.0-fold for beta-NA, 2.0-fold for alpha-NPr, 2.9-fold for alpha-NB and 1.6-fold for alpha-NC, While for S-MTB, there was nearly no difference between the two strains. Two site mutations (K14Q and N354D) with high frequency were also found by sequence analysis in the MRR strain, compared with the MSS strain. The levels of gene expression for carboxylesterase of both MRR and MSS strains were determined by real-time quantitative PCRs. Compared with the MSS strain, the relative transcription levels and gene copy numbers of the carboxylesterase were 1.99- and 4.42-fold in the MRR strain, respectively. These results indicated that the increased expression of the carboxylesterase resulted from the increased transcription levels of carboxylesterase mRNA and gene copy numbers and combined with the site mutants might play role in cotton aphid resistance to malathion.
Assuntos
Afídeos/efeitos dos fármacos , Afídeos/enzimologia , Carboxilesterase/genética , Carboxilesterase/metabolismo , Regulação Enzimológica da Expressão Gênica/genética , Resistência a Inseticidas/genética , Malation/farmacologia , Animais , Afídeos/genética , Sequência de Bases , Carboxilesterase/antagonistas & inibidores , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Dados de Sequência Molecular , Mutação , Naftalenos/farmacologia , Naftóis/farmacologia , Organotiofosfatos/farmacologia , Propionatos/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da Espécie , Tiamina Pirofosfato/farmacologia , Transcrição Gênica/genéticaRESUMO
This study evaluated the contributions of carboxyl ester lipase (CEL) and pancreatic triglyceride lipase (PTL) in lipid nutrient absorption. Results showed PTL deficiency has minimal effect on triacylglycerol (TAG) absorption under low fat dietary conditions. Interestingly, PTL(-)(/)(-) mice displayed significantly reduced TAG absorption compared with wild type mice under high fat/high cholesterol dietary conditions (80.1 +/- 3.7 versus 91.5 +/- 0.7%, p < 0.05). Net TAG absorption was reduced further to 61.1 +/- 3.8% in mice lacking both PTL and CEL. Cholesterol absorption was 41% lower in PTL(-/-) mice compared with control mice (p < 0.05), but this difference was not exaggerated in PTL(-/-), CEL(-/-) mice. Retinyl palmitate absorption was reduced by 45 and 60% in PTL(-/-) mice (p < 0.05) and PTL(-/-), CEL(-/-) mice (p < 0.01), respectively. After 15 weeks of feeding, the high fat/high cholesterol diet, wild type, and CEL(-/-) mice gained approximately 24 g of body weight. However, body weight gain was 6.2 and 8.6 g less (p < 0.01) in PTL(-/-) and PTL(-/-), CEL(-/-) mice, respectively, despite their consumption of comparable amounts of the high fat/high cholesterol diet. The decrease body weight gain in PTL(-/-) and PTL(-/-), CEL(-/-) mice was attributed to their absorption of fewer calories from the high fat/high cholesterol diet, thereby resulting in less fat mass accumulation than that observed in wild type and CEL(-/-) mice. Thus, this study documents that PTL and CEL serve complementary functions, working together to mediate the absorption of a major portion of dietary fat and fat-soluble vitamin esters. The reduced lipid absorption efficiency due to PTL and CEL inactivation also resulted in protection against diet-induced obesity.
Assuntos
Carboxilesterase/deficiência , Carboxilesterase/genética , Lipase/genética , Lipídeos/química , Pâncreas/enzimologia , Absorção , Ração Animal , Animais , Composição Corporal , Regulação da Expressão Gênica , Genótipo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/genética , Triglicerídeos/metabolismoRESUMO
Biotin affects the abundance of mRNA coding for approximately 10% of genes expressed in human-derived hepatocarcinoma (HepG2) cells. Here, we determined whether effects of biotin on gene expression are associated with changes in the abundance of distinct proteins in cell signaling and structure. HepG2 cells were cultured in media containing the following concentrations of biotin: 0.025 nmol/L (denoted "deficient"), 0.25 nmol/L ("physiological" = control), and 10 nmol/L ("pharmacological") for 10 d before harvesting. The abundance of 1009 proteins from whole-cell extracts was quantified by using high-throughput immunoblots. The abundance of 44 proteins changed by at least 25% in biotin-deficient and biotin-supplemented cells compared with physiological controls. One third of these proteins participate in cell signaling. Specifically, proteins associated with receptor tyrosine kinase-mediated signaling were identified as targets of biotin; the abundance of these proteins was greater in biotin-deficient cells than in controls. This was associated with increased DNA-binding activities of the transcription factors Fos and Jun, and increased expression of a reporter gene driven by activator protein (AP)1-binding elements in biotin-deficient cells compared with physiological controls. The abundance of selected signaling proteins was not paralleled by the abundance of mRNA, suggesting that biotin affects expression of these genes at a post-transcriptional step. Additional clusters of biotin-responsive proteins were identified that play roles in cytoskeleton homeostasis, nuclear structure and transport, and neuroscience. This study is consistent with the existence of clusters of biotin-responsive proteins in distinct biological processes, including signaling by Fos/Jun; the latter might mediate the proinflammatory and antiapoptotic effects of biotin deficiency.
Assuntos
Biotina/metabolismo , Proteínas de Neoplasias/genética , Transdução de Sinais/fisiologia , Sequência de Bases , Biotinilação , Carboxilesterase/genética , Carboxilesterase/metabolismo , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Primers do DNA , Humanos , Immunoblotting , Neoplasias Hepáticas , Proteínas de Neoplasias/metabolismo , Sondas de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Intensive chemical treatments have led to the development of a number of insecticide resistance mechanisms in the peach-potato aphid Myzus persicae (Sulzer). Some of these mechanisms are known to be associated with negative pleiotropic effects (resistance costs). Molecular and biochemical methods were used to determine the genotypes or phenotypes associated with four insecticide resistance mechanisms in single aphids from sexually-reproducing populations in southern France. The mechanisms considered were E4 and FE4 carboxylesterase overproduction, modified acetycholinesterase, and kdr and rdl resistance-associated mutations. A new method for determining individual kdr genotypes is presented. Almost all resistant individuals overproduced FE4 carboxylesterase, whereas modified acetylcholinesterase was rare. Both the kdr and rdl resistance mutations were present at high frequencies in French sexually-reproducing populations. The frequencies of insecticide resistance genes were compared before and after sexual reproduction in one peach orchard at Avignon to evaluate the potential impact of selection on the persistence of resistance alleles in the over-wintering phase. The frequencies of the kdr and rdl mutations varied significantly between autumn and spring sampling periods. The frequency of the kdr mutation increased, probably due to pyrethroid treatments at the end of the winter. Conversely, the frequency of the rdl mutation decreased significantly during winter, probably because of a fitness cost associated with this mutation.