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1.
Mol Metab ; 34: 174-186, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32180557

RESUMO

OBJECTIVE: It is well established that the liver-specific miR-122, a bona fide tumor suppressor, plays a critical role in lipid homeostasis. However, its role, if any, in amino acid metabolism has not been explored. Since glutamine (Gln) is a critical energy and anaplerotic source for mammalian cells, we assessed Gln metabolism in control wild type (WT) mice and miR-122 knockout (KO) mice by stable isotope resolved metabolomics (SIRM) studies. METHODS: Six-to eight-week-old WT and KO mice and 12- to 15-month-old liver tumor-bearing mice were injected with [U-13C5,15N2]-L-Gln, and polar metabolites from the liver tissues were analyzed by nuclear magnetic resonance (NMR) imaging and ion chromatography-mass spectrometry (IC-MS). Gln-metabolism was also assessed in a Gln-dependent hepatocellular carcinoma (HCC) cell line (EC4). Expressions of glutaminases (Gls and Gls2) were analyzed in mouse livers and human primary HCC samples. RESULTS: The results showed that loss of miR-122 promoted glutaminolysis but suppressed gluconeogenesis in mouse livers as evident from the buildup of 13C- and/or 15N-Glu and decrease in glucose-6-phosphate (G6P) levels, respectively, in KO livers. Enhanced glutaminolysis is consistent with the upregulation of expressions of Gls (kidney-type glutaminase) and Slc1a5, a neutral amino acid transporter in KO livers. Both Gls and Slc1a5 were confirmed as direct miR-122 targets by the respective 3'-UTR-driven luciferase assays. Importantly, expressions of Gls and Slc1a5 as well as glutaminase activity were suppressed in a Gln-dependent HCC (EC4) cell line transfected with miR-122 mimic that resulted in decreased 13C-Gln, 13C-á-ketoglutarate, 13C-isocitrate, and 13C-citrate levels. In contrast, 13C-phosphoenolpyruvate and 13C-G6P levels were elevated in cells expressing ectopic miR-122, suggesting enhanced gluconeogenesis. Finally, The Cancer Genome Atlas-Liver Hepatocellular Carcinoma (TCGA-LIHC) database analysis showed that expression of GLS is negatively correlated with miR-122 in primary human HCCs, and the upregulation of GLS RNA is associated with higher tumor grade. More importantly, patients with higher expressions of GLS or SLC1A5 in tumors exhibited poor survival compared with those expressing lower levels of these proteins. CONCLUSIONS: Collectively, these results show that miR-122 modulates Gln metabolism both in vitro and in vivo, implicating the therapeutic potential of miR-122 in HCCs that exhibit relatively high GLS levels.

2.
Environ Toxicol ; 34(8): 928-940, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31067004

RESUMO

Bioactive components of dietary phytochemicals have been reported to possess antitumor activities. Evidences suggested key role of stress responsive p38MAPK in the induction of nutraceuticals mediated apoptosis in hepatocellular carcinoma (HCC). Current study demonstrated detailed molecular bagatelle associated with p38 MAPK mediated effective suppression of cell growth both in HepG2 and chemically induced liver carcinoma after S-allyl cysteine (SAC) treatment. SAC promoted p38MAPK activity responsible for p53 phosphorylation, its stabilization followed by nuclear translocation leading to induction in expression and oligomerization of Fas protein. Distinctive p38MAPK-p53 axis dependent Fas-FasL-FADD mediated caspase activities along with perturbed cell cycling became normalized with continuation of SAC treatment for another month to diethylnitrosamine induced liver carcinoma. Co-treatment with SB203580, the p38MAPK inhibitor, prevented pro-apoptotic effect of SAC by altering p53 phosphorylation and death inducing signaling complex conformation in HepG2 and induced HCC. Collectively study suggested significant contribution of p38MAPK-p53-DISC-Caspase pathway in the regulation of anti-neoplastic activity of SAC against HCC.


Assuntos
Antineoplásicos/farmacologia , Cisteína/análogos & derivados , Neoplasias Hepáticas Experimentais/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Antineoplásicos/uso terapêutico , Caspases/metabolismo , Cisteína/farmacologia , Cisteína/uso terapêutico , Proteína Ligante Fas/metabolismo , Células Hep G2 , Humanos , Imidazóis/farmacologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/enzimologia , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Proteína Supressora de Tumor p53/metabolismo , Receptor fas/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
3.
Environ Toxicol Pharmacol ; 68: 120-132, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30889542

RESUMO

Andrographolide regimen in single or in combination with anticancer drugs is a promising new strategy to reverse chemoresistance in heaptocellular carcinoma. Apoptosis inducing factor (AIF) may regulate a complementary, cooperative or redundant pathway, along with caspase cascades. Despite these findings, mechanisms underlying caspase-dependent and-independent signaling pathways in andrographolide -induced apoptosis in cisplatin-resistant human hepatocellular carcinoma cell line (HepG2CR) remain unclear. Andrographolide treatment effectively reduced NF-κß nuclear localization by modulating protein kinase A- protein phosphatase 2 A- Iκß kinase (PKA/PP2 A/IKK) axis that in turn maintains initiator caspase8 activity. Lysosomal distribution of tBid stimulates cytosolic cathepsin B resulting accumulation of truncated-AIF with induction in scramblase mediated phosphatidylserine exposure in HepG2CR cells. Andrographolide treatment thereby switch on subG1 phase arrest by modulating cellular check points (cyclin A, B, cyclin dependent kinase-1) cueing to the apoptosis event. Collectively, this study suggested antineoplastic potential of andrographolide through PKA/PP2 A/IKK pathway in HepG2CR cells.


Assuntos
Antineoplásicos/farmacologia , Catepsina B/metabolismo , Cisplatino/farmacologia , Diterpenos/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Transferência de Fosfolipídeos/metabolismo , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Hep G2 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo
4.
Int J Mol Sci ; 20(2)2019 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-30669284

RESUMO

Autophagy and the (PI3K-Akt/mTOR) signaling pathway play significant roles in glioblastoma multiforme (GBM) cell death and survival. Curcumin (Cur) has been reported to prevent several cancers, including GBM. However, the poor solubility and limited bioavailability of natural Cur limits its application in preventing GBM growth. Previously, we have shown the greater apoptotic and anti-carcinogenic effects of solid lipid Cur particles (SLCP) than natural Cur in cultured GBM cells. Here, we compared the autophagic responses on cultured U-87MG, GL261, F98, C6-glioma, and N2a cells after treatment with Cur or SLCP (25 µM for 24 h). Different autophagy, mitophagy, and chaperone-mediated autophagy (CMA) markers, along with the PI3K-AKkt/mTOR signaling pathway, and the number of autophagy vacuoles were investigated after treatment with Cur and or SLCP. We observed increased levels of autophagy and decreased levels of mitophagy markers, along with inhibition of the PI3K-Akt/mTOR pathway after treatments with Cur or SLCP. Cell survival markers were downregulated, and cell death markers were upregulated after these treatments. We found greater effects in the case of SCLP-treated cells in comparison to Cur. Given that fewer effects were observed on C-6 glioma and N2a cells. Our results suggest that SLCP could be a safe and effective means of therapeutically modulating autophagy in GBM cells.


Assuntos
Autofagia/efeitos dos fármacos , Curcumina/química , Curcumina/farmacologia , Lipídeos/química , Nanopartículas/química , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Biomarcadores Ambientais , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
5.
Apoptosis ; 22(11): 1362-1379, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28836036

RESUMO

Cancer cells often have faulty apoptotic pathways resulting in sustenance of survivability, tumour metastasis and resistance to anticancer drugs. Alternate strategies are sought to improve therapeutic efficacy and therefore HepG2 cells were treated with S-allyl-cysteine (SAC) and berberine (BER) to analyze their mechanistic impact upon necroptosis along with its interacting relationship to apoptosis. In the present study we observed that SAC and BER exposure reduced NFκß nuclear translocation through adenylate cyclase-cAMP-protein kinaseA axis and eventually evaded c-FLIP inhibition. Effective RIP1 k63-polyubiquitination and persistent MKK3/MKK6 expression during drug treatment potentiated caspase8 activity via p53-DISC conformation. Resultant tBid associated lysosomal protease mediated AIF truncation induced DNA fragmentation and persuaded effector caspase mediated scramblase activation resulting induction of necroptosis in parallel to apoptotic events. SAC+BER effectively reduced Rb-phosphorylation resulting insignificant nuclear E2F presence led to ending of cell proliferation. Therefore necroptosis augmented the drug response and may be targeted alongside cell proliferation inhibition in formation of efficient therapeutics against liver cancer.


Assuntos
Adenilil Ciclases/genética , Berberina/farmacologia , Cisteína/análogos & derivados , MAP Quinase Quinase 3/genética , MAP Quinase Quinase 6/genética , Transdução de Sinais , Adenilil Ciclases/metabolismo , Apoptose/efeitos dos fármacos , Fator de Indução de Apoptose/genética , Fator de Indução de Apoptose/metabolismo , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cisteína/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Combinação de Medicamentos , Regulação da Expressão Gênica , Células Hep G2 , Humanos , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , MAP Quinase Quinase 3/metabolismo , MAP Quinase Quinase 6/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Necrose/induzido quimicamente , Necrose/genética , Necrose/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Transporte Proteico , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
6.
Curr Microbiol ; 71(4): 483-9, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26198412

RESUMO

A population of cold-tolerant Antarctic bacteria was screened for their ability to tolerate other environmental stress factors. Besides low temperature, they were predominantly found to be tolerant to alkali. Attempt was also made to postulate a genetic basis of their multistress-tolerance. Transposon mutagenesis of an isolate Pseudomonas syringae Lz4W was performed, and mutants with delayed growth at low temperature were further screened for sensitivity to some other stress factors. A number of multistress-sensitive mutants were isolated. The mutated gene in one of the mutants sensitive to low temperature, acid and alkali was found to encode citrate synthase. Possible role of citrate synthase in conferring multistress-tolerance was postulated.


Assuntos
Álcalis/toxicidade , Bactérias/efeitos dos fármacos , Bactérias/efeitos da radiação , Temperatura Baixa , Estresse Fisiológico , Regiões Antárticas , Bactérias/genética , Bactérias/isolamento & purificação , Citrato (si)-Sintase/genética , Elementos de DNA Transponíveis , DNA Bacteriano/química , DNA Bacteriano/genética , Microbiologia Ambiental , Técnicas de Inativação de Genes , Genes Bacterianos , Dados de Sequência Molecular , Mutagênese Insercional , Análise de Sequência de DNA
7.
Environ Toxicol ; 30(4): 396-410, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23997012

RESUMO

Sequelae of chronic lead (Pb(2+) ) toxicity includes anemia that is partially due to early death of erythrocytes characterized by excess accumulation of ROS and downregulation of antioxidant system causing oxidative stress and externalization of phosphatidylserine. In this study, pathophysiological based therapeutic application of garlic was evaluated against erythrocyte death. Results suggest that garlic administration prevents oxidative stress, restored the antioxidant balance in erythrocytes of Pb(2+) exposed mice. Moreover, in vitro studies revealed that activity of both scramblase and aminophospholipid translocase could be changed by modifying the critical sulfhydryl groups in presence of dithiothreitol during Pb(2+) exposure. Data also indicated that garlic treatment in Pb(2+) exposed mice exhibited sharp decline in PS exposure and increase in erythrocyte membrane thiol group followed by increase in aminophospholipid translocase activity and decline in scramblase activity. Findings indicated that garlic has the ability to restore the lifespan of erythrocytes during Pb(2+) exposure.


Assuntos
Eritrócitos/efeitos dos fármacos , Alho , Chumbo/toxicidade , Extratos Vegetais/farmacologia , Compostos de Sulfidrila/análise , Compostos de Sulfidrila/metabolismo , Anemia/induzido quimicamente , Anemia/prevenção & controle , Animais , Antioxidantes/metabolismo , Membrana Eritrocítica/química , Eritrócitos/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo , Fagocitose/efeitos dos fármacos , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo
8.
Infect Immun ; 82(6): 2637-48, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24711565

RESUMO

Prevotella intermedia is an oral bacterium implicated in a variety of oral diseases. Although internalization of this bacterium by nonphagocytic host cells is well established, the molecular players mediating the process are not well known. Here, the properties of a leucine-rich repeat (LRR) domain protein, designated AdpF, are described. This protein contains a leucine-rich region composed of 663 amino acid residues, and molecular modeling shows that it folds into a classical curved solenoid structure. The cell surface localization of recombinant AdpF (rAdpF) was confirmed by electron and confocal microscopy analyses. The recombinant form of this protein bound fibronectin in a dose-dependent manner. Furthermore, the protein was internalized by host cells, with the majority of the process accomplished within 30 min. The internalization of rAdpF was inhibited by nystatin, cytochalasin, latrunculin, nocodazole, and wortmannin, indicating that microtubules, microfilaments, and signal transduction are required for the invasion. It is noteworthy that preincubation of eukaryotic cells with AdpF increased P. intermedia 17 internalization by 5- and 10-fold for HeLa and NIH 3T3 fibroblast cell lines, respectively. The addition of the rAdpF protein was also very effective in inducing bacterial internalization into the oral epithelial cell line HN4, as well as into primary cells, including human oral keratinocytes (HOKs) and human umbilical vein endothelial cells (HUVECs). Finally, cells exposed to P. intermedia 17 internalized the bacteria more readily upon reinfection. Taken together, our data demonstrate that rAdpF plays a role in the internalization of P. intermedia 17 by a variety of host cells.


Assuntos
Proteínas de Bactérias/fisiologia , Células Eucarióticas/microbiologia , Prevotella intermedia/fisiologia , Proteínas/fisiologia , Análise de Variância , Fibroblastos/microbiologia , Fibronectinas/metabolismo , Regulação Bacteriana da Expressão Gênica , Células HeLa/microbiologia , Humanos , Prevotella intermedia/genética , Prevotella intermedia/patogenicidade
9.
Biochim Biophys Acta ; 1840(1): 219-44, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23999088

RESUMO

BACKGROUND: Diethylnitrosamine (DEN) and carbon tetrachloride (CCl4) have been used as initiator and promoter respectively to establish an animal model for investigating molecular events appear to be involved in development of liver cancer. Use of herbal medicine in therapeutics to avoid the recurrence of hepatocarcinoma has already generated considerable interest among oncologists. In this context studies involving S-allyl-cysteine (SAC) and berberine have come up with promising results. Here we have determined the individual effect of SAC and berberine on the biomolecules associated with DEN+CCl4 induced hepatocarcinoma. Effective therapeutic value of combined treatment has also been estimated. METHODS: ROS accumulation was analyzed by FACS following DCFDA incubation. Bcl2-Bax and HDAC1-pMdm2 interaction were demonstrated by co-immunoprecipitation. Immunosorbent assay was performed to analyze PP2A and caspase3 activities. MMP was determined cytofluorimetrically by investigating JC-1 fluorescence. AnnexinV binding was demonstrated by labeling the cells with AnV-FITC followed by flow cytometry. RESULTS: CytochromeP4502E1 mediated bioactivation of DEN+CCl4 induced Akt dependent pMdm2-HDAC1 interaction that led to p53 deacetylation, probable cause of its degradation. In parallel, oxidative stress dependent Nrf2-HO1 activation increased Bcl2 expression which in turn stimulated cell proliferation. SAC in combination with berberine inhibited Akt mediated cell proliferation. Activation of PP2A as well as inhibition of JNK resulted in induction of apoptosis after 30 days of treatment. Extension of combined treatment reverted tissue physiology towards control. Co-treated group displayed normal tissue structure. CONCLUSION AND GENERAL SIGNIFICANCE: SAC and berberine mediated HDAC1/Akt inhibition implicates the efficacy of combined treatment in the amelioration of DEN+CCl4 induced hepatocarcinoma.


Assuntos
Antineoplásicos/farmacologia , Berberina/farmacologia , Tetracloreto de Carbono/toxicidade , Carcinoma Hepatocelular/prevenção & controle , Cisteína/análogos & derivados , Dietilnitrosamina/toxicidade , Neoplasias Hepáticas/prevenção & controle , Alquilantes/toxicidade , Animais , Apoptose/efeitos dos fármacos , Western Blotting , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/metabolismo , Caspase 3/metabolismo , Células Cultivadas , Cisteína/farmacologia , Citocromo P-450 CYP2E1/metabolismo , Citocromos c/metabolismo , Citometria de Fluxo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Histona Desacetilase 1/metabolismo , Técnicas Imunoenzimáticas , Imunoprecipitação , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Proteínas de Transporte da Membrana Mitocondrial , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
10.
J Biosci ; 38(2): 409-12, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23660675

RESUMO

The adaptability of bacteria to extreme cold environments has been demonstrated from time to time by various investigators. Metabolic activity of bacteria at subzero temperatures is also evidenced. Recent studies indicate that bacteria continue both catabolic and anabolic activities at subzero temperatures. Implications of these findings are discussed.


Assuntos
Adaptação Fisiológica , Bactérias/metabolismo , Temperatura Baixa , Gelo , Estresse Fisiológico
12.
Infect Immun ; 78(6): 2385-96, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20308299

RESUMO

The oral bacterium Prevotella intermedia attaches to and invades gingival epithelial cells, fibroblasts, and endothelial cells. Several genes encoding proteins that mediate both the adhesion and invasion processes are carried on the genome of this bacterium. Here, we characterized one such protein, AdpC, belonging to the leucine-rich repeat (LRR) protein family. Bioinformatics analysis revealed that this protein shares similarity with the Treponema pallidum LRR (LRR(TP)) family of proteins and contains six LRRs. Despite the absence of a signal peptide, this protein is localized on the bacterial outer membrane, indicating that it is transported through an atypical secretion mechanism. The recombinant form of this protein (rAdpC) was shown to bind fibrinogen. In addition, the heterologous host strain Escherichia coli BL21 expressing rAdpC (V2846) invaded fibroblast NIH 3T3 cells at a 40-fold-higher frequency than control E. coli BL21 cells expressing a sham P. intermedia 17 protein. Although similar results were obtained by using human umbilical vein endothelial cells (HUVECs), only a 3-fold-increased invasion of V2846 into oral epithelial HN4 cells was observed. Thus, AdpC-mediated invasion is cell specific. This work demonstrated that AdpC is an important invasin protein of P. intermedia 17.


Assuntos
Proteínas de Bactérias/fisiologia , Prevotella intermedia/patogenicidade , Proteínas/fisiologia , Proteínas de Bactérias/genética , Linhagem Celular , Células Endoteliais , Escherichia coli/genética , Escherichia coli/patogenicidade , Fibrinogênio/metabolismo , Fibroblastos/microbiologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Prevotella intermedia/genética , Ligação Proteica , Proteínas/genética , Treponema pallidum/genética
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