Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 26
Filtrar
1.
J Physiol Biochem ; 2024 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-39289323

RESUMO

Palmitic acid is the most abundant saturated fatty acid in circulation and causes hepatocyte toxicity and inflammation. As saturated fatty acid can also disrupt the circadian rhythm, the present work evaluated the connection between clock genes and NAD+ dependent Sirtuins in protecting hepatocytes from lipid-induced damage. Hepatocytes (immortal cells PH5CH8, hepatoma cells HepG2) treated with higher doses of palmitic acid (400-600µM) showed typical features of steatosis accompanied with growth inhibition and increased level of inflammatory markers (IL-6 IL-8, IL-1α and IL-1ß) together with decline in NAD+ levels. Palmitic acid treated hepatocytes showed significant decline in not only the protein levels of SIRT2 but also its activity as revealed by the acetylation status of its downstream targets (Tubulin and NF-ƙB). Additionally, the circadian expression of both SIRT2 and BMAL1 was inhibited in presence of palmitic acid in only the non-cancerous hepatocytes, PH5CH8 cells. Clinical specimens obtained from subjects with NASH-associated fibrosis, ranging from absent (F0) to cirrhosis (F4), showed a significant decline in levels of SIRT2 and BMAL1, especially in the cirrhotic liver. Ectopic expression of BMAL1 or activating SIRT2 by supplementation with nicotinamide riboside (precursor of NAD+) dampened the palmitic acid induced lipoinflammation and lipotoxicity more effectively in PH5CH8 cells as compared to HepG2 cells. Mechanistically, palmitic acid caused transcriptional suppression of SIRT2 by disrupting the chromatin occupancy of BMAL1 at its promoter site. Overall, the work suggested that SIRT2 is a clock-controlled gene that is transcriptionally regulated by BMAL1. In conclusion the activation of the BMAL1-NAD+-SIRT2 axis shows hepatoprotective effects by preventing lipotoxicity and dampening inflammation.

2.
ACS Appl Bio Mater ; 7(10): 6332-6342, 2024 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-39305253

RESUMO

Infections associated with medical implants due to bacterial adhesion and biofilm formation are a serious problem, leading to acute health risks to patients by compromising their immune system. Therefore, suppressing biofilm formation on biomedical implants is a challenging task, especially for overcoming the drug resistance of bacterial biofilms. Herein, a synergistic efficient surface coating method was developed to inhibit biofilm formation on a model medical implant by combining the antimicrobial property of trimethyl chitosan (TMC) with either 2D material graphene oxide (GO) or black phosphorus (BP) sheets using layer-by-layer (LbL) self-assembly. The multilayer coatings of TMC/GO and TMC/BP were optimized on the glass surface (a model implant) and characterized by using spectroscopic and microscopy techniques. Next, we investigated the antibiofilm formation properties of the TMC/GO and TMC/BP coatings on glass surfaces against both Gram-negative, Escherichia coli (E. coli), and Gram-positive, Bacillus subtilis (B. subtilis), bacteria. The antibiofilm formation was studied using crystal violet (CV) and live/dead assays. Both the live/dead and the CV assays confirmed that the TMC/2D material (2DM)-coated surfaces prevented biofilm formation much more effectively compared to the uncoated surfaces. Scanning electron microscopy analyses revealed that the bacteria were affected physically by incubating with TMC/2DM-coated surfaces due to membrane perturbation, thereby preventing cell attachment and biofilm formation. Further, BP composite coatings (TMC/BP) showed a much better ability to thwart biofilm formation than GO composite coatings (TMC/GO). Also, multilayer coatings showed superior cytocompatibility with human foreskin fibroblast (HFF). Our results demonstrate that the developed coatings TMC/2DMs could be potential candidates for thwarting biofilm formation on medical implants.


Assuntos
Antibacterianos , Bacillus subtilis , Biofilmes , Materiais Revestidos Biocompatíveis , Escherichia coli , Teste de Materiais , Testes de Sensibilidade Microbiana , Próteses e Implantes , Biofilmes/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Antibacterianos/síntese química , Escherichia coli/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Bacillus subtilis/efeitos dos fármacos , Tamanho da Partícula , Propriedades de Superfície , Quitosana/química , Quitosana/farmacologia , Humanos , Grafite/química , Grafite/farmacologia , Fósforo/química
3.
J Biomol Struct Dyn ; : 1-20, 2023 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-37965748

RESUMO

Substituted ethoxy phthalimide pyrazole derivatives (6a-e) have been produced using a one-pot synthesis technique. Spectral analysis was used to establish the molecular structure of the synthesized compounds, and they were examined in silico and in vitro for their ability to bind to and inhibit replication of the AD-169 strain, the Davis strain of CMV, the OKA strain and the 07/1 strain of Varicella-Zoster virus (VZV). Molecular Docking was used to estimate the binding mechanism and energy of compounds 4, 6a-e to their respective target proteins, thymidine kinase (TK), Varicella-Zoster protease (VZP) of VZV and tegument protein pp71 (TPpp71) of Cytomegalovirus (CMV). The MIC50 and EC50 were utilized to evaluate the antiviral and cytotoxic activities of test compounds in human embryonic lung (HEL) cells against the two reference medicines, Ganciclovir and Acyclovir. The chemicals studied showed a high affinity for binding sites and near binding sites of target proteins by generating H-bonds, carbon-hydrogen bonds, π-anion, π-sulfur, π-sigma, alkyl and π-alkyl interactions. All of the test compounds (6a-e) had higher binding energy than the standard medications. The ADME/T data suggests that these potential inhibitors are less toxic. Drug-protein complexes are structurally compact and demonstrate minimal conformational change in molecular dynamics (MDs) simulations, indicating stability and stiffness. MM-PBSA and post-simulation analysis can predict lead compound active cavity binding stability. By inhibiting multitargeted proteins, these synthetic compounds may improve antiviral therapy. Our research suggests that these unique synthesized chemicals may be useful and accessible adjuvant antiviral therapy for Varicella Zoster and CMV. HighlightsTwo components synthesis of substituted ethoxy phthalimide pyrazole derivatives (6a-e).Tested compounds (6a-e) have antiviral and cytotoxicity activity against CMV and Varicella-Zoster virus (VZV) in HEL cells.Compounds bind to TK, Varicella-Zoster protease (VZP) of VZV, and modeled TPpp71 of Cytomegalovirus (CMV).In comparison to reference drugs, compounds have strong binding free energy and interactions with VZV and CMV protein complexes.The RMSD, RMSF, Rg, residual correlative motion (RCM), No. of hydrogen bonds, protein secondary structure content, per-residue protein secondary structure and MM/PBSA energy calculated for the selected compound with thymidine kinase (TK), VZP of VZV, and modeled tegument protein pp71 (TPpp71) of CMV through MD simulation studies for 50 ns.In comparison to the two reference drugs, ligands/compounds were found to meet the Lipinski rule of five and to have strong biological activity.Communicated by Ramaswamy H. Sarma.

4.
Sci Rep ; 13(1): 16420, 2023 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-37775650

RESUMO

Cardiac rhythm regulated by micro-macroscopic structures of heart. Pacemaker abnormalities or disruptions in electrical conduction, lead to arrhythmic disorders may be benign, typical, threatening, ultimately fatal, occurs in clinical practice, patients on digitalis, anaesthesia or acute myocardial infarction. Both traditional and genetic animal models are: In-vitro: Isolated ventricular Myocytes, Guinea pig papillary muscles, Patch-Clamp Experiments, Porcine Atrial Myocytes, Guinea pig ventricular myocytes, Guinea pig papillary muscle: action potential and refractory period, Langendorff technique, Arrhythmia by acetylcholine or potassium. Acquired arrhythmia disorders: Transverse Aortic Constriction, Myocardial Ischemia, Complete Heart Block and AV Node Ablation, Chronic Tachypacing, Inflammation, Metabolic and Drug-Induced Arrhythmia. In-Vivo: Chemically induced arrhythmia: Aconitine antagonism, Digoxin-induced arrhythmia, Strophanthin/ouabain-induced arrhythmia, Adrenaline-induced arrhythmia, and Calcium-induced arrhythmia. Electrically induced arrhythmia: Ventricular fibrillation electrical threshold, Arrhythmia through programmed electrical stimulation, sudden coronary death in dogs, Exercise ventricular fibrillation. Genetic Arrhythmia: Channelopathies, Calcium Release Deficiency Syndrome, Long QT Syndrome, Short QT Syndrome, Brugada Syndrome. Genetic with Structural Heart Disease: Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia, Dilated Cardiomyopathy, Hypertrophic Cardiomyopathy, Atrial Fibrillation, Sick Sinus Syndrome, Atrioventricular Block, Preexcitation Syndrome. Arrhythmia in Pluripotent Stem Cell Cardiomyocytes. Conclusion: Both traditional and genetic, experimental models of cardiac arrhythmias' characteristics and significance help in development of new antiarrhythmic drugs.


Assuntos
Antiarrítmicos , Fibrilação Atrial , Humanos , Animais , Cobaias , Cães , Antiarrítmicos/farmacologia , Antiarrítmicos/uso terapêutico , Fibrilação Ventricular/tratamento farmacológico , Cálcio , Fibrilação Atrial/tratamento farmacológico , Músculos Papilares , Modelos Animais
5.
Antioxidants (Basel) ; 12(2)2023 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-36829837

RESUMO

Cyclobutane pyrimidine dimers (CPDs) are ultraviolet radiation (UV)-induced carcinogenic DNA photoproducts that lead to UV signature mutations in melanoma. Previously, we discovered that, in addition to their incident formation (iCPDs), UV exposure induces melanin chemiexcitation (MeCh), where UV generates peroxynitrite (ONOO-), which oxidizes melanin into melanin-carbonyls (MCs) in their excited triplet state. Chronic MeCh and energy transfer by MCs to DNA generates CPDs for several hours after UV exposure ends (dark CPD, dCPDs). We hypothesized that MeCh and the resulting dCPDs can be inhibited using MeCh inhibitors, and MC and ONOO- scavengers. Here, we investigated the efficacy of Acetyl Zingerone (AZ), a plant-based phenolic alkanone, and its chemical analogs in inhibiting iCPDs and dCPDs in skin fibroblasts, keratinocytes, and isogenic pigmented and albino melanocytes. While AZ and its methoxy analog, 3-(4-Methoxy-benzyl)-Pentane-2,4-dione (MBPD) completely inhibited the dCPDs, MBPD also inhibited ~50% of iCPDs. This suggests the inhibition of ~80% of total CPDs at any time point post UV exposure by MBPD, which is markedly significant. MBPD downregulated melanin synthesis, which is indispensable for dCPD generation, but this did not occur with AZ. Meanwhile, AZ and MBPD both upregulated the expression of nucleotide excision repair (NER) pathways genes including Xpa, Xpc, and Mitf. AZ and its analogs were non-toxic to the skin cells and did not act as photosensitizers. We propose that AZ and MBPD represent "next-generation skin care additives" that are safe and effective for use not only in sunscreens but also in other specialized clinical applications owing to their extremely high efficacy in blocking both iCPDs and dCPDs.

6.
Biochem Biophys Res Commun ; 643: 129-138, 2023 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-36603530

RESUMO

There is an alarming increase in incidence of fatty liver disease worldwide. The fatty liver disease spectrum disease ranges from simple steatosis (NAFL) to steatohepatitis (NASH) which culminates in cirrhosis and cancer. Altered metabolism is a hallmark feature associated with fatty liver disease and palmitic acid is the most abundant saturated fatty acid, therefore, the aim of this study was to compare metabolic profiles altered in hepatocytes treated with palmitic acid and also the differentially expressed plasma metabolites in spectrum of nonalcoholic fatty liver. The metabolites were analyzed by liquid chromatography-mass spectrometry (LC-MS) platform. Hepatocyte cell lines PH5CH8 and HepG2 cells when treated with 400 µM dose of palmitic acid showed typical features of steatosis. Metabolomic analysis of lipid treated hepatocyte cell lines showed differential changes in phenylalanine and tyrosine pathways, fatty acid metabolism and bile acids. The key metabolites tryptophan, kynurenine and carnitine differed significantly between subjects with NAFL, NASH and those with cirrhosis. As the tryptophan-kynurenine axis is also involved in denovo synthesis of NAD+, we found significant alterations in the NAD+ related metabolites in both palmitic acid treated and also fatty liver disease with cirrhosis. The study underscores the importance of amino acid and NAD+supplementation as promising strategies in fatty liver disorder.


Assuntos
Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/metabolismo , NAD/metabolismo , Aminoácidos/metabolismo , Palmitatos/metabolismo , Cinurenina/metabolismo , Triptofano/metabolismo , Hepatócitos/metabolismo , Cirrose Hepática/patologia , Ácido Palmítico/farmacologia , Ácido Palmítico/metabolismo , Fígado/metabolismo
7.
Biomed Tech (Berl) ; 68(2): 147-163, 2023 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-36583250

RESUMO

This work proposes a variational mode decomposition (VMD) and binary grey wolf optimization (BGWO) based seizure classification framework. VMD decomposes the EEG signal into band-limited intrinsic mode function (BL-IMFs) non-recursively. The frequency domain, time domain, and information theory-based features are extracted from the BL-IMFs. Further, an optimal feature subset is selected using BGWO. Finally, the selected features were utilized for classification using six different supervised machine learning algorithms. The proposed framework has been validated experimentally by 58 test cases from the CHB-MIT scalp EEG and the Bonn University database. The proposed framework performance is quantified by average sensitivity, specificity, and accuracy. The selected features, along with Bayesian regularized shallow neural networks (BR-SNNs), resulted in maximum accuracy of 99.53 and 99.64 for 1 and 2 s epochs, respectively, for database 1. The proposed framework has achieved 99.79 and 99.84 accuracy for 1 and 2 s epochs, respectively, for database 2.


Assuntos
Epilepsia , Convulsões , Humanos , Teorema de Bayes , Algoritmos , Redes Neurais de Computação , Eletroencefalografia/métodos , Processamento de Sinais Assistido por Computador
8.
J Proteomics ; 264: 104614, 2022 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-35595057

RESUMO

Constitutively active K-Ras oncogene mutation at G12V changes the proteome of cells and activates macroautophagy for cell advantage. Inhibition of macroautophagy impairs K-Ras mediated tumor progression to a limited extent with increase of spontaneous tumors due to poorly understood mechanisms. Here, we show that inhibition of macroautophagy in K-Ras G12V mouse embryonic fibroblasts (MEFs) hyper activates chaperon mediated autophagy (CMA). Quantitative identification of CMA substrates through co-immunoprecipitation of CMA component heat shock cognate 70 (Hsc70) demonstrates a shift of proteins from macroautophagy to CMA mediated degradation. However, macroautophagy impairment show significant inhibition on proliferation and CMA hyper activation provides a basal support to macroautophagy-inhibited MEFs for survival. On the other hand, K-Ras G12V MEFs impaired of CMA reduces number of Hsc70 clients but activated macroautophagy significantly compensated CMA loss. Nonetheless, co-inhibition of CMA and macroautophagy had a synergistic detrimental effect on both proliferation and survival of MEFs expressing K-Ras G12V mutant. Our results point to K-Ras G12V MEFs dependency on macroautophagy and CMA partly compensates its loss for survival but not hyper-proliferation; implicating that targeting both macroautophagy and CMA as a promising therapeutic target in G12V mutation associated K-Ras cancers. SIGNIFICANCE: The present study provides a framework of Hsc70 interacting proteins, which differentially interact with Hsc70 in response to autophagy alterations. The role of proteins accumulation and induced proteo-toxicity could be underlying factor in macroautophagy and CMA co-inhibited K-Ras G12V MEFs phenotype. Our study provides rational for adaptive mechanisms in K-Ras tumors inhibited with different autophagy pathways and also supports targeting both macroautophagy and CMA simultaneously as therapeutic target. At the same time current study will help in characterizing the underlying cellular processes that may play a role in escaping tutor suppressor role CMA and macroautophagy in cancers harboring K-Ras G12V mutation that may be further utilized to identify molecular targets for K-Ras-driven cancers.


Assuntos
Autofagia Mediada por Chaperonas , Neoplasias , Animais , Proliferação de Células , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSC70/metabolismo , Humanos , Lisossomos/metabolismo , Macroautofagia , Camundongos , Chaperonas Moleculares/metabolismo , Neoplasias/metabolismo
9.
Cell Tissue Bank ; 23(1): 79-92, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33768473

RESUMO

Biological detergents like sodium deoxycholate, sodium dodecyl sulphate and Triton X-100 impairs the collagenous and non-collagenous proteins, glycosaminoglycans and growth factors. Further, certain chemical and enzymes are responsible for residual cytotoxicity in the decellularized extracellular matrix. The main focus of this study was to explore the decellularization property of soap nut pericarp extract (SPE) for development of decellularized tubular esophageal scaffold. For this 2.5, 5.0 and 10% concentrations of SPE were used for decellularization of caprine esophageal tissues. Histological analysis of hematoxylin and eosin and Masson's trichrome stained tissue samples confirmed decellularization with preservation of extracellular matrix microarchitecture. Scanning electron microscopic images of luminal surface of decellularized esophageal matrix showed randomly oriented collagen fibres with large interconnected pores and cells were absent. However, the external surface was more textured with fibrous structures and collagen fibres were well preserved. DAPI stained decellularized tissues revealed complete removal of nuclear components, verified by DNA content measurement and SDS-PAGE. The FTIR spectra of decellularized esophagus shows absorption peaks of amide A, B, I, II and III. Elastic modulus of the decellularized esophagus scaffolds increased (P > 0.05) as compared to native tissues. Histological and scanning electron microscopic evaluation of in vitro seeded scaffolds showed attachment and growth of primary chicken embryo fibroblasts over and within the decellularized scaffolds. It was concluded that 5% SPE is ideal for preparation of cytocompatible decellularized caprine esophageal scaffold with well-preserved extracellular matrix architecture and, may be used as an alternative to biological detergents and other chemicals.


Assuntos
Sapindus , Engenharia Tecidual , Animais , Embrião de Galinha , Esôfago , Matriz Extracelular , Frutas , Cabras , Extratos Vegetais , Engenharia Tecidual/métodos , Alicerces Teciduais/química
10.
J Cell Physiol ; 236(11): 7801-7813, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33899236

RESUMO

Signaling pathways that regulate homeostasis and regeneration are found to be deregulated in various human malignancies. Accordingly, attempts have been made to target them at the protein level with little success. However, studies using high-throughput sequencing technologies suggest that only about 2% of the genome translates into proteins, whereas about 75% of the genome is transcribed into noncoding RNAs. Among noncoding RNAs, long noncoding RNAs (lncRNAs) have received tremendous attention in recent years as a crucial player in the regulation of almost all cellular processes involved in tissue homeostasis as well as in the development of various malignancies, including intestinal cancer. Emerging evidence suggests that lncRNAs play an instrumental role in the regulation of intestinal stem cells, injury-induced regeneration, and initiation and progression of intestinal tumors. Here, we summarize the recently discovered lncRNAs during intestinal homeostasis, regeneration, and tumorigenesis. We further present lncRNAs as diagnostic and therapeutic markers in intestinal pathologies.


Assuntos
Biomarcadores Tumorais/metabolismo , Mucosa Intestinal/metabolismo , Neoplasias Intestinais/metabolismo , RNA Longo não Codificante/metabolismo , Regeneração , Animais , Biomarcadores Tumorais/genética , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Homeostase , Humanos , Mucosa Intestinal/patologia , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , RNA Longo não Codificante/genética , Transdução de Sinais
11.
Micron ; 142: 102997, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33388519

RESUMO

The aim of this study is to develop a novel decellularization method using aqueous extract of soap nut pericarp (SPE) and its evaluation using hematoxylin-eosin staining, scanning electron microscopy, diamidino-2-phenylindol (DAPI) staining, mechanical testing, sodium dodecyl sulfate polyacrylamide gel electrophoresis and DNA quantification. The presently available decellularization agent raises some concerns due to the potential for presence of residual cytotoxic agents in the extracellular matrix. Histological analysis of hematoxylin and eosin and masson's trichrome stained processed aortic samples shows complete decellularization with preservation of extracellular matrix microarchitecture at 120 h. Further, staining of tissue samples with DAPI demonstrates complete removal of DNA fragments. Quantitative evaluation of DNA in the decellularized aorta tissues demonstrated a significant (P < 0.01) decrease in DNA content as compared to native tissues. Collagen quantification assay indicate no significant (P> 0.05) difference in its content between native and decellularized caprine aorta. Tensile strength of the decellularized scaffolds decreased non-significantly (P > 0.05) when compared to native tissues. There was no significant (P > 0.05) difference in young's modulus of elasticity, stiffness and stretch ratio between native aortic tissues and decellularized aortic scaffolds. Histological and scanning electron microscopic examination of in vitro cultured scaffold demonstrated the cell viability and proliferation of primary chicken embryo fibroblasts. SPE treatment is thus capable of producing cytocompatible decellularized caprine aorta scaffold with preservation of extracellular matrix architecture for vascular tissue engineering and could be applied widely as one of the decellularization agent.


Assuntos
Aorta/citologia , Separação Celular/métodos , Extratos Vegetais , Sapindus , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Fenômenos Biomecânicos , Sobrevivência Celular , Embrião de Galinha , Colágeno , Matriz Extracelular , Fibroblastos/metabolismo , Frutas/química , Cabras , Histocompatibilidade , Microscopia Eletrônica de Varredura , Extratos Vegetais/química , Medicina Regenerativa , Sapindus/química
12.
Methods Mol Biol ; 2031: 313-322, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31473968

RESUMO

Ultraviolet rays induce interstrand and intrastrand DNA cross-links, usually thymine-thymine cyclobutane dimer (T-T) and thymine-thymine pyrimidine-pyrimidone (6-4) photoproduct (T (6-4) T). These DNA cross-links, if left unrepaired, increase the risk of these mutation being incorporated in the genetic material (i.e., DNA). Numerous studies have reported the mutagenic potential of above mentioned DNA adducts in prokaryotes, yeast and mammalian cells. Different techniques have been developed to identify such DNA adducts such as immuno-Southern blotting. This is a routinely used quantitative method to determine especially the amount of thymine dimers formed, following irradiation. In this chapter, the detailed methodology to identify thymine dimers formation is provided, using specific antibody against these adducts.


Assuntos
Southern Blotting/métodos , Dano ao DNA/efeitos da radiação , Dímeros de Pirimidina/análise , Raios Ultravioleta/efeitos adversos , Animais , Técnicas de Cultura de Células/métodos , Humanos
13.
World J Nucl Med ; 18(3): 310-313, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31516379

RESUMO

Small bowel hemangioma is a rare benign tumor in the pediatric population. The usual presentation of these tumors is melena, anemia, or hematochezia. Our case demonstrates the usefulness of Meckel's/Tc-99m pertechnetate scan with single-photon emission computerized tomography/computerized tomography in diagnosing a vascular lesion in the small bowel in a child presenting with melena, unresponsive to medical management. We present a case of incidentally detected jejunal hemangioma during Tc-99m pertechnetate scintigraphy which would help the nuclear medicine physician and surgeon, to be cognizant of this atypical presentation in their clinical practice.

14.
Cancer Discov ; 6(3): 300-15, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26732095

RESUMO

UNLABELLED: We have identified previously undiscovered BRAF in-frame deletions near the αC-helix region of the kinase domain in pancreatic, lung, ovarian, and thyroid cancers. These deletions are mutually exclusive with KRAS mutations and occur in 4.21% of KRAS wild-type pancreatic cancer. siRNA knockdown in cells harboring BRAF deletions showed that the MAPK activity and cell growth are BRAF dependent. Structurally, the BRAF deletions are predicted to shorten the ß3/αC-helix loop and hinder its flexibility by locking the helix in the active αC-helix-in conformation that favors dimer formation. Expression of L485-P490-deleted BRAF is able to transform NIH/3T3 cells in a BRAF dimer-dependent manner. BRAF homodimer is confirmed to be the dominant RAF dimer by proximity ligation assays in BRAF deletion cells, which are resistant to the BRAF inhibitor vemurafenib and sensitive to LY3009120, a RAF dimer inhibitor. In tumor models with BRAF deletions, LY3009120 has shown tumor growth regression, whereas vemurafenib is inactive. SIGNIFICANCE: This study discovered oncogenic BRAF deletions with a distinct activation mechanism dependent on the BRAF dimer formation in tumor cells. LY3009120 is active against these cells and represents a potential treatment option for patients with cancer with these BRAF deletions, or other atypical BRAF mutations where BRAF functions as a dimer.


Assuntos
Antineoplásicos/farmacologia , Resistencia a Medicamentos Antineoplásicos , Deleção de Genes , Compostos de Fenilureia/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Pirimidinas/farmacologia , Animais , Antineoplásicos/química , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Modelos Animais de Doenças , Expressão Ectópica do Gene , Expressão Gênica , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas B-raf/química , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Cancer Cell ; 28(3): 384-98, 2015 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-26343583

RESUMO

LY3009120 is a pan-RAF and RAF dimer inhibitor that inhibits all RAF isoforms and occupies both protomers in RAF dimers. Biochemical and cellular analyses revealed that LY3009120 inhibits ARAF, BRAF, and CRAF isoforms with similar affinity, while vemurafenib or dabrafenib have little or modest CRAF activity compared to their BRAF activities. LY3009120 induces BRAF-CRAF dimerization but inhibits the phosphorylation of downstream MEK and ERK, suggesting that it effectively inhibits the kinase activity of BRAF-CRAF heterodimers. Further analyses demonstrated that LY3009120 also inhibits various forms of RAF dimers including BRAF or CRAF homodimers. Due to these unique properties, LY3009120 demonstrates minimal paradoxical activation, inhibits MEK1/2 phosphorylation, and exhibits anti-tumor activities across multiple models carrying KRAS, NRAS, or BRAF mutation.


Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Compostos de Fenilureia/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Pirimidinas/farmacologia , Proteínas ras/genética , Linhagem Celular Tumoral , Dimerização , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação/efeitos dos fármacos , Mutação/genética , Neoplasias/genética , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Isoformas de Proteínas/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-raf/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
16.
PLoS One ; 10(4): e0125028, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25915038

RESUMO

It has previously been observed that a loss of ß-catenin expression occurs with melanoma progression and that nuclear ß-catenin levels are inversely proportional to cellular proliferation, suggesting that activation of the Wnt/ß-catenin pathway may provide benefit for melanoma patients. In order to further probe this concept we tested LY2090314, a potent and selective small-molecule inhibitor with activity against GSK3α and GSK3ß isoforms. In a panel of melanoma cell lines, nM concentrations of LY2090314 stimulated TCF/LEF TOPFlash reporter activity, stabilized ß-catenin and elevated the expression of Axin2, a Wnt responsive gene and marker of pathway activation. Cytotoxicity assays revealed that melanoma cell lines are very sensitive to LY2090314 in vitro (IC50 ~10 nM after 72hr of treatment) in contrast to other solid tumor cell lines (IC50 >10 uM) as evidenced by caspase activation and PARP cleavage. Cell lines harboring mutant B-RAF or N-RAS were equally sensitive to LY2090314 as were those with acquired resistance to the BRAF inhibitor Vemurafenib. shRNA studies demonstrated that ß-catenin stabilization is required for apoptosis following treatment with the GSK3 inhibitor since the sensitivity of melanoma cell lines to LY290314 could be overcome by ß-catenin knockdown. We further demonstrate that in vivo, LY2090314 elevates Axin2 gene expression after a single dose and produces tumor growth delay in A375 melanoma xenografts with repeat dosing. The activity of LY2090314 in preclinical models suggests that the role of Wnt activators for the treatment of melanoma should be further explored.


Assuntos
Antineoplásicos/administração & dosagem , Compostos Heterocíclicos com 3 Anéis/administração & dosagem , Maleimidas/administração & dosagem , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Via de Sinalização Wnt/efeitos dos fármacos , Animais , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Compostos Heterocíclicos com 3 Anéis/farmacologia , Humanos , Maleimidas/farmacologia , Melanoma/genética , Melanoma/metabolismo , Camundongos , Camundongos Nus , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Pharmacol Ther ; 149: 139-49, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25550229

RESUMO

Selective BRAF inhibitors have demonstrated significant clinical benefit in melanoma patients harboring oncogenic BRAF mutations. However, the majority of such patients either exhibit de novo resistance from the beginning of the treatment or acquire resistance and eventually relapse. Despite tremendous progress in understanding the underlying mechanisms of resistance, overcoming resistance to BRAF inhibitors remains an unmet medical need. Constitutive activation of cyclin-dependent kinases (CDK) 4/6 as a result of genetic aberrations including CDKN2A inactivation and CCND1 amplification is common across many cancer types and frequently co-occurs with oncogenic BRAF mutations. Also, cyclin D1 overexpression is a common feature of resistance to BRAF inhibitors. Here we review CDK4/6 as a therapeutic target in BRAF mutant cancers and discuss emerging evidence supporting a critical role of cyclin D1/CDK4/6 axis in de novo and acquired resistance to BRAF inhibitors. Co-targeting CDK4/6 and BRAF could be a more effective therapy to augment clinical response of BRAF inhibitors and overcome resistance in BRAF mutant cancers.


Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Terapia de Alvo Molecular/métodos , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Animais , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Modelos Biológicos , Mutação , Neoplasias/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas B-raf/genética
18.
Mol Cancer Ther ; 13(10): 2253-63, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25122067

RESUMO

B-RAF selective inhibitors, including vemurafenib, were recently developed as effective therapies for melanoma patients with B-RAF V600E mutation. However, most patients treated with vemurafenib eventually develop resistance largely due to reactivation of MAPK signaling. Inhibitors of MAPK signaling, including MEK1/2 inhibitor trametinib, failed to show significant clinical benefit in patients with acquired resistance to vemurafenib. Here, we describe that cell lines with acquired resistance to vemurafenib show reactivation of MAPK signaling and upregulation of cyclin D1 and are sensitive to inhibition of LY2835219, a selective inhibitor of cyclin-dependent kinase (CDK) 4/6. LY2835219 was demonstrated to inhibit growth of melanoma A375 tumor xenografts and delay tumor recurrence in combination with vemurafenib. Furthermore, we developed an in vivo vemurafenib-resistant model by continuous administration of vemurafenib in A375 xenografts. Consistently, we found that MAPK is reactivated and cyclin D1 is elevated in vemurafenib-resistant tumors, as well as in the resistant cell lines derived from these tumors. Importantly, LY2835219 exhibited tumor growth regression in a vemurafenib-resistant model. Mechanistic analysis revealed that LY2835219 induced apoptotic cell death in a concentration-dependent manner in vemurafenib-resistant cells whereas it primarily mediated cell-cycle G1 arrest in the parental cells. Similarly, RNAi-mediated knockdown of cyclin D1 induced significantly higher rate of apoptosis in the resistant cells than in parental cells, suggesting that elevated cyclin D1 activity is important for the survival of vemurafenib-resistant cells. Altogether, we propose that targeting cyclin D1-CDK4/6 signaling by LY2835219 is an effective strategy to overcome MAPK-mediated resistance to B-RAF inhibitors in B-RAF V600E melanoma.


Assuntos
Aminopiridinas/farmacologia , Benzimidazóis/farmacologia , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Nus , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas/farmacologia , Transfecção , Regulação para Cima/efeitos dos fármacos , Vemurafenib , Ensaios Antitumorais Modelo de Xenoenxerto
19.
PLoS One ; 9(5): e98293, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24874471

RESUMO

The Cancer Genome Atlas (TCGA) projects have advanced our understanding of the driver mutations, genetic backgrounds, and key pathways activated across cancer types. Analysis of TCGA datasets have mostly focused on somatic mutations and translocations, with less emphasis placed on gene amplifications. Here we describe a bioinformatics screening strategy to identify putative cancer driver genes amplified across TCGA datasets. We carried out GISTIC2 analysis of TCGA datasets spanning 16 cancer subtypes and identified 486 genes that were amplified in two or more datasets. The list was narrowed to 75 cancer-associated genes with potential "druggable" properties. The majority of the genes were localized to 14 amplicons spread across the genome. To identify potential cancer driver genes, we analyzed gene copy number and mRNA expression data from individual patient samples and identified 42 putative cancer driver genes linked to diverse oncogenic processes. Oncogenic activity was further validated by siRNA/shRNA knockdown and by referencing the Project Achilles datasets. The amplified genes represented a number of gene families, including epigenetic regulators, cell cycle-associated genes, DNA damage response/repair genes, metabolic regulators, and genes linked to the Wnt, Notch, Hedgehog, JAK/STAT, NF-KB and MAPK signaling pathways. Among the 42 putative driver genes were known driver genes, such as EGFR, ERBB2 and PIK3CA. Wild-type KRAS was amplified in several cancer types, and KRAS-amplified cancer cell lines were most sensitive to KRAS shRNA, suggesting that KRAS amplification was an independent oncogenic event. A number of MAP kinase adapters were co-amplified with their receptor tyrosine kinases, such as the FGFR adapter FRS2 and the EGFR family adapters GRB2 and GRB7. The ubiquitin-like ligase DCUN1D1 and the histone methyltransferase NSD3 were also identified as novel putative cancer driver genes. We discuss the patient tailoring implications for existing cancer drug targets and we further discuss potential novel opportunities for drug discovery efforts.


Assuntos
Biologia Computacional , Amplificação de Genes , Genômica , Neoplasias/genética , Oncogenes/genética , Transformação Celular Neoplásica/genética , Mapeamento Cromossômico , Biologia Computacional/métodos , Bases de Dados de Ácidos Nucleicos , Conjuntos de Dados como Assunto , Epigênese Genética , Dosagem de Genes , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Sistema de Sinalização das MAP Quinases , Neoplasias/tratamento farmacológico , Proteínas , Proteínas Proto-Oncogênicas/genética
20.
PLoS One ; 7(6): e39812, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22761908

RESUMO

Prolonged usage of antifungal azoles which target enzymes involved in lipid biosynthesis invariably leads to the development of multi-drug resistance (MDR) in Candida albicans. We had earlier shown that membrane lipids and their fluidity are closely linked to the MDR phenomenon. In one of our recent studies involving comparative lipidomics between azole susceptible (AS) and azole resistant (AR) matched pair clinical isolates of C. albicans, we could not see consistent differences in the lipid profiles of AS and AR strains because they came from different patients and so in this study, we have used genetically related variant recovered from the same patient collected over a period of 2-years. During this time, the levels of fluconazole (FLC) resistance of the strain increased by over 200-fold. By comparing the lipid profiles of select isolates, we were able to observe gradual and statistically significant changes in several lipid classes, particularly in plasma membrane microdomain specific lipids such as mannosylinositolphosphorylceramides and ergosterol, and in a mitochondrial specific phosphoglyceride, phosphatidyl glycerol. Superimposed with these quantitative and qualitative changes in the lipid profiles, were simultaneous changes at the molecular lipid species levels which again coincided with the development of resistance to FLC. Reverse transcriptase-PCR of the key genes of the lipid metabolism validated lipidomic picture. Taken together, this study illustrates how the gradual corrective changes in Candida lipidome correspond to the development of FLC tolerance. Our study also shows a first instance of the mitochondrial membrane dysfunction and defective cell wall (CW) in clinical AR isolates of C. albicans, and provides evidence of a cross-talk between mitochondrial lipid homeostasis, CW integrity and azole tolerance.


Assuntos
Antifúngicos/farmacologia , Candida albicans/metabolismo , Parede Celular , Fluconazol/farmacologia , Lipídeos , Mitocôndrias/metabolismo , Candida albicans/efeitos dos fármacos , Farmacorresistência Fúngica , Membranas Mitocondriais , Reação em Cadeia da Polimerase Via Transcriptase Reversa
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA