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1.
BMC Cancer ; 19(1): 640, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31253124

RESUMO

BACKGROUND: In the management of patients with RAS wild-type metastatic colorectal cancer (mCRC), anti-epidermal growth factor receptor (EGFR) therapies have demonstrated a clinical benefit, with longer survival. However, the correlation between the emergence of circulating RAS mutations and secondary resistance to anti-EGFR therapies requires further elucidation. In this study, we aim to examine evolutionary changes in RAS mutations through liquid biopsy in patients with mCRC during and after anti-EGFR therapy. METHODS: A total of 120 patients diagnosed with RAS wild-type mCRC will be enrolled in this study. Patients will receive a cetuximab-based infusional 5-fluorouracil regimen as first-line treatment. Cetuximab-based treatment is expected to continue until disease progression, intolerable toxic effects, or withdrawal of consent. Blood samples from enrolled patients will be collected before and then every 3 months during cetuximab-based treatment and also at disease progression. These blood samples will be evaluated for RAS resistance mutations by using the MassARRAY platform. The primary endpoint is the percentage of RAS mutations detected in circulating DNA from patients during cetuximab treatment. The correlation between the tumor response and survival outcomes of these patients and the emergence of circulating RAS mutations will be further analyzed. DISCUSSION: Liquid biopsy is a powerful technology that can represent tumor heterogeneity in a relatively noninvasive manner. Because RAS mutations play a major role in resistance to anti-EGFR therapy for mCRC, examining evolutionary changes in these mutations during such treatment through liquid biopsy would be useful. After comprehensively analyzing the emergence of circulating RAS mutations and its clinical relevance in this study, our results should provide practical guidance on anti-EGFR therapy for mCRC. TRIAL REGISTRATION: The date of trial registration ( NCT03401957 ) in this study was January 17, 2018.


Assuntos
Antineoplásicos Imunológicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Cetuximab/administração & dosagem , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Proteínas ras/genética , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/efeitos adversos , Antineoplásicos Imunológicos/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Cetuximab/efeitos adversos , Protocolos Clínicos , Neoplasias Colorretais/patologia , Neoplasias Colorretais/secundário , Fluoruracila/administração & dosagem , Fluoruracila/efeitos adversos , Humanos , Biópsia Líquida , Mutação , Análise de Sobrevida , Resultado do Tratamento , Proteínas ras/sangue
2.
Biochemistry (Mosc) ; 84(5): 562-569, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31234770

RESUMO

Amplification of GC-rich regions of genomic DNA is hindered either by high stability of DNA double helix or as a result of alternative structure formation by a guanine-rich DNA strand. Such potential G-quadruplex (G4) sequences are fairly common in promoters of the human genome. The efficiency of PCR amplification of promoter sequences for several human oncogenes (MYC, NRAS, TERT, KRAS, KIT) was studied. We demonstrate that the efficiency of DNA polymerase is reduced in the presence of potassium ions. The primer-extension technique localized DNA polymerase stops at the 3'-ends of potential quadruplex sequences. The structural and thermodynamic properties of short G-rich oligonucleotides corresponding to the stops of DNA polymerase were analyzed. These oligonucleotides formed stable parallel G4 in the presence of potassium ions. Correlation between the stability of G4 structure and efficiency of DNA polymerase stops was revealed. The results provide a method for detecting new G4 structures in extended genomic sequences and also clarify the mechanism of inhibition of DNA polymerase in G-rich regions of DNA.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Quadruplex G , Potássio/metabolismo , Dicroísmo Circular , DNA Polimerase Dirigida por DNA/química , Oligonucleotídeos/metabolismo , Reação em Cadeia da Polimerase , Potássio/química , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/genética , Termodinâmica , Proteínas ras/genética
4.
J BUON ; 24(2): 709-714, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31128027

RESUMO

PURPOSE: Ovarian cancer causes considerable mortality in women across the globe. The limited availability of the efficient chemotherapeutic agents and associated side effects of the existing drugs forms a bottle neck in the treatment of ovarian cancer. In this study, the in vitro and in vivo anticancer activity of a plant derived lignan pinoresinol was investigated along with deciphering its mode of action. METHODS: The anticancer activity was evaluated by MTT cell viability assay and its effects on mitochondrial membrane potential loss was checked by flow cytometry. Effects on cell invasion were measured by Matrigel invasion assay, whereas effects on autophagy were evaluated by electron microscopy and Western blotting assay. Protein expressions of the phosphor (p)-MEK and p-ERK were measured by Western blot. RESULTS: Results revealed that Pinoresinol inhibits the growth of the ovarian SKOV-3 cancer cells and exhibits an IC50 of 20 µM. The anticancer effects were found to be due to the induction of autophagy which was associated with increase in the expression of LC3 II and Beclin and decrease in the expression of p62. Furthermore, pinoresinol also caused reduction in the mitochondrial membrane potential (MMP) of the SKOV-3 cells and inhibited their invasion capacity. The effects of pinoresinol were also investigated on the Raf/MEK/ERK signalling pathway and it was observed that pinoresinol inhibited the expression of phosphore (p)-MEK and p-ERK in a concentration-dependent manner. Finally, in vivo evaluation revealed that pinoresinol significantly inhibited the growth of xenografted tumors in mice, indicating the potential of pinoresinol in the treatment of ovarian cancer. CONCLUSIONS: Pinoresinol, as per the current study, has the potential to inhibit in vitro and in vivo cancer cell growth of SKOV-3 human ovarian cancer cells and as such could be a possible drug candidate for future research.


Assuntos
Autofagia/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Furanos/farmacologia , Lignanas/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Proteína Beclina-1/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Proteínas Associadas aos Microtúbulos/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/genética , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Proteínas de Ligação a RNA/genética , Proteínas ras/genética
5.
Neoplasia ; 21(6): 582-590, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31054497

RESUMO

BACKGROUND: Low-grade serous ovarian cancer (LGSOC) is a rare subtype of epithelial ovarian carcinoma. Limited data regarding the molecular-genetic background exist beyond mutations in the RAS signaling pathway. There is a growing need to better characterize these tumors due to chemoresistance and limited therapeutic options in advanced or recurrent disease. METHODS: We performed genome-wide copy number aberration (CNA) profiles and mutation hotspot screening (KRAS, BRAF, NRAS, ERBB2, PIK3CA, TP53) in 38 LGSOC tumor samples. RESULTS: We detected mutations in the RAS-signaling pathway in 36.8% of cases, including seven KRAS, four BRAF, and three NRAS mutations. We identified two mutations in PIK3CA and one mutation in MAP3K1, EGFR, and TP53. CNAs were detected in 86.5% of cases. None of the focal aberrations was correlated with specific clinical characteristics. The most frequently detected CNA was loss of 1p36.33 in 54.1% of cases, with a trend towards lower progression-free survival and overall survival in patients with 1p36.33 loss. CONCLUSIONS: Activating RAS mutations were dominant in our series, with supplementary detection of two PIK3CA mutations which may lead to therapeutic options. Furthermore, we detected 1p36.33 deletions in half of the cases, indicating a role in tumorigenesis, and these deletions may serve as a prognostic marker.


Assuntos
Cistadenocarcinoma Seroso/genética , Variações do Número de Cópias de DNA/genética , Recidiva Local de Neoplasia/genética , Neoplasias Ovarianas/genética , Adulto , Idoso , Cromossomos Humanos Par 1/genética , Classe I de Fosfatidilinositol 3-Quinases/genética , Cistadenocarcinoma Seroso/patologia , Receptores ErbB/genética , Feminino , Genoma Humano/genética , Genótipo , Humanos , MAP Quinase Quinase Quinase 1/genética , Pessoa de Meia-Idade , Mutação , Gradação de Tumores , Neoplasias Ovarianas/patologia , Intervalo Livre de Progressão , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína Supressora de Tumor p53/genética , Proteínas ras/genética
6.
BMC Cancer ; 19(1): 441, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088402

RESUMO

BACKGROUND: Glioblastoma is a disease with high heterogeneity that has long been difficult for doctors to identify and treat. ARHI is a remarkable tumor suppressor gene in human ovarian cancer and many other cancers. We found over-expression of ARHI can also inhibit cancer cell proliferation, decrease tumorigenicity, and induce autophagic cell death in human glioma and inhibition of the late stage of autophagy can further enhance the antitumor effect of ARHI through inducing apoptosis in vitro or vivo. METHODS: Using MTT assay to detect cell viability. The colony formation assay was used to measure single cell clonogenicity. Autophagy associated morphological changes were tested by transmission electron microscopy. Flow cytometry and TUNEL staining were used to measure the apoptosis rate. Autophagy inhibitor chloroquine (CQ) was used to study the effects of inhibition at late stage of autophagy on ARHI-induced autophagy and apoptosis. Protein expression were detected by Western blot, immunofluorescence and immunohistochemical analyses. LN229-derived xenografts were established to observe the effect of ARHI in vivo. RESULTS: ARHI induced autophagic death in glioma cells, and blocking late-stage autophagy markedly enhanced the antiproliferative activites of ARHI. In our research, we observed the inhibition of RAS-AKT-mTOR signaling in ARHI-glioma cells and blockade of autophagy flux at late stage by CQ enhanced the cytotoxicity of ARHI, caused accumulation of autophagic vacuoles and robust apoptosis. As a result, the inhibition of RAS augmented autophagy of glioma cells. CONCLUSION: ARHI may also be a functional tumor suppressor in glioma. And chloroquine (CQ) used as an auxiliary medicine in glioma chemotherapy can enhance the antitumor effect of ARHI, and this study provides a novel mechanistic basis and strategy for glioma therapy.


Assuntos
Neoplasias Encefálicas/patologia , Glioblastoma/patologia , Proteínas ras/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Autofagia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Cloroquina/farmacologia , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteínas ras/genética , Proteínas rho de Ligação ao GTP/genética
7.
Nat Commun ; 10(1): 1897, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015486

RESUMO

The cellular decision regarding whether to undergo proliferation or death is made at the restriction (R)-point, which is disrupted in nearly all tumors. The identity of the molecular mechanisms that govern the R-point decision is one of the fundamental issues in cell biology. We found that early after mitogenic stimulation, RUNX3 binds to its target loci, where it opens chromatin structure by sequential recruitment of Trithorax group proteins and cell-cycle regulators to drive cells to the R-point. Soon after, RUNX3 closes these loci by recruiting Polycomb repressor complexes, causing the cell to pass through the R-point toward S phase. If the RAS signal is constitutively activated, RUNX3 inhibits cell cycle progression by maintaining R-point-associated genes in an open structure. Our results identify RUNX3 as a pioneer factor for the R-point and reveal the molecular mechanisms by which appropriate chromatin modifiers are selectively recruited to target loci for appropriate R-point decisions.


Assuntos
Pontos de Checagem do Ciclo Celular/genética , Cromatina/química , Subunidade alfa 3 de Fator de Ligação ao Core/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Animais , Butadienos/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Subunidade alfa 3 de Fator de Ligação ao Core/antagonistas & inibidores , Subunidade alfa 3 de Fator de Ligação ao Core/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Células HEK293 , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Imidazóis/farmacologia , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 4/antagonistas & inibidores , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Nitrilos/farmacologia , Piperazinas/farmacologia , Proteínas do Grupo Polycomb/genética , Proteínas do Grupo Polycomb/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
8.
Science ; 363(6432): 1226-1230, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30872527

RESUMO

RIT1 oncoproteins have emerged as an etiologic factor in Noonan syndrome and cancer. Despite the resemblance of RIT1 to other members of the Ras small guanosine triphosphatases (GTPases), mutations affecting RIT1 are not found in the classic hotspots but rather in a region near the switch II domain of the protein. We used an isogenic germline knock-in mouse model to study the effects of RIT1 mutation at the organismal level, which resulted in a phenotype resembling Noonan syndrome. By mass spectrometry, we detected a RIT1 interactor, leucine zipper-like transcription regulator 1 (LZTR1), that acts as an adaptor for protein degradation. Pathogenic mutations affecting either RIT1 or LZTR1 resulted in incomplete degradation of RIT1. This led to RIT1 accumulation and dysregulated growth factor signaling responses. Our results highlight a mechanism of pathogenesis that relies on impaired protein degradation of the Ras GTPase RIT1.


Assuntos
Síndrome de Noonan/genética , Proteínas Oncogênicas/metabolismo , Proteólise , Fatores de Transcrição/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo , Animais , Técnicas de Introdução de Genes , Mutação em Linhagem Germinativa , Células HEK293 , Células HeLa , Humanos , Espectrometria de Massas , Camundongos , Camundongos Mutantes , Proteínas Oncogênicas/genética , Fatores de Transcrição/genética
10.
Mol Med Rep ; 19(4): 3255-3262, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816469

RESUMO

Repulsive guidance molecule a (RGMa) is a membrane­associated glycoprotein that regulates axonal guidance and inhibits axon outgrowth. In our previous study, we hypothesized that RGMa may be involved in temporal lobe epilepsy (TLE) via the repulsive guidance molecule a (RGMa)­focal adhesion kinase (FAK)­Ras signaling pathway. To investigate the role of RGMa in epilepsy, recombinant RGMa protein and FAK inhibitor 14 was intracerebroventricularly injected into a pentylenetetrazol (PTZ) kindling model and Timm staining, co­immunoprecipitation and western blotting analyses were subsequently performed. The results of the present study revealed that intracerebroventricular injection of recombinant RGMa protein reduced the phosphorylation of FAK (Tyr397) and intracerebroventricular injection of FAK inhibitor 14 reduced the interaction between FAK and p120GAP, as wells as Ras expression. Recombinant RGMa protein and FAK inhibitor 14 exerted seizure­suppressant effects; however, recombinant RGMa protein but not FAK inhibitor 14 suppressed mossy fiber sprouting in the PTZ kindling model. Collectively, these results demonstrated that RGMa may be considered as a potential therapeutic agent for epilepsy, and that RGMa may exert the aforementioned biological effects partly via the FAK­p120GAP­Ras signaling pathway.


Assuntos
Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Glicoproteínas de Membrana/metabolismo , Fibras Musgosas Hipocampais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Convulsões/metabolismo , Convulsões/fisiopatologia , Transdução de Sinais , Proteína p120 Ativadora de GTPase/metabolismo , Proteínas ras/metabolismo , Animais , Modelos Animais de Doenças , Expressão Gênica , Masculino , Glicoproteínas de Membrana/administração & dosagem , Fibras Musgosas Hipocampais/fisiopatologia , Proteínas do Tecido Nervoso/administração & dosagem , Pentilenotetrazol/efeitos adversos , Fosforilação , Ligação Proteica , Ratos , Proteínas Recombinantes , Convulsões/tratamento farmacológico , Convulsões/etiologia , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/genética
11.
EBioMedicine ; 42: 43-53, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30898653

RESUMO

BACKGROUND: Noonan syndrome (NS) is a genetic disorder characterized by short stature, a distinctive facial appearance, and heart defects. We recently discovered a novel NS gene, RIT1, which is a member of the RAS subfamily of small GTPases. NS patients with RIT1 mutations have a high incidence of hypertrophic cardiomyopathy and edematous phenotype, but the specific role of RIT1 remains unclear. METHODS: To investigate how germline RIT1 mutations cause NS, we generated knock-in mice that carried a NS-associated Rit1 A57G mutation (Rit1A57G/+). We investigated the phenotypes of Rit1A57G/+ mice in fetal and adult stages as well as the effects of isoproterenol on cardiac function in Rit1A57G/+ mice. FINDINGS: Rit1A57G/+ embryos exhibited decreased viability, edema, subcutaneous hemorrhage and AKT activation. Surviving Rit1A57G/+ mice had a short stature, craniofacial abnormalities and splenomegaly. Cardiac hypertrophy and cardiac fibrosis with increased expression of S100A4, vimentin and periostin were observed in Rit1A57G/+ mice compared to Rit1+/+ mice. Upon isoproterenol stimulation, cardiac fibrosis was drastically increased in Rit1A57G/+ mice. Phosphorylated (at Thr308) AKT levels were also elevated in isoproterenol-treated Rit1A57G/+ hearts. INTERPRETATION: The A57G mutation in Rit1 causes cardiac hypertrophy, fibrosis and other NS-associated features. Biochemical analysis indicates that the AKT signaling pathway might be related to downstream signaling in the RIT1 A57G mutant at a developmental stage and under ß-adrenergic stimulation in the heart. FUND: The Grants-in-Aid were provided by the Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development, the Japan Society for the Promotion of Science KAKENHI Grant.


Assuntos
Cardiomegalia/etiologia , Cardiomegalia/patologia , Mutação , Síndrome de Noonan/complicações , Síndrome de Noonan/genética , Proteínas ras/genética , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Agonistas Adrenérgicos beta , Alelos , Animais , Cardiomegalia/diagnóstico , Modelos Animais de Doenças , Ecocardiografia , Feminino , Fibrose , Estudos de Associação Genética , Loci Gênicos , Mutação em Linhagem Germinativa , Testes de Função Cardíaca , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Masculino , Camundongos , Camundongos Transgênicos , Síndrome de Noonan/diagnóstico , Síndrome de Noonan/mortalidade , Fenótipo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais
12.
Phytomedicine ; 59: 152895, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30913453

RESUMO

BACKGROUND: There have been some reports implicating the pharmacologic action of Dihydrosanguinarine (DHSA), but little research including the effects of it on cancer cells. PANC-1 cells have mutations in K-Ras and TP53, which respectively express mutant K-Ras and p53 protein, and the mutations in Ras/p53 have been believed with closely relationship to the occurrence of various tumors. PURPOSE: To reveal the inhibition of Dihydrosanguinarine on pancreatic cancer cells (PANC-1 and SW1990) proliferation by inducing G0/G1 and G2/M phase arrest via the downregulation of mut-p53 protein, inducing apoptosis and inhibiting invasiveness through the Ras/Mek/Erk signaling pathway. METHODS: Human pancreatic cancer cell lines were cultured with cisplatin and DHSA. Then, cell proliferation, the cell cycle and apoptosis were measured by CCK-8 and flow cytometry. The migratory and invasive abilities of pancreatic cancer cells were evaluated by transwell assay. The expression levels of mRNA and protein were measured by RT-PCR and western blotting. RESULTS: The results showed that DHSA treatment inhibited cell proliferation, migration and invasion in a time- and dose-dependent manner and led to induction of cell cycle arrest and apoptosis. G0/G1 and G2/M phase arrest inhibited the viability of PANC-1 cells by downregulating the expression of mut-p53 protein. Decreased levels of C-Raf and Erk phosphorylation in DHSA-treated PANC-1 and SW1990 cells were observed in a time- and dose-dependent manner. However, the total expression of p53 and Ras proteins had a different change in PANC-1 and SW1990 cells. CONCLUSIONS: Our findings offer the novel perspective that DHSA inhibits pancreatic cancer cells through a bidirectional regulation between mut-p53/-Ras and WT-p53/-Ras to restore the dynamic balance by Ras and p53 proteins.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Benzofenantridinas/farmacologia , Isoquinolinas/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Apoptose/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas p21(ras)/genética , Quinases raf/genética , Quinases raf/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
13.
BMC Cancer ; 19(1): 280, 2019 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-30922269

RESUMO

BACKGROUND: A first-line biologic treatment for metastatic colorectal cancer (mCRC) is still controversial. We, therefore, performed a meta-analysis to determine the efficacy of first-line cetuximab versus bevacizumab for RAS and BRAF wild-type mCRC. METHODS: In March 2018, an electronic search of the following biomedical databases was performed: PubMed, EMBASE, Cochrane Library, ClinicalTrials.gov and Web of Knowledge. Randomized controlled trials (RCTs) and prospective or observational cohort studies (OCSs) were included. Subgroup analyses of all RCTs were performed in all outcomes. All statistical analyses were performed using RevMan software 5.3. RESULTS: Two RCTs and three OCSs, involving a total 2576 patients, were included. The meta-analysis reported that cetuximab was associated with a longer overall survival (OS) [HR 0.89, 95% CI (0.81-0.98); p = 0.02], a higher ORR [RR 1.11, 95% CI (1.03-1.19); p = 0.006], higher complete response [RR 3.21, 95% CI (1.27-8.12); p = 0.01] and a greater median depth of response than bevacizumab. However, no significant difference was observed between cetuximab and bevacizumab groups for PFS, DCR, partial response, progressive disease, curative intent metastasectomy, EORR and incidence of grade 3 or higher adverse events. In the subgroup meta-analyses of the RCTs, inconsistent results compared to the main analysis, however, were found, in the ORR, DCR and curative intent metastasectomy. CONCLUSIONS: The current evidence indicates that compared to bevacizumab treatment, cetuximab provides a clinically relevant effect in first-line treatment against mCRC, at the cost of having lower stable disease.


Assuntos
Bevacizumab/uso terapêutico , Cetuximab/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/genética , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Observacionais como Assunto , Proteínas Proto-Oncogênicas B-raf/genética , Ensaios Clínicos Controlados Aleatórios como Assunto , Análise de Sobrevida , Resultado do Tratamento , Adulto Jovem , Proteínas ras/genética
14.
Int J Med Sci ; 16(3): 450-460, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30911279

RESUMO

Papillary thyroid cancer (PTC) is the most prevalent form of malignancy among all cancers of the thyroid. It is also one of the few cancers with a rapidly increasing incidence. PTC is usually contained within the thyroid gland and generally biologically indolent. Prognosis of the cancer is excellent, with less than 2% mortality at 5 years. However, more than 25% of patients with PTC developed a recurrence during a long term follow-up. The present article provides an updated condensed overview of PTC, which focuses mainly on the molecular alterations involved and recent biomarker investigations.


Assuntos
Biomarcadores Tumorais/genética , Câncer Papilífero da Tireoide/genética , Neoplasias da Glândula Tireoide/genética , Biomarcadores Tumorais/metabolismo , Feminino , Bócio Nodular/complicações , Humanos , Masculino , MicroRNAs , Recidiva Local de Neoplasia/genética , Proteômica , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-ret/genética , Câncer Papilífero da Tireoide/diagnóstico , Câncer Papilífero da Tireoide/terapia , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/terapia , Proteínas ras/genética
15.
PLoS Genet ; 15(3): e1008076, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30925167

RESUMO

Organoid cultures derived from colorectal cancer (CRC) samples are increasingly used as preclinical models for studying tumor biology and the effects of targeted therapies under conditions capturing in vitro the genetic make-up of heterogeneous and even individual neoplasms. While 3D cultures are initiated from surgical specimens comprising multiple cell populations, the impact of tumor heterogeneity on drug effects in organoid cultures has not been addressed systematically. Here we have used a cohort of well-characterized CRC organoids to study the influence of tumor heterogeneity on the activity of the KRAS/MAPK-signaling pathway and the consequences of treatment by inhibitors targeting EGFR and downstream effectors. MAPK signaling, analyzed by targeted proteomics, shows unexpected heterogeneity irrespective of RAS mutations and is associated with variable responses to EGFR inhibition. In addition, we obtained evidence for intratumoral heterogeneity in drug response among parallel "sibling" 3D cultures established from a single KRAS-mutant CRC. Our results imply that separate testing of drug effects in multiple subpopulations may help to elucidate molecular correlates of tumor heterogeneity and to improve therapy response prediction in patients.


Assuntos
Técnicas de Cultura de Células/métodos , Neoplasias Colorretais/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Linhagem Celular Tumoral , Estudos de Coortes , Neoplasias Colorretais/fisiopatologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Genes erbB-1 , Humanos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Mutação , Organoides/metabolismo , Organoides/fisiologia , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Transdução de Sinais , Proteínas ras/genética
16.
Nutrients ; 11(3)2019 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-30871060

RESUMO

Glycyrrhetinic acid (GA), a bioactive component in the human diet, has been reported to improve hyperglycemia, dyslipidemia, insulin resistance and obesity in rats with metabolic syndrome. However, GA-specific target proteins and the mechanisms involved in the downstream signaling and cross-talk to improve insulin sensitivity have not been fully elucidated. In this study, the potential targets of GA were identified by chemical proteomics strategies using serial GA probes for target fishing and cell molecular imaging. Intracellular enzyme activity evaluation and insulin resistance models were used for validating the function of the target proteins on the downstream insulin signaling pathways. Collectively, our data demonstrate that GA improved the insulin-responsive pathway and glucose consumption levels via multiple diabetogenic factors that activated the insulin signaling pathway in HepG2 cells. GA improved Glucose transporter 4(GLUT4) expression by targeting the Ras protein to regulate the mitogen-activated protein kinase (MAPK) pathway. GA exhibited a strong inhibitory effect on IRS1ser307 phosphorylation in cells treated with the Protein kinase C (PKC) activator Phorbol 12-myristate 13-acetate (PMA.) Consistently, IRS1ser307 phosphorylation was also inhibited by GA in Free fatty acid (FFA)-treated HepG2 cells. GA also inhibited the PMA-induced phosphorylation of IκB kinase α/ß (IKKα/ß), c-Jun N-terminal kinase (JNK) and p38 proteins (P38), suggesting that IKKα/ß, JNK and P38 activation is dependent on PKC activity.


Assuntos
Ácido Glicirretínico/farmacologia , Insulina/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas ras/metabolismo , Animais , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Glucose/administração & dosagem , Glucose/metabolismo , Células Hep G2 , Humanos , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas ras/genética
17.
G3 (Bethesda) ; 9(4): 1085-1102, 2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30733383

RESUMO

Wsc1p and Mid2p are transmembrane signaling proteins of cell wall stress in the budding yeast Saccharomyces cerevisiae When an environmental stress compromises cell wall integrity, they activate a cell response through the Cell Wall Integrity (CWI) pathway. Studies have shown that the cytoplasmic domain of Wsc1p initiates the CWI signaling cascade by interacting with Rom2p, a Rho1-GDP-GTP exchange factor. Binding of Rom2p to the cytoplasmic tail of Wsc1p requires dephosphorylation of specific serine residues but the mechanism by which the sensor is dephosphorylated and how it subsequently interacts with Rom2p remains unclear. We hypothesize that Wsc1p and Mid2p must be physically associated with interacting proteins other than Rom2p that facilitate its interaction and regulate the activation of CWI pathway. To address this, a cDNA plasmid library of yeast proteins was expressed in bait strains bearing membrane yeast two-hybrid (MYTH) reporter modules of Wsc1p and Mid2p, and their interacting preys were recovered and sequenced. 14 previously unreported interactors were confirmed for Wsc1p and 29 for Mid2p The interactors' functionality were assessed by cell growth assays and CWI pathway activation by western blot analysis of Slt2p/Mpk1p phosphorylation in null mutants of each interactor under defined stress conditions. The susceptibility of these strains to different stresses were tested against antifungal agents and chemicals. This study reports important novel protein interactions of Wsc1p and Mid2p that are associated with the cellular response to oxidative stress induced by Hydrogen Peroxide and cell wall stress induced by Caspofungin.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Glicoproteínas de Membrana/fisiologia , Proteínas de Membrana/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico , Caspofungina/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Peróxido de Hidrogênio/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Espectrometria de Massas , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Estresse Oxidativo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Purificação por Afinidade em Tandem , Proteínas ras/genética , Proteínas ras/metabolismo , Proteínas ras/fisiologia
18.
Mol Genet Genomic Med ; 7(4): e00581, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30784236

RESUMO

BACKGROUND: Noonan syndrome (NS) is an autosomal dominant disorder that belongs to a group of developmental disorders called RASopathies with overlapping features and multiple causative genes. The aim of the study was to identify mutations underlying this disorder in patients from Southeast Asia and characterize their clinical presentations. METHODS: Patients were identified from the hospital's Genetics clinics after assessment by attending clinical geneticists. A targeted gene panel was used for next-generation sequencing on genomic DNA extracted from the blood samples of 17 patients. RESULTS: Heterozygous missense variants were identified in 13 patients: eight were in PTPN11, three in SOS1, and one each in RIT1 and KRAS. All are known variants that have been reported in patients with NS. Of the 13 patients with identified variants, 10 had short stature, the most common feature for NS. Four of the eight patients with PTPN11 variants had atrial septal defect. Only two had pulmonary stenosis which is reported to be common for PTPN11 mutation carriers. Another two had hypertrophic cardiomyopathy, a feature which is negatively associated with PTPN11 mutations. CONCLUSIONS: Our study provides the mutation and phenotypic spectrum of NS from a new population group. The molecular testing yield of 76% is similar to other studies and shows that the targeted panel approach is useful for identifying genetic mutations in NS which has multiple causative genes. The molecular basis for the phenotypes of the remaining patients remains unknown and would need to be uncovered via sequencing of additional genes or other investigative methods.


Assuntos
Taxa de Mutação , Síndrome de Noonan/genética , Fenótipo , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Mutação de Sentido Incorreto , Síndrome de Noonan/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteína SOS1/genética , Singapura , Proteínas ras/genética
19.
PLoS Genet ; 15(2): e1007976, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30742618

RESUMO

Amino acids are among the earliest identified inducers of yeast-to-hyphal transitions in Candida albicans, an opportunistic fungal pathogen of humans. Here, we show that the morphogenic amino acids arginine, ornithine and proline are internalized and metabolized in mitochondria via a PUT1- and PUT2-dependent pathway that results in enhanced ATP production. Elevated ATP levels correlate with Ras1/cAMP/PKA pathway activation and Efg1-induced gene expression. The magnitude of amino acid-induced filamentation is linked to glucose availability; high levels of glucose repress mitochondrial function thereby dampening filamentation. Furthermore, arginine-induced morphogenesis occurs more rapidly and independently of Dur1,2-catalyzed urea degradation, indicating that mitochondrial-generated ATP, not CO2, is the primary morphogenic signal derived from arginine metabolism. The important role of the SPS-sensor of extracellular amino acids in morphogenesis is the consequence of induced amino acid permease gene expression, i.e., SPS-sensor activation enhances the capacity of cells to take up morphogenic amino acids, a requisite for their catabolism. C. albicans cells engulfed by murine macrophages filament, resulting in macrophage lysis. Phagocytosed put1-/- and put2-/- cells do not filament and exhibit reduced viability, consistent with a critical role of mitochondrial proline metabolism in virulence.


Assuntos
Candida albicans/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Prolina/metabolismo , Proteínas ras/metabolismo , Trifosfato de Adenosina/metabolismo , Aminoácidos/metabolismo , Animais , Candida albicans/genética , Candida albicans/patogenicidade , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Fúngicas/genética , Humanos , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Macrófagos/microbiologia , Camundongos , Mitocôndrias/metabolismo , Morfogênese , Prolina Oxidase/genética , Prolina Oxidase/metabolismo , Células RAW 264.7 , Transdução de Sinais , Virulência/fisiologia , Proteínas ras/genética
20.
Mol Biol Cell ; 30(9): 1108-1117, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30785867

RESUMO

HRAS, NRAS, and KRAS isoforms are almost identical proteins that are ubiquitously expressed and activate a common set of effectors. In vivo studies have revealed that they are not biologically redundant; however, the isoform specificity of Ras signaling remains poorly understood. Using a novel panel of isogenic SW48 cell lines endogenously expressing wild-type or G12V-mutated activated Ras isoforms, we have performed a detailed characterization of endogenous isoform-specific mutant Ras signaling. We find that despite displaying significant Ras activation, the downstream outputs of oncogenic Ras mutants are minimal in the absence of growth factor inputs. The lack of mutant KRAS-induced effector activation observed in SW48 cells appears to be representative of a broad panel of colon cancer cell lines harboring mutant KRAS. For MAP kinase pathway activation in KRAS-mutant cells, the requirement for coincident growth factor stimulation occurs at an early point in the Raf activation cycle. Finally, we find that Ras isoform-specific signaling was highly context dependent and did not conform to the dogma derived from ectopic expression studies.


Assuntos
Proteínas ras/genética , Proteínas ras/metabolismo , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Genes ras , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mutação , Isoformas de Proteínas , Transdução de Sinais/fisiologia
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