RESUMO
As a best-characterized epigenetic modification, DNA methylation plays an important role in mammalian development. Uhrf1 is a critical epigenetic regulator that can bind to hemimethylated DNA and recruit DNA methyltransferase 1 to maintain DNA methylation. So far, the role of Uhrf1-mediated DNA methylation in intestinal development is still unknown. In order to investigate the impact of Uhrf1 deletion in intestinal development, we have successfully constructed the epithelial-specific Uhrf1 knockout mouse model. After Uhrf1 ablation, we found the mutant mice exhibited abnormal epithlial structure with less and shorter villi and shrinked crypts compared with wild type mice via hematoxylin-eosin staining. Further analysis showed that Uhrf1 deletion in the intestinal epithelium significantly decreased the cell proliferation and induced cell apoptosis. In addition, Uhrf1 deletion inhibited the normal epithelial differentiation and the expression of intestinal stem cell marker genes. Preliminary mechanism study revealed that loss of Uhrf1 caused global DNA hypomethylation which induced DNA damage in crypt cells. Taken together, our data suggested that DNA methylation mediated by Uhrf1 is vital for the normal intestinal development. Our results enriched the in vivo role of Uhrf1 and laid the foundation for further epigenetic regulatory mechanism exploration.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/fisiologia , Metilação de DNA , Epigênese Genética , Intestinos/crescimento & desenvolvimento , Ubiquitina-Proteína Ligases/fisiologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Deleção de Genes , Camundongos , Camundongos Knockout , Ubiquitina-Proteína Ligases/genéticaRESUMO
The mammalian target of rapamycin (mTOR) signaling pathway integrates environmental cues to regulate cell growth and survival through various mechanisms. However, how mTORC1 responds to acute inflammatory signals to regulate bowel regeneration is still obscure. In this study, we investigated the role of mTORC1 in acute inflammatory bowel disease. Inhibition of mTORC1 activity by rapamycin treatment or haploinsufficiency of Rheb through genetic modification in mice impaired intestinal cell proliferation and induced cell apoptosis, leading to high mortality in dextran sodium sulfate- and 2,4,6-trinitrobenzene sulfonic acid-induced colitis models. Through bone marrow transplantation, we found that mTORC1 in nonhematopoietic cells played a major role in protecting mice from colitis. Reactivation of mTORC1 activity by amino acids had a positive therapeutic effect in mTORC1-deficient Rheb(+/-) mice. Mechanistically, mTORC1 mediated IL-6-induced Stat3 activation in intestinal epithelial cells to stimulate the expression of downstream targets essential for cell proliferation and tissue regeneration. Therefore, mTORC1 signaling critically protects against inflammatory bowel disease through modulation of inflammation-induced Stat3 activity. As mTORC1 is an important therapeutic target for multiple diseases, our findings will have important implications for the clinical usage of mTORC1 inhibitors in patients with acute inflammatory bowel disease.
Assuntos
Colite/imunologia , Proteínas Monoméricas de Ligação ao GTP/imunologia , Complexos Multiproteicos/antagonistas & inibidores , Neuropeptídeos/imunologia , Fator de Transcrição STAT3/imunologia , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Animais , Transplante de Medula Óssea , Células CACO-2 , Proliferação de Células/efeitos dos fármacos , Colite/induzido quimicamente , Colite/genética , Colite/mortalidade , Regulação da Expressão Gênica , Haploinsuficiência , Humanos , Interleucina-6/genética , Interleucina-6/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Monoméricas de Ligação ao GTP/deficiência , Proteínas Monoméricas de Ligação ao GTP/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/imunologia , Neuropeptídeos/deficiência , Neuropeptídeos/genética , Proteína Enriquecida em Homólogo de Ras do Encéfalo , Fator de Transcrição STAT3/genética , Transdução de Sinais , Dodecilsulfato de Sódio , Análise de Sobrevida , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologia , Ácido TrinitrobenzenossulfônicoRESUMO
Cowden syndrome (CS) is a difficult-to-recognize multiple hamartoma syndrome with high risks of breast, thyroid, and other cancers. Germline mutations in PTEN on 10q23 were found to cause 85% of CS when accrued from tertiary academic centers, but prospective accrual from the community over the last 12 years has revealed a 25% PTEN mutation frequency. PTEN is the phosphatase that has been implicated in a heritable cancer syndrome and subsequently in multiple sporadic cancers and developmental processes. PTEN antagonizes the AKT1/PI3K signaling pathway and has roles in cell cycle, migration, cell polarity, and apoptosis. We report that 8 of 91 (8.8%) unrelated CS individuals without germline PTEN mutations carried 10 germline PIK3CA mutations (7 missense, 1 nonsense, and 2 indels) and 2 (2.2%) AKT1 mutations. These mutations result in significantly increased P-Thr308-AKT and increased cellular PIP3. Our observations suggest that PIK3CA and AKT1 are CS susceptibility genes.
Assuntos
Síndrome do Hamartoma Múltiplo/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Classe I de Fosfatidilinositol 3-Quinases , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Humanos , PTEN Fosfo-HidrolaseRESUMO
Eight new fungal polyketides named koningiopisins A-H (1-8) and four previously known polyketides (9-12) were isolated from the endophytic fungus Trichoderma koningiopsis YIM PH 30002. Their structures were elucidated using extensive spectral data interpretation, and their antifungal and synergistic activities were also evaluated. Koningiopisin C (3) exhibited in vitro antifungal activity against the phytopathogenic fungus Plectosphaerella cucumerina with an MIC of 16 µg/mL. Although the antifungal activities of single compounds were not obvious, a mixture of six compounds (4-9) exhibited potent synergistic antifungal activity against P. cucumerina with an MIC of 16 µg/mL, and the antifungal activity of the mixture of any two compounds with a 1:1 ratio was better than that observed from the individual compound. The synergistic biological activity of the metabolites in YIM PH 30002 demonstrates the significant ecological function of the endophyte for its host plant, and provides additional insight into the search for and development of agents for biological control.
Assuntos
Antifúngicos/isolamento & purificação , Policetídeos/isolamento & purificação , Trichoderma/química , Antifúngicos/química , Antifúngicos/farmacologia , Ascomicetos/efeitos dos fármacos , Sinergismo Farmacológico , Testes de Sensibilidade Microbiana , Estrutura Molecular , Policetídeos/química , Policetídeos/farmacologiaRESUMO
Hepatocellular carcinoma (HCC) is a disease with a high incidence and mortality rate worldwide. However, the mechanisms underlying its pathogenesis are still elusive. In recent years, studies on functions of Krüppel-like factors (KLFs) in HCC have shed new light on this field. To date, five members (KLF4, KLF6, KLF8, KLF9, and KLF17) in the KLF family have been reported to function in the pathogenesis of HCC in multiple ways, which hold the potential of deepening and widening our understanding in the initiation and progression of HCC. In this review, we focus on the functions, roles, and regulatory networks of these five KLFs in HCC, summarize key pathways, and propose areas for further investigation, with the hope that this review will provide a reliable and concise reference for readers interested in this area.
Assuntos
Carcinoma Hepatocelular/genética , Fatores de Transcrição Kruppel-Like/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genética , Carcinoma Hepatocelular/patologia , Redes Reguladoras de Genes , Humanos , Fator 4 Semelhante a Kruppel , Fator 6 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
The Krüppel-like factor (KLF) family of transcription factors regulates diverse biological processes that include proliferation, differentiation, apoptosis, development, and responses to external stress. In the present study, we aim to investigate the roles of KLF2 in hepatic steatosis. Our results showed that mRNA and protein levels of KLF2 were significantly elevated in livers from obese mice. Adenoviruses-mediated overexpression of KLF2 induced accumulation of triglycerides in C57BL/6 mice, whereas KLF2 silencing ameliorates hepatosteatosis in ob/ob mice. At the molecular level, our data established CD36 as a novel transcriptional target of KLF2. KLF2 upregulated CD36 expression through a consensus binding site on its proximal promoter region. Additionally, the steatotic effect of KLF2 was dramatically inhibited in CD36-null mice. Therefore, our study reveals a novel link between KLF2-induced hepatic triglyceride accumulation and the expression of CD36.
Assuntos
Antígenos CD36/genética , Fígado Gorduroso/metabolismo , Regulação da Expressão Gênica , Fatores de Transcrição Kruppel-Like/fisiologia , Animais , Antígenos CD36/metabolismo , Fígado Gorduroso/genética , Hipertrigliceridemia/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Especificidade de Órgãos , Regiões Promotoras GenéticasRESUMO
Cowden syndrome (CS) and Bannayan-Riley-Ruvalcaba syndrome are allelic, defined by germline PTEN mutations, and collectively referred to as PTEN hamartoma tumor syndrome. To date, there are no existing criteria based on large prospective patient cohorts to select patients for PTEN mutation testing. To address these issues, we conducted a multicenter prospective study in which 3042 probands satisfying relaxed CS clinical criteria were accrued. PTEN mutation scanning, including promoter and large deletion analysis, was performed for all subjects. Pathogenic mutations were identified in 290 individuals (9.5%). To evaluate clinical phenotype and PTEN genotype against protein expression, we performed immunoblotting (PTEN, P-AKT1, P-MAPK1/2) for a patient subset (n = 423). In order to obtain an individualized estimation of pretest probability of germline PTEN mutation, we developed an optimized clinical practice model to identify adult and pediatric patients. For adults, a semiquantitative score-the Cleveland Clinic (CC) score-resulted in a well-calibrated estimation of pretest probability of PTEN status. Overall, decreased PTEN protein expression correlated with PTEN mutation status; decreasing PTEN protein expression correlated with increasing CC score (p < 0.001), but not with the National Comprehensive Cancer Network (NCCN) criteria (p = 0.11). For pediatric patients, we identified highly sensitive criteria to guide PTEN mutation testing, with phenotypic features distinct from the adult setting. Our model improved sensitivity and positive predictive value for germline PTEN mutation relative to the NCCN 2010 criteria in both cohorts. We present the first evidence-based clinical practice model to select patients for genetics referral and PTEN mutation testing, further supported biologically by protein correlation.
Assuntos
Testes Genéticos , Síndrome do Hamartoma Múltiplo/genética , PTEN Fosfo-Hidrolase/genética , Seleção de Pacientes , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Estudos de Coortes , Feminino , Mutação em Linhagem Germinativa , Síndrome do Hamartoma Múltiplo/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Quinases Ativadas por Mitógeno/análise , Proteínas Quinases Ativadas por Mitógeno/genética , Modelos Genéticos , PTEN Fosfo-Hidrolase/metabolismo , Regiões Promotoras Genéticas/genética , Estudos Prospectivos , Proteínas Proto-Oncogênicas c-akt/análise , Proteínas Proto-Oncogênicas c-akt/genética , Adulto JovemRESUMO
BACKGROUND & AIMS: Gastrointestinal polyposis is a common clinical problem, yet there is no consensus on how to best manage patients with moderate-load polyposis. Identifying genetic features of this disorder could improve management and especially surveillance of these patients. We sought to determine the prevalence of hamartomatous polyposis-associated mutations in the susceptibility genes PTEN, BMPR1A, SMAD4, ENG, and STK11 in individuals with ≥5 gastrointestinal polyps, including at least 1 hamartomatous or hyperplastic/serrated polyp. METHODS: We performed a prospective, referral-based study of 603 patients (median age: 51 years; range, 2-89 years) enrolled from June 2006 through January 2012. Genomic DNA was extracted from peripheral lymphocytes and analyzed for specific mutations and large rearrangements in PTEN, BMPR1A, SMAD4, and STK11, as well as mutations in ENG. Recursive partitioning analysis was used to determine cutoffs for continuous variables. The prevalence of mutations was compared using Fisher's exact test. Logistic regression analyses were used to determine univariate and multivariate risk factors. RESULTS: Of 603 patients, 119 (20%) had a personal history of colorectal cancer and most (n = 461 [76%]) had <30 polyps. Seventy-seven patients (13%) were found to have polyposis-associated mutations, including 11 in ENG (1.8%), 13 in PTEN (2.2%), 13 in STK11 (2.2%), 20 in BMPR1A (3.3%), and 21 in SMAD4 (3.5%). Univariate clinical predictors for risk of having these mutations included age at presentation younger than 40 years (19% vs 10%; P = .008), a polyp burden of ≥30 (19% vs 11%; P = .014), and male sex (16% vs 10%; P = .03). Patients who had ≥1 ganglioneuroma (29% vs 2%; P < .001) or presented with polyps of ≥3 histologic types (20% vs 2%; P = .003) were more likely to have germline mutations in PTEN. CONCLUSIONS: Age younger than 40 years, male sex, and specific polyp histologies are significantly associated with risk of germline mutations in hamartomatous-polyposis associated genes. These associations could guide clinical decision making and further investigations.
Assuntos
Antígenos CD/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Pólipos do Colo/genética , Mutação em Linhagem Germinativa , PTEN Fosfo-Hidrolase/genética , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/genética , Proteína Smad4/genética , Quinases Proteína-Quinases Ativadas por AMP , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Endoglina , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome de Peutz-Jeghers/genética , Estudos Prospectivos , Adulto JovemRESUMO
There is still a lack of satisfactory tumor markers for gastric cancer (GasC). This study is aimed at optimizing parameters in CE-MS based on moving reaction boundary (MRB) so as to improve its sensitivity and stability, and at searching for potential tumor markers of GasC in patients' urine samples via MRB-CE-MS. In this study, several parameters of MRB-CE-MS were investigated and optimized in order to gain optimal stability, sensitivity, and specificity, and was afterwards evaluated as valid. Subsequently, urine samples from GasC patients and control subjects were subjected to MRB-CE-MS analysis under optimized conditions, which successfully distinguished GasC patients from controls, as well as early-stage patients from advanced stage patients. Differentiation performance was evaluated by area under the curve, which showed fine differential value between GasC patients and controls, as well as between early and advanced stage patients (area under the curve value 1.0 and 0.847, respectively). In conclusion, this study established a set of feasible and useful methodology in searching potential tumor markers in urine samples from GasC patients. Moreover, several amino acids have been recognized as potential tumor markers that deserve further investigation.
Assuntos
Biomarcadores Tumorais/urina , Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Metaboloma/fisiologia , Neoplasias Gástricas/urina , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Metabolômica , Pessoa de Meia-IdadeRESUMO
BACKGROUND: Pancreatic cancer (PC) is a highly deadly malignancy with few effective therapies. We aimed to unmask the role that long non-coding RNA small nucleolar RNA host gene 6 (SNHG6) plays in PC cells by targeting far upstream element binding protein 1 (FUBP1) via microRNA-26a-5p (miR-26a-5p). METHODS: SNHG6 expression was predicted by bioinformatics, followed by verification via reverse transcription quantitative polymerase chain reaction. Then, the interactions among SNHG6, miR-26a-5p, and FUBP1 were detected through online software analysis, dual luciferase reporter assay and RNA pull-down. After that, cells were treated with different small interfering RNAs and/or mimic to determine the interactions among SNHG6, miR-26a-5p, and FUBP1 and their roles in PC cells. Finally, the role of SNHG6 in tumor growth in vivo was evaluated by measuring the growth and weight of transplanted tumors in nude mice. A t-test, one-way and two-way analysis of variance were used for data analysis. RESULTS: Compared with that in normal tissues, SNHG6 was highly expressed in PC tissues (1.00â±â0.05 vs. 1.56â±â0.06, tâ=â16.03, Pâ<â0.001). Compared with that in human pancreatic duct epithelial cells (HPDE6-C7), SNHG6 showed the highest expression in PANC-1 cells (1.00â±â0.06 vs. 3.87â±â0.13, tâ=â34.72, Pâ<â0.001) and the lowest expression in human pancreatic cancer cells (MIAPaCa-2) (1.00â±â0.06 vs. 1.41â±â0.07, tâ=â7.70, Pâ=â0.0015). Compared with the levels in the si-negative control group, SNHG6 (0.97â±â0.05 vs. 0.21â±â0.06, tâ=â16.85, Pâ<â0.001), N-cadherin (0.74â±â0.05 vs. 0.41â±â0.04, tâ=â8.93, Pâ<â0.001), Vimentin (0.55â±â0.04 vs. 0.25â±â0.03, tâ=â10.39, Pâ<â0.001), and ß-catenin (0.62â±â0.05 vs. 0.32â±â0.03, tâ=â8.91, Pâ<â0.001) were decreased, while E-cadherin (0.65â±â0.06 vs. 1.36â±â0.07, tâ=â13.34, Pâ<â0.001) was increased after SNHG6 knockdown or miR-26a-5p overexpression, accompanied by inhibited cell proliferation, migration, and invasion. SNHG6 overexpression exerted the opposite effects. SNHG6 upregulated FUBP1 expression by sponging miR-26a-5p. Silencing SNHG6 blocked the growth of PC in vivo. CONCLUSION: Silencing SNHG6 might ameliorate PC through inhibition of FUBP1 by sponging miR-26a-5p, thus providing further supporting evidence for its use in PC treatment.
Assuntos
Proteínas de Ligação a DNA , MicroRNAs , Neoplasias Pancreáticas , RNA Longo não Codificante , Proteínas de Ligação a RNA , Animais , Proliferação de Células/genética , Humanos , Camundongos , Camundongos Nus , MicroRNAs/genética , Neoplasias Pancreáticas/genética , RNA Longo não Codificante/genética , RNA Nucleolar Pequeno , Regulação para CimaRESUMO
Previous circular RNA (circRNA) microarray analyses have uncovered an abnormal expression of hsa_circ_0070963 in hepatic stellate cells (HSCs). However, the specific role of hsa_circ_0070963 in liver fibrosis remains unknown. Here, we show that hsa_circ_0070963 inhibits liver fibrosis via regulation of miR-223-3p and LEMD3. Moreover, we demonstrated that hsa_circ_0070963 levels were reduced during liver fibrosis while restoring hsa_circ_0070963 levels abolished HSC activation, with a reduction in α-SMA and type I collagen levels both in vitro and in vivo. Furthermore, hsa_circ_0070963 overexpression suppressed both cell proliferation and the cell cycle of HSCs. MiR-223-3p was confirmed as a target of hsa_circ_0070963 and was shown to be involved in the effects of hsa_circ_0070963 on HSC activation. Furthermore, LEMD3 was confirmed as a target of miR-223-3p and was shown to be responsible for the activation of HSCs. The interactions between hsa_circ_0070963, miR-223-3p, and LEMD3 were validated via bioinformatic analysis, luciferase reporter assays, and rescue experiments. Collectively, hsa_circ_0070963 appeared to function as a miR-223-3p sponge that inhibited HSC activation in liver fibrosis via regulation of miR-223-3p and LEMD3. Therefore, hsa_circ_0070963 may serve as a potential therapeutic target for liver fibrosis.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação Neoplásica da Expressão Gênica , Cirrose Hepática/etiologia , Proteínas de Membrana/genética , MicroRNAs/genética , Linhagem Celular , Predisposição Genética para Doença , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/patologia , Interferência de RNARESUMO
OBJECTIVE: To investigate the effect of N-desulfated heparin on tumor metastasis, tumor angiogenesis and basic fibroblast growth factor(bFGF) gene expression of orthotopically implanted human gastric carcinoma in NOD-SCID mice. METHODS: Human gastric cancer SGC-7901 tissues were orthotopically implanted into the stomach of the NOD-SCID mice. Twenty mice were randomly divided into two groups which received either intravenous injection of 0.9% NaCl solution(0.9%NaCl solution group) or 10 mg/kg N-desulfated heparin (N-desulfated heparin group) twice a week for three weeks. Mice were sacrificed six weeks after tumor implantation. Tissues from stomach and other organs were obtained for histopathological evaluation. The intratumoral microvessel density (MVD) in tumor was evaluated immunohistochemically. Real time PCR was used to detect bFGF mRNA expression. RESULTS: The tumor metastasis rates were 9/10 in 0.9% NaCl solution group and 2/10 in N-desulfated heparin group(P<0.05).MVD was 9.1+/-3.4 in 0.9% NaCl solution group and 4.7+/-1.8 in N-desulfated heparin group (t=3.617,P<0.05). bFGF mRNA expression was lower in N-desulfated heparin group(2.60+/-0.56%)than that in 0.9% NaCl solution group(30.65+/-6.84%). CONCLUSION: N-desulfated heparin can inhibit the metastasis of gastric cancer through inhibiting tumor bFGF gene expression and tumor angiogenesis with no obvious anticoagulant activity.
Assuntos
Fator 2 de Crescimento de Fibroblastos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Heparina/análogos & derivados , Transplante de Neoplasias , Neovascularização Patológica/tratamento farmacológico , Neoplasias Gástricas/irrigação sanguínea , Neoplasias Gástricas/genética , Animais , Regulação Neoplásica da Expressão Gênica/genética , Heparina/farmacologia , Heparina/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias Gástricas/tratamento farmacológicoRESUMO
AIM: To investigate the synergistic effect of oxymatrine (OM) and angiogenesis inhibitor NM-3 on modulating apoptosis in human gastric cancer cell lines SGC-7901, MKN-45, MKN-74. METHODS: Human gastric cancer lines SGC-7901, MKN-45, MKN-74 were treated with OM in the absence and presence of NM-3. The inhibitory rates were detected by MTT assay. Synergistic effect of OM and NM-3 on the growth of survivin, bcl-2, bax and p53 in SGC-7901 cells were examined by semiquantitative RT-PCR and Western blotting, and their growth inhibitory effects were also observed on SGC-7901 tumor xenograft in nude mice. RESULTS: OM combined with NM-3 exhibited a synergistic inhibitory effect on the growth of SGC-7901, MKN-45 and MKN-74 cells in a time-dependent manner. Twenty-four hours after treatment with OM, NM-3 alone and their combination, mRNA expression of survivin and bcl-2 in SGC-7901 cells decreased, p53 mRNA expression increased. OM (4 g/L) combined with NM-3 significantly increased the expression of p53 mRNA and decreased the expression of survivin and bcl-2 compared with either agent alone (193% +/- 34% vs 129% +/- 12%; 44% +/- 18% vs 92% +/- 18%; 36 +/- 17% vs 93% +/- 23%, P < 0.05). Western blotting showed that the synergistic effect of OM and NM-3 on protein translation of survivin, bcl-2 and p53 was in accordance with their mRNAs. Furthermore, OM/NM-3 combination obviously exhibited antitumor growth effect in xenografted human gastric cancer cells SGC-7901 compared with either agent alone. CONCLUSION: OM combined with NM-3 has synergistic inhibitory effects on human gastric cancer cells in vitro and can suppress the growth of xenografted human gastric cancer cells SGC-7901 in vivo.
Assuntos
Alcaloides/farmacologia , Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Isocumarinas/farmacologia , Quinolizinas/farmacologia , Neoplasias Gástricas/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Inibidoras de Apoptose , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Survivina , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
AIM: To investigate the effect of N-desulfated heparin on tumor metastasis and angiogenesis, and expression of vascular endothelial growth factor (VEGF) of orthotopic implantation of human gastric carcinoma in male severe combined immune deficiency (SCID) mice. METHODS: Human gastric cancer SGC-7901 cells were orthotopically implanted into the stomach of SCID mice. The mice were randomly divided into normal saline group and N-desulfated heparin group. One week after operation, the mice in N-desulfated heparin group received i.v. injections of N-desulfated heparin (Shanghai Institute of Cell Biology, Chinese Academy of Sciences, 10 mg/kg.d) twice weekly for 3 wk. The mice in normal saline group received i.v. injections of normal saline (100 microL) twice weekly for 3 wk. The mice were sacrificed six weeks after implantation. Tumor metastasis was evaluated histologically for metastasis under microscope. Intratumoral microvessel density (MVD) and VEGF expression were evaluated immuohistochemically. VEGF mRNA expression in gastric tissue of SCID mice was detected by real time PCR. RESULTS: The tumor metastasis rate was 80% in normal saline group and 20% in N-desulfated heparin group (P < 0.05). MVD was 8.0 +/- 3.1 in normal saline group and 4.3 +/- 1.8 in N-desulfated heparin group (P < 0.05). VEGF positive immunostaining was found in cytoplasm of cancer cells. The rate of VEGF positive expression was higher in normal saline group than in N-desulfated heparin treated group (90% vs 20%, P < 0.05). VEGF mRNA expression was significantly inhibited by N-desulfated heparin and was higher in normal saline group than in N-desulfated heparin group (Ct value 19.51 +/- 1.01 vs 22.55 +/- 1.36, P < 0.05). N-desulfated heparin significantly inhibited the expression of VEGF mRNA in cancer cells. No bleeding occurred in N-desulfated heparin group. CONCLUSION: N-desulfated heparin can inhibit metastasis of gastric cancer by suppressing tumor VEGF expression and tumor angiogenesis, but has no obvious anticoagulant activity.
Assuntos
Carcinoma/tratamento farmacológico , Heparina/uso terapêutico , Neovascularização Patológica/tratamento farmacológico , Neoplasias Gástricas/tratamento farmacológico , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Animais , Carcinoma/irrigação sanguínea , Carcinoma/patologia , Expressão Gênica/efeitos dos fármacos , Heparina/farmacologia , Humanos , Masculino , Camundongos , Camundongos SCID , Metástase Neoplásica/tratamento farmacológico , Neoplasias Gástricas/irrigação sanguínea , Neoplasias Gástricas/patologiaRESUMO
AIM: To investigate the effects of 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid (NM-3) alone and in combination with carboplatin on tumor growth and apoptosis in mouse models of human gastric cancer constructed by subcutaneous implantation of histologically intact tumor tissue. METHODS: Human gastric cancer SGC-7901 tissues were implanted into the dorsal subcutis of nude mice. One week after tumors reached to a volume of 50-100 mm(3) for around 1 wk, these mice were randomly divided into 8 groups (n = 10). NM-3 was injected peritoneally at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg every other day for 5 wk, combined with carboplatin (5 mg/kg) every third day for 4 wk. As controls of combined treatment, another 4 groups of mice were injected with either NM-3 at 10 mg/kg, 20 mg/kg or 40 mg/kg, or with carboplatin alone (5 mg/kg). The control mice received normal saline. Tumor weight, tumor growth inhibition (TGI), and intratumoral microvessel density (MVD) were evaluated. Apoptosis of human gastric cancer was detected by TUNEL method and flow cytometry analysis, respectively. RESULTS: The mean tumor volume (692.40 +/- 58.43 mm(3), 548.30 +/- 66.02 mm(3), 382.13 +/- 43.52 mm(3)) after treatment with carboplatin combined NM-3 at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg was lower than that after treatment with either NM-3 at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg or with carboplatin alone. Compared with the normal saline group, NM-3 administered at 10 mg/kg, 20 mg/kg or 40 mg/kg significantly reduced the tumor weight in these groups (P < 0.05). Carboplatin used alone at 5 mg/kg showed minimal effects. But NM-3 in combination with carboplatin had greater effects of tumor weight than either NM-3 or carboplatin alone. NM-3 alone at the dose 10 mg/kg or in combination with carboplatin had no obvious effects on body changes. Two mice died of diarrhea in each of the two groups treated with 40 mg/kg NM-3 or with 40 mg/kg NM-3 in combination with carboplatin. A significant increase in apoptosis was observed in the NM-3 treated groups, and the effect was more significant in the groups treated with carboplatin in combination with NM-3 at 10 mg/kg, 20 mg/kg and 40 mg/kg, than in the control group. The induction of apoptosis was positively associated with the dose of NM-3. NM-3 significantly reduced the neo-microvascular formation of gastric cancer. The MVD was lower in the groups treated with NM-3 or with NM-3 in combination with carboplatin than in the group treated with carboplatin or in the normal saline group (P < 0.05). CONCLUSION: The results suggest that the inhibitory effect of NM-3 on gastric cancer growth is mediated through decreased angiogenesis and the increased induction of apoptosis. Furthermore, NM-3 alone at the dose of 10 mg/kg or in combination with carboplatin has no obvious effects on body changes, indicating that NM-3 in combination with carboplatin may be effective in the treatment of gastric cancer. The toxicity of NM-3 needs further studies.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carboplatina/administração & dosagem , Isocumarinas/administração & dosagem , Neovascularização Patológica/tratamento farmacológico , Neoplasias Gástricas/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Neoplasias Gástricas/irrigação sanguínea , Neoplasias Gástricas/patologia , Transplante HeterólogoRESUMO
IMPORTANCE: Effective cancer prevention is based on accurate molecular diagnosis and results of genetic family screening, genotype-informed risk assessment, and tailored strategies for early diagnosis. The expanding etiology for hereditary pheochromocytomas and paragangliomas has recently included SDHA, TMEM127, MAX, and SDHAF2 as susceptibility genes. Clinical management guidelines for patients with germline mutations in these 4 newly included genes are lacking. OBJECTIVE: To study the clinical spectra and age-related penetrance of individuals with mutations in the SDHA, TMEM127, MAX, and SDHAF2 genes. DESIGN, SETTING, AND PATIENTS: This study analyzed the prospective, longitudinally followed up European-American-Asian Pheochromocytoma-Paraganglioma Registry for prevalence of SDHA, TMEM127, MAX, and SDHAF2 germline mutation carriers from 1993 to 2016. Genetic predictive testing and clinical investigation by imaging from neck to pelvis was offered to mutation-positive registrants and their relatives to clinically characterize the pheochromocytoma/paraganglioma diseases associated with mutations of the 4 new genes. MAIN OUTCOMES AND MEASURES: Prevalence and spectra of germline mutations in the SDHA, TMEM127, MAX, and SDHAF2 genes were assessed. The clinical features of SDHA, TMEM127, MAX, and SDHAF2 disease were characterized. RESULTS: Of 972 unrelated registrants without mutations in the classic pheochromocytoma- and paraganglioma-associated genes (632 female [65.0%] and 340 male [35.0%]; age range, 8-80; mean [SD] age, 41.0 [13.3] years), 58 (6.0%) carried germline mutations of interest, including 29 SDHA, 20 TMEM127, 8 MAX, and 1 SDHAF2. Fifty-three of 58 patients (91%) had familial, multiple, extra-adrenal, and/or malignant tumors and/or were younger than 40 years. Newly uncovered are 7 of 63 (11%) malignant pheochromocytomas and paragangliomas in SDHA and TMEM127 disease. SDHA disease occurred as early as 8 years of age. Extra-adrenal tumors occurred in 28 mutation carriers (48%) and in 23 of 29 SDHA mutation carriers (79%), particularly with head and neck paraganglioma. MAX disease occurred almost exclusively in the adrenal glands with frequently bilateral tumors. Penetrance in the largest subset, SDHA carriers, was 39% at 40 years of age and is statistically different in index patients (45%) vs mutation-carrying relatives (13%; P < .001). CONCLUSIONS AND RELEVANCE: The SDHA, TMEM127, MAX, and SDHAF2 genes may contribute to hereditary pheochromocytoma and paraganglioma. Genetic testing is recommended in patients at clinically high risk if the classic genes are mutation negative. Gene-specific prevention and/or early detection requires regular, systematic whole-body investigation.
Assuntos
Neoplasias das Glândulas Suprarrenais/genética , Predisposição Genética para Doença , Neoplasias de Cabeça e Pescoço/genética , Segunda Neoplasia Primária/genética , Paraganglioma Extrassuprarrenal/genética , Feocromocitoma/genética , Adolescente , Neoplasias das Glândulas Suprarrenais/diagnóstico por imagem , Adulto , Idade de Início , Idoso , Idoso de 80 Anos ou mais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Criança , Análise Mutacional de DNA , Detecção Precoce de Câncer/métodos , Complexo II de Transporte de Elétrons/genética , Feminino , Testes Genéticos , Genótipo , Mutação em Linhagem Germinativa , Neoplasias de Cabeça e Pescoço/diagnóstico por imagem , Humanos , Estudos Longitudinais , Imageamento por Ressonância Magnética , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Proteínas Mitocondriais/genética , Paraganglioma Extrassuprarrenal/diagnóstico por imagem , Penetrância , Feocromocitoma/diagnóstico por imagem , Estudos Prospectivos , Sistema de Registros , Adulto JovemRESUMO
AIM: To study the differential gene expression profiles of target cells in primary gastric cancer and its metastatic lymph nodes using laser microdissection (LMD) in combination with cDNA microarray. METHODS: Normal gastric tissue samples from 30 healthy individuals, 36 cancer tissue samples from primary gastric carcinoma and lymph node metastasis tissue samples from 58 patients during gastric cancer resection were obtained using LMD in combination with cDNA microarray independently. After P27-based amplification, aRNA from 36 of 58 patients (group 1) with lymph node metastasis and metastatic tissue specimens from the remaining 22 patients (group 2) were applied to cDNA microarray. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical assay verified the results of microarray in group 2 and further identified genes differentially expressed in the progression of gastric cancer. RESULTS: The expression of 10 genes was up-regulated while the expression of 15 genes was down-regulated in 22 gastric carcinoma samples compared with that of genes in the normal controls. The results were confirmed at the level of mRNA and protein, and suggested that four genes (OPCML, RNASE1, YES1 and ACK1) could play a key role in the tumorigenesis and metastasis of gastric cancer. The expression pattern of 3 genes (OPCML, RNASE1 and YES1) was similar to tumor suppressor genes. For example, the expression level of these genes was the highest in normal gastric epithelium, which was decreased in primary carcinoma, and further decreased in metastatic lymph nodes. On the contrary, the expression pattern of gene ACK1 was similar to that of oncogene. Four genes were further identified as differentially expressed genes in the majority of the cases in the progression of gastric cancer. CONCLUSION: LMD in combination with cDNA microarray provides a unique support foe the identification of early expression profiles of differential genes and the expression pattern of 3 genes (OPCML, RNASE1 and YES1) associated with the progression of gastric cancer. Further study is needed to reveal the molecular mechanism of lymph node metastasis in patients with gastric cancer.
Assuntos
Perfilação da Expressão Gênica/métodos , Lasers , Metástase Linfática/genética , Microdissecção/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Neoplasias Gástricas/genética , Idoso , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , DNA de Neoplasias/genética , Progressão da Doença , Feminino , Proteínas Ligadas por GPI , Regulação Neoplásica da Expressão Gênica/genética , Genes Neoplásicos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-yes/genética , Proteínas Proto-Oncogênicas c-yes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonuclease Pancreático/genética , Ribonuclease Pancreático/metabolismo , Neoplasias Gástricas/metabolismo , Regulação para Cima/genéticaRESUMO
BACKGROUND: Biocontrol agents are regarded as promising and environmental friendly approaches as agrochemicals for phytodiseases that cause serious environmental and health problems. Trichoderma species have been widely used in suppression of soil-borne pathogens. In this study, an endophytic fungus, Trichoderma gamsii YIM PH30019, from healthy Panax notoginseng root was investigated for its biocontrol potential. METHODS: In vitro detached healthy roots, and pot and field experiments were used to investigate the pathogenicity and biocontrol efficacy of T. gamsii YIM PH30019 to the host plant. The antagonistic mechanisms against test phytopathogens were analyzed using dual culture, scanning electron microscopy, and volatile organic compounds (VOCs). Tolerance to chemical fertilizers was also tested in a series of concentrations. RESULTS: The results indicated that T. gamsii YIM PH30019 was nonpathogenic to the host, presented appreciable biocontrol efficacy, and could tolerate chemical fertilizer concentrations of up to 20%. T. gamsii YIM PH30019 displayed antagonistic activities against the pathogenic fungi of P. notoginseng via production of VOCs. On the basis of gas chromatography-mass spectrometry, VOCs were identified as dimethyl disulfide, dibenzofuran, methanethiol, ketones, etc., which are effective ingredients for antagonistic activity. T. gamsii YIM PH30019 was able to improve the seedlings' emergence and protect P. notoginseng plants from soil-borne disease in the continuous cropping field tests. CONCLUSION: The results suggest that the endophytic fungus T. gamsii YIM PH30019 may have a good potential as a biological control agent against notoginseng phytodiseases and can provide a clue to further illuminate the interactions between Trichoderma and phytopathogens.
RESUMO
Four new fungal polyketides named koninginins N-Q (1-4), together with four known analogues (5-8), were isolated from the endophytic fungus Trichoderma koningiopsis YIM PH30002 harbored in Panax notoginseng. Their structures were determined on the basis of spectral data interpretation. These compounds were evaluated for their antifungal activity, nitric oxide inhibition, and anticoagulant activity.
RESUMO
AIM: To investigate the role of P-selectin, intercellular adhesion molecule-1 (ICAM-1) and dendritic cells (DCs) in liver/kidney of rats with hepatic/renal ischemia-reperfusion injury and the preventive effect of anti-P-selectin lectin-EGF domain monoclonal antibody (anti-PsL-EGFmAb) on the injury. METHODS: Rat models of hepatic and renal ischemia-reperfusion were established. The rats were then divided into two groups, one group treated with anti-PsL-EGFmAb (n = 20) and control treated with saline (n = 20). Both groups were subdivided into four groups according to reperfusion time (1, 3, 6 and 24 h). The sham-operated group (n = 5) served as a control group. DCs were observed by the microscopic image method, while P-selectin and ICAM-1 were analyzed by immunohistochemistry. RESULTS: P-selectin increased significantly in hepatic sinusoidal endothelial cells and renal tubular epithelial cells 1 h after ischemia-reperfusion, and the expression of ICAM-1 was up-regulated in hepatic sinusoid and renal vessels after 6 h. CD1a(+)CD80(+)DCs gradually increased in hepatic sinusoidal endothelium and renal tubules and interstitium 1 h after ischemia-reperfusion, and there was the most number of DCs in 24-h group. The localization of DCs was associated with rat hepatic/renal function. These changes became less significant in rats treated with anti-PsL-EGFmAb. CONCLUSION: DCs play an important role in immune pathogenesis of hepatic/renal ischemia-reperfusion injury. Anti-PsL-EGFmAb may regulate and inhibit local DC immigration and accumulation in liver/kidney.