RESUMO
BACKGROUND: Epithelial-mesenchymal transition (EMT) is an essential component of metastasis. Our previous study demonstrated that cancer-associated fibroblasts (CAFs) induce EMT in lung cancer cells. In recent years, many studies have demonstrated that CAFs induce metastasis and drug resistance in cancer cells via exosomes. AIM: We sought to discover the mechanism underlying how CAFs induce EMT in lung cancer cells, unveiling the role of exosomes in lung cancer progression. DESIGN: We cultured lung cancer cell (i) with control medium, normal fibroblasts (NFs) or CAFs; (ii) with SNAI1-transfected or NC (negative control)-transfected CAFs; (iii) with exosomes extracted from NF- or CAF-conditioned medium; (iv) with exosomes released by SNAI1 or NC-transfected CAFs; (v) with CAF-conditioned medium or exosome-depleted CAF-conditioned medium. METHODS: qRT-PCR was conducted to examine the expression of CDH1 (gene of E-cadherin) and VIM (gene of Vimentin), western blotting was conducted to examine E-cadherin and vimentin levels in lung cancer cells. RESULTS: Exosomes released by CAFs-promoted EMT in lung cancer cells. Interestingly, SNAI1 levels in exosomes secreted from CAFs were correlated with SNAI1 expression in CAFs. Furthermore, the level of SNAI1 in exosomes was crucial for inducing EMT in lung cancer cells. Finally, treatment of CAFs with GW4869, an inhibitor of exosome release, noticeably inhibited their EMT-inducing effect on recipient epithelial cells. CONCLUSIONS: The molecular mechanism underlying how CAFs induce EMT in cancer cells may be that CAFs deliver SNAI1 to recipient cancer cells via exosomes.
Assuntos
Fibroblastos Associados a Câncer/metabolismo , Exossomos/metabolismo , Neoplasias Pulmonares/patologia , Fatores de Transcrição da Família Snail/metabolismo , Compostos de Anilina/farmacologia , Compostos de Benzilideno/farmacologia , Fibroblastos Associados a Câncer/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Transição Epitelial-Mesenquimal , Exossomos/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Fatores de Transcrição da Família Snail/genéticaRESUMO
Objective: To study the inducing factors and clinical characteristics of patients hospitalized for asthma exacerbation in China. Methods: Patients hospitalized for asthma exacerbation at 29 hospitals in China were retrospectively recruited during 2013-2014. Results: Clinical data of 3 240 asthmatic patients were collected and analyzed including 1 369(42.3%) males and 1 871(57.7%) females. The patients hospitalized for asthma exacerbation counted for 2.95% (6 375/215 955) of all patients hospitalized during the same period. The leading six inducing factors, in sequence, were acute upper respiratory tract infection [42.3%(1 370/3 240)], changes of weather [22.8%(738/3 240)], noxious gas [(4.3%(140/3 240), allergy challenges [3.5%(115/3 240)], strenuous exercise [1.8%(57/3 240)], and air pollution [1.5%(49/3 240)]. In older patients, more exacerbations were induced by weather changes, yet less sensitive to allergy challenges. As to middle-aged patients, they were less sensitive to upper respiratory tract infections, however the difference was not statistically significant (P>0.05). In winter more asthma patients were induced by upper respiratory tract infections, while in autumn more patients were induced by weather changes, strenuous exercise and air pollution. In spring and summer more patients were induced by allergy challenges, but the differences failed to achieve statistical significance (P>0.05). In northern cities more patients were induced by upper respiratory infections, whereas in southern cities more by noxious gases. Allergy challenges and air pollution tended to affect more patients in northern cities, but the difference was of no significance (P>0.05). The differences of inducing factors among patients of different gender, with or without a smoking history, and with different exacerbation severity didn't show any statistical significance. The patients with severe and life-threatening exacerbations counted for 20.1% (652/3 240). The percentage of patients older than 60 years was higher in patients with severe or life-threatening exacerbations than in whose with mild or moderate exacerbations, so did the percentage of male patients, of patients with disease duration longer than 10 years, with smoking history, and with a history of hospitalization or emergency department visits due to asthma exacerbation during the last year. Conclusion: The acute upper respiratory tract infection ranks top among all the inducing factors. Senility, male gender, long duration of disease, smoking history, and a history of frequent hospital visits might be the risk factors for severe or life-threatening asthma exacerbations.
Assuntos
Antiasmáticos/uso terapêutico , Asma/tratamento farmacológico , Hospitalização , Infecções Respiratórias/complicações , Poluição do Ar/efeitos adversos , Antiasmáticos/administração & dosagem , Asma/diagnóstico , Asma/epidemiologia , Asma/etiologia , China/epidemiologia , Feminino , Humanos , Hipersensibilidade , Masculino , Pessoa de Meia-Idade , Infecções Respiratórias/epidemiologia , Estudos Retrospectivos , Fatores de Risco , Estações do Ano , Fatores SexuaisRESUMO
BACKGROUND: Afatinib 40 mg/day is approved for first-line treatment of EGFR mutation-positive non-small-cell lung cancer (NSCLC). In the case of drug-related grade ≥3 or selected prolonged grade 2 adverse events (AEs), the dose can be reduced by 10 mg decrements to a minimum of 20 mg. Here, we evaluate the influence of afatinib dose reduction on AEs, pharmacokinetics and progression-free survival (PFS) in the phase III LUX-Lung 3 and 6 (LL3/6) trials. PATIENTS AND METHODS: Treatment-naïve patients with advanced EGFR mutation-positive NSCLC in LL3 (global) and LL6 (China, Thailand, South Korea) were randomized to afatinib or chemotherapy. All afatinib-treated patients (LL3, n = 229; LL6, n = 239) were included in the post hoc analyses. Incidence and severity of common AEs before and after afatinib dose reduction were assessed. Afatinib plasma concentrations were compared in patients who reduced to 30 mg versus those remaining at 40 mg. PFS was compared between patients who dose reduced within the first 6 months of treatment and those who did not. RESULTS: Dose reductions occurred in 53.3% (122/229) and 28.0% (67/239) of patients in LL3 and LL6, respectively; most (86.1% and 82.1%) within the first 6 months of treatment. Dose reduction led to decreases in the incidence of drug-related AEs, and was more likely in patients with higher afatinib plasma concentrations. On day 43, patients who dose reduced to 30 mg (n = 59) had geometric mean afatinib plasma concentrations of 23.3 ng/ml, versus 22.8 ng/ml in patients who remained on 40 mg (n = 284). The median PFS was similar in patients who dose reduced during the first 6 months versus those who did not {LL3: 11.3 versus 11.0 months [hazard ratio (HR) 1.25]; LL6: 12.3 versus 11.0 months (HR 1.00)}. CONCLUSIONS: Tolerability-guided dose adjustment is an effective measure to reduce afatinib-related AEs without affecting therapeutic efficacy. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov identifiers: NCT00949650 and NCT0112393.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Quinazolinas/administração & dosagem , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adenocarcinoma de Pulmão , Adulto , Afatinib , Idoso , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , China , Intervalo Livre de Doença , Relação Dose-Resposta a Droga , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/patologia , Feminino , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Inibidores de Proteínas Quinases/administração & dosagem , Quinazolinas/efeitos adversos , República da CoreiaRESUMO
OBJECTIVE: To investigate the role and mechanism of miR-155 in invasion and metastasis of lung adenocarcinoma A549 cells. METHODS: Real-time PCR and fluorescence in situ hybridization were used to detect the miR-155 expression in patients' lung adenocarcinoma and adjacent tissue and lymph nodes. Scratch test and Transwell migration assay were used to assess the effect of miR-155 on the A549 cell migration and invasion capability. Bioinformatics software was used to predict the target genes of miR-155, and using luciferase to assay the target gene. Western blot and real-time PCR were performed to confirm the role of miR-155 expression in the regulation of target gene PTEN. RESULTS: The real-time quantitative PCR showed that the miR-155 expression levels in adjacent normal tissue, lung adenocarcinoma and metastatic lymph nodes were 4.1±0.5, 9.6±3.1 and 7.8±2.2, respectively. The in situ hybridization showed that the expression rates of miR-155 in the adjacent normal tissue, lung adenocarcinoma and metastatic lymph nodes were (23.2±15.3)%, (75.4±20.2)% and (60.4±25.1)%, respectively. The Scratch assay showed that the wound healing rates in the miR-155 mimics group, miR-155 mimics NC group, miR-155 inhibitor group and miR-155 inhibitor NC group at 24 h were (43.2±2.2)%, (21.3±4.2)%, (24.3±5.3)%, and (35.2±5.1)%, and that at 48 h were (75.2±4.5)%, (52.6±5.2)%, (39.4±4.2)%, and (51.5±4.3)%, respectively. Dual luciferase reporter gene assay showed that the value of the luciferase in the miR-155 mimics group co-transfected with PTEN 3'UTR-containing wild-type and mutant plasmids were 4.7±0.5 and 7.3±0.7, and the miR-155 mimics luciferase values of the control group co-transfected with PTEN 3'UTR-containing wild-type and mutant plasmids were 7.8±0.9 and 7.5±0.8, respectively. The real-time quantitative fluorescence PCR showed that the relative expression of PTEN protein in the miR-155 mimics group, miR-155 mimics control group, miR-155 mimics inhibitor group, and miR-155 inhibitor control group were 0.5±0.3, 1.0±0.1, 2.2±0.2 and 1.2±0.1, respectively. The Western blot assay detected that the relative expression of PTEN protein levels in the miR-155 mimics group, miR-155 mimics control group, miR-155 inhibitor group and miR-155 inhibitor control group were 0.4±0.1, 1.0±0.3, 2.8±0.2 and 1.4±0.1, respectively. The differences in PTEN mRNA and protein expressions of the four groups were statistically significant (P<0.05 for all). CONCLUSIONS: miR-155 may promote the invasion and metastasis of lung adenocarcinoma through reducing the target PTEN gene expression.
Assuntos
Adenocarcinoma/patologia , Adenocarcinoma/secundário , Neoplasias Pulmonares/patologia , MicroRNAs/fisiologia , PTEN Fosfo-Hidrolase/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Expressão Gênica , Genes Reporter , Humanos , Hibridização in Situ Fluorescente , Luciferases , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , MicroRNAs/metabolismo , Invasividade Neoplásica , PTEN Fosfo-Hidrolase/metabolismo , Plasmídeos , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , TransfecçãoRESUMO
AIM: The aim of this study was to investigate the mode of action of the lavender essential oil (LV) on antimicrobial activity against multi-drug-resistant Escherichia coli J53 R1 when used singly and in combination with piperacillin. METHOD AND RESULTS: In the time-kill analysis, a complete killing of bacteria was observed based on colony counts within 4 h when LV was combined with piperacillin during exposure at determined FIC concentrations. Analysis of the membrane permeabilizing effects of LV on treated cultures through their stability against sodium dodecyl sulphate revealed that the LV played a role in disrupting the bacterial cell membrane. The finding is further supported by scanning electron microscopy analysis and zeta potential measurement. In addition, reduction in light production expression of E. coli [pSB1075] by the LV showed the presence of potential quorum sensing (QS) inhibitors. CONCLUSIONS: These results indicated that the LV has the potential to reverse bacterial resistance to piperacillin in E. coli J53 R1. It may operate via two mechanisms: alteration of outer membrane permeability and inhibition of bacterial QS. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings offer a novel approach to develop a new option of phytopharmaceuticals against multi-drug-resistant E. coli.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Óleos Voláteis/farmacologia , Óleos de Plantas/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla , Sinergismo Farmacológico , Escherichia coli/genética , Escherichia coli/ultraestrutura , Lavandula , Óleos Voláteis/química , Óleos de Plantas/química , Plasmídeos/genética , Percepção de Quorum/efeitos dos fármacosRESUMO
Earlier detection and intervention for chronic renal allograft injury (CRAI) remain major challenges for transplantation physicians. Endocan plays a key role in the regulation of cell adhesion, inflammatory disorders, and tumor progression. We conducted this cross-sectional study of 97 renal transplant (RT) recipients with mean RT duration of 7.0 ± 5.7 years to determine whether Endocan could be a diagnostic and prognostic marker. The patients' mean age was 43.6 ± 13.2 years, and 55.7% (54/97) were male. Higher Endocan levels were found in more advanced chronic kidney disease (CKD) stages in a dose-dependent manner. Interestingly, the Endocan ≥ 643.19 pg/mL group had higher creatinine (Cr; 1.2 ± 0.4 vs 1.6 ± 1.1 mg/dL; P = .029) and lower estimated glomerular filtration rate (eGFR; 67.8 ± 23.8 mL/min vs 54.4 ± 22.0; P = .006) than the Endocan <643.19 pg/mL group after 3 months of follow-up, respectively. Linear regression analysis found tumor necrosis factor (TNF)-α correlated well with Endocan. To elucidate the response of endothelium activation, we stimulated human umbilical vein endothelial cells (HUVECs) with TNF-α in vitro, and found the levels of Endocan (P = .022) and transforming growth factor (TGF)-ß1 (P = .034) increased with time, but interleukin (IL)-10 decreased (P = .013). In summary, Endocan may reflect the degree of endothelial cell injury in renal allografts, and showed a trend of elevation in late-stage CKD. An in vitro study demonstrated TNF-α-activated HUVECs secreted high levels of Endocan and TGF-ß1, which could lead to a better understanding of the role of endothelium in immune balance. In conclusion, Endocan may have potential as a useful long-term indicator of CRAI in RT recipients, but further study is needed to verify our findings.
Assuntos
Falência Renal Crônica/sangue , Transplante de Rim , Proteínas de Neoplasias/sangue , Proteoglicanas/sangue , Adulto , Feminino , Taxa de Filtração Glomerular , Células Endoteliais da Veia Umbilical Humana , Humanos , Falência Renal Crônica/cirurgia , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: The OPTIMAL study found that erlotinib improved progression-free survival (PFS) versus standard chemotherapy in Chinese patients with advanced EGFR mutation-positive non-small-cell lung cancer (NSCLC). This report describes the quality of life (QoL) and updated PFS analyses from this study. PATIENTS AND METHODS: Chinese patients ≥ 18 years with histologically confirmed stage IIIB or IV NSCLC and a confirmed activating mutation of EGFR (exon 19 deletion or exon 21 L858R point mutation) received erlotinib (150 mg/day; n = 82) or gemcitabine-carboplatin (n = 72). The primary efficacy end point was PFS; QoL was assessed using the Functional Assessment of Cancer Therapy-Lung (FACT-L) questionnaire, Trial Outcome Index (TOI) and Lung Cancer Subscale (LCS). RESULTS: Patients receiving erlotinib experienced clinically relevant improvements in QoL compared with the chemotherapy group in total FACT-L, TOI and LCS (P < 0.0001 for all scales). Erlotinib scored better than chemotherapy for all FACT-L subscales from baseline to cycles 2 and 4 (non-significant). In the updated analysis, PFS was significantly longer for erlotinib than chemotherapy (median PFS 13.7 versus 4.6 months; HR = 0.164, 95% CI = 0.105-0.256; P < 0.0001), which was similar to the previously reported primary analysis. CONCLUSION: Erlotinib improves QoL compared with standard chemotherapy in the first-line treatment of patients with EGFR mutation-positive advanced NSCLC.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Receptores ErbB/genética , Mutação/genética , Qualidade de Vida , Adulto , Idoso , Carcinoma Pulmonar de Células não Pequenas/psicologia , Intervalo Livre de Doença , Cloridrato de Erlotinib , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores de Proteínas Quinases/administração & dosagem , Qualidade de Vida/psicologia , Quinazolinas/administração & dosagem , Inquéritos e QuestionáriosRESUMO
OBJECTIVE: To evaluate the effect of Wenshen Xiaozhng Tang (WXT) on ectopic endometrial growth and on angiogenesis in endometrial implants in a rat model. METHODS: Sprague-Dawley rats with endometriotic implants were randomly treated with low-dose WXT, high-dose WXT, or vehicle (negative control) for 28 days. Cell proliferation and vascular density in the lesions were assessed by immunohistochemistry. The levels of VEGF in peritoneal fluid were determined by ELISA. The mRNA expression of HIF-1α and Flk-1 in the endometriotic lesions was evaluated by real-time PCR. RESULTS: WXT treatment significantly decreased the lesion size and inhibited cell proliferation in the endometriotic lesions. Lowered vascular density and reduced mRNA expression of HIF-1α in the endometriotic lesions, associated with decreased concentration of VEGF in peritoneal fluid, were also observed in WXT-treated rats. CONCLUSIONS: These results suggest that WXT could be effective to suppress the growth of endometriosis, partially through its antiangiogenic activity.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Medicamentos de Ervas Chinesas/uso terapêutico , Endometriose/tratamento farmacológico , Inibidores da Angiogênese/administração & dosagem , Animais , Líquido Ascítico/metabolismo , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Endometriose/metabolismo , Feminino , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Neovascularização Patológica/metabolismo , Neovascularização Patológica/prevenção & controle , Antígeno Nuclear de Célula em Proliferação/análise , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Chronic hepatitis B virus (HBV) infection is closely related to the development of severe liver complications, including hepatocellular carcinoma. In previous studies, we reported that in vivo long-term HBV suppression in transgenic mice can be achieved without apparent toxicity by short hairpin RNA sequentially delivered using adeno-associated viral (AAV) vectors of different serotypes. Our goal herein was to address the clinical utility of this delivery system and, in particular, to determine whether RNA interference (RNAi) and its ability to induce long-term HBV suppression will modulate the development of HBV-associated liver pathology. As a model system, we used a unique HBV transgenic mouse model, containing a 1.3 times over length of the HBV genome, on the ICR mouse background. These transgenic mice produce high serum HBV titers comparable with human chronic HBV patients, and, importantly, manifest characteristic HBV-associated pathology, including progressive hepatocellular injury and the development of hepatocellular adenoma. Using this system, we injected animals with AAV vectors expressing either HBV-specific or a control luciferase-specific short hairpin RNA and followed animals for a total of 18 months. We report herein that AAV-mediated RNAi therapy profoundly inhibits HBV replication and gene expression, with a significant reduction in hepatic regeneration, liver enzymes and, importantly, the appearance of liver adenomas. Indeed, the therapeutic effect of RNAi correlated with the reduction in HBV titers. Our data demonstrate that appropriately designed RNAi therapy has the potential to prevent formation of HBV-associated hepatocellular adenoma.
Assuntos
Adenoma de Células Hepáticas/terapia , Regulação Viral da Expressão Gênica , Vírus da Hepatite B/patogenicidade , Neoplasias Hepáticas/terapia , Interferência de RNA , RNA Viral/genética , Adenoma de Células Hepáticas/sangue , Adenoma de Células Hepáticas/patologia , Adenoma de Células Hepáticas/virologia , Animais , Northern Blotting , Dependovirus/genética , Dependovirus/metabolismo , Feminino , Técnicas de Transferência de Genes , Antígenos de Superfície da Hepatite B/análise , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/genética , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/sangue , Hepatite B Crônica/patologia , Hepatite B Crônica/terapia , Hepatite B Crônica/virologia , Hepatócitos/citologia , Hepatócitos/metabolismo , Hepatócitos/virologia , Humanos , Neoplasias Hepáticas/sangue , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/virologia , Neoplasias Hepáticas Experimentais , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA Viral/metabolismo , Transgenes , Carga Viral , Replicação ViralRESUMO
Transforming growth factor beta(1) (TGFbeta(1)) has been purported to protect tissues from ischemia-reperfusion (I-R) injury. This study was designed to examine if overexpression of TGFbeta(1) using adeno-associated virus type 2 (AAV) protects cardiomyocytes from reoxygenation injury. TGFbeta(1) was overexpressed in cultured HL-1 mouse cardiomyocytes by transfection with AAV/TGFbeta(1)(Latent) or with AAV/TGFbeta(1)(ACT) (active TGFbeta(1)). TGFbeta(1) upregulation reduced cardiomyocyte apoptosis and necrosis induced by 24 h of hypoxia followed by 3 h of reoxygenation concomitant with reduction in reactive oxygen species release, activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and NF-kappaB expression. Transfection with AAV/TGFbeta(1)(ACT) was superior to that with AAV/TGFbeta(1)(Latent). To determine if AAV/TGFbeta(1)(ACT) upregulation in vivo would induce cardioprotection from I-R injury, rat hearts were injected with AAV/TGFbeta(1)(ACT) or phosphate-buffered saline (PBS). Six weeks later, TGFbeta(1)(ACT) was upregulated throughout the myocardium. Following I-R, AAV/TGFbeta(1)(ACT)-overexpressing rats had much smaller infarct size (P<0.01 vs PBS group), which was also related to reduced activation of NADPH oxidase and NF-kappaB, and lower levels of malondialdehyde in I-R tissues. These data demonstrate that overexpression of TGFbeta(1) by AAV can protect cardiac tissues from reperfusion injury, possibly via antioxidant mechanism. These findings suggest potential of TGFbeta(1)(ACT) gene therapy for cardioprotection from I-R injury.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Fator de Crescimento Transformador beta1/genética , Animais , Apoptose , Biomarcadores/análise , Células Cultivadas , Vetores Genéticos/genética , Masculino , Malondialdeído/análise , Camundongos , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/química , Miocárdio/patologia , NADPH Oxidases/análise , NF-kappa B/análise , Ratos , Ratos Endogâmicos , Espécies Reativas de Oxigênio/análise , Regulação para CimaRESUMO
RNA interference (RNAi) was reported to block hepatitis B virus (HBV) gene expression and replication in vitro and in vivo. However, it remains a technical challenge for RNAi-based therapy to achieve long-term and complete inhibition effects in chronic HBV infection, which presumably requires more extensive and uniform transduction of the whole infected hepatocytes. To increase the in vivo transfection efficiency in liver, we used a double-stranded adeno-associated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA. HBV transgenic mice were used as an animal model to evaluate the inhibition effects of the RNAi-based gene therapy. A single administration of dsAAV2/8 vector, carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2-3 log(10) decrease in HBV load in the circulation. Significant HBV suppression sustained for at least 120 days after vector administration. The therapeutic effect of shRNA was target sequence dependent and did not involve activation of interferon. These results underscore the potential for developing RNAi-based therapy by dsAAV2/8 vector to treat HBV chronic infection, and possibly other persistent liver infections as well.
Assuntos
Dependovirus/genética , Terapia Genética/métodos , Vírus da Hepatite B/fisiologia , Hepatite B Crônica/terapia , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Animais , Linhagem Celular , Engenharia Genética , Vetores Genéticos/administração & dosagem , Hepatite B Crônica/virologia , Hepatócitos/imunologia , Hepatócitos/virologia , Camundongos , Camundongos Transgênicos , RNA de Cadeia Dupla/administração & dosagem , Fatores de TempoRESUMO
OBJECTIVE: To investigate the telomerase activity in human non-small-cell lung cancer (NSCLC) and to investigate the possibility of telomerase as a tumor biological marker. METHODS: The semi-quantitative assay and expression of PCR-based telomeric repeats amplification Protocol ELISA (TRAP-PCR-ELISA) and PAGE (TRAP-PCR-PAGE) were detected in 15 NSCLC tissues and 15 tumor-adjacent tissues, 3 normal lung tissues. RESULTS: No telomerase activity in 15 tumor-adjacent tissues and 3 normal lung tissues was detected, However, of 15 NSCLC tissues, 13 were positive for telomerase activity with a 86.7% positive rate, telomerase activity was significantly higher in NSCLC than in Tumor-adjacent tissues and normal lung tissues (P < 0.001). CONCLUSION: The results suggest that telomerase is a tumor specific gene marker.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Neoplasias Pulmonares/enzimologia , Telomerase/metabolismo , Adenocarcinoma/enzimologia , Carcinoma de Células Escamosas/enzimologia , HumanosRESUMO
The hepatitis B virus (HBV) genome is known to contain four conserved and overlapped open reading frames (ORFs) encoding the viral core, polymerase (P), surface (S), and X proteins. Whether HBV encodes other proteins has long been a major interest in the field. Using (32)P-labeling of an introduced protein kinase A site attached to the N- or C-terminus of the HBV polymerase gene, a 43-kDa P-S fusion protein was detected in cell lysate, secreted virions, and 22-nm subviral particles. Immunobiochemical studies showed that the 43-kDa protein contains the epitopes of the N-terminus of polymerase and most parts of the surface proteins. This 43-kDa protein was shown to be a glycoprotein, similar to the surface protein. RT-PCR and sequence analyses identified a spliced mRNA which was derived from pregenomic RNA with a deletion of 454 nucleotides (nt) from nt 2447 to 2902. This splice event creates a P-S fusion ORF. This finding is consistent with the result obtained from an immunobiochemical study. Mutations at the splice donor or acceptor site on the HBV genome abrogated the production of the 43-kDa protein. These mutants had no effect on viral replication in transfected HuH-7 cells. However, this P-S fusion protein is able to substitute for the LS protein in virion maturation. On the basis of these results, we conclude that the 43-kDa protein is a polymerase-surface fusion protein encoded by a spliced RNA. Similar to the LS protein, the 43-kDa P-S fusion protein is a structural protein of HBV and might play a role in the HBV life cycle.
Assuntos
Vírus da Hepatite B/genética , Splicing de RNA , Proteínas Virais de Fusão/genética , Proteínas Estruturais Virais/genética , Ácido Aspártico Endopeptidases/genética , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Epitopos , Produtos do Gene pol/genética , Glicoproteínas/genética , Glicoproteínas/metabolismo , Glicosilação , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Humanos , Peso Molecular , Fases de Leitura Aberta , Testes de Precipitina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais de Fusão/metabolismo , Proteínas Estruturais Virais/metabolismoRESUMO
Human lung squamous carcinoma cell line(TUL cell) was treated with 5 x 10(-6) mol.L-1 all-trans retinoic acid(ATRA). Cell numbers were estimated by 0.4% trypon blue stain. MTT test was used for determining the percentage of cell growth inhibition(GI). DNA syntheses of cells were evaluated by 3H-thymidine incorporation. Cell ultrastructure was examined by transmission electron microscope(TEM). TUL cells were treated with or without ATRA, and transplanted in cutaneous of Balb/c nude mice. The results showed that: 1. The speed of cell growth in study group was smaller than that in control group. 2. The GI of cell lines TUL was 42.3%. 3. The percentage of 3H-thymidine incorporation of TUL cells was declined significantly. 4. Ultrastructure under TME showed that TUL cells treated with ATRA was suppressed. These results suggest that ATRA has the effects on growth-inhibition and differentiation-induction of TUL cell.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/patologia , Neoplasias Pulmonares/patologia , Tretinoína/farmacologia , Animais , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células Tumorais CultivadasRESUMO
Hepatocellular carcinoma (HCC) is one of the major causes of human cancer deaths worldwide. To identify alterations of the genetic program associated with human HCC, we designed a new protocol based on the high-density replica method to analyze protein kinase gene expression in normal liver, HCC, and HCC-derived cell lines. RNA was prepared for reverse transcription and cDNA was used for PCR amplification of the conserved catalytic domain of protein kinase genes. Initially, from a pair of HCC and the adjacent noncancerous tissues, we sequenced 228 samples and identified 26 genes that represent different tyrosine kinase subfamilies. High-density grid filters were then prepared to assist the identification, by hybridization, of genes that are differentially expressed in normal vs HCC samples. Eleven tyrosine kinase genes were tested, and positive signals were reliably scored by doubly offset duplicates and by two independent gene-specific probes. Of the 11 genes tested, PDGF receptor-beta, MEKK-3, axl, and FGFR-4 are preferentially expressed in tumor samples. Additionally, we analyzed protein kinase gene expression in five HCC cell lines and identified distinct kinase gene expression patterns in different cell lines. Our results suggest that multiple kinases are activated in different tumors and confirm that there is molecular heterogeneity in the mechanisms sustaining autonomous cell growth in liver tumor formation.
Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Hibridização In Situ/métodos , Neoplasias Hepáticas/genética , MAP Quinase Quinase Quinase 1 , Proteínas Tirosina Quinases/genética , Northern Blotting , Western Blotting , Carcinoma Hepatocelular/enzimologia , Biblioteca Gênica , Humanos , Fígado/enzimologia , Neoplasias Hepáticas/enzimologia , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Análise de Sequência de DNA , Transcrição Gênica , Células Tumorais Cultivadas , Receptor Tirosina Quinase AxlRESUMO
A mutation within the Schizosaccharomyces pombe cdc24(+) gene was identified previously in a screen for cell division cycle mutants and the cdc24(+) gene was determined to be essential for S phase in this yeast. We have isolated the cdc24(+) gene by complementation of a new temperature-sensitive allele of the gene, cdc24-G1. The DNA sequence predicts the presence of an open reading frame punctuated by six introns which encodes a pioneer protein of 58 kD. A cdc24 null mutant was generated by homologous recombination. Haploid cells lacking cdc24(+) are inviable, indicating that cdc24(+) is an essential gene. The transcript of cdc24(+) is present at constant levels throughout the cell cycle. Cells lacking cdc24(+) function show a checkpoint-dependent arrest with a 2N DNA content, indicating a block late in S phase. Arrest is accompanied by a rapid loss of viability and chromosome breakage. An S. pombe homolog of the replicative DNA helicase DNA2 of S. cerevisiae suppresses cdc24. These results suggest that Cdc24p plays a role in the progression of normal DNA replication and is required to maintain genomic integrity.
Assuntos
Proteínas de Ciclo Celular/genética , Fatores de Troca do Nucleotídeo Guanina , Proteínas Proto-Oncogênicas/genética , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Alelos , Sequência de Aminoácidos , Sequência de Bases , Ciclo Celular/genética , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/química , Divisão Celular/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Teste de Complementação Genética , Genótipo , Íntrons , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/química , Recombinação Genética , Proteínas Repressoras/química , Proteínas Repressoras/genética , Mapeamento por Restrição , Schizosaccharomyces/crescimento & desenvolvimento , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TemperaturaRESUMO
d-Dicentrine, a naturally occurring aporphine type isoquinoline alkaloid, isolated from the root of Lindera megaphylla Hemsl. (Lauraceae), was evaluated for its potential anti-cancer activity. We found d-dicentrine significantly inhibited the growth of human hepatoma cell line HuH-7 by delaying its doubling time in tissue culture. An in vitro colony forming assay showed that d-dicentrine decreased the colony formation efficiency in both hepatoma cell lines, HuH-7 and MS-G2, used in our study. Biosyntheses of the macromolecules DNA and RNA were also strongly inhibited. An MTT assay in 21 tumor cell lines also revealed that d-dicentrine was most cytotoxic to esophageal carcinoma HCE-6, lymphoma cell lines Molt-4 and CESS, leukemia cell lines HL60 and K562, and hepatoma cell line MS-G2. An in vitro tumor growing assay in the Severe Combined immunodeficiency (SCID) mice showed that intraperitoneal injection of d-dicentrine at the dose of 100 micrograms twice a week for 4 weeks significantly inhibited the tumor incidence of leukemia cell line K562 in SCID mice. All these data indicated that d-dicentrine has potential anti-tumor applications.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Aporfinas/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Plantas Medicinais/química , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Raízes de Plantas , Células Tumorais CultivadasRESUMO
Platelet-derived growth factors (PDGF) are important mitogens for mesenchyme-derived cells. Neither PDGF nor PDGF receptors (PDGFR) are expressed in epithelial cells under normal physiological conditions. However, we have found that PDGF-BB induces c-jun expression and promotes the growth of the human esophageal carcinoma cell line CE48T/VGH. Scatchard analysis revealed the presence of 6 x 10(5) binding sites for PDGF-BB per cell, with a Kd of 9.7 nM. Furthermore, our data indicate that CE48T/VGH expresses beta type PDGFR (PDGFRbeta) with in vitro auto-kinase activity. We have also found that CE48T/VGH expresses the mRNA of the PDGF-A and PDGF-B chains and secretes PDGF molecules. Addition of anti-PDGF neutralizing antibody significantly decreased cell numbers of CE48T/VGH under serum-free conditions. The detached cells underwent apoptosis characterized by micronucleation. These results suggest that expression of the PDGF autocrine system may not only provide the growth advantage but also prevent the apoptosis for CE48T/VGH.
Assuntos
Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Apoptose , Becaplermina , Linhagem Celular , Neoplasias Esofágicas , Humanos , Fator de Crescimento Derivado de Plaquetas/biossíntese , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/fisiologia , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-sis , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Receptores do Fator de Crescimento Derivado de Plaquetas/biossíntese , Receptores do Fator de Crescimento Derivado de Plaquetas/fisiologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transcrição Gênica , Células Tumorais CultivadasRESUMO
We have previously reported the establishment of well-differentiated BALB/c mouse liver (ML) cell lines. Transfection of these cell lines with hepatitis B virus (HBV) DNA led to the expression of HBV-specific antigens and integration of HBV sequences in the cellular genome. Two cloned HBV-transfected ML cell lines, ML-2(HBV) and ML-3(HBV), expressed viral antigens and were highly tumorigenic in nude mice. However, the tumorigenicity of the two cell lines was significantly reduced in BALB/c mice. Southern blot analyses showed that the integrated HBV sequences were retained in tumors growing in nude mice but deleted in tumors growing in BALB/c mice. Furthermore, the deletion of HBV DNA was accompanied by deletion of chromosomal sequences flanking the HBV integration sites. In ML-2(HBV) cells, a significant reduction in chromosomal number was also observed. These results suggest that the immune response of BALB/c mice selected against hepatocellular carcinoma (HCC) cells expressing viral antigens and led to the proliferation of cells with deleted HBV sequences and concomitant chromosome aberrations. By using this mechanism, HCC cells escape the immune surveillance and gain the advantage of cell growth.