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BACKGROUND: To investigate the effects of osimertinib on hepatocellular carcinoma (HCC) and angiogenesis, and its combinatory effects with venetoclax in HCC. METHODS: Viability was assessed by flow cytometry of Annexin V in multiple HCC cell lines after drug treatment. In vitro angiogenesis assay was performed using primary human liver tumor associated endothelial cell (HLTEC). HCC-bearing model was generated by subcutaneous implantation of Hep3B cells to investigate the efficacy of osimertinib alone and its combination with venetoclax. RESULTS: Osimertinib significantly induced apoptosis in a panel of HCC cell lines regardless of EGFR expression level. It inhibited capillary network formation and induced apoptosis in HLTEC. Using HCC xenograft mouse model, we further showed that osimertinib at non-toxic dose inhibited tumor growth by ~ 50% and remarkably decreased blood vessel in tumor. Mechanism studies demonstrated that osimertinib acted on HCC cells in an EGFR-independent manner. It decreased level of VEGF and Mcl-1 in HCC cells via suppressed phosphorylation of eIF4E, thus leading to inhibition of eIF4E-mediated translation. Mcl-1 overexpression reversed pro-apoptotic effect of osimertinib, suggesting an important role of Mcl-1 in osimertinib's action in HCC cells. Of note, the combination of osimertinib and venetoclax achieved approximately complete HCC cell death and tumor growth in mice. CONCLUSIONS: We provide pre-clinical evidence that osimertinib is a promising candidate for the treatment of HCC via targeting tumor cells and angiogenesis. The combination of osimertinib and venetoclax is synergistic in inhibiting HCC.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Animales , Ratones , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/uso terapéutico , Factor 4E Eucariótico de Iniciación , Apoptosis , Receptores ErbB , Línea Celular Tumoral , Ensayos Antitumor por Modelo de Xenoinjerto , Proliferación CelularAsunto(s)
Vacunas contra la COVID-19/efectos adversos , Vacunas contra la COVID-19/inmunología , Hepatitis B Crónica/inmunología , Inmunogenicidad Vacunal , Vacunas de Productos Inactivados/efectos adversos , Vacunas de Productos Inactivados/inmunología , Femenino , Humanos , Masculino , Encuestas y CuestionariosRESUMEN
BACKGROUND: The dynamic changes of lymphocyte subsets and cytokines profiles of patients with novel coronavirus disease (COVID-19) and their correlation with the disease severity remain unclear. METHODS: Peripheral blood samples were longitudinally collected from 40 confirmed COVID-19 patients and examined for lymphocyte subsets by flow cytometry and cytokine profiles by specific immunoassays. FINDINGS: Of the 40 COVID-19 patients enrolled, 13 severe cases showed significant and sustained decreases in lymphocyte counts [0·6 (0·6-0·8)] but increases in neutrophil counts [4·7 (3·6-5·8)] than 27 mild cases [1.1 (0·8-1·4); 2·0 (1·5-2·9)]. Further analysis demonstrated significant decreases in the counts of T cells, especially CD8+ T cells, as well as increases in IL-6, IL-10, IL-2 and IFN-γ levels in the peripheral blood in the severe cases compared to those in the mild cases. T cell counts and cytokine levels in severe COVID-19 patients who survived the disease gradually recovered at later time points to levels that were comparable to those of the mild cases. Moreover, the neutrophil-to-lymphocyte ratio (NLR) (AUC=0·93) and neutrophil-to-CD8+ T cell ratio (N8R) (AUC =0·94) were identified as powerful prognostic factors affecting the prognosis for severe COVID-19. INTERPRETATION: The degree of lymphopenia and a proinflammatory cytokine storm is higher in severe COVID-19 patients than in mild cases, and is associated with the disease severity. N8R and NLR may serve as a useful prognostic factor for early identification of severe COVID-19 cases. FUNDING: The National Natural Science Foundation of China, the National Science and Technology Major Project, the Health Commission of Hubei Province, Huazhong University of Science and Technology, and the Medical Faculty of the University of Duisburg-Essen and Stiftung Universitaetsmedizin, Hospital Essen, Germany.
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Betacoronavirus/inmunología , Infecciones por Coronavirus/inmunología , Citocinas/sangre , Recuento de Leucocitos , Subgrupos Linfocitarios/inmunología , Neumonía Viral/inmunología , Adulto , Anciano , Linfocitos T CD8-positivos/inmunología , COVID-19 , China/epidemiología , Comorbilidad , Infecciones por Coronavirus/sangre , Infecciones por Coronavirus/complicaciones , Infecciones por Coronavirus/epidemiología , Síndrome de Liberación de Citoquinas/etiología , Síndrome de Liberación de Citoquinas/inmunología , Femenino , Citometría de Flujo , Humanos , Recuento de Linfocitos , Linfopenia/etiología , Masculino , Persona de Mediana Edad , Neutrófilos/inmunología , Pandemias , Neumonía Viral/sangre , Neumonía Viral/complicaciones , Neumonía Viral/epidemiología , Pronóstico , SARS-CoV-2 , Factores de TiempoRESUMEN
AIM: To establish a new model for predicting survival in acute-on-chronic liver failure (ACLF) patients treated with an artificial liver support system. METHODS: One hundred and eighty-one ACLF patients who were admitted to the hospital from January 1, 2012 to December 31, 2014 and were treated with an artiï¬cial liver support system were enrolled in this retrospective study, including a derivation cohort (n = 113) and a validation cohort (n = 68). Laboratory parameters at baseline were analyzed and correlated with clinical outcome. In addition to standard medical therapy, ACLF patients underwent plasma exchange (PE) or plasma bilirubin adsorption (PBA) combined with plasma exchange. For the derivation cohort, Kaplan-Meier methods were used to estimate survival curves, and Cox regression was used in survival analysis to generate a prognostic model. The performance of the new model was tested in the validation cohort using a receiver-operator curve. RESULTS: The mean overall survival for the derivation cohort was 441 d (95%CI: 379-504 d), and the 90- and 270-d survival probabilities were 70.3% and 58.3%, respectively. The mean survival times of patients treated with PBA plus PE and patients treated with PE were 531 d (95%CI: 455-605 d) and 343 d (95%CI: 254-432 d), respectively, which were significantly different (P = 0.012). When variables with bivariate significance were selected for inclusion into the multivariate Cox regression model, number of complications, age, scores of the model for end-stage liver disease (MELD) and type of artiï¬cial liver support system were defined as independent risk factors for survival in ACLF patients. This new prognostic model could accurately discriminate the outcome of patients with different scores in this cohort (P < 0.001). The model also had the ability to assign a predicted survival probability for individual patients. In the validation cohort, the new model remained better than the MELD. CONCLUSION: A novel model was constructed to predict prognosis and accurately discriminate survival in ACLF patients treated with an artiï¬cial liver support system.
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Insuficiencia Hepática Crónica Agudizada/terapia , Técnicas de Apoyo para la Decisión , Hígado Artificial , Insuficiencia Hepática Crónica Agudizada/sangre , Insuficiencia Hepática Crónica Agudizada/diagnóstico , Insuficiencia Hepática Crónica Agudizada/mortalidad , Adulto , Anciano , Anciano de 80 o más Años , Área Bajo la Curva , Bilirrubina/sangre , Femenino , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Análisis Multivariante , Intercambio Plasmático , Valor Predictivo de las Pruebas , Modelos de Riesgos Proporcionales , Curva ROC , Reproducibilidad de los Resultados , Estudios Retrospectivos , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
This study examined the effect of subcellular localization of P21 on the proliferation and apoptosis of HepG2 cells. The coding genes of the wild and the mutant P21 were amplified by mega primer PCR from the plasmid pCEP-WAF1 which contains human P21 cDNA in the nuclear localizational signal (NLS) sequence, and then inserted into the eukaryotic expression vector pDsRed1-C1. The recombinants were transfected into HepG2 cells. The transcription and expression of P21 were determined by RT-PCR and fluorescence microscopy. The cell proliferation was measured by MTT, and the cell cycle and apoptosis of HepG2 cells by flow cytometry. The results of restriction analysis, DNA sequencing and fluorescence microscopy confirmed the construction of the wild and the mutant P21 in the eukaryotic expression plasmid. The plasmid containing the mutant P21 was found to accelerate cell proliferation and the wild P21 plasmid to inhibit cell proliferation. Cell cycle analysis showed that the cell ratio of G(0)/G(1) in the wild type group was significantly increased as compared with that in the mutant type group, and cell apoptosis analysis revealed that the apoptosis rate in the wild type group was much higher than that in the mutant type group. It was concluded that the subcellular localization of P21 may contribute to the development of hepatic cancer.
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Apoptosis/fisiología , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Citoplasma/metabolismo , Secuencia de Bases , Núcleo Celular/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Células Hep G2 , Humanos , Datos de Secuencia Molecular , Distribución TisularRESUMEN
This study examined the effect of subcellular localization of P21 on the proliferation and apoptosis of HepG2 cells.The coding genes of the wild and the mutant P21 were amplified by mega primer PCR from the plasmid pCEP-WAF1 which contains human P21 cDNA in the nuclear localizational signal (NLS) sequence,and then inserted into the eukaryotic expression vector pDsRed1-C1.The recombinants were transfected into HepG2 cells.The transcription and expression of P21 were determined by RT-PCR and fluorescence microscopy.The cell proliferation was measured by MTT,and the cell cycle and apoptosis of HepG2 cells by flow cytometry.The results of restriction analysis,DNA sequencing and fluorescence microscopy confirmed the construction of the wild and the mutant P21 in the eukaryotic expression plasmid.The plasmid containing the mutant P21 was found to accelerate cell proliferation and the wild P21 plasmid to inhibit cell proliferation.Cell cycle analysis showed that the cell ratio of G0/G1 in the wild type group was significantly increased as compared with that in the mutant type group,and cell apoptosis analysis revealed that the apoptosis rate in the wild type group was much higher than that in the mutant type group.It was concluded that the subcellular localization of P21 may contribute to the development of hepatic cancer.
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This study investigated the influence of silencing TRAF6 with shRNA on lipopolysaccharide (LPS)/toll-like receptor (TLR)-4 signaling pathway in vitro. Four plasmids (pGCsi-TRAF6-shRNA1, 2, 3, 4) containing different shRNA sequences were designed and synthesized. The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay. Inflammatory cellular models were established by LPS stimulation. Levels of TNF-alpha, IL-1beta and TGF-beta1 in the supernatants, mRNA expressions of TRAF6, IL-6 and COX-2, protein expression of TRAF6 and translocation of NF-kappaB were assayed by ELISA, real-time quantitative PCR and Western blotting, respectively. The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h. The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1, 2 groups than in the TRAF6-shRNA3, 4 groups. Therefore, pGCsi-TRAF6-shRNA1, 2 were selected for the subsequent experiments. Our results still showed that pGCsi-TRAF6-shRNA1, 2 could significantly reduce the production of pro-inflammatory cytokines and mediators including TNF-alpha, IL-1beta, IL-6 and COX-2, and inhibit NF-kappaB nuclear translocation. Moreover, pGCsi-TRAF6-shRNA1, 2 could suppress the release of TGF-beta1 at the protein level. It was concluded that the recombinant plasmid pTRAF6-shRNA can, to some extent, inhibit inflammatory response stimulated by LPS at the initial phase. TRAF6 may become the potential therapeutic target of many inflammation-related diseases.
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Macrófagos/citología , Interferencia de ARN , ARN Interferente Pequeño/genética , Factor 6 Asociado a Receptor de TNF/genética , Receptor Toll-Like 4/metabolismo , Animales , Línea Celular , Inflamación/inducido químicamente , Inflamación/genética , Lipopolisacáridos , Ratones , Transducción de Señal , TransfecciónRESUMEN
This study investigated the influence of silencing TRAF6 with shRNA on lipopolysac-charide (LPS)/toll-like receptor (TLR)-4 signaling pathway in vitro. Four plasmids (pGCsi-TRAF6-shRNA 1, 2, 3, 4) containing different shRNA sequences were designed and synthesized. The proliferation of RAW264.7 cells after transfected with these plasmids was measured by MTT assay. Inflammatory cellular models were established by LPS stimulation. Levels of TNF-α, IL-1β and TGF-β1 in the supernatants, mRNA expressions of TRAF6, IL-6 and COX-2, protein expression of TRAF6 and translocation of NF-κB were assayed by ELISA, real-time quantitative PCR and Western blotting, respectively. The results showed that the TRAF6 gene knockdown by RNAi hardly inhibited the proliferation of RAW264.7 cells within 72 h. The mRNA and protein expression of TRAF6 was lower in the TRAF6-shRNA1, 2 groups than in the TRAF6-shRNA3, 4 groups. Therefore, pGCsi-TRAF6-shRNA1, 2 were selected for the subsequent experiments. Our results still showed that pGCsi-TRAF6-shRNA 1, 2 could significantly reduce the production of pro-inflammatory cyto-kines and mediators including TNF-α, IL-1β, IL-6 and COX-2, and inhibit NF-κB nuclear transloca-tion. Moreover, pGCsi-TRAF6-shRNA1, 2 could suppress the release of TGF-β1 at the protein level. It was concluded that the recombinant plasmid pTRAF6-shRNA can, to some extent, inhibit inflam-matory response stimulated by LPS at the initial phase. TRAF6 may become the potential therapeutic target of many inflammation-related diseases.
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Quercetin and praziquantel were used to treat mice with hepatic fibrosis due to Schistosoma japonicum infection. Quercetin treatment obviously relieved the degree of hepatic fibrosis, significantly reduced the expression of immediate early gene, tissue inhibitor of metalloproteinase 1 (TIMP 1), types I and III collagen compared to the control. The expression of c-jun mRNA, type I and type III collagen were reduced significantly compared to the group treated with praziquantel, whereas no difference in the expression of c-fos mRNA and TIMP1 between the two groups, indicating that quercetin may have better effect on schistosomal liver fibrosis than praziquantel in the long term.