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Ground glass hepatocytes (GGHs) have been associated with hepatocellular carcinoma (HCC) recurrence and poor prognosis. We previously demonstrated that pre-S expression in some GGHs is resistant to current hepatitis B virus (HBV) antiviral therapies. This study aimed to investigate whether integrated HBV DNA (iDNA) is the primary HBV DNA species responsible for sustained pre-S expression in GGH after effective antiviral therapy. We characterized 10 sets of micro-dissected, formalin-fixed-paraffin-embedded, and frozen GGH, HCC, and adjacent hepatitis B surface antigen-negative stained tissues for iDNA, pre-S deletions, and the quantity of covalently closed circular DNA. Eight patients had detectable pre-S deletions, and nine had detectable iDNA. Interestingly, eight patients had integrations within the TERT and CCNE1 genes, which are known recurrent integration sites associated with HCC. Furthermore, we observed a recurrent integration in the ABCC13 gene. Additionally, we identified variations in the type and quantity of pre-S deletions within individual sets of tissues by junction-specific PacBio long-read sequencing. The data from long-read sequencing indicate that some pre-S deletions were acquired following the integration events. Our findings demonstrate that iDNA exists in GGH and can be responsible for sustained pre-S expression in GGH after effective antiviral therapy.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Virus de la Hepatitis B/genética , ADN Viral/genética , Neoplasias Hepáticas/genética , Hepatocitos , Mutación , Antivirales/uso terapéuticoRESUMEN
Alzheimer's disease (AD) is a progressive neurodegenerative disease with a multifactorial etiology. A multitarget treatment that modulates multifaceted biological functions might be more effective than a single-target approach. Here, the therapeutic efficacy of combination treatment using anti-Aß antibody NP106 and curcumin analog TML-6 versus monotherapy was investigated in an APP/PS1 mouse model of AD. Our data demonstrate that both combination treatment and monotherapy attenuated brain Aß and improved the nesting behavioral deficit to varying degrees. Importantly, the combination treatment group had the lowest Aß levels, and insoluble forms of Aß were reduced most effectively. The nesting performance of APP/PS1 mice receiving combination treatment was better than that of other APP/PS1 groups. Further findings indicate that enhanced microglial Aß phagocytosis and lower levels of proinflammatory cytokines were concurrent with the aforementioned effects of NP106 in combination with TML-6. Intriguingly, combination treatment also normalized the gut microbiota of APP/PS1 mice to levels resembling the wild-type control. Taken together, combination treatment outperformed NP106 or TML-6 monotherapy in ameliorating Aß pathology and the nesting behavioral deficit in APP/PS1 mice. The superior effect might result from a more potent modulation of microglial function, cerebral inflammation, and the gut microbiota. This innovative treatment paradigm confers a new avenue to develop more efficacious AD treatments.
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Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/deficiencia , Anticuerpos Monoclonales/farmacología , Curcumina/farmacología , Presenilina-1/deficiencia , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Animales , Conducta Animal/efectos de los fármacos , Biomarcadores , Curcumina/análogos & derivados , Manejo de la Enfermedad , Modelos Animales de Enfermedad , Susceptibilidad a Enfermedades , Relación Dosis-Respuesta a Droga , Quimioterapia Combinada , Inmunohistoquímica , Ratones , Ratones Noqueados , Microbiota/efectos de los fármacos , Microglía/efectos de los fármacos , Microglía/metabolismo , Terapia Molecular Dirigida , Placa Amiloide/tratamiento farmacológico , Placa Amiloide/patologíaRESUMEN
Drugs for the treatment of Alzheimer's disease (AD) are in urgent demand due to the unmet need and the social burden associated with the disease. Curcumin has been historically considered as a beneficial product for anti-aging and AD. However, many efforts to develop curcumin for clinical use are hindered mainly due to its poor bioavailability. Recent development in drug delivery and structural design has resolved these issues. In this study, we identified a small molecule, TML-6, as a potential drug candidate for AD through screening a panel of curcumin derivatives using six biomarker platforms related to aging biology and AD pathogenesis. The structural modification of TML-6 is designed to improve the stability and metabolism of curcumin. Cell biological studies demonstrated that TML-6 could inhibit the synthesis of the ß-amyloid precursor protein and ß-amyloid (Aß), upregulate Apo E, suppress NF-κB and mTOR, and increase the activity of the anti-oxidative Nrf2 gene. In the 3x-Tg AD animal model, TML-6 treatment resulted in significant improvement in learning, suppression of the microglial activation marker Iba-1, and reduction in Aß in the brain. Although TML-6 exhibited a greater improvement in bioavailability as compared to curcumin, formulation optimization and toxicological studies are under development to assure its druggability. Taken together, TML-6 meets the current strategy to develop therapeutics for AD, targeting the combination of the Aß cascade and aging-related biology processes.
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Enfermedad de Alzheimer/tratamiento farmacológico , Curcumina/farmacología , Inflamación/tratamiento farmacológico , Factor 2 Relacionado con NF-E2/genética , Placa Amiloide/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Curcumina/análogos & derivados , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/genética , Inflamación/patología , Ratones , Microglía/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Placa Amiloide/genética , Placa Amiloide/patologíaRESUMEN
Dysfunction of degradation machineries causes cancers, including hepatocellular carcinoma (HCC). Overexpression of cyclin D1 in HCC has been reported. We previously reported that autophagy preferentially recruits and degrades the oncogenic microRNA (miR)-224 to prevent HCC. Therefore, in the present study, we attempted to clarify whether cyclin D1 is another oncogenic factor selectively regulated by autophagy in HCC tumorigenesis. Initially, we found an inverse correlation between low autophagic activity and high cyclin D1 expression in tumors of 147 HCC patients and three murine models, and these results taken together revealed a correlation with poor overall survival of HCC patients, indicating the importance of these two events in HCC development. We found that increased autophagic activity leads to cyclin D1 ubiquitination and selective recruitment to the autophagosome (AP) mediated by a specific receptor, sequestosome 1 (SQSTM1), followed by fusion with lysosome and degradation. Autophagy-selective degradation of ubiquitinated cyclin D1 through SQSTM1 was confirmed using cyclin D1/ubiquitin binding site (K33-238 R) and phosphorylation site (T286A) mutants, lentivirus-mediated silencing autophagy-related 5 (ATG5), autophagy-related 7 (ATG7), and Sqstm1 knockout cells. Functional studies revealed that autophagy-selective degradation of cyclin D1 plays suppressive roles in cell proliferation, colony, and liver tumor formation. Notably, an increase of autophagic activity by pharmacological inducers (amiodarone and rapamycin) significantly suppressed tumor growth in both the orthotopic liver tumor and subcutaneous tumor xenograft models. Our findings provide evidence of the underlying mechanism involved in the regulation of cyclin D1 by selective autophagy to prevent tumor formation. CONCLUSION: Taken together, our data demonstrate that autophagic degradation machinery and the cell-cycle regulator, cyclin D1, are linked to HCC tumorigenesis. We believe these findings may be of value in the development of alternative therapeutics for HCC patients. (Hepatology 2018;68:141-154).
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Autofagia , Carcinoma Hepatocelular/metabolismo , Ciclina D1/metabolismo , Neoplasias Hepáticas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Autofagosomas/metabolismo , Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/mortalidad , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Humanos , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/mortalidad , Masculino , Ratones SCID , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Persona de Mediana Edad , Ratas Sprague-Dawley , Proteína Sequestosoma-1/metabolismo , Taiwán/epidemiología , UbiquitinaciónRESUMEN
Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S2 mutant large HBV surface antigen (LHBS) is highly associated with HCC. This study analyzed the expression of the large form of surface protein in tumors and evaluated the LHBS with mutations within the pre-S2 region as a high-risk recurrence marker in HCC patients after curative hepatic resection. By analyses using immunohistochemical staining (n = 12) and western blotting (n = 22), the HBV surface protein, which is mainly comprised of the major form of HBV surface antigen, was greatly diminished in the tumors. However, LHBS was not significantly decreased in tumorous regions, suggesting that LHBS maintains its expression in cancer development. A cohort of 175 patients with HBV-related HCC who underwent curative hepatic resection was analyzed for pre-S gene mutations using Pre-S Gene Chip. Results of the multivariate regression analysis showed that the serum pre-S2 mutant level and the American Joint Committee on Cancer stage were the two main independent high-risk factors for recurrence. A Cox proportional hazards analysis also revealed a prediction model, which indicated the recurrence-free survival rate along with the time after surgery; this was developed and further validated in an independent HCC cohort. Receiver operating characteristic curve analysis revealed that the model showed close sensitivities in the main and validation cohorts (area under the curve values, 0.741 and 0.704, respectively). Conclusion: Unlike the major HBV surface antigen, LHBS is mostly expressed in the tumorous regions of HBV-induced HCC, indicating that it plays a unique role in tumor progression; the relative level of pre-S2 mutant in serum is, independently of tumor stage, an important high-risk marker for HCC recurrence after primary hepatic resection. (Hepatology 2018).
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Carcinoma Hepatocelular/sangre , Antígenos de Superficie de la Hepatitis B/sangre , Neoplasias Hepáticas/sangre , Recurrencia Local de Neoplasia/sangre , Precursores de Proteínas/sangre , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/cirugía , Estudios de Cohortes , Femenino , Antígenos de Superficie de la Hepatitis B/genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/cirugía , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Precursores de Proteínas/genética , Adulto JovenRESUMEN
Hepatitis B virus (HBV) is an aetiological factor for liver cirrhosis and hepatocellular carcinoma (HCC). Despite current antiviral therapies that successfully reduce the viral load in patients with chronic hepatitis B, persistent hepatitis B surface antigen (HBsAg) remains a risk factor for HCC. To explore whether intrahepatic viral antigens contribute directly to hepatocarcinogenesis, we monitored the mitotic progression of HBV-positive cells. Cytokinesis failure was increased in HBV-positive HepG2.2.15 and 1.3ES2 cells, as well as in HuH-7 cells transfected with a wild-type or X-deficient HBV construct, but not in cells transfected with an HBsAg-deficient construct. We show that expression of viral large surface antigen (LHBS) was sufficient to induce cytokinesis failure of immortalized hepatocytes. Premitotic defects with DNA damage and G2 /M checkpoint attenuation preceded cytokinesis in LHBS-positive cells, and ultimately resulted in hyperploidy. Inhibition of polo-like kinase-1 (Plk1) not only restored the G2 /M checkpoint in these cells, but also suppressed LHBS-mediated in vivo tumourigenesis. Finally, a positive correlation between intrahepatic LHBS expression and hepatocyte hyperploidy was detected in >70% of patients with chronic hepatitis B. We conclude that HBV LHBS provokes hyperploidy by inducing DNA damage and upregulation of Plk1; the former results in atypical chromatin structures, and the latter attenuates the function of the G2 /M DNA damage checkpoint. Our data uncover a mechanism by which genomic integrity of hepatocytes is disrupted by viral LHBS. These findings highlight the role of intrahepatic surface antigen as an oncogenic risk factor in the development of HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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Carcinoma Hepatocelular/virología , Citocinesis , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/metabolismo , Hepatitis B Crónica/virología , Hepatocitos/virología , Neoplasias Hepáticas/virología , Ploidias , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular/metabolismo , Transformación Celular Viral , Daño del ADN , Modelos Animales de Enfermedad , Puntos de Control de la Fase G2 del Ciclo Celular , Células Hep G2 , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B de la Marmota/genética , Virus de la Hepatitis B de la Marmota/metabolismo , Virus de la Hepatitis B/genética , Hepatitis B Crónica/genética , Hepatitis B Crónica/metabolismo , Hepatitis B Crónica/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Hepatocitos/trasplante , Interacciones Huésped-Patógeno , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Marmota , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Quinasa Tipo Polo 1RESUMEN
BACKGROUND: In addition to messenger RNA (mRNA), noncoding RNAs (ncRNAs) are essential components in cellular machineries for translation and splicing. Besides their housekeeping functions, ncRNAs are involved in cell type-specific regulation of translation, mRNA stability, genome structure, and accessibility. To have a comprehensive understanding of the identities and functions of different cell types, a method to comprehensively quantify both mRNA and ncRNA in a sensitive manner is highly desirable. METHODS: Here we tried to develop a system capable of concurrently profiling both mRNA and ncRNA by polyadenylating RNA in samples before reverse transcription. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. The single-tube amplification (STA) system was applied to single to 100 cells of 293T cells, human pluripotent stem cells (hPSCs) and their differentiated endothelial progenies to validate its quantitative power and sensitivity by qPCR and high-throughput sequencing. RESULTS: Using microRNA (miRNA) as an example, we showed that complementary DNA (cDNA) from ncRNAs could be amplified and specifically detected from a few cells within a single tube. The sensitivity of the system was maximized by avoiding purification from cell lysis to amplified cDNA and by optimizing the buffer conditions. With 100 human embryonic stem cells (hESCs) and their differentiated endothelial cells as input for high-throughput sequencing, the single-tube amplification (STA) system revealed both well-known and other miRNAs selectively enriched in each cell type. The selective enrichment of the miRNAs was further verified by qPCR with 293FT cells and a human induced pluripotent stem cell (hiPSC) line. In addition, the detection of other non-miRNA transcripts indicated that the STA target was not limited to miRNA, but extended to other ncRNAs and mRNAs as well. Finally, the STA system was capable of detecting miRNA and mRNA expression down to single cells, albeit with some loss of sensitivity and power. CONCLUSIONS: Overall, STA offered a simple and sensitive way to concurrently quantify both mRNA and ncRNA expression in low-cell-number samples for both qPCR and high-throughput sequencing.
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Endotelio/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , ARN/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Transcriptoma/genética , Tampones (Química) , Recuento de Células , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Endotelio/efectos de los fármacos , Secuenciación de Nucleótidos de Alto Rendimiento , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/efectos de los fármacos , Células Madre Embrionarias Humanas/metabolismo , Humanos , Límite de Detección , Magnesio/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Nucleótidos/farmacología , Células Madre Pluripotentes/efectos de los fármacos , Poliadenilación/efectos de los fármacos , ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcripción Reversa/efectos de los fármacos , Análisis de la Célula Individual , Transcriptoma/efectos de los fármacosRESUMEN
Curcumin has been proven to be a potent agent in colon cancer treatment. However, its hydrophobicity and low oral bioavailability hampered its clinical application. These limitations could be improved through appropriate formulations such as using polyelectrolyte complexes (PECs). PECs were self-assembled with polycations and polyanions in polar solvents. In this study, a novel pectin-type B gelatin PEC was developed for use in curcumin formulation. At pH 4.0, natural polyanions pectin and polycations type B gelatin spontaneously formed PECs in ethanol/water solution, whereas under mimetic gastrointestinal tract (GI tract) conditions, at pH 2.0 and 8.0, pectin and type B gelatin were electrically neutralized, and the PECs swelled to allow payload release. After being transferred to pH 7.0 condition, as in the colon environment, PECs were internalized into colon carcinomas. Thus, pectin-type B gelatin PECs were successfully prepared, and their constituent ratio and drug-loading process were also optimized. The optimum particle size of the PECs was 264.0 ± 3.1 nm and they could swell as the zeta potential was altered at either pH 2.0 or 8.0. The optimum drug content and loading efficiency were 40% and 53%, respectively. At pH 2.0, curcumin was rapidly released from curcumin-loaded PECs, whereas at pH 8.0, curcumin-loaded PECs showed a sustained-release of curcumin. The bare PECs showed very low toxicity toward human normal cells, whereas curcumin-loaded PECs, after incubation at pH 2.0 for 2 h and at pH 8.0 for 4 h, induced cell cycle arrest and exhibited cytotoxic effect to HCT116 human colon cancer cells, even though these loaded PECs were pretreated with mimetic GI tract conditions. Our pectin-type B gelatin PECs were shown to be a promising oral formulation for curcumin delivery in anticancer therapy.
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Antineoplásicos/farmacología , Curcumina/farmacología , Sistemas de Liberación de Medicamentos , Gelatina/química , Pectinas/química , Polielectrolitos/química , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Liberación de Fármacos , Células HCT116 , Humanos , Concentración de Iones de Hidrógeno , Tamaño de la Partícula , Electricidad EstáticaRESUMEN
AIMS: Previously, we reported an association between Epstein-Barr virus (EBV)-positive Hodgkin lymphoma (HL), older age, and poorer prognosis. The aim of this study was to investigate the mechanisms underlying this association. METHODS AND RESULTS: Transfection of HL cell lines with EBV latent membrane protein-1 (LMP1) resulted in up-regulation of many cytokine genes as assessed by the use of oligonucleotide microarrays. The up-regulation of cytokines was validated by using an inflammatory cytokine protein array: macrophage inflammatory protein (MIP)-1α, MIP-1ß, and interleukin (IL)-13. Immunostaining of HL samples (n = 104) showed that expression of MIP-1α, MIP-1ß and IL-13 correlated with EBV infection and LMP1 expression. Combined expression of these cytokines was more common in patients aged >60 years (P < 0.001), and was associated with a poorer prognosis (P = 0.042). In another cohort, serum levels of MIP-1α, MIP-1ß and IL-13 were increased in HL patients (n = 53) and highest in EBV-positive HL patients as compared with healthy controls (n = 40). Xenograft mice injected with EBV-positive HL cells had higher serum levels of MIP-1α, MIP-1ß and IL-13 than mice injected with EBV-negative HL cells, although there was no difference in growth. CONCLUSIONS: EBV infection appears to promote the release of cytokines in HL patients, and negatively impacts on patient survival. Physiological immunosenescence probably explains the association between EBV infection and older age. Cytokine modulation is a potential therapeutic target for EBV-positive HL patients.
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Citocinas/biosíntesis , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/inmunología , Enfermedad de Hodgkin/virología , Proteínas de la Matriz Viral/metabolismo , Adulto , Envejecimiento , Animales , Ensayo de Inmunoadsorción Enzimática , Femenino , Xenoinjertos , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/mortalidad , Humanos , Immunoblotting , Inmunohistoquímica , Hibridación in Situ , Estimación de Kaplan-Meier , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , Análisis de Matrices Tisulares , Regulación hacia ArribaRESUMEN
Metabolic syndrome has closely linked to the development of human hepatocellular carcinoma (HCC). By using the hepatitis B virus (HBV) X (HBx) transgenic mouse model, we studied the dynamic evolution of serum and liver profiles of lipids and global cDNA expression at different stages of HBx tumorigenesis. We observed that the lipid (triglycerides, cholesterol, and fatty acids) profiles revealed a biphasic response pattern during the progression of HBx tumorigenesis: a small peak at early phase and a large peak or terminal switch at the tumor phase. By analyzing cDNA microarray data, the early peak correlated to the oxidative stress and pro-inflammatory response, which then resolved at the middle phase and were followed by the terminal metabolic switch in the tumor tissues. Five lipid metabolism-related genes, the arachidonate 5-lipoxygenase, lipoprotein lipase, fatty acid binding protein 4, 1-acylglycerol-3-phosphate O-acyltransferase 9, and apolipoprotein A-IV were identified to be significantly activated in HBx transgenic HCCs and further validated in human HBV-related HCCs. Inhibition of these lipid genes could reverse the effect of HBx on lipid biosynthesis and suppress HBx-induced cell proliferation in vitro. Our results support the concept that metabolic syndrome plays an important role in HBV tumorigenesis. The dysregulation of lipid metabolic genes may predict the disease progression to HCC in chronic hepatitis B patients.
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Carcinoma Hepatocelular/etiología , Carcinoma Hepatocelular/metabolismo , Transformación Celular Viral , Metabolismo de los Lípidos , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/metabolismo , Metabolómica , Transactivadores/genética , Animales , Carcinoma Hepatocelular/patología , Proliferación Celular , Transformación Celular Viral/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Ácidos Grasos/sangre , Ácidos Grasos/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Metabolismo de los Lípidos/genética , Lípidos/sangre , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/patología , Metaboloma , Metabolómica/métodos , Ratones , Ratones Transgénicos , Estadificación de Neoplasias , Proteínas Reguladoras y Accesorias ViralesRESUMEN
UNLABELLED: The development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) has been found to be associated with disturbed lipid metabolism. To elucidate the role of lipid metabolism in HBV tumorigenesis, we investigated the dynamic pattern of lipid metabolism in HBV pre-S2 mutant-induced tumorigenesis. Lipid and gene expression profiles were analyzed in an in vitro culture system and in transgenic mouse livers harboring HBV pre-S2 mutant. The pre-S2 mutant transgenic livers showed a biphasic pattern of lipid accumulation, starting from mild fatty change in early (1 month) transgenic livers, which subsided and then, remarkably, increased in HCC tissues. This biphasic pattern was synchronized with ATP citrate lyase (ACLY) activation. Further analyses revealed that the pre-S2 mutant initiated an endoplasmic reticulum (ER) stress-dependent mammalian target of rapamycin (mTOR) signalling cascade. The pre-S2 mutant-induced mTOR signal activated the sterol regulatory element binding transcription factor 1 (SREBF1) to upregulate ACLY, which then activated the fatty acid desaturase 2 (FADS2), mediated through ACLY-dependent histone acetylation. Such an ER stress-dependent mTOR signal cascade also is important for the proliferation of hepatocytes in vitro and is further validated in HBV-related HCC tissues. IMPORTANCE: Aberrations of lipid metabolism frequently occur in chronic HBV infection. Our results provide a potential mechanism of disturbed lipid metabolism in HBV pre-S2 mutant-induced tumorigenesis, which should be valuable for the design of HCC chemoprevention in high-risk HBV carriers.
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ATP Citrato (pro-S)-Liasa/biosíntesis , Carcinogénesis , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Interacciones Huésped-Patógeno , Metabolismo de los Lípidos , Precursores de Proteínas/genética , Serina-Treonina Quinasas TOR/metabolismo , Animales , Línea Celular , Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Hepatocitos/virología , Hígado/patología , Hígado/virología , Ratones Transgénicos , Proteínas Mutantes/genéticaRESUMEN
UNLABELLED: Human enterovirus A71 (EV-A71) belongs to the Enterovirus A species in the Picornaviridae family. Several vaccines against EV-A71, a disease causing severe neurological complications or even death, are currently under development and being tested in clinical trials, and preventative vaccination programs are expected to start soon. To characterize the potential for antigenic change of EV-A71, we compared the sequences of two antigenically diverse genotype B4 and B5 strains of EV-A71 and identified substitutions at residues 98, 145, and 164 in the VP1 capsid protein as antigenic determinants. To examine the effects of these three substitutions on antigenicity, we constructed a series of recombinant viruses containing different mutation combinations at these three residues with a reverse genetics system and then investigated the molecular basis of antigenic changes with antigenic cartography. We found that a novel EV-A71 mutant, containing lysine, glutamine, and glutamic acid at the respective residues 98, 145, and 164 in the VP1 capsid protein, exhibited neutralization reduction against patients' antisera and substantially increased virus binding ability to human cells. These observations indicated that this low-neutralization-reactive EV-A71 VP1-98K/145Q/164E mutant potentially increases viral binding ability and that surveillance studies should look out for these mutants, which could compromise vaccine efficacy. IMPORTANCE: Emerging and reemerging EV-A71 viruses can cause severe neurological etiology, primarily affecting children, especially around Asia-Pacific countries. We identified a set of mutations in EV-A71 that both reduced neutralization activity against humoral immunity in antisera of patients and healthy adults and greatly increased the viral binding ability to cells. These findings provide important insights for EV-A71 antigenic determinants and emphasize the importance of continuous surveillance, especially after EV-A71 vaccination programs begin.
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Variación Antigénica/inmunología , Proteínas de la Cápside/inmunología , Enterovirus Humano A/inmunología , Infecciones por Enterovirus/prevención & control , Epítopos/inmunología , Vacunas Virales/inmunología , Adulto , Sustitución de Aminoácidos/genética , Sustitución de Aminoácidos/inmunología , Anticuerpos Antivirales/sangre , Variación Antigénica/genética , Secuencia de Bases , Evolución Biológica , Proteínas de la Cápside/genética , Línea Celular Tumoral , Preescolar , Enterovirus Humano A/clasificación , Enterovirus Humano A/genética , Infecciones por Enterovirus/inmunología , Mapeo Epitopo , Epítopos/genética , Humanos , Datos de Secuencia MolecularRESUMEN
Although hepatitis B virus (HBV) has been established to cause hepatocellular carcinoma (HCC), the exact mechanism remains to be clarified. Type II ground glass hepatocytes (GGHs) harbouring the HBV pre-S2 mutant large surface protein (LHBS) have been recognized as a morphologically distinct hallmark of HCC in the advanced stages of chronic HBV infection. Considering its preneoplastic nature, we hypothesized that type II GGH may exhibit high genomic instability, which is important for the carcinogenic process in chronic HBV carriers. In this study we found that pre-S2 mutant LHBS directly interacted with importin α1, the key factor that recognizes cargos undergoing nuclear transportation mediated by the importin α/ß-associated nuclear pore complex (NPC). By interacting with importin α1, which inhibits its function as an NPC factor, pre-S2 mutant LHBS blocked nuclear transport of an essential DNA repair and recombination factor, Nijmegen breakage syndrome 1 (NBS1), upon DNA damage, thereby delaying the formation of nuclear foci at the sites of DNA double-strand breaks (DSBs). Pre-S2 mutant LHBS was also found to block NBS1-mediated homologous recombination repair and induce multi-nucleation of cells. In addition, pre-S2 mutant LHBS transgenic mice showed genomic instability, indicated by increased global gene copy number variations (CNVs), which were significantly higher than those in hepatitis B virus X mice, indicating that pre-S2 mutant LHBS is the major viral oncoprotein inducing genomic instability in HBV-infected hepatocytes. Consistently, the human type II GGHs in HCC patients exhibited increased DNA DSBs representing significant genomic instability. In conclusion, type II GGHs harbouring HBV pre-S2 mutant oncoprotein represent a high-risk marker for the loss of genome integrity in chronic HBV carriers and explain the complex chromosome changes in HCCs. Mouse array CGH raw data: GEO Accession No. GSE61378 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE61378).
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Carcinoma Hepatocelular/genética , Transformación Celular Viral/genética , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Hepatitis B Crónica/genética , Neoplasias Hepáticas/genética , Precursores de Proteínas/genética , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Hibridación Genómica Comparativa , Roturas del ADN de Doble Cadena , Variaciones en el Número de Copia de ADN , Daño del ADN , Reparación del ADN , Femenino , Inestabilidad Genómica , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/metabolismo , Hepatitis B Crónica/metabolismo , Hepatitis B Crónica/patología , Hepatitis B Crónica/virología , Hepatocitos/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación , Precursores de Proteínas/metabolismo , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
UNLABELLED: In hepatocellular carcinoma (HCC), dysregulated expression of microRNA-224 (miR-224) and impaired autophagy have been reported separately. However, the relationship between them has not been explored. In this study we determined that autophagy is down-regulated and inversely correlated with miR-224 expression in hepatitis B virus (HBV)-associated HCC patient specimens. These results were confirmed in liver tumors of HBV X gene transgenic mice. Furthermore, miR-224 was preferentially recruited and degraded during autophagic progression demonstrated by real-time polymerase chain reaction and miRNA in situ hybridization electron microscopy after extraction of autophagosomes. Our in vitro study demonstrated that miR-224 played an oncogenic role in hepatoma cell migration and tumor formation through silencing its target gene Smad4. In HCC patients, the expression of low-Atg5, high-miR-224, and low-Smad4 showed significant correlation with HBV infection and a poor overall survival rate. Autophagy-mediated miR-224 degradation and liver tumor suppression were further confirmed by the autophagy inducer amiodarone and miR-224 antagonist using an orthotopic SD rat model. CONCLUSION: A noncanonical pathway links autophagy, miR-224, Smad4, and HBV-associated HCC. These findings open a new avenue for the treatment of HCC.
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Autofagia/fisiología , Carcinogénesis/metabolismo , Carcinoma Hepatocelular/metabolismo , Virus de la Hepatitis B/fisiología , Neoplasias Hepáticas/metabolismo , MicroARNs/metabolismo , Amiodarona/farmacología , Animales , Autofagia/efectos de los fármacos , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Hepatitis B/complicaciones , Humanos , Técnicas In Vitro , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , MicroARNs/genética , Microscopía Electrónica , Ratas , Ratas Sprague-Dawley , Proteína Smad4/metabolismoRESUMEN
PURPOSE: A phase II trial was conducted to evaluate the therapeutic efficacy and safety profiles of frontline concurrent chemoradiotherapy (CCRT) plus consolidation chemotherapy for patients with stage I/II nasal natural killer/T-cell lymphoma (NKTCL). PATIENTS AND METHODS: Patients with newly diagnosed, measurable stage I/II nasal NKTCL were eligible. The CCRT included two cycles of the DEP regimen (dexamethasone, etoposide, and cisplatin) every 4 wk with concurrent 5040 cGy radiation in 28 fractions for 5 wk. Patients without disease progression after CCRT were subjected to two cycles of DVIP consisted of dexamethasone, etoposide, ifosphamide, mesna, and cisplatin every 4 wk. The primary endpoint was tumor response rate, and secondary endpoints were survival and toxicities. This phase II study has been registered in the ClinicalTrials.gov (NCT00292695). RESULTS: Thirty-three patients received CCRT, and 29 patients received two cycles of consolidation DVIP after CCRT. Among the 32 evaluable patients, 20 achieved complete response and 6 achieved partial response. The overall and complete response rate was 81% (95% CI, 68-95%) and 63% (95% CI, 46-79%), respectively. The 2-yr and 5-yr progression-free survival rate for intention-to-treat population was 64% (95% CI, 47-80%) and 60% (95% CI, 39-73%), respectively; while the corresponding overall survival rate was 73% (95% CI, 57-88%) and 66% (95% CI, 50-83%), respectively. The most common treatment-related grade 3/4 adverse event was leukopenia (85%). CONCLUSION: Frontline CCRT plus consolidation chemotherapy is feasible and effective for treating localized nasal NKTCL.
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Quimioradioterapia , Linfoma Extranodal de Células NK-T/patología , Linfoma Extranodal de Células NK-T/terapia , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Quimioradioterapia/efectos adversos , Terapia Combinada , Quimioterapia de Consolidación , Femenino , Humanos , Linfoma Extranodal de Células NK-T/mortalidad , Masculino , Cumplimiento de la Medicación , Persona de Mediana Edad , Estadificación de Neoplasias , Resultado del Tratamiento , Adulto JovenRESUMEN
Taiwan is a relatively isolated island, serving as a mixing vessel for colonization by different waves of ethnic and migratory groups over the past 4 centuries. The potential transmission pattern of Mycobacterium tuberculosis in different ethnic and migratory populations remains to be elucidated. By using mycobacterial tandem repeat sequences as genetic markers, the prevalence of M. tuberculosis strains in Taiwan revealed a close link to the historical migration. Interestingly, the M. tuberculosis strain in the aborigines of Eastern and Central Taiwan had a dominance of the Haarlem (Dutch) strain while those in Southern Taiwan had a dominance of the East-African Indian (EAI) strain. The prevalence of different M. tuberculosis strains in specific ethnic populations suggests that M. tuberculosis transmission is limited and restricted to close contact. The prevalence of the Beijing modern strain in the young population causes a concern for M. tuberculosis control, because of high virulence and drug resistance. Furthermore, our data using molecular genotyping should provide valuable information on the historical study of the origin and migration of aborigines in Taiwan.
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Genotipo , Mycobacterium tuberculosis/genética , Tuberculosis/etnología , Pueblo Asiatico , Farmacorresistencia Bacteriana Múltiple , Emigración e Inmigración , Humanos , Taiwán/etnología , Tuberculosis/microbiologíaRESUMEN
PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) was compared with culture for pathogen detection in peritoneal dialysis (PD)-related peritonitis. Of 21 samples of PD effluent, PCR/ESI-MS identified microorganisms in 18 (86%) samples, including Mycobacterium tuberculosis in 1 culture-negative sample. Of 15 double-positive samples, PCR/ESI-MS and culture reached levels of agreement of 100% (15/15) and 87.5% (7/8) at the genus and species levels, respectively. PCR/ESI-MS can be used for rapid pathogen detection in PD-related peritonitis.
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Bacterias/aislamiento & purificación , Infecciones Bacterianas/diagnóstico , Candida/aislamiento & purificación , Candidiasis/diagnóstico , Soluciones para Diálisis , Técnicas Microbiológicas/métodos , Peritonitis/diagnóstico , Adulto , Anciano , Infecciones Bacterianas/microbiología , Candidiasis/microbiología , Niño , Femenino , Humanos , Masculino , Persona de Mediana Edad , Diálisis Peritoneal/efectos adversos , Peritonitis/microbiología , Reacción en Cadena de la Polimerasa/métodos , Espectrometría de Masa por Ionización de Electrospray/métodos , Adulto JovenRESUMEN
Chronic hepatitis B virus (HBV) infection is a major cause of hepatocellular carcinoma (HCC) worldwide. The pre-S2 mutant large HBV surface protein (Δ2 LHBS), which contains an in-frame deletion of approximately 17 amino acids in LHBS, is highly associated with risks and prognoses of HBV-induced HCC. It was previously reported that Δ2 LHBS interacts with the Jun activation domain-binding protein 1 (JAB1), a zinc metalloprotease. This promotes the degradation of the cell cycle regulator p27(Kip1) and is believed to be the major mechanism for Δ2 LHBS-induced HCC. In this study, it was found that the interaction between JAB1 and Δ2 LHBS is facilitated by divalent metal Zn(2+) ions. The binding of JAB1 to Δ2 LHBS requires the JAB1/CSN5 MPN metalloenzyme (JAMM) motif and residue H138 that binds to Zn(2+) ions in JAB1. Isothermal titration calorimetry showed that Δ2 LHBS binds directly to Zn(2+) ions in a two-site binding mode. Residues H71 and H116 in Δ2 LHBS, which also contact Zn(2+) ions, are also indispensable for Δ2 LHBS-mediated p27(Kip1) degradation in human HuH7 cells. These results suggest that developing drugs that interrupt interactions between Δ2 LHBS and JAB1 can be used to mitigate Δ2 LHBS-associated risks for HCC.
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Carcinoma Hepatocelular/enzimología , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/metabolismo , Hepatitis B Crónica/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Hepáticas/enzimología , Péptido Hidrolasas/metabolismo , Precursores de Proteínas/metabolismo , Zinc/metabolismo , Secuencias de Aminoácidos , Complejo del Señalosoma COP9 , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Hepatitis B Crónica/virología , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Hepáticas/virología , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Unión Proteica , Precursores de Proteínas/genéticaRESUMEN
Chronic hepatitis B virus (HBV) infection can cause hepatocellular carcinoma (HCC). Several hypotheses have been proposed to explain the mechanisms of HBV tumorigenesis, including inflammation and liver regeneration associated with cytotoxic immune injuries and transcriptional activators of mutant HBV gene products. The mutant viral oncoprotein-driven tumorigenesis is prevailed at the advanced stage or anti-HBe-positive phase of chronic HBV infection. Besides HBx, the pre-S2 (deletion) mutant protein represents a newly recognized oncoprotein that is accumulated in the endoplasmic reticulum (ER) and manifests as type II ground glass hepatocytes (GGH). The retention of pre-S2 mutant protein in ER can induce ER stress and initiate an ER stress-dependent VEGF/Akt/mTOR and NFκB/COX-2 signal pathway. Additionally, the pre-S2 mutant large surface protein can induce an ER stress-independent pathway to transactivate JAB-1/p27/RB/cyclin A,D pathway, leading to growth advantage of type II GGH. The pre-S2 mutant protein-induced ER stress can also cause DNA damage, centrosome overduplication, and genomic instability. In 5-10% of type II GGHs, there is co-expression of pre-S2 mutant protein and HBx antigen which exhibited enhanced oncogenic effects in transgenic mice. The mTOR signal cascade is consistently activated throughout the course of pre-S2 mutant transgenic livers and in human HCC tissues, leading to metabolic disorders and HCC tumorigenesis. Clinically, the presence of pre-S2 deletion mutants in sera frequently develop resistance to nucleoside analogues anti-virals and predict HCC development. The pre-S2 deletion mutants and type II GGHs therefore represent novel biomarkers of HBV-related HCCs. A versatile DNA array chip has been developed to detect pre-S2 mutants in serum. Overall, the presence of pre-S2 mutants in serum has implications for anti-viral treatment and can predict HCC development. Targeting at pre-S2 mutant protein-induced, ER stress-dependent, mTOR signal cascade and metabolic disorders may offer potential strategy for chemoprevention or therapy in high risk chronic HBV carriers.
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Carcinoma Hepatocelular , Transformación Celular Viral/genética , Virus de la Hepatitis B , Neoplasias Hepáticas , Eliminación de Secuencia , Animales , Secuencia de Bases , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , RatonesRESUMEN
BACKGROUND: Clinical manifestations of enterovirus 71 (EV71) range from herpangina, hand-foot-and-mouth disease (HFMD), to severe neurological complications. Unlike the situation of switching genotypes seen in EV71 outbreaks during 1998-2008 in Taiwan, genotype B5 was responsible for two large outbreaks in 2008 and 2012, respectively. In China, by contrast, EV71 often persists as a single genotype in the population and causes frequent outbreaks. To investigate genetic changes in viral evolution, complete EV71 genome sequences were used to analyze the intra-genotypic evolution pattern in Taiwan, China, and the Netherlands. RESULTS: Genotype B5 was predominant in Taiwan's 2008 outbreak and was re-emergent in 2012. EV71 strains from both outbreaks were phylogenetically segregated into two lineages containing fourteen non-synonymous substitutions predominantly in the non-structural protein coding region. In China, genotype C4 was first seen in 1998 and caused the latest large outbreak in 2008. Unlike shifting genotypes in Taiwan, genotype C4 persisted with progressive drift through time. A majority of non-synonymous mutations occurred in residues located in the non-structural coding region, showing annual increases. Interestingly, genotype B1/B2 in the Netherlands showed another stepwise evolution with dramatic EV71 activity increase in 1986. Phylogeny of the VP1 coding region in 1971-1986 exhibited similar lineage turnover with genotype C4 in China; however, phylogeny of the 3D-encoding region indicated separate lineage appearing after 1983, suggesting that the 3D-encoding region of genotype B2 was derived from an unidentified ancestor that contributed to intra-genotypic evolution in the Netherlands. CONCLUSIONS: Unlike VP1 coding sequences long used for phylogenetic study of enteroviruses due to expected host immune escape, our study emphasizes a dominant role of non-synonymous mutations in non-structural protein regions that contribute to (re-)emergent genotypes in continuous stepwise evolution. Dozens of amino acid substitutions, especially in non-structural proteins, were identified via genetic changes driven through intra-genotypic evolution worldwide. These identified substitutions appeared to increase viral fitness in the population, affording valuable insights not only for viral evolution but also for prevention, control, and vaccine against EV71 infection.