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In both normal turnover of the hepatic tissue and acute hepatic injury, the liver predominantly activates terminally differentiated hepatocytes to proliferate and repair. However, in chronic and severe chronic injury, this capacity fails, and liver progenitor cells (LPCs) can give rise to hepatocytes to restore both hepatic architecture and liver metabolic function. Although the promotion of LPC-to-hepatocyte differentiation to acquire a considerable number of functional hepatocytes could serve as a potentially new therapeutic option for patients with end-stage liver disease, its development first requires the identification of the molecular mechanisms driving this process. Here, we found that the epithelial cell adhesion molecule (EpCAM), a progenitor cell marker, regulates the differentiation of LPCs into hepatocytes through Notch1 signaling pathway. Western blotting (WB) revealed a consistent expression pattern of EpCAM and Notch1 during LPC-to-hepatocyte differentiation in vitro. Additionally, overexpression of EpCAM blocked LPC-to-hepatocyte differentiation, which was in consistent with the repressive role of Notch signaling during hepatic differentiation. WB and immunofluorescence data also showed that the upregulation of EpCAM expression increased the generation of Notch intracellular domain (N1ICD), indicating the promotion of Notch1 activity. Our results established the EpCAM-Notch1 signaling axis as an inhibitory mechanism preventing LPC-to-hepatocyte differentiation in vitro.
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Potential biomarkers that can be used to determine prognosis and perform targeted therapies are urgently needed to treat patients with hepatocellular carcinoma (HCC). To meet this need, we performed a screen to identify functional genes associated with hepatocellular carcinogenesis and its progression at the transcriptome and proteome levels. We identified aldehyde dedydrogenase-2 (ALDH2) as a gene of interest for further study. ALDH2 levels were significantly lower at the mRNA and protein level in tumor tissues than in normal tissues, and they were even lower in tissues that exhibited increased migratory capacity. A study of clinical associations showed that ALDH2 is correlated with survival and multiple migration-associated clinicopathological traits, including the presence of metastasis and portal vein tumor thrombus. The result of overexpressing or knocking down ALDH2 showed that this gene inhibited migration and invasion both in vivo and in vitro. We also found that ALDH2 altered the redox status of cells by regulating acetaldehyde levels and that it further activated the AMP-activated protein kinase (AMPK) signaling pathway. CONCLUSION: Decreased levels of ALDH2 may indicate a poor prognosis in HCC patients, while forcing the expression of ALDH2 in HCC cells inhibited their aggressive behavior in vitro and in mice largely by modulating the activity of the ALDH2-acetaldehyde-redox-AMPK axis. Therefore, identifying ALDH2 expression levels in HCC might be a useful strategy for classifying HCC patients and for developing potential therapeutic strategies that specifically target metastatic HCC. (Hepatology 2017;65:1628-1644).
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Aldehído Deshidrogenasa Mitocondrial/metabolismo , Carcinoma Hepatocelular/enzimología , Neoplasias Hepáticas Experimentales/enzimología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , China/epidemiología , Expresión Génica Ectópica , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Hígado/patología , Neoplasias Hepáticas Experimentales/mortalidad , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones Desnudos , Persona de Mediana Edad , Metástasis de la Neoplasia , Oxidación-Reducción , Distribución AleatoriaRESUMEN
UNLABELLED: Solid tumors often suffer from suboptimal oxygen and nutrient supplies. This stress underlies the requirement for metabolic adaptation. Aberrantly activated de novo lipogenesis is critical for development and progression of human hepatocellular carcinoma (HCC). However, whether de novo lipogenesis influences biological behaviors of HCCs under conditions of metabolic stress are still poorly understood. Here, we show that HCCs display distinct levels of glucose-derived de novo lipogenesis, which are positively correlated with their survival responses to glucose limitation. The enhanced lipogenesis in HCCs is characterized by an increased expression of rate-limiting enzyme acetyl-coenzyme A carboxylase alpha (ACCα). ACCα-mediated fatty acid (FA) synthesis determines the intracellular lipid content that is required to maintain energy hemostasis and inhibit cell death by means of FA oxidation (FAO) during metabolic stress. In accord, overexpression of ACCα facilitates tumor growth. ACCα forms a complex with carnitine palmitoyltransferase 1A (CPT1A) and prevents its mitochondria distribution under nutrient-sufficient conditions. During metabolic stress, phosphorylation of ACCα leads to dissociation of the complex and mitochondria localization of CPT1A, thus promoting FAO-mediated cell survival. Therefore, ACCα could provide both the substrate and enzyme storage for FAO during glucose deficiency. Up-regulation of ACCα is also significantly correlated with poorer overall survival and disease recurrence postsurgery. Multivariate Cox's regression analysis identified ACCα as an effective predictor of poor prognosis. CONCLUSION: These results present novel mechanistic insight into a pivotal role of ACCα in maintaining HCC survival under metabolic stress. It could be exploited as a novel diagnostic marker and therapeutic target.
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Acetil-CoA Carboxilasa/metabolismo , Carcinoma Hepatocelular/enzimología , Glucosa/metabolismo , Neoplasias Hepáticas/enzimología , Estrés Oxidativo , Acetil-CoA Carboxilasa/genética , Animales , Apoptosis/genética , Carcinoma Hepatocelular/mortalidad , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Ácido Graso Sintasas/metabolismo , Ácidos Grasos/metabolismo , Regulación Neoplásica de la Expresión Génica , Silenciador del Gen , Xenoinjertos , Humanos , Estimación de Kaplan-Meier , Metabolismo de los Lípidos/fisiología , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/patología , Ratones , Valor Predictivo de las Pruebas , Pronóstico , Modelos de Riesgos Proporcionales , Análisis de Supervivencia , Regulación hacia ArribaRESUMEN
Tumor tissue has great clinical and scientific value which relies highly on the proper preservation of primary materials. Conventional tumor tissue cryopreservation using slow-freezing method has yielded limited success, leading to significant cell loss and morphological damage. Here we report a standardized vitrification-based cryopreservation method, by which we have successfully vitrified and warmed 35 intrahepatic cholangiocarcinoma (ICC) tissues with up to 80% viability of the fresh tumor tissues. Cryopreserved ICC tissue could generate patient-derived xenografts (PDXs) with take rates of 68.2% compared to 72.7% using fresh tumor tissues. Histological and genetic analyses showed that no significant alterations in morphology and gene expression were introduced by this cryopreservation method. Our procedure may facilitate collection, long-time storage and propagation of cholangiocarcinoma or other tumor specimens for (pre)clinical studies of novel therapies or for basic research.
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Colangiocarcinoma/patología , Criopreservación/métodos , Trasplante de Neoplasias/métodos , Vitrificación , Animales , Supervivencia Celular , Congelación , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante Heterólogo/métodos , Células Tumorales CultivadasRESUMEN
BACKGROUND & AIMS: Hepatocellular carcinoma (HCC) develops in response to chronic hepatic injury. Although induced cell death is regarded as the major component of p53 tumor-suppressive activity, we recently found that sustained p53 activation subsequent to DNA damage promotes inflammation-associated hepatocarcinogenesis. Here we aim at exploring the mechanism linking p53 activation and hepatic inflammation during hepatocarcinogenesis. METHODS: p53(-/-) hepatocytes expressing inducible p53 and primary wild type hepatocytes were treated to induce p53 expression. The supernatants were collected and analyzed for the presence of released inflammatory cytokines. Ethyl pyruvate was used in a rat model of carcinogen-induced hepatocarcinogenesis to examine its effect on p53-dependent chronic hepatic injury, inflammation, and tumorigenesis. RESULTS: Here we show that cytoplasmic translocation and circulating levels of potent inflammatory molecule high-mobility group protein 1 (HMGB1) were greater in wild type rats than in p53(+/-) rats following carcinogen administration. Restoration of p53 expression in p53-null hepatocytes or induction of endogenous p53 in wild type hepatocytes gives rise to the release of HMGB1. Administration of the HMGB1 release inhibitor ethyl pyruvate, which does not affect p53-mediated hepatic apoptosis, substantially prevented carcinogen-induced cirrhosis and tumorigenesis in rat livers. CONCLUSIONS: These results suggest that although p53 is usually regarded as a tumor suppressor, its constant activation can promote pro-tumorigenic inflammation, at least in part, via inducing HMGB1 release. Application of HMGB1 inhibitors when restoring p53 in cancer therapy might protect against pro-tumorigenic effects while leaving p53-mediated clearance of malignant cells intact.
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Genes p53 , Proteína HMGB1/metabolismo , Neoplasias Hepáticas Experimentales/etiología , Animales , Línea Celular , Dietilnitrosamina/toxicidad , Técnicas de Inactivación de Genes , Hepatitis Crónica/etiología , Hepatitis Crónica/metabolismo , Hepatitis Crónica/patología , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Neoplasias Hepáticas Experimentales/patología , Ratas , Activación Transcripcional , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
UNLABELLED: Hepatitis B virus X (HBx) protein is implicated in hepatitis B virus (HBV)-associated liver carcinogenesis. However, it remains unclear whether HBx-expressing hepatic progenitor cells (HPCs) are attributed to liver tumor formation. In this study, by using HBx transgenic mice and a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver injury model, the relationship between HBx expression and tumorigenicity of HPCs was analyzed. Compared with control mice, an elevated number of EpCAM(+) cells with characteristics of HPCs was observed in HBx mice after 1 month and 4 months of DDC diet feeding. All HBx transgenic mice developed liver tumors characterized by histological features of both hepatocellular carcinoma (HCC) and cholangiocarcinoma after 7 months of DDC feeding. Notably, EpCAM(+) HPCs isolated from premalignant HBx mice exposed to a DDC diet for 4 months formed subcutaneous mixed-lineage tumors (four out of six) in nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice, and none of the cells from wildtype (WT) induced tumor, indicating that HBx may induce malignant transformation of HPCs that contributes to tumorigenesis. We also found higher titers of circulating interleukin (IL)-6, activities of IL-6/STAT3, and Wnt/ß-catenin signaling pathways in HBx transgenic mice, suggesting HBx may induce intrinsic changes in HPCs by way of the above signaling that enables HPCs with tumorigenicity potential. Finally, clinical evidence showed that high HBx expression in human HBV-related HCC was statistically associated with expansion of EpCAM(+) or OV6(+) tumor cells and aggressive clinicopathologic features. CONCLUSION: HBx induces intrinsic cellular transformation promoting the expansion and tumorigenicity of HPCs in DDC-treated mice, which may be a possible origin for liver cancer induced by chronic hepatitis infection.
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Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/virología , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/virología , Piridinas/toxicidad , Transactivadores/genética , Animales , Neoplasias de los Conductos Biliares/inducido químicamente , Neoplasias de los Conductos Biliares/fisiopatología , Neoplasias de los Conductos Biliares/virología , Conductos Biliares Intrahepáticos , Carcinoma Hepatocelular/fisiopatología , Transformación Celular Neoplásica/inducido químicamente , Colangiocarcinoma/inducido químicamente , Colangiocarcinoma/fisiopatología , Colangiocarcinoma/virología , Modelos Animales de Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Neoplasias Hepáticas Experimentales/fisiopatología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Ratones Transgénicos , Persona de Mediana Edad , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Madre/efectos de los fármacos , Células Madre/fisiología , Células Madre/virología , Transactivadores/metabolismo , Proteínas Reguladoras y Accesorias ViralesRESUMEN
BACKGROUND: CD24+CK19+/CD24+SOX9+ resident liver cells are activated and expanded after chronic liver injury in a ductular reaction. However, the sources and functions of these cells in liver damage remain disputed. RESULTS: The current study combined genetic lineage tracing with in vitro small-molecule-based reprogramming to define liver progenitor cells (LPCs) derived from hepatic parenchymal and non-parenchymal tissues. tdTom+ hepatocytes were isolated from ROSA26tdTomato mice following AAV8-Tbg-Cre-mediated recombination, EpCAM+ biliary epithelial cells (BECs) from wild-type intrahepatic bile ducts and ALB/GFP-EpCAM- cells were isolated from AlbCreERT/R26GFP mice. A cocktail of small molecules was used to convert the isolated cells into LPCs. These in vitro cultured LPCs with CD24 and SOX9 expression regained the ability to proliferate. Transcriptional profiling showed that the in-vitro cultured LPCs derived from the resident LPCs in non-parenchymal tissues expressed Lipocalin-2 (Lcn2) at high levels. Accordingly, endogenous Cd24a+Lcn2+ LPCs were identified by integration of sc-RNA-sequencing and pathological datasets of liver dysfunction which indicates that LPCs produced by ductular reactions might also originate from the resident LPCs. Transplantation of in-vitro cultured Cd24a+Lcn2+ LPCs into CCl4-induced fibrotic livers exacerbated liver damage and dysfunction, possibly due to LCN2-dependent macrophage inflammatory response. CONCLUSIONS: CD24+LCN2+ LPCs constituted the expanding ductular reaction and contributed to macrophage-mediated inflammation in chronic liver damage. The current findings highlight the roles of LPCs from distinct origins and expose the possibility of targeting LPCs in the treatment of chronic hepatic diseases.
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The p53 tumor suppressor gene is highly mutated in human cancers. Individuals who inherit one p53 mutant allele are susceptible to a wide range of tumor types, including breast cancer and sarcoma. We recently generated p53 knockout rats through gene targeting in embryonic stem cells. Here we show that rats homozygous for the null allele are prone to early onset spontaneous sarcomas and lymphoma with high incidence of metastases. Heterozygous rats are also highly predisposed to cancer, but with a delayed onset and a wider spectrum of tumor types compared with homozygotes. Importantly, up to 20% of female heterozygotes developed breast cancer and about 70% of the tumors were positive for estrogen receptor. Exposing p53-deficient rats to a low dose of the carcinogen diethylnitrosamine dramatically decreased the latency for sarcoma development and survival time compared with equivalently treated wild-type rats. These unique features make this knockout line a valuable model for investigating human malignancy and in vivo carcinogenicity of chemicals and therapeutic compounds.
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Técnicas de Inactivación de Genes , Genes p53 , Modelos Animales , Alelos , Animales , Neoplasias de la Mama/genética , Transformación Celular Neoplásica/genética , Femenino , Heterocigoto , Homocigoto , Linfoma/genética , Quinolinas , Ratas , Sarcoma/genéticaRESUMEN
BACKGROUND & AIMS: Due to its anatomic connection, the liver is constantly exposed to gut-derived bacterial products or metabolites. Disruption of gut homeostasis is associated with many human diseases. The aim of this study was to determine the role of gut homeostasis in initiation and progression of hepatocellular carcinoma (HCC). METHODS: Disruption of intestinal homeostasis by penicillin or dextran sulfate sodium (DSS) and its restoration by probiotics were applied in a diethylnitrosamine (DEN) model of rat hepatocarcinogenesis. RESULTS: Patients with liver cirrhosis and HCC had significantly increased serum endotoxin levels. Chronic DEN treatment of rats was associated with an imbalance of subpopulations of the gut microflora including a significant suppression of Lactobacillus species, Bifidobacterium species and Enterococcus species as well as intestinal inflammation. Induction of enteric dysbacteriosis or intestinal inflammation by penicillin or DSS, respectively, significantly promoted tumor formation. Administration of probiotics dramatically mitigated enteric dysbacteriosis, ameliorated intestinal inflammation, and most importantly, decreased liver tumor growth and multiplicity. Interestingly, probiotics not only inhibited the translocation of endotoxin, which bears pathogen-associated molecular patterns (PAMPs) but also the activation of damage-associated molecular patterns (DAMPs) such as high-mobility group box 1 (HMGB1). As a result, the production of pro- and anti-inflammatory cytokines was skewed in favor of a reduced tumorigenic inflammation in the liver. CONCLUSIONS: The data highlights the importance of gut homeostasis in the pathogenesis of HCC. Modulation of the gut microbiota by probiotics may represent a new avenue for therapeutic intervention to treat or prevent HCC development.
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Carcinoma Hepatocelular/patología , Endotoxinas/metabolismo , Tracto Gastrointestinal/microbiología , Homeostasis , Neoplasias Hepáticas Experimentales/patología , Probióticos/farmacología , Alquilantes/farmacología , Animales , Antibacterianos/farmacología , Bifidobacterium/efectos de los fármacos , Carcinoma Hepatocelular/sangre , Carcinoma Hepatocelular/etiología , Citocinas/biosíntesis , Sulfato de Dextran/farmacología , Dietilnitrosamina/farmacología , Dietilnitrosamina/toxicidad , Progresión de la Enfermedad , Endotoxinas/sangre , Enterococcus/efectos de los fármacos , Gastroenteritis/inducido químicamente , Gastroenteritis/tratamiento farmacológico , Gastroenteritis/metabolismo , Tracto Gastrointestinal/fisiopatología , Proteína HMGB1/metabolismo , Homeostasis/efectos de los fármacos , Humanos , Lactobacillus/efectos de los fármacos , Cirrosis Hepática/sangre , Cirrosis Hepática/complicaciones , Neoplasias Hepáticas Experimentales/inducido químicamente , Neoplasias Hepáticas Experimentales/microbiología , Masculino , Penicilinas/farmacología , Probióticos/uso terapéutico , Ratas , Ratas Sprague-DawleyRESUMEN
BACKGROUND & AIMS: Accumulating evidence suggests the involvement of tumor-initiating cells (T-ICs) in cancer genesis, but whether liver T-ICs contribute to HCC invasion and metastasis remains unclear. METHODS: OV6(+) T-ICs were isolated from SMMC7721 and HuH7 cell lines by magnetic sorting. Characteristics of T-ICs were assessed by in vitro and mouse xenograft assays. Expression of OV6 was determined by immunostaining in specimens from 218 HCC patients, and Kaplan-Meier survival analysis was used to determine the correlation of OV6 expression with prognosis. RESULTS: OV6(+) T-ICs isolated from HCC cell lines not only possess a higher capacity to form tumor spheroids in vitro, but also had a greater potential to form tumors when implanted in non-obese diabetic/severe combined immunodeficient mice, suggesting their elevated self-renewal capacity and tumorigenicity. Moreover, OV6(+) T-ICs exhibited more invasive and metastatic potentials both in vitro and in vivo. Patients with more OV6(+) tumor cells were associated with aggressive clinicopathologic features and poor prognosis. CXCR4 is expressed at higher levels in OV6(+) cells. Recombinant stromal cell-derived factor-1 (SDF-1) treatment expanded the OV6(+) HCC T-ICs population, by sustaining the stem cell property of OV6(+) cells. The SDF-1 effect was blocked by a specific CXCR4 inhibitor, AMD3100, or transfection of siRNA targeting CXCR4. CONCLUSIONS: OV6(+) HCC cells may represent a subpopulation of T-ICs with augmented invasion and metastasis potential, which contribute to progression and metastasis of HCC. The SDF-1/CXCR4 axis also provides therapeutic targets for elimination of liver T-ICs.
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Antígenos de Diferenciación/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/secundario , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/metabolismo , Células Madre Neoplásicas/metabolismo , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Bencilaminas , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Movimiento Celular , Quimiocina CXCL12/metabolismo , Ciclamas , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Molécula de Adhesión Celular Epitelial , Femenino , Técnicas de Silenciamiento del Gen , Glicoproteínas/metabolismo , Compuestos Heterocíclicos/farmacología , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Análisis Multivariante , Invasividad Neoplásica , Péptidos/metabolismo , Pronóstico , ARN Interferente Pequeño , Receptores CXCR4/efectos de los fármacos , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Esferoides CelularesRESUMEN
Models considering hepatocellular carcinoma (HCC) complexity cannot be accurately replicated in routine cell lines or animal models. We aimed to evaluate the practicality of tissue slice culture by combining it with a cryopreservation technique. We prepared 0.3mmthick tissue slices by a microtome and maintained their cell viability using a cryopreservation technique. Slices were cultured individually in the presence or absence of regorafenib (REG) for 72 h. Alterations in morphology and gene expression were assessed by histological and genetic analysis. Overall viability was also analyzed in tissue slices by CCK8 quantification assay and fluorescent staining. Tissue morphology and cell viability were evaluated to quantify drug effects. Histological and genetic analyses showed that no significant alterations in morphology and gene expression were induced by the vitrificationbased cryopreservation method. The viability of warmed HCC tissues was up to 90% of the fresh tissues. The viability and proliferation could be retained for at least four days in the filter culture system. The positive drug responses in precisioncut slice culture in vitro were evaluated by tissue morphology and cell viability. In summary, the successful application of precisioncut HCC slice culture combined with a cryopreservation technique in a systematic drug screening demonstrates the feasibility and utility of slice culture method for assessing drug response.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Animales , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Supervivencia Celular , Criopreservación , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genéticaRESUMEN
Cancer cells are addicted to glutamine. However, cancer cells often suffer from glutamine starvation, which largely results from the fast growth of cancer cells and the insufficient vascularization in the interior of cancer tissues. Herein, based on clinical samples, patient-derived cells (PDCs), and cell lines, it is found that liver cancer cells display stem-like characteristics upon glutamine shortage due to maintaining the stemness of tumor initiating cells (TICs) and even promoting transformation of non-TICs into stem-like cells by glutamine starvation. Increased expression of glutamine synthetase (GS) is essential for maintaining and promoting stem-like characteristics of liver cancer cells during glutamine starvation. Mechanistically, glutamine starvation activates Rictor/mTORC2 to induce HDAC3-mediated deacetylation and stabilization of GS. Rictor is significantly correlated with the expression of GS and stem marker OCT4 at tumor site, and closely correlates with poor prognosis of hepatocellular carcinomas. Inhibiting components of mTORC2-HDAC3-GS axis decrease TICs and promote xenografts regression upon glutamine-starvation therapy. Collectively, the data provides novel insights into the role of Rictor/mTORC2-HDAC3 in reprogramming glutamine metabolism to sustain stemness of cancer cells. Targeting Rictor/HDAC3 may enhance the efficacy of glutamine-starvation therapy and limit the rapid growth and malignant progression of tumors.
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Neoplasias Hepáticas , Línea Celular , Glutamato-Amoníaco Ligasa , Glutamina/deficiencia , Glutamina/metabolismo , Histona Desacetilasas , Humanos , Diana Mecanicista del Complejo 2 de la Rapamicina/metabolismo , Factores de TranscripciónRESUMEN
UNLABELLED: Increasing evidence suggests that the presence of endotoxemia is of substantial clinical relevance to patients with cirrhosis, but it is unclear whether and how gut-derived LPS amplifies the tumorigenic response of the liver. We found that the circulating levels of LPS were elevated in animal models of carcinogen-induced hepatocarcinogenesis. Reduction of LPS using antibiotics regimen in rats or genetic ablation of its receptor Toll-like receptor 4 (TLR4) in mice prevented excessive tumor growth and multiplicity. Additional investigation revealed that TLR4 ablation sensitizes the liver to carcinogen-induced toxicity via blocking NF-κB activation and sensitizing the liver to reactive oxygen species (ROS)-induced toxicity, but lessens inflammation-mediated compensatory proliferation. Reconstitution of TLR4-expressing myeloid cells in TLR4-deficient mice restored diethylnitrosamine (DEN)-induced hepatic inflammation and proliferation, indicating a paracrine mechanism of LPS in tumor promotion. Meanwhile, deletion of gut-derived endotoxin suppressed DEN-induced cytokine production and compensatory proliferation, whereas in vivo LPS pre-challenge promotes hepatocyte proliferation. CONCLUSION: Our data indicate that sustained LPS accumulation represents a pathological mediator of inflammation-associated hepatocellular carcinoma (HCC) and manipulation of the gut flora to prevent pathogenic bacterial translocation and endotoxin absorption may favorably influence liver function in patients with cirrhosis who are at risk of developing HCC.
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Apoptosis/efectos de los fármacos , Carcinógenos/farmacología , Carcinoma Hepatocelular/etiología , Lipopolisacáridos/farmacología , Neoplasias Hepáticas/etiología , Receptor Toll-Like 4/fisiología , Animales , Carcinoma Hepatocelular/patología , Proliferación Celular/efectos de los fármacos , Dietilnitrosamina/toxicidad , Intestinos/microbiología , Lipopolisacáridos/sangre , Cirrosis Hepática/complicaciones , Masculino , Ratones , FN-kappa B/antagonistas & inhibidores , Neomicina/farmacología , Polimixina B/farmacología , Ratas , Especies Reactivas de Oxígeno/farmacologíaRESUMEN
Rationale: We developed a cocktail of soluble molecules mimicking the in vivo milieu supporting liver regeneration that could convert mature hepatocytes to expandable liver progenitor-like cells in vitro. This study aimed to induce endogenous liver progenitor cells by the administration of the soluble molecules to provide an alternative approach for the resolution of liver fibrosis. Methods:In vitro cultured hepatocyte-derived liver progenitor-like cells (HepLPCs) were transplanted into CCL4-treated mice to investigate the therapeutic effect against liver fibrosis. Next, we used HGF in combination with a cocktail of small molecules (Y-27632, A-83-01, and CHIR99021 (HACY)) to induce endogenous CD24+ liver progenitor cells and to inhibit the activation of hepatic stellate cells (HSCs) during CCL4-induced hepatic injury. RNA sequencing was performed to further clarify the features of HACY-induced CD24+ cells compared with CCL4-induced CD24+ cells and in vitro derived HepLPCs. Finally, we evaluated the expansion of HACY-induced CD24+ cells in human hepatocyte-spheroids from fibrotic liver tissues. Results: HepLPCs exhibited the capacity to alleviate liver fibrosis after transplantation into CCL4-treated mice. The in vivo administration of HACY not only induced the conversion of mature hepatocytes (MHs) to CD24+ progenitor cells but prevented the activation of HSCs, thus leading to enhanced improvement of liver fibrosis in CCL4-treated mice. Compared to CD24+ cells induced by CCL4 alone, HACY-induced CD24+ cells retained an enhanced level of hepatic function and could promote the restoration of liver function that exhibited comparable gene expression profiles with HepLPCs. CD24+ cells were also observed in human liver fibrotic tissues and were expanded in three-dimensional (3D) hepatic spheroids in the presence of HACY in vitro. Conclusions: Hepatocyte-derived liver progenitor-like cells are crucial for liver regeneration during chronic hepatic injuries. The administration of HACY, which allowed the induction of endogenous CD24+ progenitor cells and the inactivation of HSCs, exerts beneficial effects in the treatment of liver fibrosis by re-establishing a balance favoring liver regeneration while preventing fibrotic responses.
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Células Estrelladas Hepáticas/efectos de los fármacos , Cirrosis Hepática/tratamiento farmacológico , Hígado/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Células Madre/efectos de los fármacos , Amidas/farmacología , Animales , Antígeno CD24/metabolismo , Tetracloruro de Carbono/farmacología , Células Cultivadas , Expresión Génica/efectos de los fármacos , Células Estrelladas Hepáticas/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Hígado/metabolismo , Cirrosis Hepática/metabolismo , Regeneración Hepática/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Piridinas/farmacología , Pirimidinas/farmacología , Células Madre/metabolismoRESUMEN
Globally, about two million people die from liver diseases every year. Liver transplantation is the only reliable therapy for severe end-stage liver disease, however, the shortage of organ donors is a huge limitation. Human hepatocytes derived liver progenitor-like cells (HepLPCs) have been reported as a novel source of liver cells for development of in vitro models, cell therapies, and tissue-engineering applications, but their functionality as transplantation donors is unclear. Here, a 3-dimensional (3D) co-culture system using HepLPCs and human umbilical vein endothelial cells (HUVECs) was developed. These HepLPC spheroids mimicked the cellular interactions and architecture of mature hepatocytes, as confirmed through ultrastructure morphology, gene expression profile and functional assays. HepLPCs encapsulated in alginate beads are able to mitigate liver injury in mice treated with carbon tetrachloride (CCL4), while alginate coating protects the cells from immune attack. We confirmed these phenomena due to HUVECs producing glial cell line-derived neurotrophic factor (GDNF) to promote HepLPCs maturation and enhance HepLPCs tight junction through MET phosphorylation. Our results display the efficacy and safety of the alginate microencapsulated spheroids in animal model with acute liver injury (ALF), which may suggest a new strategy for cell therapy.
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Local recurrence is a therapeutic challenge for radiofrequency ablation (RFA) in treatment of small solid focal malignancies. Here we show that RFA induced heat shock proteins (HSPs) expression and high mobility group box-1 (HMGB1) translocation in xenografted melanoma, which might create a proinflammatory microenvironment that favors tumor antigen presentation and activation of the effector T cells. On this basis, we investigate whether a prime-boost strategy combining a prime with heat-shocked tumor cell lysate-pulsed dendritic cell (HT-DC) followed by an in situ boost with radiofrequency thermal ablation can prevent local tumor recurrence. The combination treatment with HT-DC and RFA showed potent antitumor effects, with >or=90% of tumor recurrence abrogated following RFA treatment. By contrast, prevaccination with unheated tumor lysate-pulsed DC had little effect on tumor relapse. Analysis of the underlying mechanism revealed that splenocytes from mice treated with HT-DC plus RFA contained significantly more tumor-specific, IFN-gamma-secreting T cells compared with control groups. Moreover, adoptive transfer of splenocytes from successfully treated tumor-free mice protected naive animals from tumor recurrence following RFA, and this was mediated mainly by CD8(+) T cells. Therefore, the optimal priming for the DC vaccination before RFA is important for boosting antigen-specific T cell responses and prevention of cancer recurrence.
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Vacunas contra el Cáncer/inmunología , Ablación por Catéter , Células Dendríticas/inmunología , Recurrencia Local de Neoplasia/prevención & control , Neoplasias Experimentales/terapia , Traslado Adoptivo , Animales , Western Blotting , Linfocitos T CD8-positivos/inmunología , Terapia Combinada , Citotoxicidad Inmunológica , Células Dendríticas/trasplante , Citometría de Flujo , Proteínas de Choque Térmico/metabolismo , Calor , Interferón gamma/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Recurrencia Local de Neoplasia/inmunología , Neoplasias Experimentales/inmunologíaRESUMEN
Living tumors are of great scientific value for clinical medicine and basic research, especially for drug testing. An increasing number of drug tests fail due to the use of imperfect models. The aim of the present study was to develop a novel method combining vitrificationbased cryopreservation of tumor biopsies and precisioncut slice cultivation for the assessment of anticancer drug responses. Biological characteristics of rectal cancer liver metastasis biopsies could be retained by vitrificationbased cryopreservation. The patientderived xenograft models were successfully established using both fresh and warmed biopsy tissues. Precisioncut slicing provided a similar threedimensional architecture and heterogeneity to the original tumor. The positive drug responses in the xenograft model were consistent with those in precisioncut slice cultures in vitro. The present study demonstrated that live tumor biopsies could be preserved using vitrificationbased cryopreservation. The warmed tissues developed xenograft tumors, which were also useful for either in vivo or in vitro anticancer drug testing. Precisioncut slices derived from the warmed tissues provided an efficient tool to assess anticancer drug response in vitro.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/patología , Neoplasias del Recto/tratamiento farmacológico , Neoplasias del Recto/patología , Técnicas de Cultivo de Tejidos/métodos , Animales , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Biopsia , Criopreservación , Femenino , Humanos , Neoplasias Hepáticas/secundario , Masculino , Ratones , Persona de Mediana Edad , Resultado del Tratamiento , Vitrificación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Clinical advancement of the bioartificial liver is hampered by the lack of expandable human hepatocytes and appropriate bioreactors and carriers to encourage hepatic cells to function during extracorporeal circulation. We have recently developed an efficient approach for derivation of expandable liver progenitor-like cells from human primary hepatocytes (HepLPCs). Here, we generated immortalized and functionally enhanced HepLPCs by introducing FOXA3, a hepatocyte nuclear factor that enables potentially complete hepatic function. When cultured on macroporous carriers in an air-liquid interactive bioartificial liver (Ali-BAL) support device, the integrated cells were alternately exposed to aeration and nutrition and grew to form high-density three-dimensional constructs. This led to highly efficient mass transfer and supported liver functions such as albumin biosynthesis and ammonia detoxification via ureagenesis. In a porcine model of drug overdose-induced acute liver failure (ALF), extracorporeal Ali-BAL treatment for 3 hours prevented hepatic encephalopathy and led to markedly improved survival (83%, n = 6) compared to ALF control (17%, n = 6, P = 0.02) and device-only (no-cell) therapy (0%, n = 6, P = 0.003). The blood ammonia concentrations, as well as the biochemical and coagulation indices, were reduced in Ali-BAL-treated pigs. Ali-BAL treatment attenuated liver damage, ameliorated inflammation, and enhanced liver regeneration in the ALF porcine model and could be considered as a potential therapeutic avenue for patients with ALF.
Asunto(s)
Fallo Hepático Agudo , Hígado Artificial , Albúminas , Animales , Hepatocitos , Humanos , Hígado , Fallo Hepático Agudo/terapia , PorcinosRESUMEN
Recognition of double-stranded RNA (dsRNA) activates interferon-regulatory factor 3 (IRF3)-dependent expression of anti-viral factors. The innate immune system recognizes viral dsRNA through two distinct pathways. First, the Toll-like receptor 3 (TLR3) detects dsRNA phagocytosed in endosomes. In addition, the helicases retinoic acid induced protein I (RIG-I)/melanoma differentiation associated gene 5 (MDA5) binds cytoplasmic dsRNA generated during viral replication. Both RIG-I/MDA5 and TLR3 can bind polyriboinosinic:polyribocytidylic acid (poly(I:C)), the synthetic analog of viral dsRNA, and mediate type I IFN production. Here we show that signal regulatory protein (SIRP) alpha negatively regulates both TLR3- and RIG-1/MDA5-dependent anti-viral pathways. Suppression of SIRPalpha expression by RNA interference results in enhanced activation of IRF3 and MAPK pathways after poly(I:C) treatment, coupled with the up-regulation of IFN-beta and IFN-beta-inducible gene transcriptional activation. The requirement of phosphoinositide 3-kinase (PI3K) activity for the induction of IFN-beta and IFN-beta-inducible genes by dsRNA is supported by the observation that a PI3K inhibitor failed to activate IFN-beta and IFN-beta-inducible gene expression. PI3K, whose activity is essential for activation of IRF3, is recruited to the phosphorylated tyrosine residues of SIRPalpha upon poly(I:C) stimulation, which lead to a reduction in the activity of the downstream kinase AKT. Thus SIRPalpha may accomplish its inhibitory function in type I IFN induction, in part, through its association and sequestration of the signal transducer PI3K.
Asunto(s)
Antígenos de Diferenciación/inmunología , Citoplasma/metabolismo , Interferón beta/inmunología , Receptores Inmunológicos/inmunología , Transducción de Señal , Receptor Toll-Like 3/inmunología , Secuencias de Aminoácidos , Animales , Antígenos de Diferenciación/química , Línea Celular , Citoplasma/efectos de los fármacos , Humanos , Factor 3 Regulador del Interferón/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Poli I-C/farmacología , ARN Bicatenario/farmacología , Receptores Inmunológicos/química , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 3/genética , Activación Transcripcional/efectos de los fármacosRESUMEN
The most essential tools for studying drug hepatotoxicity, liver diseases, and bioartificial livers have always been models that can recapitulate liver physiology in vitro. The liver progenitor cell line HepaRG represents an effective surrogate of the primary hepatocyte. However, the differentiation of HepaRG relies on long-term induction using a high concentration of dimethyl sulfoxide (DMSO), which may compromise the research of drug metabolism and restrict the applicability of this hepatic model. Here, we present a novel hepatic maturation medium (HMM) for the differentiation of HepaRG, which is based on a cocktail of soluble molecules that mimick the in vivo environment. We showed that HMM could rapidly (about nine days) induce HepaRG differentiation into polarized hepatocytes with maturely metabolic functions. In addition, under three-dimensional culture conditions, the hepatic spheroids showed multiple liver functions and toxicity profiles close to those of primary human hepatocytes (PHH). Our work demonstrates the utility of HMM as an alternative to the DMSO-dependent differentiation protocol for HepaRG; moreover, these results facilitate the application of HepaRG.