RESUMEN
The incidence of advanced hepatocellular carcinoma (HCC) is increasing worldwide, and its prognosis is extremely poor. Interferon-alpha (IFN-α)/5-fluorouracil (5-FU) therapy is reportedly effective in some HCC patients. In the present study, to improve HCC prognosis, we identified the genes that are sensitizing to these agents. The screening strategy was dependent on the concentration of ribozymes that rendered HepG2 cells resistant to 5-FU by the repeated transfection of ribozymes into the cells. After 10 cycles of transfection, which was initiated by 5,902,875 sequences of a ribozyme library, three genes including protein kinase, adenosine monophosphate (AMP)-activated, gamma 2 non-catalytic subunit (PRKAG2); transforming growth factor-beta receptor II (TGFBR2); and exostosin 1 (EXT1) were identified as 5-FU-sensitizing genes. Adenovirus-mediated transfer of TGFBR2 and EXT1 enhanced IFN-α/5-FU-induced cytotoxicity as well as 5-FU, although the overexpression of these genes in the absence of IFN-α/5-FU did not induce cell death. This effect was also observed in a tumor xenograft model. The mechanisms of TGFBR2 and EXT1 include activation of the TGF-ß signal and induction of endoplasmic reticulum stress, resulting in apoptosis. In HCC patients treated with IFN-α/5-FU therapy, the PRKAG2 mRNA level in HCC tissues was positively correlated with survival period, suggesting that PRKAG2 enhances the effect of IFN-α/5-FU and serves as a prognostic marker for IFN-α/5-FU therapy. In conclusion, we identified three genes that chemosensitize the effects of 5-FU and IFN-α/5-FU on HCC cells and demonstrated that PRKAG2 mRNA can serve as a prognostic marker for IFN-α/5-FU therapy.
Asunto(s)
Antimetabolitos Antineoplásicos/uso terapéutico , Carcinoma Hepatocelular/genética , Fluorouracilo/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Factores Inmunológicos/uso terapéutico , Interferón-alfa/uso terapéutico , Neoplasias Hepáticas/genética , Proteínas Quinasas Activadas por AMP/genética , Adulto , Anciano , Animales , Antimetabolitos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/tratamiento farmacológico , Supervivencia Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Femenino , Fluorouracilo/farmacología , Células Hep G2 , Humanos , Factores Inmunológicos/farmacología , Interferón-alfa/farmacología , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/tratamiento farmacológico , Masculino , Ratones , Ratones SCID , Persona de Mediana Edad , N-Acetilglucosaminiltransferasas/genética , Pronóstico , Proteínas Serina-Treonina Quinasas/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta , Receptores de Factores de Crecimiento Transformadores beta/genética , Resultado del TratamientoRESUMEN
UNLABELLED: Human bone marrow-derived mesenchymal stem cells (BM-MSCs) are expected to be a potential source of cells for transplantation. Although recent reports have shown that isolated MSCs can differentiate into hepatocytes, the efficiency of differentiation is insufficient for therapeutic application. To circumvent this problem, it is necessary to understand the mechanisms of hepatic differentiation of human BM-MSCs. Hepatocyte nuclear factor 3beta (HNF3beta), a forkhead/winged helix transcription factor, is essential for liver development. In the present study, we established a tetracycline (Tet)-regulated expression system for HNF3beta in UE7T-13 BM-MSCs. HNF3beta expression significantly enhanced expression of albumin, alpha-fetoprotein (AFP), tyrosine amino transferase (TAT) and epithelial cell adhesion molecule (EpCAM) genes. The differentiated cells showed hepatocyte-specific functions including glycogen production and urea secretion. During treatment with the Tet-on system for 8 days, over 80% of UE7T-13 cells turned out to express albumin. Furthermore, the combination of Tet with basic fibroblast growth factor (bFGF) efficiently induced the genes such as albumin and TAT, which are associated with maturity of hepatocytes; however, it suppressed genes such as AFP and EpCAM, which are associated with immaturity of hepatocytes, suggesting that Tet-induced HNF3beta expression sensitizes BM-MSCs to bFGF signals. Finally, the results of the present study suggest that down-regulation of Wnt/beta-catenin signals caused by translocation of beta-catenin to cytoplasmic membrane is associated with hepatic differentiation of human BM-MSCs. CONCLUSION: HNF3beta expression induced efficient differentiation of UE7T-13 human BM-MSCs.
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Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Factor Nuclear 3-beta del Hepatocito/metabolismo , Hígado/citología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Tetraciclina/farmacología , Albúminas/metabolismo , Células de la Médula Ósea/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Citocinas/farmacología , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Factor 2 de Crecimiento de Fibroblastos/farmacología , Expresión Génica/efectos de los fármacos , Glucógeno/biosíntesis , Factor Nuclear 3-beta del Hepatocito/genética , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Células Madre Mesenquimatosas/efectos de los fármacos , Transducción de Señal/fisiología , Tetraciclina/administración & dosificación , Transfección , Urea/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMEN
Retinoids play an important role in the regulation of cell growth and death. Synthetic retinoid CD437 reportedly induces apoptosis in various cancer cell lines. However, the mechanism of inducing apoptosis in hepatocellular carcinoma (HCC) cells by this agent remains to be clarified. In this study, we investigated the signaling pathway by which CD437 induces apoptosis in HCC cell lines. Apoptosis of six human HCC cell lines was induced by treatment with CD437. Caspase-3 and -9 were activated by CD437, suggesting that the apoptosis is mediated by mitochondrial pathways. Consistent with these findings, the treatment with CD437 upregulated Bax protein, downregulated Bcl-2 protein and released cytochrome c into the cytoplasm. Moreover, rhodamine123 staining revealed mitochondrial depolarization in the cells treated with CD437. These data of the present study suggest that CD437 induces apoptosis in HCC cells via mitochondrial pathways.
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Apoptosis , Carcinoma Hepatocelular/metabolismo , Proliferación Celular/efectos de los fármacos , Neoplasias Hepáticas/metabolismo , Mitocondrias/efectos de los fármacos , Retinoides/farmacología , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Línea Celular Tumoral , Humanos , Mitocondrias/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
A synthetic retinoid, CD437, has been shown to exert potent anti-tumor activity against various types of cancer cell lines, regardless of their sensitivities to natural retinoids. We herein demonstrate that CD437 induces endoplasmic reticulum (ER) stress, including the up-regulation of CHOP, BIP and GADD34 mRNA through ER stress transducer (PERK and IRE1alpha) activation in an ovarian adenocarcinoma cell line, SKOV3. It was also shown that CD437 induced the CHOP and GADD34 expressions in another four ovarian adenocarcinoma cell lines, indicating that CD437 functions as an ER stress inducer in these cell lines. Moreover, the siRNA-mediated knockdown of inducible CHOP expression prevented the cytotoxic effect of CD437. These results suggest that ER stress plays an important role in the mechanism by which CD437 induces apoptosis in ovarian adenocarcinoma cells.
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Adenocarcinoma/metabolismo , Apoptosis/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Neoplasias Ováricas/metabolismo , Retinoides/administración & dosificación , Transducción de Señal/efectos de los fármacos , Línea Celular Tumoral , Retículo Endoplásmico/efectos de los fármacos , Femenino , Humanos , Estrés Oxidativo/efectos de los fármacosRESUMEN
BACKGROUND/AIMS: Although the importance of reactive oxygen species (ROS) in the pathogenesis of various diseases is stressed, clinical significance of the markers reflecting DNA oxidation such as 8-hydroxy-2'-deoxyguanosine (8-OHdG) remains to be clarified. METHODOLOGY: To examine clinical usefulness of 8-OHdG in healthy individuals in comparison with liver disease patients, urinary excretion of 8-OHdG was measured in 336 healthy individuals and 110 patients with liver disease. RESULTS: In healthy persons, the 8-OHdG excretion was increased in an age-dependent manner. It was positively correlated with cigarettes smoked a day and negatively correlated with body mass index (BMI) (P < 0.05, each). Age, smoking and BMI were independent predictors of urinary 8-OHdG excretion (P < 0.01, P < 0.01 and P < 0.05, respectively). In liver disease, the excretion of 8-OHdG was not changed, as compared with healthy individuals. However, the liver disease patients under the age of 40 had higher values of 8-OHdG than healthy persons. In addition, the urinary excretion of 8-OHdG was higher in patients with hepatitis C virus (HCV) infection than those with hepatitis B virus (HBV) infection. CONCLUSIONS: The results of the present study suggest that measurement of urinary 8-OHdG excretion is useful in assessing DNA oxidation caused by aging, smoking, body composition and liver disease.
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Desoxiguanosina/análogos & derivados , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/orina , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Índice de Masa Corporal , ADN/metabolismo , Desoxiguanosina/orina , Femenino , Humanos , Neoplasias Hepáticas/diagnóstico , Masculino , Persona de Mediana Edad , Oxígeno/metabolismo , Especies Reactivas de Oxígeno , Valores de Referencia , Reproducibilidad de los Resultados , FumarRESUMEN
Human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) are expected to be an excellent source of cells for transplantation. In addition, the stem cell plasticity of human UCBMSCs, which can transdifferentiate into hepatocytes, has been reported. However, the mechanisms involved remain to be clarified. To identify the genes and/or signals that are important in specifying the hepatic fate of human UCBMSCs, we analyzed gene expression profiles during the hepatic differentiation of UCBMSCs with human telomerase reverse transcriptase, UCBMSCs immortalized by infection with a retrovirus carrying telomerase reverse transcriptase, but whose differentiation potential remains unchanged. Efficient differentiation was induced by 5-azacytidine (5-aza)/hepatocyte growth factor (HGF)/oncostatin M (OSM)/fibroblast growth factor 2 (FGF2) treatment in terms of function as well as protein expression: 2.5-fold increase in albumin, 4-fold increase in CCAAT enhancer-binding protein alpha, 1.5-fold increase in cytochrome p450 1A1/2, and 8-fold increase in periodic acid-Schiff staining. Consequently, we found that the expression of Wnt/beta-catenin-related genes downregulated, and the translocation of beta-catenin was observed along the cell membrane and in the cytoplasm, although some beta-catenin was still in the nucleus. Downregulation of Wnt/beta-catenin signals in the cells by Fz8-small interference RNA treatment, which was analyzed with a Tcf4 promoter-luciferase assay, resulted in similar hepatic differentiation to that observed with 5-azacytidine/HGF/OSM/FGF2. In addition, the subcellular distribution of beta-catenin was similar to that of cells treated with 5-azacytidine/HGF/OSM/FGF2. In conclusion, the suppression of Wnt/beta-catenin signaling induced the hepatic differentiation of UCBMSCs, suggesting that Wnt/beta-catenin signals play an important role in the hepatic fate specification of human UCBMSCs.
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Diferenciación Celular/fisiología , Sangre Fetal/fisiología , Hígado/citología , Hígado/fisiología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Proteínas Wnt/fisiología , beta Catenina/fisiología , Cartilla de ADN , Sangre Fetal/citología , Humanos , Recién Nacido , Reacción en Cadena de la Polimerasa , Transducción de Señal , Urea/análisisRESUMEN
AIM: Bone marrow-derived mesenchymal stem cells (MSC) are expected to be an excellent source of cells for transplantation. We aimed to study the culture conditions and involved genes to differentiate MSC into hepatocytes. METHODS: The culture conditions to induce the efficient differentiation of human bone marrow-derived UE7T-13 cells were examined using cytokines, hormones, 5-azacytidine and type IV collagen. RESULTS: We found that combination of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) with type IV collagen coating induced hepatic differentiation of UE7T-13 cells at over 30% frequency, where expression of albumin mRNA was increased over 20-fold. The differentiated cells had functions of albumin production, glycogen synthesis and urea secretion as well as expressing hepatocyte-specific genes. In addition, these cellshave binuclear and cuboidal morphology, which is a characteristic feature of hepatocytes. During hepatic differentiation, UE7T-13 cells showed depressed expression of WISP1 and WISP2 genes, members of the CCN family. Conversely, knockdown of WISP1 or WISP2 gene by siRNA stimulated hepatic differentiation. The effect of aFGF/bFGF/HGF/type IV collagen coating and WISP1-siRNA on hepatic differentiation was additive. CONCLUSION: The present study suggests that aFGF/bFGF/HGF/type IV collagen coating is the efficient condition for hepatic differentiation of UE7T-13 cells, and that WISP1 and WISP2 play an important role in hepatic transdifferentiation of these cells.
RESUMEN
BACKGROUND/AIMS: To rescue patients with severe liver injury, it is critical to develop the efficient regulatory system of hepatic stem cell proliferation in vitro. Our aims are to examine whether combination of adenovirus-mediated hepatocyte growth factor (HGF) gene transfer with signal transduction inhibitors can regulate cell proliferation of oval cells. METHODOLOGY: We examined the effects of treatment with adenoviral mediated HGF gene transfer and signal transduction inhibitors including LY294002, rapamycin and U0126 on proliferation OC/CDE22 hepatic oval cells and expression of signal transduction molecules. RESULTS: Infection with pAxCAHGF expanded the cells by 8-fold at 2 days, by 18-fold at 3 days and by 55-fold at 4 days. The addition of inhibitors inhibited pAxCAHGF-induced cell proliferation by LY294002 or rapamycin (P < 0.01, each). U0126 also inhibited growth of hepatic oval cells (P < 0.01). pAxCAHGF treatment induced phosphorylation of AKT. Treatment with rapamycin resulted in enhanced phosphorylation of AKT, and phosphorylation of AKT was induced by pAxCAHGF plus U0126. CONCLUSIONS: Autocrine expression of HGF with signal transduction inhibitors can regulate proliferation of OC/CDE22 hepatic oval cells. In addition, the AKT pathway is important for HGF-stimulated hepatic oval cell proliferation.
Asunto(s)
Técnicas de Cultivo de Célula , Proliferación Celular , Factor de Crecimiento de Hepatocito/genética , Hepatocitos/fisiología , Adenoviridae/genética , Butadienos/farmacología , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Técnicas de Transferencia de Gen , Hepatocitos/efectos de los fármacos , Humanos , Morfolinas/farmacología , Nitrilos/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , beta-Galactosidasa/análisis , beta-Galactosidasa/metabolismoRESUMEN
The mechanisms of prevention of the development of liver cancer by NIK-333, an acyclic retinoid (ACR), were investigated. The transgenic mice expressing the dominant negative form of retinoic acid receptor alpha (RARE mice), that produce reactive oxygen species and lead to development of liver tumor were used. The effect of NIK-333 on hepatocarcinogenesis in RARE mice was studied. The RARE mice were examined after feeding 0.03% and 0.06% NIK-333 diets at 12 months of age. In the mice fed 0.06% NIK-333 diet, tumor incidence was greatly suppressed, compared to that of wild type mice (0/9 versus 5/9, P<0.05), but not in the mice fed 0.03% NIK-333 diet. In addition, expression of cytochrome p450 4a14 and acyl-CoA oxidase was normalized, and the percentages of positive cells for 8-hydroxy-2'-deoxyguanosine, 4-hydroxy-2-nonenal and proliferating cell nuclear antigen were decreased. Furthermore, expression of beta-catenin and cyclin D1 was also depressed. These data suggest that NIK-333 suppressed liver tumor in association with repression of oxidative stress.