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Interleukin-6 (IL-6) is a pleiotropic cytokine that has many biological activities, including inflammation, hematopoiesis, bone metabolism, embryonic development, and other fundamental processes. Recently, IL-6 has been widely recognized as an important pro-inflammatory cytokine involved in cytokine storm pathogenesis during severe inflammatory diseases, such as coronavirus disease 2019 (COVID-19). Therefore, IL-6 is considered to be a therapeutic target for inhibiting cytokine storm. In the present study, we investigated the suppressive effect of isofraxidin, a major coumarin compound of Acanthopanax senticosus, on the overexpression of IL-6 and its molecular mechanism. The expression of IL-6 mRNA was measured using quantitative real-time PCR, and intracellular signaling molecules were detected using western blotting. When the HuH-7 human hepatocellular carcinoma cell line and HepG2 human hepatoblastoma cell line were treated with 12-O-tetradecanoylphorbol 13-acetate (TPA), a marked induction of IL-6 mRNA expression was observed in HuH-7 cells compared with HepG2 cells. Isofraxidin significantly suppressed TPA-induced IL-6 mRNA expression in HuH-7 cells in a dose-dependent manner. Furthermore, isofraxidin inhibited TPA-induced phosphorylation of ERK1/2 in a dose-dependent manner. Similarly, the MAPK/ERK inhibitor U0126 suppressed TPA-induced IL-6 mRNA expression. However, isofraxidin had no effects on TPA-induced phosphorylation of SAPK/JNK, Akt (Ser473), and STAT3 (Tyr705), nuclear translocation of NF-κB p65, and degradation of IκB. Taken together, isofraxidin suppresses TPA-induced overexpression of IL-6 mRNA by selectively inhibiting the activation of the MAPK/ERK pathway in HuH-7 cells, indicating that isofraxidin may be an effective anti-inflammatory agent for treating cytokine storm.
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Introduction: Extramammary Paget's disease is an eczematous skin condition that affects the vulva and perineum. Extramammary Paget's disease secondary to urothelial carcinoma is a rare condition that is typically treated with invasive surgical resection of the lesion. Case presentation: An 80-year-old woman with a 7-year history of urothelial carcinoma presented with erythema of the labia majora. Immunostaining of skin biopsy specimens suggested extramammary Paget's disease secondary to urothelial carcinoma. The patient did not consent to resection of the lesion. Nine cycles of first-line platinum-based chemotherapy for metastatic urothelial carcinoma were administered. As tumor cells remained after systemic chemotherapy, pembrolizumab will be administered to the patient for treating residual extramammary Paget's disease. Conclusion: Platinum-based chemotherapy can control extramammary Paget's disease secondary to urothelial carcinoma.
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Cytochrome P450 (CYP) enzymes, especially CYP3A4 play a major role in the metabolism of xenobiotics in human liver. CYP3A4-expressing human liver or hepatoma cell lines may be good cell substitutes of human hepatocytes for drug metabolism studies. However, there are only a few cell lines expressing high levels of CYP3A4. The aim of this study is to investigate the expression of CYP3A4 and its mechanism in an immortalized non-tumorigenic human liver epithelial cell line, THLE-5b in differentiation-inducing conditions. When THLE-5b cells were cultivated in culture medium supplemented with hepatocytic differentiation-inducing factors, they showed hepatocytic morphology. In addition, elevated levels of expression not only of α1-antitrypsin (AAT) and albumin (ALB) mRNAs, but also of CYP3A4 mRNA, which are functional hepatocyte markers, were observed compared with the control. Among hepatocytic differentiation-inducing factors, dexamethasone (DEX) and insulin-transferrin-sodium selenite (ITS) seemed to be involved in elevation of expression of CYP3A4 mRNA. The mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126 or the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 reduced CYP3A4 mRNA levels of THLE-5b cells. Furthermore, the CpG site of the CYP3A4 promoter region in THLE-5b cells was found to be unmethylated, although in low CYP3A4-expressing HepG2 cells, the site was methylated. In conclusion, THLE-5b cells, which are unmethylated at the CpG site of the CYP3A4 promoter region, express CYP3A4 mRNA through the MEK/ERK1/2 and PI3K/Akt signaling pathways and acquire hepatocytic functions in differentiation-inducing conditions. Thus, THLE-5b cells could be a useful cell system for the study of drug metabolism.
Asunto(s)
Diferenciación Celular/genética , Citocromo P-450 CYP3A/genética , Citocromo P-450 CYP3A/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Expresión Génica , Hígado/citología , Hígado/metabolismo , Regulación hacia Arriba/genética , Línea Celular , Humanos , Sistema de Señalización de MAP Quinasas , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de SeñalRESUMEN
We compared brain metastasis of renal cell carcinoma (RCC) in patients who received tyrosine kinase inhibitors (TKIs) ; 17 received local therapy, while 16 did not. The median survival rate was 19 months in the local therapy group and 11 months in the tyrosine kinase inhibitor alone group, showing no significant difference (pï¼0.52). Symptoms such as paralysis, headache, and dysarthria occurred due to brain metastasis. These symptoms improved in 8 out of 10 patients in the local therapy group. There were no cases of grade 3 or higher complications due to local therapy. Although local therapy did not prolong the overall survival, an improvement in symptoms was observed, suggesting that it is acceptable as palliative treatment.
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Neoplasias Encefálicas , Carcinoma de Células Renales , Neoplasias Renales , Neoplasias Encefálicas/tratamiento farmacológico , Carcinoma de Células Renales/tratamiento farmacológico , Humanos , Neoplasias Renales/tratamiento farmacológico , Metástasis de la Neoplasia , Inhibidores de Proteínas Quinasas , Estudios Retrospectivos , Tasa de SupervivenciaRESUMEN
Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of biliary atresia (BA) may contain stem/progenitor cells, and clusters of hepatocyte-like cells appear via hepatocyte growth factor/c-Met signaling in primary cultures of NPCs. BA is a rare and serious liver disease, and procurement of BA cells is difficult. Therefore, cryopreservation of BA liver cells is an unavoidable challenge. In this study, we examined the appearance and liver function of hepatocyte-like cells in cultures of BA liver-derived NPC fractions after cryopreservation for 1 or 6 mo using a chemically defined cryopreservation solution, STEM-CELLBANKER. Although a decrease in cell viability was observed in recovered cells after 1 mo of cryopreservation, clusters of hepatocyte-like cells appeared in the culture of cells that had been cryopreserved for 1 or 6 mo, similar to non-cryopreserved cells. In addition, these hepatocyte-like cells expressed hepatocyte-related mRNAs and demonstrated albumin production and glycogen storage. The present results suggest that hepatic stem/progenitor cells in NPC fractions may be efficiently cryopreserved, as demonstrated by the appearance of hepatocyte-like cells that show various hepatic functions even after cryopreservation. This study may lead to future BA cell therapy using the patient's own cells.
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Atresia Biliar/patología , Técnicas de Cultivo de Célula/métodos , Criopreservación/métodos , Cirrosis Hepática/patología , Albúminas/genética , Atresia Biliar/complicaciones , Supervivencia Celular , Regulación de la Expresión Génica , Factor Nuclear 4 del Hepatocito/genética , Hepatocitos/citología , Hepatocitos/fisiología , Humanos , Células Madre/citología , Factores de TiempoRESUMEN
Biliary atresia (BA) is a rare and serious liver disease in newborn infants. Previously, we reported that non-parenchymal cell (NPC) fractions from cirrhotic liver of BA may contain hepatic stem/progenitor cells in primary culture of NPC fractions. In this study, NPC fractions were subjected to primary or passage culture and found that clusters of hepatocyte-like cells appear even without adding hepatocyte growth factor (HGF) to the culture medium, but not in their passage culture used as a control. Based on these findings, conditioned media (CMs) were collected and soluble factors in the CMs were analyzed in order to elucidate the mechanism of the appearance of hepatocyte-like cells or their clusters. A large amount of active HGF consisting of α and ß chains was detected in CMs derived from primary culture, but not in CMs from passage culture, as determined by western blot analysis, bone morphogenetic protein (BMP)-4, oncostatin M (OSM), and transforming growth factor (TGF)-ß1 were not detected in any of the CMs. The number of hepatocyte-like cells in primary culture tended to decrease following treatment with the HGF receptor c-Met inhibitor, SU11274 in a dose-dependent manner. Furthermore, the clusters of hepatocyte-like cells tended to increase in size and number when freshly isolated NPC fractions were cultured in the presence of 10% of CMs collected after 3-4 wk of primary culture. In conclusion, these findings indicate that CMs derived from primary culture of NPC fractions of BA liver contain a large amount of active HGF, which may activate hepatic stem/progenitor cells and promote the appearance of hepatocyte-like cells or their clusters through HGF/c-Met signaling. The present study would lead to cell therapy using the patient's own cells for the treatment of BA.
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Atresia Biliar/metabolismo , Medios de Cultivo Condicionados/química , Factor de Crecimiento de Hepatocito/aislamiento & purificación , Cirrosis Hepática/metabolismo , Proteínas Proto-Oncogénicas c-met/genética , Atresia Biliar/patología , Medios de Cultivo Condicionados/metabolismo , Factor de Crecimiento de Hepatocito/química , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Humanos , Indoles/farmacología , Hígado/citología , Hígado/crecimiento & desarrollo , Cirrosis Hepática/patología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Transducción de Señal , Sulfonamidas/farmacologíaRESUMEN
The in vivo repopulation of hepatocytes depends on donor cell growth potential and recipient conditioning. We herein demonstrate the successful cell transplantation of a human hepatocyte cell line, THLE-5b, into the SCID mouse liver by means of a rather mild conditioning using a 55% hepatectomy and p21 transfection. Adult human liver-derived cells, THLE-5b, are SV40 T antigen-immortalized epithelial cells. A phenotypic examination of THLE-5b showed they expressed hepatic stem cell markers such as EpCAM, OCT3/4, and Thy-1, thus indicating the immature nature of the cells. A three-dimensional aggregate culture of THLE-5b showed a higher expression level of liver-specific genes such as albumin, α1-antitrypsin, and CYP3A4, thus suggesting that THLE-5b possess the capability to differentiate into hepatocytes. In a cell transplantation experiment, the cell cycle regulator p21 was transfected with adenoviral vector into the SCID mouse liver. On the next day, 8 × 10(5) cells of GFP-transfected THLE-5b were injected intrasplenically, together with the intraperitoneal administration of anti-asialo GM1 antibodies. The following day, a partial hepatectomy was performed. The GFP-THLE-5b cells were observed to have migrated and become integrated into the liver parenchyma 14 days after transplantation. The present protocol is thus considered to be a novel experimental model to elucidate the mechanism of hepatocyte repopulation and to develop efficient stem cell therapy in the liver.
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Hepatocitos/citología , Hígado/metabolismo , Adenoviridae/genética , Animales , Antígenos de Neoplasias/metabolismo , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Movimiento Celular , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Citocromo P-450 CYP3A/metabolismo , Molécula de Adhesión Celular Epitelial , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Hepatocitos/metabolismo , Hepatocitos/trasplante , Humanos , Hígado/patología , Masculino , Ratones , Ratones SCID , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Albúmina Sérica/metabolismo , Antígenos Thy-1/metabolismo , Transfección , Trasplante Heterólogo , alfa 1-Antitripsina/metabolismoRESUMEN
We sought to determine whether hepatic progenitor cells can be isolated from cirrhotic liver using epithelial cell adhesion molecule (EpCAM) or Thy-1 markers. Liver tissue with cirrhosis secondary to biliary atresia (BA) was collagenase digested, and nonparenchymal cells (NPCs) were cultivated for 24 h. Noncirrhotic NPCs derived from patients with carbamyl phosphate synthetase and ornithine transcarbamylase deficiencies were used as controls. Flow cytometric analysis demonstrated that the percentages of EpCAM- and Thy-1-positive cells were significantly higher in NPC populations derived from BA liver than in those derived from control liver. Reverse transcription polymerase chain reaction analysis revealed that EpCAM-positive sorted cells expressed EpCAM, Thy-1, albumin, and CK-19, whereas Thy-1-positive sorted cells expressed Thy-1, albumin, and CK-19. These findings indicate that EpCAM- or Thy-1-positive hepatic progenitor cells can be more efficiently isolated from BA liver than from control liver and suggest that the properties of EpCAM-positive cells are somewhat different from those of Thy-1-positive cells.
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The existence of hepatic stem cells in human livers is controversial. We investigated whether the side population (SP) cells derived from an immortalized human liver epithelial cell line THLE-5b possess the properties of hepatic stem-like cells. SP cells derived from THLE-5b were isolated using flow cytometry and were assayed for the expression of phenotypic markers by reverse transcription polymerase chain reaction and immunostaining. THLE-5b SP cells retained the capacity to generate both SP and non-SP cells, showed a capacity for self-renewal, and were more efficient in colony formation than non-SP cells. Neither the SP nor the non-SP cells formed tumors when transplanted into athymic nude mice or severe combined immunodeficient mice. The expression level of stem cell-associated markers such as an ATP-binding cassette membrane transporter, epithelial cell adhesion molecule, c-kit, Thy-1, and octomer binding transcription factor 4 was higher in SP cells than in non-SP cells. When cultivated as rotation-mediated aggregates, the expression of liver-specific genes including tryptophan oxygenase and CYP3A4 was up-regulated in SP cells, suggesting that THLE-5b SP cells have the ability to differentiate into a hepatocyte phenotype. One of the clonal cell lines derived from the SP cells expressed stem cell-associated markers. These results indicate that SP cells derived from THLE-5b possess hepatic stem-like cell properties and suggest that THLE-5b can be used as a model of normal human liver progenitor or stem cell line.
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7-Hydroxy-6,8-dimethoxy-2H-1-benzopyran-2-one (isofraxidin) is a major coumarin component isolated from the stem bark of Acanthopanax senticosus, a widely used Chinese medicinal herb. We investigated isofraxidin in its anti-tumor effects on human hepatoma cell lines HuH-7 and HepG2. Isofraxidin significantly inhibited hepatoma cell invasion, without affecting cell attachment or growth. Expression of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced matrix metalloproteinase-7 (MMP-7) in hepatoma cells was inhibited by isofraxidin at the both mRNA and protein levels. This inhibition tended to be greater in cells inoculated at low density than in those at high density. Isofraxidin showed an inhibitory effect on the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in hepatoma cells, whereas activator protein-1 (AP-1) DNA binding activity, nuclear factor-kappa B (NF-κB) nuclear translocation, and inhibitory kappa B (IκB) degradation were affected very little. These results indicate that isofraxidin inhibits expression of MMP-7 and in vitro cell invasion at a non-toxic level through inhibiting ERK1/2 phosphorylation in hepatoma cell lines, which suggest isofraxidin might become an effective agent for suppressing hepatoma cell invasion.
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Antineoplásicos Fitogénicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Cumarinas/farmacología , Eleutherococcus/química , Neoplasias Hepáticas/tratamiento farmacológico , Inhibidores de la Metaloproteinasa de la Matriz , Extractos Vegetales/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Carcinoma Hepatocelular/secundario , Línea Celular Tumoral , Cumarinas/uso terapéutico , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Invasividad Neoplásica/prevención & control , Fosforilación , Fitoterapia , Corteza de la Planta , Extractos Vegetales/uso terapéutico , Tallos de la Planta , Acetato de TetradecanoilforbolRESUMEN
The scattered cell clusters that can differentiate into hepatocytes or biliary epithelial cells have been isolated from primary cultures of adult porcine livers. We have generated 11 clonal cell lines from this system and identified liver progenitor cells (LPCs) among the clonal lines. These clonal lines expressed c-kit, HNF-1, HNF-6, and/or CK19 mRNA. An immunocytochemical study of the clonal lines indicated that clonal line CL-11 expressed liver epithelial cell markers CK14, vimentin, CK18, and BD-1. The expression of albumin and alpha1-antitrypsin (alpha1-AT) mRNA was only upregulated in CL-11 among the clonal lines when they were grown as aggregates. Under these conditions, CL-11 also exhibited ammonia metabolic activity and several indicators that suggest hepatocytic differentiation, including the upregulation of liver-specific genes such as dipeptidyl peptidase IV, CYP1A1, and CYP3A4 mRNA, and the downregulation of biliary cell markers such as gamma-glutamyltrans-peptidase (GGT), CK19, and HNF6 mRNA. After culturing CL-11 in Matrigel, the expression of GGT and HNF6 mRNA was upregulated. These results indicate that CL-11 has dual potential: the ability to differentiate as hepatocytes or as bile duct cells. The isolation of scattered cells could provide a simple method to generate LPC lines from adult livers.
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Hepatocitos , Células Madre , Animales , Conductos Biliares/citología , Biomarcadores/análisis , Técnicas de Cultivo de Célula , Diferenciación Celular , Células Clonales , Citocromo P-450 CYP3A/biosíntesis , Sistema Enzimático del Citocromo P-450/biosíntesis , Dipeptidil Peptidasa 4/biosíntesis , Células Epiteliales/citología , Células Epiteliales/fisiología , Factor Nuclear 4 del Hepatocito/biosíntesis , Factor Nuclear 6 del Hepatocito/biosíntesis , Hepatocitos/citología , Hepatocitos/fisiología , Células Madre/citología , Células Madre/fisiología , Sus scrofaRESUMEN
Side-population (SP) cells have been shown to be highly enriched stem cells. We investigated whether an immortalized, nontumorigenic human liver cell line, THLE-5b, contains SP cells. Flow cytometry analysis after Hoechst 33342 staining demonstrated that the THLE-5b line contained a small component of SP cells. These SP cells were essentially eliminated by treatment with verapamil and expressed higher levels of ABCG2 mRNA than non-SP cells. In addition, the level of these SP cells detected by Hoechst 33342 staining was affected by the experimental conditions including the incubation medium. This is the first report of the presence of SP cells in the immortalized, nontumorigenic human liver cell line.
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Línea Celular , Células Epiteliales/citología , Hígado/citología , Células Madre/citología , Animales , Bencimidazoles/metabolismo , Células Cultivadas , Colorantes Fluorescentes/metabolismo , Humanos , Ratones , Células Madre/fisiologíaRESUMEN
Previously, we showed that dexamethasone (DEX) inhibited the expression of inflammatory cytokines, matrix metalloproteinase-1, and cyclooxygenase-2 messenger ribonucleic acid in SW982 cells. In this study, the effect of DEX on the transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) was examined in SW982 cells by electrophoretic mobility shift assay (EMSA). Both NF-kappaB and AP-1 deoxyribonucleic acid binding activities were detectable in SW982 cells by EMSA, and they were induced by interleukin-1beta treatment. DEX inhibited NF-kappaB binding activity at 10 microM as well as at 100 microM, although the inhibition was only partial. However, DEX had little effect on AP-1 activity. These results suggest that DEX reduces the expression of inflammatory cytokines and other proteins in SW982 cells by inhibiting NF-kappaB.
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Dexametasona/farmacología , FN-kappa B/metabolismo , Factor de Transcripción AP-1/metabolismo , Línea Celular Tumoral , Humanos , FN-kappa B/efectos de los fármacos , Sarcoma Sinovial , Factor de Transcripción AP-1/efectos de los fármacosRESUMEN
SW982 cells are characterized by expression of inflammatory cytokine and matrix metalloproteinase (MMP) genes and by their response to dexamethasone at different cell densities. They express genes encoding interleukin (IL)-1 beta; IL-6; transforming growth factor-beta; intercellular adhesion molecule-1; cycloxygenase (COX)-2; and MMPs, including MMP-1, MMP-2, MMP-13, and MT1-MMP; tissue inhibitor of metalloproteinase-2; and a disintegrin and metalloproteinase with thrombospondin motifs-4. Expression of all the genes examined was induced with 2 ng/ml IL-1 beta at low cell density. The cells, however, failed to express tumor necrosis factor-alpha, COX-1, and MMP-9, regardless of the presence of IL-1 beta. Dexamethasone significantly reduced IL-1 beta, IL-6, COX-2, and MMP-1 expression at high cell density. The results suggest that SW982 cells are a useful tool for studying the expression of inflammatory cytokine or MMP genes.
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Línea Celular Tumoral/efectos de los fármacos , Citocinas/genética , Dexametasona/farmacología , Glucocorticoides/farmacología , Metaloproteinasas de la Matriz/genética , Sarcoma Sinovial/patología , Línea Celular Tumoral/inmunología , Citocinas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Metaloproteinasas de la Matriz/metabolismo , Fenotipo , Sarcoma Sinovial/genética , Sarcoma Sinovial/inmunologíaRESUMEN
Previously, we reported the presence of certain nonparenchymal epithelial cells (NPECs) in adult porcine livers that demonstrate differentiation patterns including an emergence of duct-like structures (DLSs) in the colonies. In the present study, we examined the effect of supplements to the NAIR-1 medium (Dulbecco modified Eagle medium [DMEM]-F12 containing 5% fetal bovine serum [FBS] and 11 supplements) used in these cultures on formation of DLSs-emerged colonies (type I colonies). No type I colonies were observed in the cultures of the nonparenchymal cell fraction when Roswell Park Memorial Institute-1640 medium or DMEM-F12 (1:1) supplemented with 5% FBS was used as the culture medium. NAIR-1 medium without each component did not produce any significant results. No type I colonies were formed when epidermal growth factor, and hydrocortisone and insulin mixture (A) or nicotinamide and l-ascorbic acid phosphate magnesium salt (Asc2P) mixture (B) was added to the DMEM-F12 medium supplemented with 5% FBS. However, when a combination of A and B was added, colonies were formed at a significant level. Together, the number of type I colonies was increased in the combination of A and B containing a higher concentration of Asc2P. We conclude that NPECs need a mixture of Asc2P and other components as supplements for type 1 colony formation.