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1.
Life Sci ; 251: 117607, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32240679

RESUMO

BACKGROUND: Arsenic trioxide (ATO) can bind directly to the human promyelocytic leukemia (PML) protein, leading to modification of PML by SUMOs. UBC9 is the only known E2-conjugating enzyme involved in SUMOylation. PML degradation via RNF4, an E3 ubiquitin ligases family member. PML is key organizer of nuclear bodies (NBs) that regulate many biological processes such as senescence, and DNA damage. ATO can activate the TGFß/Smad signaling pathway, causing liver fibrosis. However, the roles of PML Sumoylation in ATO-induced liver fibrosis remain unclear. OBJECTIVE: This study aimed to investigate the role of PML Sumoylation in the ATO-induced HSCs activation and to improve the mechanism of ATO-induced liver fibrosis. METHODS: Hepatic stellate cells (HSCs) were treated with 2 µmol/L ATO. Cell viability was detected by CCK-8 analysis. Immunoblot analysis and real-time quantitative PCR were used to detect the expression of IL-1ß, TNF-α, TGF-ß1, p-Smad2/3, α-SMA, Collagen I and PML SUMOylation after silencing PML, UBC9, and RNF4, respectively. The formation of PML-NBs was observed by immunofluorescence staining. RESULTS: 2 and 5 µmol/L ATO intervention increased HSCs cell viability. ATO was able to significantly trigger PML SUMOylation and the formation of PML-NBs. Inhibition of SUMOylated PML by silencing UBC9, subsequently preventing the downregulation of HSCs activation indicators induced by ATO (P < 0.05). Conversely, enhancing SUMOylated PML accumulation by silencing RNF4, activating TGFß/Smad signaling pathway, eventually promoting the induction of liver fibrosis. CONCLUSION: These results indicated that PML SUMOylation plays a critical role in the development of liver fibrosis induced by ATO.


Assuntos
Trióxido de Arsênio/toxicidade , Células Estreladas do Fígado/patologia , Cirrose Hepática/patologia , Proteína da Leucemia Promielocítica/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Inativação Gênica , Humanos , Proteínas Nucleares/genética , Sumoilação , Fatores de Transcrição/genética , Enzimas de Conjugação de Ubiquitina/genética
2.
Life Sci ; 251: 117595, 2020 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-32240681

RESUMO

AIMS: The activation of hepatic stellate cells (HSCs) plays a central role in liver fibrosis progression. Phospholipase D (PLD) enzymes participate in multiple cellular activities. However, whether and how PLD regulates HSCs activation remain elusive. MAIN METHODS: The expression of intrahepatic PLD1 and PLD2 was determined in CCl4-induced mouse liver fibrosis models by western blot and immunohistochemistry. Cell model of liver fibrogenesis was constructed using rat HSCs line (HSC-T6) treated with recombinant transforming growth factor ß1 (TGFß1). Fibrogenesis was evaluated on the aspects of proliferation, expression of pro-fibrogenic markers and migration. The effects mediated by PLD1-mTOR axis on TGFß1-induced fibrogenesis were evaluated using HSC-T6 treated with small-molecular PLD1 inhibitors, PLD1-SiRNA, rapamycin (mTOR inhibitor) and MHY1485 (mTOR activator). KEY FINDINGS: Significant increase of PLD1, not PLD2 was documented in CCl4-induced cirrhotic compared to normal liver tissues. Suppression of PLD1 activities by PLD inhibitors or down-regulation of PLD1 expression in HSC-T6 could significantly restrain TGFß1-induced fibrogenesis, as reflected by decreased cell proliferation and reduced expression of pro-fibrogenic markers. Besides, either PLD1 inhibitor or PLD1-SiRNA significantly inhibited mTOR activity of HSC-T6. Moreover, PLD1 inhibitors not only exhibited similar effects with rapamycin in TGFß1-induced fibrogenesis, but also blunted MHY1485 enhanced cell proliferation of HSC-T6. SIGNIFICANCE: The PLD1-mTOR axis of HSCs could be therapeutically targeted in advanced liver fibrosis.


Assuntos
Células Estreladas do Fígado/metabolismo , Cirrose Hepática/fisiopatologia , Fosfolipase D/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Linhagem Celular , Proliferação de Células/fisiologia , Modelos Animais de Doenças , Progressão da Doença , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos
3.
Chem Biol Interact ; 323: 109054, 2020 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-32217109

RESUMO

BACKGROUND AND AIMS: Non-alcoholic steatohepatitis (NASH) has been associated with fibrosis that may progress to cirrhosis. The purpose of this study was to examine hepatocytes and perisinusoidal cells in liver biopsies of 3 families (3 males and 4 females) with non-cirrhotic and cirrhotic NASH to determine unique histological changes during a period of 2-7 years from diagnosis. METHODS: In this study, hepatocytes, stellate cells and Kupffer cells were analyzed using light and electron microscopy, and immunohistochemistry with specific anti-macrophage antibody staining of liver biopsies. RESULTS: Body mass index of all patients was over 28, and all viral, metabolic markers were negative. Alcohol consumption was insignificant. In all liver biopsies, diffuse, non-zonal macrovesicular steatosis involved 40-70% of liver samples. The lobular hepatocytes showed prominent ballooning hepatocyte degeneration. No Mallory Denk hyaline bodies (MDBs) were observed in three of the patients. MDBs developed in ballooned hepatocytes of four individuals that also presented foci of lobular inflammation. The apoptotic bodies were stained by cytokeratin 18. The trichrome stain revealed portal to portal bridging fibrosis. In one family, there was a three-fold increase in relative numbers of perisinusoidal macrophages in the older sister with NASH compared to livers of the younger siblings. The special finding in livers of patients with NASH was accumulation of groups of perisinusoidal macrophages, which was not associated with focal necrosis. CONCLUSION: Perisinusoidal macrophages appear to accumulate in NASH. It is possible that collections of macrophages are a response to chronic portal endotoxemia or lipotoxic activation of immuno-mediators. The persistent activation of these macrophages could lead to the chronic release of pro-inflammatory cytokines and contribute to chronic inflammation, fibrosis and cirrhosis leading to HCC.


Assuntos
Carcinoma Hepatocelular/etiologia , Neoplasias Hepáticas/etiologia , Hepatopatia Gordurosa não Alcoólica/complicações , Adulto , Biópsia , Carcinoma Hepatocelular/patologia , Feminino , Células Estreladas do Fígado/patologia , Humanos , Macrófagos do Fígado/patologia , Gotículas Lipídicas/metabolismo , Fígado/patologia , Fígado/ultraestrutura , Neoplasias Hepáticas/patologia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/patologia
4.
Zhonghua Gan Zang Bing Za Zhi ; 28(2): 135-140, 2020 Feb 20.
Artigo em Chinês | MEDLINE | ID: mdl-32164064

RESUMO

Objective: To investigate the mechanism of occurrence and development of zinc-alpha-2-glycoprotein (AZGP1) in the activated hepatic stellate cells (HSCs) and liver fibrosis. Methods: The activated human hepatic stellate cell line LX2 was induced by the stimulation of transforming growth factor - ß1 to construct carbon tetrachloride liver fibrosis mice model. The situation expression of AZGP1 in liver cells and tissues were observed. Plasmid transfection method was used to detect the activation, proliferation, apoptotic functions and changes in related factors of LX2 cells, respectively, after the overexpression and inhibition of AZGP1expression. Univariate analysis of variance was used for multiple group comparison. Results: The results of immunofluorescence staining showed that AZGP1 protein was decreased and α-smooth muscle actin was increased in the activated LX2 cells, and the two were negatively correlated. AZGP1 gene and protein were significantly under-expressed in activated LX2 cells and liver tissues of mice with carbon tetrachloride liver fibrosis. Collagen I, matrix metalloproteinase-2, and α-smooth muscle actin genes and proteins were significantly down-regulated in LX2 cells after over-expression of AZGP1. Cell fluorescence showed that AZGP1-overexpressing cells were activated and α-smooth muscle actin protein was reduced. In addition, the proliferative activity and G1/S-specific cyclin D1 protein of LX2 cells were significantly reduced after overexpression of AZGP1, while cell cycle experiments showed that the proportion of cells overexpressing AZGP1 was significantly increased in the G0/G1 phase, and the proportion of S phase was significantly reduced. AZGP1 had no significant effect on the apoptosis of LX2 cells. Conclusion: AZGP1 can reverse liver fibrosis by inhibiting the activation and proliferation of hepatic stellate cells, and thereby overexpression of AZGP1 is expected to become a new target for liver fibrosis treatment.


Assuntos
Tetracloreto de Carbono/toxicidade , Proliferação de Células/efeitos dos fármacos , Glicoproteínas/metabolismo , Células Estreladas do Fígado/efeitos dos fármacos , Actinas , Animais , Glicoproteínas/genética , Humanos , Fígado/efeitos dos fármacos , Fígado/patologia , Cirrose Hepática , Metaloproteinase 2 da Matriz , Camundongos , Zinco
5.
PLoS One ; 15(2): e0228729, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32053633

RESUMO

BACKGROUND: There is a correlation between the endocannabinoid system and hepatic fibrosis based on the activation of CB1 and CB2 receptors; where CB1 has profibrogenic effects. Gene therapy with a plasmid carrying a shRNA for CB1 delivered by hydrodynamic injection has the advantage of hepatic tropism, avoiding possible undesirable effects of CB1 pharmacological inhibition. OBJECTIVE: To evaluate hydrodynamics-based liver transfection in an experimental model of liver cirrhosis of a plasmid with the sequence of a shRNA for CB1 and its antifibrogenic effects. METHODS: Three shRNA (21pb) were designed for blocking CB1 mRNA at positions 877, 1232 and 1501 (pshCB1-A, B, C). Sequences were cloned in the pENTR™/U6. Safety was evaluated monitoring CB1 expression in brain tissue. The silencing effect was determined in rat HSC primary culture and CCl4 cirrhosis model. Hydrodynamic injection in cirrhotic liver was through iliac vein and with a dose of 3mg/kg plasmid. Serum levels of liver enzymes, mRNA levels of TGF-ß1, Col IA1 and α-SMA and the percentage of fibrotic tissue were analyzed. RESULTS: Hydrodynamic injection allows efficient CB1 silencing in cirrhotic livers and pshCB1-B (position 1232) demonstrated the main CB1-silencing. Using this plasmid, mRNA level of fibrogenic molecules and fibrotic tissue considerably decrease in cirrhotic animals. Brain expression of CB1 remained unaltered. CONCLUSION: Hydrodynamics allows a hepatotropic and secure transfection in cirrhotic animals. The sequence of the shCB1-B carried in a plasmid or any other vector has the potential to be used as therapeutic strategy for liver fibrosis.


Assuntos
Inativação Gênica , Hidrodinâmica , Cirrose Hepática/patologia , RNA Interferente Pequeno/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Actinas/genética , Actinas/metabolismo , Alanina Transaminase/sangue , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/sangue , Aspartato Aminotransferases/metabolismo , Encéfalo/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Fígado/metabolismo , Masculino , Plasmídeos/metabolismo , RNA Interferente Pequeno/administração & dosagem , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB1 de Canabinoide/genética , Transfecção , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo
6.
Anticancer Res ; 40(2): 743-749, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32014916

RESUMO

BACKGROUND/AIM: The hepatic stellate cells (HSCs) have relationship to cancer progression. The aim of this study is to investigate the effect of HSCs and the role of IL-6/Stat3 pathway on hepatocellular carcinoma (HCC) progression. MATERIALS AND METHODS: HCCs were co-cultured with HSCs. The viability and migration ability of cancer cells were detected. Epithelial-mesenchymal transition (EMT) marker (E-cadherin), stem cell marker (CD44) and p-signal transducer and activator of transcription 3 (p-STAT3) of cancer cells were evaluated. Finally, interleukin-6 (IL-6) neutralization was performed. RESULTS: Co-culture of HCCs with HSCs increased cancer cell viability and migration ability. EMT and stemness of cancer cells increased with HSCs. Following IL-6 neutralization, phospho-STAT3 activation, cancer cell viability and migration, as well as EMT, and stemness of cancer cells decreased. CONCLUSION: HSCs promoted HCC progression through the IL-6/STAT3 pathway.


Assuntos
Carcinoma Hepatocelular/metabolismo , Células Estreladas do Fígado/metabolismo , Interleucina-6/metabolismo , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/patologia , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Sobrevivência Celular/fisiologia , Técnicas de Cocultura , Células Hep G2 , Células Estreladas do Fígado/patologia , Humanos , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais
7.
Adv Exp Med Biol ; 1234: 43-56, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32040854

RESUMO

Hepatocellular carcinoma and intrahepatic cholangiocarcinoma are the most common types of primary liver cancers. Moreover, the liver is the second most frequently involved organ in cancer metastasis after lymph nodes. The tumor microenvironment is crucial for the development of both primary and secondary liver cancers. The hepatic microenvironment consists of multiple cell types, including liver sinusoidal endothelial cells, Kupffer cells, natural killer cells, liver-associated lymphocytes, and hepatic stellate cells (HSCs). The microenvironment of a normal liver changes to a tumor microenvironment when tumor cells exist or tumor cells migrate to and multiply in the liver. Interactions between tumor cells and non-transformed cells generate a tumor microenvironment that contributes significantly to tumor progression. HSCs play a central role in the tumor microenvironment crosstalk. As this crosstalk is crucial for liver carcinogenesis and liver-tumor development, elucidating the mechanism underlying the interaction of HSCs with the tumor microenvironment could provide potential therapeutic targets for liver cancer.


Assuntos
Células Estreladas do Fígado , Neoplasias Hepáticas/patologia , Microambiente Tumoral , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/patologia , Humanos , Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico
8.
Chem Biol Interact ; 317: 108945, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31935363

RESUMO

Liver fibrosis is a common pathological consequence of liver injury, which increases liver failure-related morbidity and mortality. Hence, anti-fibrotic treatment is urgently needed. Oxidative stress plays a pivotal role in the progression of liver fibrosis. Thus, targeting ROS may be an effective strategy for liver fibrosis treatment. In this study, we investigated four benzoquinones derivatives, including 5-isopropyl-2-methyl-1,4-benzoquinone (TQ), 2-tert-butyl-1,4-benzoquinone (tBu-Q) 2,5-dimethyl-p-benzoquinone (Dime-Q) and p-benzoquinone (Ph-Q), as well as the evaluation of their antioxidant activity and anti-fibrotic effects on activated hepatic stellate cells and TAA-induced mice. Electrochemical analysis showed that all compounds possessed antioxidant property. The result was first confirmed by in vitro experiments, which revealed potential anti-fibrotic activity of all four compounds at the cellular level. Benzoquinone derivatives act as ROS-scavenging molecules, which modulated the TLR4-CD14 signaling pathway to inhibit the expression of procaspase-1 and IL-1ß in cells, induced apoptosis via a mitochondrial pathway by upregulating the ratio of Bax/Bcl-2 and by activating caspase-3, as well as inhibited the expression of the anti-apoptotic proteins FLIP and XIAP in activated LX-2 cells. In addition, a TAA (Thioacetamide)-induced mouse model was used to further validate the results. Treatment with benzoquinone derivatives significantly decreased the levels of liver injury markers and lipid peroxidation caused by excessive ROS, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), and malondialdehyde (MDA). Moreover, treatment with benzoquinone derivatives significantly inhibited extracellular matrix (ECM) deposition and downregulated the mRNA and protein expression of liver fibrosis markers, such as collagen I, alpha-smooth muscle actin (α-SMA), and TIMP-1. In summary, these results indicate that benzoquinone derivatives may act as potential therapeutic drugs against liver fibrosis.


Assuntos
Antioxidantes/farmacologia , Benzoquinonas/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Tioacetamida/toxicidade , Actinas/genética , Actinas/metabolismo , Animais , Biomarcadores , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Inflamação/prevenção & controle , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Estresse Oxidativo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo
9.
Nat Commun ; 11(1): 240, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932588

RESUMO

Farnesoid X receptor (FXR) is a promising target for nonalcoholic steatohepatitis (NASH) and fibrosis. Although various FXR agonists have shown anti-fibrotic effects in diverse preclinical animal models, the response rate and efficacies in clinical trials were not optimum. Here we report that prophylactic but not therapeutic administration of obeticholic acid (OCA) prevents hepatic stellate cell (HSC) activation and fibrogenesis. Activated HSCs show limited response to OCA and other FXR agonists due to enhanced FXR SUMOylation. SUMOylation inhibitors rescue FXR signaling and thereby increasing the efficacy of OCA against HSC activation and fibrosis. FXR upregulates Perilipin-1, a direct target gene of FXR, to stabilize lipid droplets and thereby prevent HSC activation. Therapeutic coadministration of OCA and SUMOylation inhibitors drastically impedes liver fibrosis induced by CCl4, bile duct ligation, and more importantly NASH. In conclusion, we propose a promising therapeutic approach by combining SUMOylation inhibitors and FXR agonists for liver fibrosis.


Assuntos
Cirrose Hepática/tratamento farmacológico , Receptores Citoplasmáticos e Nucleares/agonistas , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/antagonistas & inibidores , Sumoilação , Animais , Células Cultivadas , Ácido Quenodesoxicólico/administração & dosagem , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacologia , Modelos Animais de Doenças , Quimioterapia Combinada , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/patologia , Humanos , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/patologia , Cirrose Hepática/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/patologia , Perilipina-1/genética , Perilipina-1/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ativação Transcricional/efeitos dos fármacos , Resultado do Tratamento
10.
Mol Cell Biochem ; 465(1-2): 115-123, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31893334

RESUMO

Increasing studies have indicated that hypoxia serves as a pivotal microenvironmental factor that facilitates activation of hepatic stellate cells (HSCs). However, the mechanism by which hypoxia activates HSCs is not clear. Here, we demonstrated that plasmacytoma variant translocation 1 (PVT1) and autophagy were overexpressed in liver fibrotic specimens. In primary mouse HSCs, both PVT1 and autophagy were induced by hypoxia. Further study showed that hypoxia-induced autophagy depended on expression of PVT1 and miR-152 in HSCs. Luciferase reporter assay indicated that autophagy-related gene 14 (ATG14) was a direct target of miR-152. In addition, inhibition of autophagy by 3-methyladenine and Beclin-1 siRNA impeded activation of HSCs cultured in 1% O2. Taken together, autophagy induction via the PVT1-miR-152-ATG14 signaling pathway contributes to activation of HSCs under hypoxia condition.


Assuntos
Morte Celular Autofágica , Proteínas Relacionadas à Autofagia/metabolismo , Células Estreladas do Fígado/metabolismo , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Proteínas de Transporte Vesicular/metabolismo , Animais , Hipóxia Celular , Células Estreladas do Fígado/patologia , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos
11.
Am J Pathol ; 190(3): 586-601, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31953035

RESUMO

Galanin (Gal) is a peptide with a role in neuroendocrine regulation of the liver. In this study, we assessed the role of Gal and its receptors, Gal receptor 1 (GalR1) and Gal receptor 2 (GalR2), in cholangiocyte proliferation and liver fibrosis in multidrug resistance protein 2 knockout (Mdr2KO) mice as a model of chronic hepatic cholestasis. The distribution of Gal, GalR1, and GalR2 in specific liver cell types was assessed by laser-capture microdissection and confocal microscopy. Galanin immunoreactivity was detected in cholangiocytes, hepatic stellate cells (HSCs), and hepatocytes. Cholangiocytes expressed GalR1, whereas HSCs and hepatocytes expressed GalR2. Strategies were used to either stimulate or block GalR1 and GalR2 in FVB/N (wild-type) and Mdr2KO mice and measure biliary hyperplasia and hepatic fibrosis by quantitative PCR and immunostaining of specific markers. Galanin treatment increased cholangiocyte proliferation and fibrogenesis in both FVB/N and Mdr2KO mice. Suppression of GalR1, GalR2, or both receptors in Mdr2KO mice resulted in reduced bile duct mass and hepatic fibrosis. In vitro knockdown of GalR1 in cholangiocytes reduced α-smooth muscle actin expression in LX-2 cells treated with cholangiocyte-conditioned media. A GalR2 antagonist inhibited HSC activation when Gal was administered directly to LX-2 cells, but not via cholangiocyte-conditioned media. These data demonstrate that Gal contributes not only to cholangiocyte proliferation but also to liver fibrogenesis via the coordinate activation of GalR1 in cholangiocytes and GalR2 in HSCs.


Assuntos
Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Colestase/metabolismo , Galanina/metabolismo , Cirrose Hepática/metabolismo , Receptor Tipo 1 de Galanina/metabolismo , Receptor Tipo 2 de Galanina/metabolismo , Animais , Ductos Biliares/metabolismo , Proliferação de Células , Colestase/patologia , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Feminino , Galanina/genética , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/patologia , Camundongos , Camundongos Knockout , Receptor Tipo 1 de Galanina/genética , Receptor Tipo 2 de Galanina/genética
12.
Gut ; 69(2): 365-379, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31076403

RESUMO

OBJECTIVE: Hepatocellular carcinoma (HCC), mostly developed in fibrotic/cirrhotic liver, exhibits relatively low responsiveness to immune checkpoint blockade (ICB) therapy. As myeloid-derived suppressor cell (MDSC) is pivotal for immunosuppression, we investigated its role and regulation in the fibrotic microenvironment with an aim of developing mechanism-based combination immunotherapy. DESIGN: Functional significance of MDSCs was evaluated by flow cytometry using two orthotopic HCC models in fibrotic liver setting via carbon tetrachloride or high-fat high-carbohydrate diet and verified by clinical specimens. Mechanistic studies were conducted in human hepatic stellate cell (HSC)-peripheral blood mononuclear cell culture systems and fibrotic-HCC patient-derived MDSCs. The efficacy of single or combined therapy with anti-programmed death-1-ligand-1 (anti-PD-L1) and a clinically trialled BET bromodomain inhibitor i-BET762 was determined. RESULTS: Accumulation of monocytic MDSCs (M-MDSCs), but not polymorphonuclear MDSCs, in fibrotic livers significantly correlated with reduced tumour-infiltrating lymphocytes (TILs) and increased tumorigenicity in both mouse models. In human HCCs, the tumour-surrounding fibrotic livers were markedly enriched with M-MDSC, with its surrogate marker CD33 significantly associated with aggressive tumour phenotypes and poor survival rates. Mechanistically, activated HSCs induced monocyte-intrinsic p38 MAPK signalling to trigger enhancer reprogramming for M-MDSC development and immunosuppression. Treatment with p38 MAPK inhibitor abrogated HSC-M-MDSC crosstalk to prevent HCC growth. Concomitant with patient-derived M-MDSC suppression by i-BET762, combined treatment with anti-PD-L1 synergistically enhanced TILs, resulting in tumour eradication and prolonged survival in the fibrotic-HCC mouse model. CONCLUSION: Our results signify how non-tumour-intrinsic properties in the desmoplastic microenvironment can be exploited to reinstate immunosurveillance, providing readily translatable combination strategies to empower HCC immunotherapy.


Assuntos
Carcinoma Hepatocelular/terapia , Imunoterapia/métodos , Neoplasias Hepáticas/terapia , Animais , Antígeno B7-H1/antagonistas & inibidores , Carcinoma Hepatocelular/etiologia , Carcinoma Hepatocelular/imunologia , Reprogramação Celular/imunologia , Ciclopropanos/farmacologia , Ciclopropanos/uso terapêutico , Células Estreladas do Fígado/imunologia , Humanos , Tolerância Imunológica , Cirrose Hepática/complicações , Cirrose Hepática/patologia , Neoplasias Hepáticas/etiologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas Experimentais/etiologia , Neoplasias Hepáticas Experimentais/imunologia , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/terapia , Masculino , Camundongos Endogâmicos C57BL , Monócitos/imunologia , Células Supressoras Mieloides/imunologia , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Transdução de Sinais/fisiologia , Células Tumorais Cultivadas , Microambiente Tumoral , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia
13.
Cancer Sci ; 111(2): 406-417, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31785057

RESUMO

STMN1 has been regarded as an oncogene and its upregulation is closely associated with malignant behavior and poor prognosis in multiple cancers. However, the detailed functions and underlying mechanisms of STMN1 are still largely unknown in hepatocellular carcinoma (HCC) development. Herein, we analyzed STMN1 expression and the related clinical significance in HCC by using well-established Protein Atlas, The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) cancer databases. Analysis indicated that STMN1 was highly expressed in HCC and closely associated with vascular invasion, higher histological grade, advanced clinical grade and shorter survival time in HCC patients. Overexpressing and silencing STMN1 in HCC cell lines showed that STMN1 could regulate cell proliferation, migration, drug resistance, cancer stem cell properties in vitro as well as tumor growth in vivo. Further experiments showed that STMN1 mediated intricate crosstalk between HCC and hepatic stellate cells (HSC) by triggering the hepatocyte growth factor (HGF)/MET signal pathway. When HSC were cocultured with HCC cells, HSC secreted more HGF to stimulate the expression of STMN1 in HCC cells. Mutually, STMN1 upregulation in HCC cells facilitated HSC activation to acquire cancer-associated fibroblast (CAF) features. The MET inhibitor crizotinib significantly blocked this crosstalk and slowed tumor growth in vivo. In conclusion, our findings shed new insight on STMN1 function, and suggest that STMN1 may be used as a potential marker to identify patients who may benefit from MET inhibitor treatment.


Assuntos
Carcinoma Hepatocelular/patologia , Células Estreladas do Fígado/citologia , Neoplasias Hepáticas/patologia , Transdução de Sinais , Estatmina/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos , Retroalimentação Fisiológica , Regulação Neoplásica da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Gradação de Tumores , Transplante de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Regulação para Cima
14.
Toxicol Lett ; 320: 1-8, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31756458

RESUMO

With the spread of hexavalent chromium [Cr(VI)] contamination, risk of exposure in non-occupational populations is increasing. The liver is the main target organ for Cr(VI) accumulation; however, the effect of long-term Cr(VI) exposure on liver toxicity is largely unknown. In this study, we investigated the effect of chronic Cr(VI) exposure on liver fibrosis and its possible mechanism. Mice were injected with Cr(VI) for two months, and our results showed Cr(VI) treatment caused liver toxicity characterized by liver structure disorganization, liver dysfunction, and antioxidant enzyme system inhibition. The development of liver fibrosis was also found via the emergence of collagen fibril deposition, increased expression of extracellular matrix-related genes, activation of hepatic stellate cells (HSCs) and increase the expression levels of Hedgehog (Hh) signaling pathway-related molecules. To demonstrate the role of Hh signaling in the regulation of Cr(VI)-induced liver fibrosis, genetically modified mice with heterozygous deficiency of Shh (Shh+/-) were used. In the Shh+/- mice, Hh signaling, HSCs activation and liver fibrosis development were all ameliorated. In conclusion, we demonstrated that Cr(VI)-induced liver fibrosis development resulted from Hh pathway-mediated HSCs activation. Our findings strongly suggest that inhibition of Hh pathway may help in the development of new strategies for Cr(VI)-associated liver fibrosis.


Assuntos
Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Cromo , Proteínas Hedgehog/metabolismo , Células Estreladas do Fígado/metabolismo , Cirrose Hepática Experimental/metabolismo , Fígado/metabolismo , Dicromato de Potássio , Transdução de Sinais , Animais , Doença Hepática Induzida por Substâncias e Drogas/genética , Doença Hepática Induzida por Substâncias e Drogas/patologia , Modelos Animais de Doenças , Proteínas Hedgehog/deficiência , Proteínas Hedgehog/genética , Células Estreladas do Fígado/patologia , Fígado/patologia , Cirrose Hepática Experimental/induzido quimicamente , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout
15.
J Biochem Mol Toxicol ; 34(1): e22413, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31714634

RESUMO

Hepatic diseases leading to fibrosis affect millions of individuals worldwide and are a major public health challenge. Although, there have been many advances in understanding hepatic fibrogenesis, an effective therapy remains elusive. Studies focus primarily on activation of the hepatic stellate cells (HSCs), the principal fibrogenic cells in the liver; however, fewer numbers of studies have examined molecular mechanisms that deactivate HSC, controlling the profibrogenic phenotype. In the present study, we evaluated cellular and molecular actions of the chemical triclosan (TCS) in reverting activated HSCs to a quiesced phenotype. We demonstrated that the inhibition of the enzyme fatty acid synthase by TCS in activated HSCs promotes survival of the cells and triggers cellular and molecular changes that promote cellular phenotypic reversion, offering potentially new therapeutic directions.


Assuntos
Inibidores da Síntese de Ácidos Graxos/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Triclosan/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ácido Graxo Sintases/antagonistas & inibidores , Células Estreladas do Fígado/citologia , Humanos
16.
Food Chem Toxicol ; 135: 111044, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31830547

RESUMO

Hemistepsin A (HsA), isolated from Hemistepta lyrata (Bunge) Bunge, has the ability to ameliorate hepatitis in mice. However, the effects of H. lyrata and HsA on other types of liver disease have not been explored. In this report, we investigated the effects of H. lyrata and HsA on liver fibrosis and the underlying molecular mechanisms in activated hepatic stellate cells (HSCs). Based on cell viability-guided isolation, we found HsA was the major natural product responsible for H. lyrata-mediated cytotoxicity in LX-2 cells. HsA significantly decreased the viability of LX-2 cells and primary activated HSCs, increased the binding of Annexin V, and altered the expression of apoptosis-related proteins, suggesting that HsA induces apoptosis in activated HSCs. HsA reduced the phosphorylation of IKKε and the transactivation of nuclear factor-κB (NF-κB). Moreover, HsA decreased the phosphorylation of Akt and its downstream signaling molecules. Transfection experiments suggested that inhibition of NF-κB or Akt is essential for HsA-induced apoptosis of HSCs. In a CCl4-induced liver fibrosis model, HsA administration significantly decreased ALT and AST activities. Furthermore, HsA attenuated CCl4-mediated collagen deposits and profibrogenic genes expression in hepatic tissue. Thus, HsA may serve as a natural product for managing liver fibrosis through inhibition of NF-κB/Akt-dependent signaling.


Assuntos
Apoptose/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Lactonas/farmacologia , Cirrose Hepática/prevenção & controle , Sesquiterpenos/farmacologia , Animais , Linhagem Celular Transformada , Clorofórmio/farmacologia , Células Estreladas do Fígado/metabolismo , Humanos , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , NF-kappa B/metabolismo , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos
17.
Chemosphere ; 242: 124959, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31669990

RESUMO

Long-term exposure to arsenic can cause liver injury and fibrosis. The activation of hepatic stellate cells (HSCs) plays an essential role in the process of liver fibrosis. We found that NaAsO2 caused liver damage and fibrosis in vivo, accompanied by excessive collagen deposition and HSCs activation. In addition, NaAsO2 upregulated autophagy flux, elevated the level of cytoplasmic cathepsin B (CTSB), and activated the NOD-like receptors containing pyrin domain 3 (NLRP3) inflammasome in a subtle way. Consistent with these findings in vivo, we demonstrated that NaAsO2-induced activation of HSCs depended on CTSB-mediated NLRP3 inflammasome activation in HSC-t6 cells and rats primary HSCs. Moreover, inhibition of autophagy decreased the cytoplasmic CTSB and alleviated the activation of the NLRP3 inflammasome, thereby attenuating the NaAsO2-induced HSCs activation. In summary, these results indicated that NaAsO2 induced HSCs activation via autophagic-CTSB-NLRP3 inflammasome pathway. These findings may provide a novel insight into the potential mechanism of NaAsO2-induced liver fibrosis.


Assuntos
Arsênico/toxicidade , Autofagia , Catepsina B/metabolismo , Células Estreladas do Fígado/metabolismo , Inflamassomos/fisiologia , Cirrose Hepática/induzido quimicamente , Animais , Arsênico/metabolismo , Inflamassomos/metabolismo , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/patologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ratos
18.
Acta Biochim Biophys Sin (Shanghai) ; 52(1): 18-25, 2020 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-31828297

RESUMO

As a highly malignant tumor, hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. In most HCC patients, the development of HCC begins with hepatitis, which is followed by fibrosis and cirrhosis before progressing to HCC. Cancer-associated fibroblasts (CAFs), which are generally believed to be derived from activated hepatic stellate cells (HSCs), are highly involved in the development of HCC through the secretion of cytokines and angiogenic factors. The results of our study showed that a considerable number of CAFs highly expressed CD90 and were enriched in HCC tissues. Bioinformatics analysis of the transcriptome of HCC tissues revealed that placental growth factor (PlGF) is significantly correlated with CD90 expression. The isolated primary CAFs and activated HSCs overexpressed PlGF and CD90. In addition, the results of gene expression profiling interactive analysis based on The Cancer Genome Atlas showed that high levels of both PlGF and CD90 are correlated with tumor angiogenesis markers (CD31, CD34, and CD105) and predict poor HCC patient prognosis. In summary, our results suggest that CAFs can generate PlGF and may provide an effective target for CAFs-regulated neoangiogenesis in HCC.


Assuntos
Carcinoma Hepatocelular/irrigação sanguínea , Fibroblastos/metabolismo , Neoplasias Hepáticas/irrigação sanguínea , Neovascularização Patológica/metabolismo , Fator de Crescimento Placentário/metabolismo , Actinas/metabolismo , Antígenos CD34/metabolismo , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Endoglina/metabolismo , Regulação Neoplásica da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Humanos , Neoplasias Hepáticas/genética , Fator de Crescimento Placentário/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Prognóstico , Antígenos Thy-1/genética , Antígenos Thy-1/metabolismo , Transcriptoma/genética
19.
Chem Biol Interact ; 316: 108917, 2020 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-31838050

RESUMO

Stearoyl-CoA desaturase (SCD) generates monounsaturated fatty acids (MUFAs) which contribute to cell growth, survival, differentiation, metabolic regulation and signal transduction. Overexpression of SCD is evident and implicated in metabolic diseases such as diabetes and non-alcoholic fatty liver disease. SCD also stimulates canonical Wnt pathway and YAP activation in support of stemness and tumorigenesis. SCD facilitates metabolic reprogramming in cancer which is mediated, at least in part, by regulation of AKT, AMPK, and NF-kB via MUFAs. Our research has revealed the novel positive loop to amplify Wnt signaling through stabilization of LRP5/6 in both hepatic stellate cells and liver tumor-initiating stem cell-like cells. As such, this loop is pivotal in promoting liver fibrosis and liver tumor development. This review summarizes the mechanisms of SCD-mediated tumor promotion described by recent studies and discusses the future prospect for SCD-mediated signaling crosstalk as a potential therapeutic target for cancer.


Assuntos
Carcinogênese , Estearoil-CoA Dessaturase/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Estearoil-CoA Dessaturase/genética , Via de Sinalização Wnt
20.
Proc Natl Acad Sci U S A ; 117(1): 454-463, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871210

RESUMO

Liver fibrosis interferes with normal liver function and facilitates hepatocellular carcinoma (HCC) development, representing a major threat to human health. Here, we present a comprehensive perspective of microRNA (miRNA) function on targeting the fibrotic microenvironment. Starting from a murine HCC model, we identify a miRNA network composed of 8 miRNA hubs and 54 target genes. We show that let-7, miR-30, miR-29c, miR-335, and miR-338 (collectively termed antifibrotic microRNAs [AF-miRNAs]) down-regulate key structural, signaling, and remodeling components of the extracellular matrix. During fibrogenic transition, these miRNAs are transcriptionally regulated by the transcription factor Pparγ and thus we identify a role of Pparγ as regulator of a functionally related class of AF-miRNAs. The miRNA network is active in human HCC, breast, and lung carcinomas, as well as in 2 independent mouse liver fibrosis models. Therefore, we identify a miRNA:mRNA network that contributes to formation of fibrosis in tumorous and nontumorous organs of mice and humans.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Cirrose Hepática/patologia , Neoplasias Hepáticas/genética , MicroRNAs/genética , PPAR gama/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Hepatocelular/patologia , Ilhas de CpG/genética , Metilação de DNA , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Epigênese Genética , Matriz Extracelular/patologia , Feminino , Células Estreladas do Fígado/patologia , Humanos , Fígado/citologia , Fígado/patologia , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Camundongos , Cultura Primária de Células , Regiões Promotoras Genéticas/genética , RNA-Seq , Microambiente Tumoral/genética
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