RESUMO
Environmental antineoplastics such as sorafenib may pose a risk to humans through water recycling, and the increased risk of cardiotoxicity is a clinical issue in sorafenib users. Thus, developing strategies to prevent sorafenib cardiotoxicity is an urgent work. Empagliflozin, as a sodium-glucose co-transporter-2 (SGLT2) inhibitor for type 2 diabetes control, has been approved for heart failure therapy. Still, its cardioprotective effect in the experimental model of sorafenib cardiotoxicity has not yet been reported. Real-time quantitative RT-PCR (qRT-PCR), immunoblot, and immunohistochemical analyses were applied to study the effect of sorafenib exposure on cardiac SGLT2 expression. The impact of empagliflozin on cell viability was investigated in the sorafenib-treated cardiomyocytes using Alamar blue assay. Immunoblot analysis was employed to delineate the effect of sorafenib and empagliflozin on ferroptosis/proinflammatory signaling in cardiomyocytes. Ferroptosis/DNA damage/fibrosis/inflammation of myocardial tissues was studied in mice with a 28-day sorafenib ± empagliflozin treatment using histological analyses. Sorafenib exposure significantly promoted SGLT2 upregulation in cardiomyocytes and mouse hearts. Empagliflozin treatment significantly attenuated the sorafenib-induced cytotoxicity/DNA damage/fibrosis in cardiomyocytes and mouse hearts. Moreover, GPX4/xCT-dependent ferroptosis as an inducer for releasing high mobility group box 1 (HMGB1) was also blocked by empagliflozin administration in the sorafenib-treated cardiomyocytes and myocardial tissues. Furthermore, empagliflozin treatment significantly inhibited the sorafenib-promoted NFκB/HMGB1 axis in cardiomyocytes and myocardial tissues, and sorafenib-stimulated proinflammatory signaling (TNF-α/IL-1ß/IL-6) was repressed by empagliflozin administration. Finally, empagliflozin treatment significantly attenuated the sorafenib-promoted macrophage recruitments in mouse hearts. In conclusion, empagliflozin may act as a cardioprotective agent for humans under sorafenib exposure by modulating ferroptosis/DNA damage/fibrosis/inflammation. However, further clinical evidence is required to support this preclinical finding.
RESUMO
INTRODUCTION: Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, often accompanied by obesity, diabetes, and increased risks of depression and anxiety. Currently, there are no FDA-approved drugs to treat NAFLD and its related systemic symptoms. Previously, we identified a new barbituric acid derivative (BA-5) that expressed effectiveness against fibrosis and drug-resistant hepatocellular carcinoma. AIMS: This study investigated the potential of BA-5 against high-fat diet (HFD)-induced NAFLD and mood disorders in mice. MAIN METHODS: Six-weeks-old male C57BL/6 mice were fed with a 45 % HFD for 8 weeks to induce NAFLD and associated metabolic disorders. Mice were treated with a BA-5 and the therapeutic effects and the underlying molecular mechanisms were investigated. KEY FINDINGS: Administration of BA-5 significantly reduced serum levels of alanine aminotransferase (ALT), low-density lipoprotein (LDL), fatty acids (FA), and triglycerides (TG) in HFD-fed mice. BA-5 treatment decreased expressions of hepatic lipogenesis-related markers (acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and ATP-citrate lyase (ACLY)), increased fatty acid oxidation markers (carnitine palmitoyltransferase 1A (CPT1A) and acyl-CoA oxidase 1 (ACOX1)), and attenuated hepatic fat accumulation in HFD-fed mice. Moreover, HFD-induced adipocyte size enlargement and activation of lipolysis markers such as phosphorylated (p)-hormone-sensitive lipase (HSL) 565, p-HSL 660, and perilipin were inhibited in BA-5-treated mice. Notably, HFD-induced anxiety- and depression-like behaviors significantly improved in the BA-5 treated group through enhanced anti-inflammatory responses in the hippocampus. SIGNIFICANCE: This study provides new insights into clinical therapeutic strategies of barbituric acid derivatives for HFD-induced NAFLD and associated mood disturbances.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Masculino , Animais , Camundongos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/etiologia , Hepatopatia Gordurosa não Alcoólica/metabolismo , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Dieta Hiperlipídica/efeitos adversosRESUMO
Hepatocellular carcinoma (HCC) is the most common primary malignant tumor. Although sorafenib and regorafenib have been approved for first-line and second-line treatment, respectively, of patients with advanced HCC, long-term treatment often results in acquired resistance. Given that glycolysis-mediated lactate production can contribute to drug resistance and impair HCC treatment efficacy, we investigated the effects of ketone body treatment on the metabolic shift in sorafenib-resistant HCC cells. We discovered differential expression of 3-hydroxymethyl glutaryl-CoA synthase 2 (HMGCS2) and the ketone body D-ß-hydroxybutyrate (ß-HB) in four sorafenib-resistant HCC cell lines. In sorafenib-resistant HCC cells, lower HMGCS2 and ß-HB levels were correlated with more glycolytic alterations and higher lactate production. ß-HB treatment enhanced pyruvate dehydrogenase (PDH) expression and decreased lactate dehydrogenase (LDHA) expression and lactate production in sorafenib-resistant HCC cells. Additionally, ß-HB combined with sorafenib or regorafenib promoted the antiproliferative and antimigratory abilities of sorafenib-resistant HCC cells by inhibiting the B-raf/mitogen-activated protein kinase pathway and mesenchymal N-cadherin-vimentin axis. Although the in vivo ß-HB administration did not affect tumor growth, the expression of proliferative and glycolytic proteins was inhibited in subcutaneous sorafenib-resistant tumors. In conclusion, exogenous ß-HB treatment can reduce lactate production and reverse sorafenib resistance by inducing a glycolytic shift; it can also synergize with regorafenib for treating sorafenib-resistant HCC.
Assuntos
Antineoplásicos , Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Sorafenibe/farmacologia , Sorafenibe/uso terapêutico , Carcinoma Hepatocelular/patologia , Ácido 3-Hidroxibutírico , Neoplasias Hepáticas/patologia , Resistencia a Medicamentos Antineoplásicos , Glicólise , Lactatos/farmacologia , Linhagem Celular Tumoral , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
Plasticizers are considered as environmental pollution released from medical devices and increased potential oncogenic risks in clinical therapy. Our previous studies have shown that long-term exposure to di-ethylhexyl phthalate (DEHP)/mono-ethylhexyl phthalate (MEHP) promotes chemotherapeutic drug resistance in colorectal cancer. In this study, we investigated the alteration of glycosylation in colorectal cancer following long-term plasticizers exposure. First, we determined the profiles of cell surface N-glycomes by using mass spectrometry and found out the alterations of α2,8-linkages glycans. Next, we analyzed the correlation between serum DEHP/MEHP levels and ST8SIA6 expression from matched tissues in total 110 colorectal cancer patients. Moreover, clinical specimens and TCGA database were used to analyze the expression of ST8SIA6 in advanced stage of cancer. Finally, we showed that ST8SIA6 regulated stemness in vitro and in vivo. Our results revealed long-term DEHP/MEHP exposure significantly caused cancer patients with poorer survival outcome and attenuated the expression of ST8SIA6 in cancer cells and tissue samples. As expected, silencing of ST8SIA6 promoted cancer stemness and tumorigenicity by upregulating stemness-associated proteins. In addition, the cell viability assay showed enhanced drug resistance in ST8SIA6 silencing cells treated with irinotecan. Besides, ST8SIA6 was downregulated in the advanced stage and positively correlated with tumor recurrence in colorectal cancer. Our results imply that ST8SIA6 potentially plays an important role in oncogenic effects with long-term phthalates exposure.
Assuntos
Neoplasias Colorretais , Dietilexilftalato , Humanos , Plastificantes/análise , Dietilexilftalato/análise , Glicosilação , Sialiltransferases/metabolismoRESUMO
Leukocyte cell-derived chemotaxin 2 (LECT2) acts as a tumor suppressor in hepatocellular carcinoma (HCC). However, the antineoplastic mechanism of LECT2, especially its influence on hepatic cancer stem cells (CSCs), remains largely unknown. In The Cancer Genome Atlas cohort, LECT2 mRNA expression was shown to be associated with stage, grade, recurrence, and overall survival in human HCC patients, and LECT2 expression was downregulated in hepatoma tissues compared with the adjacent nontumoral liver. Here, we show by immunofluorescence and immunoblot analyses that LECT2 was expressed at lower levels in tumors and in poorly differentiated HCC cell lines. Using functional assays, we also found LECT2 was capable of suppressing oncogenic behaviors such as cell proliferation, anchorage-independent growth, migration, invasiveness, and epithelial-mesenchymal transition in hepatoma cells. Moreover, we show exogenous LECT2 treatment inhibited CSC functions such as tumor sphere formation and drug efflux. Simultaneously, hepatic CSC marker expression was also downregulated, including expression of CD133 and CD44. This was supported by infection with adenovirus encoding LECT2 (Ad-LECT2) in HCC cells. Furthermore, in animal experiments, Ad-LECT2 gene therapy showed potent efficacy in treating HCC. We demonstrate LECT2 overexpression significantly promoted cell apoptosis and reduced neovascularization/CSC expansion in rat hepatoma tissues. Mechanistically, we showed using immunoblot and immunofluorescence analyses that LECT2 inhibited ß-catenin signaling via the suppression of the hepatocyte growth factor/c-MET axis to diminish CSC properties in HCC cells. In summary, we reveal novel functions of LECT2 in the suppression of hepatic CSCs, suggesting a potential alternative strategy for HCC therapy.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Animais , Humanos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/terapia , Linhagem Celular Tumoral , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/uso terapêutico , Neoplasias Hepáticas/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Ratos , Terapia GenéticaRESUMO
Objective: Keloid is an abnormal scar that often develops in high-tension skin. It is caused by excessive fibroblast proliferation and collagen deposition. Nonmuscle myosin IIA (NM-IIA) is an important motor protein that regulates the mechanical transduction of cells. However, the role of NM-IIA in keloid pathogenesis remains unclear. Approach: NM-IIA expression was examined and compared in keloid skin and normal skin by immunofluorescence. The organization of smooth muscle actin (SMA)-mediated stress fibers in normal and keloid fibroblasts (NFs and KFs, respectively) were determined. Cell proliferation and cell contractility were measured in fibroblasts derived from normal and keloids. The NM-II pharmacological inhibitor (blebbistatin) and RNA interference were applied to block NM-IIA and investigate its regulatory role in SMA-mediated stress fibers, cell contractility, and cell proliferation after NM-IIA inhibition. Results: NM-IIA expression is increased in keloid tissue. Inhibition of NM-II by blebbistatin or targeting NM-IIA by RNA interference reduced transforming growth factor beta (TGF-ß)-mediated SMA-mediated stress fiber formation, cell proliferation, and cell contractility of NFs and KFs. Although TGF-ß failed to mediate phosphorylation of myosin light chain (pMLC, the activator of NM-II), pMLC can interact with SMA-mediated stress fiber. Finally, inhibition of NM-II by blebbistatin also reduced NF and KF proliferation after TGF-ß stimulation. Innovation: NM-IIA synergizes with TGF-ß to regulate fibroblast proliferation, contraction activity, and myofibroblasts differentiation. Conclusion: NM-IIA might be one of the therapeutic targets in keloids.
Assuntos
Diferenciação Celular/genética , Proliferação de Células/genética , Queloide/metabolismo , Miofibroblastos/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Células Cultivadas , Colágeno/metabolismo , Feminino , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Queloide/patologia , Masculino , Pessoa de Meia-Idade , Miofibroblastos/citologia , Miosina não Muscular Tipo IIA/antagonistas & inibidores , Miosina não Muscular Tipo IIA/genética , Interferência de RNA , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Pele/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
Non-alcoholic fatty liver disease (NAFLD) is currently the most frequently occurring liver disorder in the world. However, a specific drug for the treatment of patients with NAFLD is not available. Therefore, the discovery of novel compounds for the treatment of NAFLD and elucidation of the underlying mechanisms of therapeutic drugs that can be used to treat this disease are urgently needed. 1,2,3,4,6 penta-O-galloyl-ß-d-glucose (PGG) is known to exert anti-inflammatory, antidiabetic, and hepatoprotective effects. However, little is known about the therapeutic potential of PGG in NAFLD. In this study, we investigated the effects of PGG on a high-fat diet (HFD)-induced mouse model of NAFLD. PGG was co-administered along with an HFD to C57BL/6 mice. After eight weeks of treatment, serum biochemistry, liver steatosis, and lipid metabolism-related genes were examined. The results showed that PGG treatment significantly reduced HFD-induced gain in body weight, liver steatosis, and leukocyte infiltration in a dose-dependent manner. Furthermore, PGG treatment markedly reduced serum triglyceride and glucose levels in HFD mice. Moreover, alterations in the mRNA expression of genes involved in lipid metabolism, including Hmgcr, Acc1, Abca1, Mttp, and Cd36, observed in the livers of HFD-treated mice were significantly reversed by PGG treatment. PGG significantly reduced HFD-induced protein expression of CD36, which is associated with fatty acid uptake, insulin resistance, hyperinsulinemia, and increased hepatic steatosis, in the liver of HFD mice. These results suggest that PGG inhibits HFD-induced hepatic steatosis and reverses HFD-induced alterations of gene expression in lipid metabolism. PGG has been shown to be well tolerated; therefore, it has potential uses in NAFLD treatment.
Assuntos
Dieta Hiperlipídica , Taninos Hidrolisáveis/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Animais , Biomarcadores/sangue , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Homeostase , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Fígado/patologia , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
Characterized with a high recurrence rate and low detection rate, prevention is the best approach to reduce mortality in hepatocellular carcinoma (HCC). The overexpression of Phosphatidylinositol-3,4,5-Trisphosphate Dependent Rac Exchange Factor 2 (PREX2) is observed in various tumors, including HCC; and the frequent PREX2 mutations in melanoma are associated with invasiveness. We sought to identify somatic mutations and the functional changes in mutational signatures of PREX2. Genomic DNA sequencing was performed in 68 HCC samples with three types of hepatitis viral infection status: HBs Ag-positive, anti-HCV Ab-positive, and negative for any hepatitis B or C markers. Stabilities and interactions of proteins as well as cell proliferation and migration were evaluated. Fourteen non-silent point mutations in PREX2 were detected, with 16 of 68 HCC patients harboring at least one non-silent mutation. All mutant forms of PREX2, except for K400f, had an extended half-life compared with wild-type PREX2. Moreover, only the half-life of S1113R was twice that of the wild-type. PREX2 mutant-S1113R also promoted migration and activated the AKT pathway as well as impaired HectH9-mediated ubiquitination. Our study identified a gain-of-function mutation of PREX2 - S1113R in HCC. Such mutation enhanced PREX2 protein stability, promoted cell proliferation, and was associated with aggressiveness of HCC.
Assuntos
Carcinoma Hepatocelular/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Neoplasias Hepáticas/genética , Mutação , Adulto , Idoso , Proliferação de Células , Feminino , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Vírus de Hepatite , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNARESUMO
Glycine N-methyltransferase (GNMT) is abundantly expressed in normal livers and plays a protective role against tumor formation. GNMT depletion leads to progression of hepatocellular carcinoma (HCC). In this study, we investigated the activity of ectopic GNMT delivered using recombinant adeno-associated virus (AAV) gene therapy in mouse models of liver cirrhosis and HCC. Injection of AAV serotype 8 (AAV8) vector carrying the GNMT gene (AAV8-GNMT) in Gnmt-/- mice increased GNMT expression and downregulated pro-inflammatory responses, resulting in reduced liver damage and incidence of liver tumors. Moreover, AAV8-GNMT resulted in the amelioration of carbon tetrachloride (CCl4)-induced liver fibrosis in BALB/c mice. We showed that AAV8-GNMT protected hepatocytes from CCl4-induced liver damage. AAV8-GNMT significantly attenuated the levels of pro-fibrotic markers and increased efficiency of hepatocyte proliferation. These results suggest that correction of hepatic GNMT by gene therapy of AAV8-mediated gene enhancement may provide a potential strategy for preventing and delaying development of liver diseases.
Assuntos
Carcinoma Hepatocelular/terapia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Terapia Genética/métodos , Glicina N-Metiltransferase/biossíntese , Neoplasias Hepáticas/terapia , Animais , Tetracloreto de Carbono/efeitos adversos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Proliferação de Células/fisiologia , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Metilação de DNA , Dependovirus/enzimologia , Dependovirus/genética , Progressão da Doença , Regulação para Baixo , Feminino , Glicina N-Metiltransferase/genética , Glicina N-Metiltransferase/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Glycine N-methyltransferase (GNMT) is a tumor suppressor for HCC. It is down-regulated in HCC, but the mechanism is not fully understood. MicroRNA-224 (miR-224) acts as an onco-miR in HCC. This study is the first to investigate miR-224 targeting the coding region of GNMT transcript. The GNMT-MT plasmid containing a miR-224 binding site silent mutation of the GNMT coding sequence can escape the suppression of miR-224 in HEK293T cells. Expression of both exogenous and endogenous GNMT was suppressed by miR-224, while miR-224 inhibitor enhanced GNMT expression. miR-224 counteracts the effects of GNMT on the reduction of cell proliferation and tumor growth. The levels of miR-224 and GNMT mRNA showed a significant inverse relationship in tumor specimens from HCC patients. Utilizing CCl4-treated hepatoma cells and mice as a cell damage of inflammatory or liver injury model, we observed that the decreased expression levels of GNMT were accompanied with the elevated expression levels of miR-224 in hepatoma cells and mouse liver. Finally, hepatic AAV-mediated GNMT also reduced CCl4-induced miR-224 expression and liver fibrosis. These results indicated that AAV-mediated GNMT has potential liver protection activity. miR-224 can target the GNMT mRNA coding sequence and plays an important role in GNMT suppression during liver tumorigenesis.
Assuntos
Aciltransferases/genética , Carcinoma Hepatocelular/enzimologia , Regulação para Baixo/fisiologia , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/enzimologia , MicroRNAs/fisiologia , Regiões 3' não Traduzidas , Animais , Carcinoma Hepatocelular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Células HEK293 , Humanos , Neoplasias Hepáticas/genética , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , RNA Mensageiro/genéticaRESUMO
Plants containing aristolochic acids (AA) are nephrotoxins. Glycine N-methyltransferase (GNMT) acts to bind environmental toxins such as benzo(a)pyrene and aflatoxin B1, translocate into nucleus, and alter hepatic metabolism. This study aims to determine the role of GNMT in AA-induced nephropathy. We established an AA nephropathy mouse model and found that AA type I (AAI)-induced nephropathy at a lower concentration in male than in female mice, implying sex differences in AAI resistance. Microarray analysis and AAI-treated mouse models showed that GNMT moderately reduced AAI-induced nephropathy by lowering the upregulated level of NQO1 in male, but significantly improved the nephropathy additionally by increasing Cyp3A44/3A41 in female. The protective effects of GNMT were absent in female GNMT knockout mice, in which re-expression of hepatic GNMT significantly decreased AAI-induced nephropathy. Mechanism-wise, AAI enhanced GNMT nuclear translocation, resulting in GNMT interaction with the promoter region of the genes encoding Nrf2 and CAR/PXR, the transcription factors for NQO1 and CYP3A44/3A41, respectively. Unlike the preference for Nrf2/NQO1 transcriptions at lower levels of GNMT, overexpression of GNMT preferred CAR/PXR/CYP3A44/3A41 transcriptions and alleviated kidney injury upon AAI treatment. In summary, hepatic GNMT protected mice from AAI nephropathy by enhancing CAR/PXR/CYP3A44/3A41 transcriptions and reducing Nrf2/NQO1 transcriptions.
Assuntos
Ácidos Aristolóquicos/efeitos adversos , Sistema Enzimático do Citocromo P-450/genética , Glicina N-Metiltransferase/metabolismo , Nefropatias/induzido quimicamente , NAD(P)H Desidrogenase (Quinona)/genética , Ativação Transcricional , Animais , Regulação para Baixo , Feminino , Glicina N-Metiltransferase/genética , Hepatócitos/metabolismo , Hepatócitos/patologia , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Proteção , Fatores Sexuais , Regulação para CimaRESUMO
The pathogenesis of hepatocellular carcinoma (HCC) involves many molecular pathways. Glycine N-methyltransferase (GNMT) is downregulated in almost all HCC and its gene knockout mice developed HCC with high penetrance. We identified PREX2, a novel PTEN inhibitor, as a GNMT-interacting protein. Such interaction enhanced degradation of PREX2 through an E3 ligase HectH9-mediated proteasomal ubiquitination pathway. Depletion of GNMT or HectH9 resulted in AKT activation in a PREX2 dependent manner and enhanced cell proliferation. An elevated PREX2 protein expression accompanied by activation of AKT was observed in the liver of Gnmt knockout mice. PREX2 protein expression was upregulated in 54.9% of human HCC samples, while its mRNA level was comparable in tumor and tumor-adjacent tissue, suggesting a post-translational alteration of PREX2 expression. Higher level of PREX2 in the tumor tissues was associated with poorer survival. These results reveal a novel mechanism in which GNMT participates in AKT signaling and HCC tumorigenesis by promoting HectH9-mediated PREX2 degradation.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/patologia , Transformação Celular Neoplásica/patologia , Glicina N-Metiltransferase/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neoplasias Hepáticas/patologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Western Blotting , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Feminino , Seguimentos , Glicina N-Metiltransferase/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pessoa de Meia-Idade , Estadiamento de Neoplasias , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Prognóstico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Primary hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third leading cause of cancer-related death. It is important to identify new targets for early diagnosis and treatment of HCC. Niemann-Pick type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in HCC tumorigenesis. In this study, we showed that NPC2 is abundantly expressed in normal liver, but is downregulated in human HCC tissues. The patients with NPC2 downregulation expressed much higher α-fetoprotein, multiple tumor type, vascular invasion, later pathological stage and shorter survival rate. Knockdown NPC2 in liver cancer cell lines promote cell proliferation, migration and xenograft tumorigenesis. In contrast, NPC2 overexpression inhibits HuH7 promoted tumor growth. Furthermore, administration of hepatotropic adeno-associated virus 8 (AAV8) delivered NPC2 decreased the inflammatory infiltration, the expression of two early HCC markers-glypican 3 and survivin and suppressed the spontaneous HCC development in mice. To identify the NPC2-dependent mechanism, we emphasized on the status of MAPK/ERK signaling. MEK1/2 inhibitor treatment demonstrated that the expression of NPC2 affected the activation of ERK1/2 but not MEK1/2. In addition, cholesterol trafficking inhibitor treatment did not alter the cell proliferation and the activation of MEK/ERK. In conclusion, our study demonstrates that NPC2 may play an important role in negatively regulate cell proliferation and ERK1/2 activation that were independent of cholesterol accumulation. AAV-NPC2 may thus represent a new treatment strategy for liver cancer.
Assuntos
Proteínas de Transporte/genética , Glicoproteínas/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Sistema de Sinalização das MAP Quinases/genética , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Progressão da Doença , Regulação para Baixo/genética , Feminino , Células HEK293 , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Taxa de Sobrevida , Proteínas de Transporte Vesicular , alfa-Fetoproteínas/genéticaRESUMO
Glycine N-methyltransferase (GNMT) is known for its function as a tumor suppressor gene. Since 100% of female Gnmt(-/-) mice developed hepatocellular carcinoma, we hypothesized that Gnmt(-/-) mice may have defective immune surveillance. In this study, we examined the immune modulation of GNMT in T-cell responses using experimental autoimmune encephalomyelitis (EAE). The results showed that EAE severity was reduced significantly in Gnmt(-/-) mice. Pathological examination of the spinal cords revealed that Gnmt(-/-) mice had significantly lower levels of mononuclear cell infiltration and demyelination than the wild-type mice. In addition, quantitative real-time PCR showed that expression levels of proinflammatory cytokines, including interferon (IFN)-γ and interleukin (IL)-17A, were much lower in the spinal cord of Gnmt(-/-) than in that of wild-type mice. Accordingly, myelin oligodendrocyte glycoprotein (MOG)-specific T-cell proliferation and induction of T-helper (Th)1 and Th17 cells were markedly suppressed in MOG(35-55)-induced Gnmt(-/-) mice. Moreover, the number of regulatory T (Treg) cells was increased significantly in these mice. When the T-cell receptor was stimulated, the proliferative capacity and the activation status of mTOR-associated downstream signaling were decreased significantly in Gnmt(-/-) CD4(+) T cells via an IL-2- and CD25-independent manner. Moreover, GNMT deficiency enhanced the differentiation of Treg cells without affecting the differentiation of Th1 and Th17 cells. Furthermore, the severity of EAE in mice adoptive transferred with GNMT-deficient CD4(+) T cells was much milder than in those with wild-type CD4(+) T cells. In summary, our findings suggest that GNMT is involved in the pathogenesis of EAE and plays a crucial role in the regulation of CD4(+) T-cell functions.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Glicina N-Metiltransferase/imunologia , Linfócitos T/imunologia , Animais , Citocinas/imunologia , Feminino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Serina-Treonina Quinases TOR/imunologiaRESUMO
A relevant animal model is critical for investigating the pathogenic mechanisms underlying hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC). Mice are not naturally infected by HBV, presumably due to the lack of HBV receptors on mouse hepatocytes. To bypass this entry step of HBV infection, we report generation of a novel HBV model in immunocompetent mice by hepatic delivery of the HBV genome using trans-splicing adeno-associated viral vectors (AAV/HBV). We confirmed production of HBV virions and proteins in the liver and circulation in all AAV/HBV-transduced mice in all four immunocompetent mouse strains tested. These mice produced antigen and antibody profiles similar to that observed in chronic HBV patients. Importantly, 12-16 months later, all 12 AAV/HBV-transduced mice developed macroscopically visible liver-tumor nodules. Ten of the twelve tumors were characterized with typical HCC features. This AAV/HBV-transduced murine HCC model provides a useful instrument for studying the pathogenesis of HBV-associated HCC and the development of HCC therapeutic interventions.
Assuntos
Carcinoma Hepatocelular/virologia , Dependovirus/genética , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Hepatite B Crônica/complicações , Neoplasias Hepáticas/virologia , Animais , Sequência de Bases , Carcinoma Hepatocelular/patologia , Vírus da Hepatite B/genética , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Trans-Splicing , Carga ViralRESUMO
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most difficult cancers to treat. The interleukin (IL)-12 family cytokines, including IL-12, IL-23 and IL-27, display overlapping, but not redundant, roles in regulating lymphocyte subpopulations. IL-12 is known as a potent antitumor cytokine, whereas the results of the antitumor effect of IL-23 and IL-27 are inconsistent. The present study aimed to directly compare the relative antitumor efficacy of these three IL-12 family cytokines on HCC. METHODS: A murine orthotopic BNL HCC model, in which the tumor is located in an environment heavily populated with different lymphocyte subsets, was established. The hepatotropic adeno-associated virus serotype 8 (AAV8) vector was used to deliver the cytokine genes aiming to achieve sustained cytokine expression in the liver. RESULTS: AAV8/IL-12 treatment significantly reduced hepatic metastases and prolonged survival time, whereas treatment with AAV8/IL-23 or AAV8/IL-27 had only moderate antitumor effects at a high dose. The antitumor efficacy of these cytokines was positively correlated with their ability to regulate hepatic T cells, natural killer cells and natural killer T cells, with IL-12 greatly increasing the number and activation status of these cells, whereas IL-27 had no effect and IL-23 had a negative effect. AAV8/IL-12 treatment also resulted in a marked decrease in tumor vessel density, which was not observed with AAV8/IL-23 and AAV8/IL-27 treatment. CONCLUSIONS: The data obtained in the present study highlight the importance of local lymphocytes and anti-angiogenesis for influencing the antitumor activity of these three IL-12 family cytokines and suggest that IL-12 is the best candidate for treating HCC.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Interleucina-12/imunologia , Interleucina-12/uso terapêutico , Interleucina-17/uso terapêutico , Interleucina-23/uso terapêutico , Neoplasias Hepáticas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/patologia , Contagem de Células , Dependovirus/genética , Ensaios de Seleção de Medicamentos Antitumorais , Vetores Genéticos/genética , Imuno-Histoquímica , Interleucina-12/farmacologia , Interleucina-17/imunologia , Interleucina-17/farmacologia , Interleucina-23/imunologia , Interleucina-23/farmacologia , Fígado/efeitos dos fármacos , Fígado/imunologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/imunologia , Metástase Neoplásica/prevenção & controle , Neovascularização Patológica/tratamento farmacológicoRESUMO
Using a hepatitis B virus (HBV) transgenic mouse model, we previously showed that a single dose of double-stranded adeno-associated virus (dsAAV) vector serotype 8 carrying a small hairpin RNA (shRNA) effectively reduces HBV replication and gene expression, but the effect gradually decreases with time. In this report, we compared the anti-HBV RNA interference (RNAi) effect of dsAAV8 with those of dsAAV7 and dsAAV9, two other hepatotropic AAV vectors, and examined whether the sequential use of these heterologous AAV vectors could prolong the anti-HBV effect. Our results showed that shRNA delivered by each of the three dsAAV vectors profoundly reduced the serum HBV titer and liver HBV mRNA and DNA levels in the transgenic mice for up to 22 weeks, with dsAAV8 having the greatest inhibitory effect, followed by dsAAV9 and dsAAV7. The potency of dsAAV8 correlated with the presence of higher levels of vector DNA and anti-HBV shRNA in the liver. An in vivo cross-administration experiment showed that preexisting anti-AAV8 antibody completely blocked the anti-HBV RNAi effect of dsAAV8, but had no effect on the potency of dsAAV7 and dsAAV9. Moreover, we demonstrated that a longer anti-HBV effect could be achieved by the sequential use of dsAAV8 and dsAAV9. These results indicate that effective and persistent HBV suppression might be achieved by a combination of the power of RNAi silencing effect and multiple treatments with different AAV serotypes.Molecular Therapy (2009) 17 2, 352-359 doi:10.1038/mt.2008.245.