RESUMO
Acute myeloid leukemia (AML) is a clinically and molecularly heterogeneous hematopoietic disorder. To effectively eradicate AML, it is urgent to develop new therapeutic approaches and identify novel molecular targets. In silico analysis indicated that the expression of cysteine-rich intestinal protein 1 (CRIP1) was significantly elevated in AML cells and correlated with worse overall survival of the AML patients. However, its specific roles in AML remain elusive. Here we demonstrated that CRIP1 acted as a key oncogene to support AML cell survival and migration. Using a loss-of-function analysis, we found that CRIP1 silencing in U937 and THP1 cells by lentivirus-mediated shRNAs resulted in a decrease in cell growth, migration and colony formation, and an increase in chemosensitivity to Ara-C. CRIP1 silencing induced cell apoptosis and G1/S transition arrest. Mechanically, CRIP1 silencing caused inactivation of Wnt/ß-catenin pathway through upregulating axin1 protein. The Wnt/ß-catenin agonist SKL2001 markedly rescued the cell growth and migration defect induced by CRIP1 silencing. Our findings reveals that CRIP1 may contribute to AML-M5 pathogenesis and represent a novel target for AML-M5 treatment.
Assuntos
Leucemia Mieloide Aguda , beta Catenina , Humanos , Linhagem Celular Tumoral , beta Catenina/genética , beta Catenina/metabolismo , Via de Sinalização Wnt , Leucemia Mieloide Aguda/tratamento farmacológico , Proliferação de Células , Apoptose , Proteínas de Transporte , Proteínas com Domínio LIMRESUMO
Context: Tamoxifen (TAM) ameliorates D-galactosamine/lipopolysaccharide (Gal/LPS)-induced acute liver failure (ALF) through its antioxidative effect; thus, this study was designed to determine whether the effectiveness of TAM is related to nuclear factor-κB (NF-κB) reactivation. Materials and methods: Experimental mice were injected with TAM once daily for 3 consecutive days intraperitoneally (i.p). Twelve hours after pretreatment, Gal/LPS was given to mice (i.p) for ALF induction. In the positive control group, N-acetylcysteine (NAC) was administered immediately after ALF establishment. Except for survival observation, other animals were sacrificed 7 h after Gal/LPS treatment. Survival and hepatic failure were evaluated. For the oxidation assessment, the reduced/oxidized glutathione (GSH/GSSG) ratio and hepatic superoxide dismutase (SOD) activity were analyzed using both colorimetry and Western blotting. Lastly, hepatic NF-κB activation was measured through Western blot analysis of p65 and IκBα. Results: The results indicated that pretreatment with TAM dramatically attenuated Gal/LPS-induced ALF, as demonstrated by improved survival (70%), decreased transaminase levels, and reversed histopathological manifestation. In addition, the hepatic GSH/GSSG ratio and SOD activity were decreased in the ALF model. However, to some degree, TAM and NAC effectively prevented this undesirable phenomenon in contrast to the ALF model. Western blotting revealed that compared with mice in the ALF model group, mice treated with TAM or NAC showed reactivation of hepatic NF-κB. Conclusions: Taking the results together with those of other studies, we conclude that TAM may attenuate Gal/LPS-induced ALF by antagonizing oxidative stress through NF-κB reactivation.
Assuntos
Galactosamina/toxicidade , Lipopolissacarídeos/toxicidade , Falência Hepática Aguda , Fígado/metabolismo , NF-kappa B/metabolismo , Tamoxifeno/farmacologia , Animais , Modelos Animais de Doenças , Fígado/patologia , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/tratamento farmacológico , Falência Hepática Aguda/metabolismo , Falência Hepática Aguda/patologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Oxidative stress is considered one of the major mechanisms underlying lipopolysaccharide (LPS)-induced acute liver failure (ALF). Tamoxifen has been reported to ameliorate LPS-induced ALF via the induction of monocyte to macrophage differentiation-associated 2 (Mmd-2). Whether antioxidant effects are involved remains unknown. Mice were given tamoxifen (TAM) once a day for 3 days. Twelve hours later, d-galactosamine (GaIN) and LPS were injected intraperitoneally to induce ALF. N-Acetylcysteine (NAC) was administered immediately after ALF induction as a positive control. The results showed that serum transaminases increased and hepatic antioxidants decreased significantly in the model group. ALF was alleviated markedly by TAM or NAC treatment. This demonstrated that ALF may be associated with excessive oxidative stress caused by decreased expression of antioxidant enzymes. Both TAM and NAC increased the levels and activity of these antioxidant enzymes significantly (p < 0.05). Hepatic Mmd-2 expression was downregulated in the control group while remaining stable or exhibiting elevated levels in the TAM or NAC groups. The results indicate that TAM may protect mice from GaIN/LPS-induced ALF through increased activity of antioxidant enzymes and upregulation of Mmd-2 expression.
Assuntos
Antígenos de Diferenciação/biossíntese , Antioxidantes/uso terapêutico , Falência Hepática Aguda/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Tamoxifeno/uso terapêutico , Acetilcisteína/administração & dosagem , Alanina Transaminase/sangue , Animais , Aspartato Aminotransferases/sangue , Catalase/metabolismo , Galactosamina/toxicidade , Glutationa Peroxidase/metabolismo , Lipopolissacarídeos/toxicidade , Falência Hepática Aguda/induzido quimicamente , Camundongos , Camundongos Endogâmicos BALB C , Superóxido Dismutase/metabolismo , Regulação para CimaRESUMO
Bacterial lipopolysaccharide (LPS)-induced acute liver failure (ALF) is a common severe clinical syndrome in intensive care unit. No other methods are available for its prevention apart from supportive treatment and liver transplantation. Tamoxifen (TAM) was reported to attenuate ALF induced by excessive acetaminophen, while its effect on LPS-induced ALF remained unknown. For this, in the present study, we comprehensively assessed whether TAM can attenuate ALF induced by LPS/galactosamine (GaIN). Mice were given TAM once a day for three times. Twelve hours after the last treatment, mice were given LPS/GaIN (intraperitoneally [i.p.]). Survival, plasma transaminases, and histopathology were examined. Serum TNF-α and IL-1ß were analyzed by ELISA. Hepatic apoptosis was analyzed by TUNEL and caspase-3 Western blotting, respectively. Compared to the model group, ALF induced by LPS/GaIN was alleviated remarkably following TAM administration, as evidenced by the improvement of survival (87.5% vs. 37.5%), hepatic swell, moderate transaminases, slightly increased serum TNF-α, IL-1ß (P < 0.05), and moderate histopathology. In respect of apoptosis, severe hepatocellular apoptosis was reduced notably by TAM treatment confirmed by less TUNEL-positive hepatocytes and decreased caspase-3 cleavage. The results demonstrated that TAM could attenuate LPS/GaIN-induced ALF effectively, probably due to hepatic inflammation and apoptosis antagonism. Furthermore, it was the first report about the effect of TAM on LPS/GaIN-induced ALF.
Assuntos
Apoptose/efeitos dos fármacos , Doença Hepática Induzida por Substâncias e Drogas/tratamento farmacológico , Falência Hepática Aguda/tratamento farmacológico , Fígado/efeitos dos fármacos , Tamoxifeno/uso terapêutico , Animais , Caspase 3/metabolismo , Galactosamina/imunologia , Humanos , Interleucina-1beta/sangue , Lipopolissacarídeos/imunologia , Fígado/imunologia , Fígado/patologia , Falência Hepática Aguda/induzido quimicamente , Camundongos , Camundongos Endogâmicos BALB C , Transaminases/sangue , Fator de Necrose Tumoral alfa/sangueRESUMO
We developed a novel trimeric sTNFRII fusion protein, named sTNFRII-gAD, which exhibited a higher in vitro antagonistic efficacy for TNFα in comparison with sTNFRII-Fc. This study aimed to investigate the arthritic protection of sTNFRII-gAD in a rat collagen-induced arthritis (CIA). The rats were injected intradermally with collagen type II at days 0 and 7. Three days after the second injection (day 10), the rats were intraperitoneally given sTNFRII-gAD or sTNFRII-Fc, or PBS. Effects of treatments were examined with respect of CIA incidence, severity and pathological changes. Serum TNFα, IL-17A and regulatory T cell (Treg) in periphery were determined at days 10 and 16, respectively. Our results showed that sTNFRIIgAD significantly reduced CIA incidence and severity (p < 0.05); meanwhile it led to a dramatic improvement in cartilage and bone damage. Moreover, the increase in serum anti-CII and IL-17A, and the reduction in Treg population were inhibited (p < 0.05) by sTNFRII-gAD or sTNFRII-Fc. Serum TNFα was found to be accumulated in the groups treated with sTNFRII-gAD or sTNFRII-Fc compared with the group treated with PBS (p < 0.05). It is noteworthy that sTNFRII-gAD displayed a better efficacy than sTNFRII-Fc in CIA incidence, pathological changes in cartilage and the elevation of anti-CII antibody, indicating that sTNFRII-gAD is potentially a more efficacious anti-TNFα agent for rheumatoid arthritis.
Assuntos
Artrite Experimental/tratamento farmacológico , Receptores Tipo II do Fator de Necrose Tumoral/uso terapêutico , Animais , Anticorpos/sangue , Artrite Experimental/sangue , Artrite Experimental/patologia , Cartilagem/patologia , Colágeno Tipo II/imunologia , Colágeno Tipo II/toxicidade , Avaliação Pré-Clínica de Medicamentos , Feminino , Injeções Intraperitoneais , Interleucina-17/sangue , Articulação do Joelho/patologia , Contagem de Linfócitos , Ratos , Ratos Wistar , Receptores Tipo II do Fator de Necrose Tumoral/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/uso terapêutico , Solubilidade , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Fator de Necrose Tumoral alfa/análiseRESUMO
CONTEXT: Tumor necrosis factor (TNF) α plays a key role in acute liver injury (ALI) induced by injection of d-galactosamine (D-Gal)/lipopolysaccharide (LPS). A novel recombinant trimeric sTNFRII, sTNFRII-gAD, has been tested to be effective in ameliorating ALI, when administered prior to ALI establishment. This study aims to validate the protective effect of sTNFRII-gAD when given after ALI setup and further explore its effect on hepatic apoptosis. MATERIALS AND METHODS: The treatments were carried out concomitantly with ALI establishment with clinically approved sTNFRII-Fc (the dimeric sTNFRII) as a positive control. Lethality, liver weight, and serum alanine transaminase were measured, and histological analysis was performed to evaluate liver injury induced by D-Gal/LPS. Additionally, Terminal-deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) and Western blot analyses of caspase-3 were used to examine hepatocellular apoptosis. RESULTS: sTNFRII-gAD given after D-Gal/LPS injection turned out to attenuate animal mortality significantly (p < 0.01), and had better hepatic protection. In terms of apoptosis, both sTNFRII-gAD and sTNFRII-Fc displayed noticeable improvement of apoptosis evidenced by dramatic decline of active caspase-3 compared to the control group. CONCLUSIONS: The results demonstrated that sTNFRII-gAD therapeutically diminished the lethality induced by D-Gal/LPS, possibly through blocking hepatic apoptosis initiated by TNFα. Of note, sTNFRII-gAD was superior to sTNFRII-Fc in some respects, indicating a promising alternative for the therapeutic strategy against the diseases associated with excessive TNFα.
Assuntos
Apoptose/efeitos dos fármacos , Falência Hepática Aguda/tratamento farmacológico , Receptores Tipo II do Fator de Necrose Tumoral/farmacologia , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Apoptose/imunologia , Caspase 3/imunologia , Feminino , Galactosamina/toxicidade , Lipopolissacarídeos/toxicidade , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Fator de Necrose Tumoral alfa/imunologiaRESUMO
TNF α plays a central role in the pathogenesis of inflammatory diseases such as rheumatoid arthritis and murine acute liver injury induced by injection of D-galactosamine and subsequent LPS. Recombinant Fc-fused soluble TNF receptor II (sTNFRII-Fc) has been used in the treatment of rheumatoid arthritis for a decade. We have recently constructed a novel fusion protein sTNFRII-gAD, which is composed of a soluble TNF receptor II and a globular domain of adiponectin. Utilizing the inclination of gAD to form homologous trimer naturally, we sought to explore TNFα antagonism of the novel trimerized sTNFRII-gAD and meantime compare TNFα-neutralizing effects in vitro and in vivo between sTNFRII-Fc and sTNFRII-gAD. Here, we evaluated the TNFα-antagonizing activity of sTNFRII-gAD with TNFα-induced L929 cytotoxicity assay. Furthermore, sTNFRII-Fc or sTNFRII-gAD was administered simultaneously with d-galactosamine 1h prior to LPS injection in the murine model of acute liver injury. Serum TNFα and TNFα-sTNFRII-gAD complex were measured by ELISA and the liver injury was assessed through alanine transaminase measurement and liver histological analysis. sTNFRII-gAD was shown to have higher TNFα-neutralizing activity than sTNFRII-Fc (p<0.05) in the L929 cytotoxicity assay. With a significant attenuation of murine lethality (p<0.05), sTNFRII-gAD showed more protective effects than sTNFRII-Fc in the murine model of acute liver injury. These results demonstrated that sTNFRII-gAD was more efficacious than sTNFRII-Fc as a TNFα antagonist, highlighting the potential of sTNFRII-gAD for the treatment of diseases associated with excessive TNFα.