RESUMEN
Steroidogenic acute regulatory protein (StAR), a mitochondrial cholesterol delivery protein, plays a beneficial role in hyperlipidemia, NAFLD, and endothelial inflammation. Elevated circulating fatty acids and low grade inflammation are known as key risk factors of insulin resistance and type 2 diabetes. In the present study, C57BL/6J mice were fed with HFD and infected with recombinant adenovirus expressing StAR by tail-vein injection. Intraperitoneal glucose/insulin tolerance test was performed to assess the insulin sensitivity. Morphological analysis and intramuscular lipid determination were used to illustrate the adipose hypertrophy and ectopic fat accumulation in skeletal muscle. The levels of inflammatory factor and nitric oxide were determined by ELISA and classic Griess reagent methods, respectively. The fatty acids composition was analysis using gas chromatography-mass spectrometry (GC-MS). The expression of genes associated with inflammation and insulin resistance were determined by Western blotting and qPCR to elucidate the underlying mechanism. We demonstrated that StAR overexpression ameliorated insulin resistance and systemic inflammatory response with the reduction of adipose hypertrophy and intramuscular lipid in HFD-fed mice. In addition, StAR overexpression increased serum unsaturated fatty acids (UFAs) and PPARγ expression in muscle and adipose tissue of obese mice. In conclusion, StAR may activate PPARγ by increasing UFAs, which leads to a protective role in systemic inflammation and insulin resistance in obese mice. J. Cell. Biochem. 118: 3932-3942, 2017. © 2017 Wiley Periodicals, Inc.
Asunto(s)
Grasas de la Dieta/efectos adversos , Resistencia a la Insulina , Obesidad/metabolismo , Fosfoproteínas/biosíntesis , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Grasas de la Dieta/farmacología , Ácidos Grasos Insaturados/sangre , Inflamación , Masculino , Ratones , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Ácido Nítrico/sangre , Obesidad/inducido químicamente , Obesidad/genética , Obesidad/patología , PPAR gamma/genética , PPAR gamma/metabolismo , Fosfoproteínas/genéticaRESUMEN
Non-alcoholic fatty liver disease (NAFLD) covers a wide spectrum of liver pathology. Intracellular lipid accumulation is the first step in the development and progression of NAFLD. Steroidogenic acute regulatory protein (StAR) plays an important role in the synthesis of bile acid and intracellular lipid homeostasis and cholesterol metabolism. We hypothesize that StAR is involved in non-alcoholic fatty liver disease (NAFLD) pathogenesis. The hypothesis was identified using free fatty acid (FFA)-overloaded NAFLD in vitro model and high-fat diet (HFD)-induced NAFLD mouse model transfected by recombinant adenovirus encoding StAR (StAR). StAR expression was also examined in pathology samples of patients with fatty liver by immunohistochemical staining. We found that the expression level of StAR was reduced in the livers obtained from fatty liver patients and NAFLD mice. Additionally, StAR overexpression decreased the levels of hepatic lipids and maintained the hepatic glucose homeostasis due to the activation of farnesoid x receptor (FXR). StAR overexpression attenuated the impairment of insulin signaling in fatty liver. This protective role of StAR was owing to a reduction of intracellular diacylglycerol levels and the phosphorylation of PKCε. Furthermore, FXR inactivation reversed the observed beneficial effects of StAR. The present study revealed that StAR overexpression can reduce hepatic lipid accumulation, regulate glucose metabolism and attenuate insulin resistance through a mechanism involving the activation of FXR. Our study suggests that StAR may be a potential therapeutic target for NAFLD.
Asunto(s)
Regulación de la Expresión Génica , Metabolismo de los Lípidos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Fosfoproteínas/metabolismo , Animales , Diglicéridos/genética , Diglicéridos/metabolismo , Femenino , Humanos , Resistencia a la Insulina , Masculino , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Enfermedad del Hígado Graso no Alcohólico/terapia , Fosfoproteínas/genéticaRESUMEN
Hydroxysteroid sulfotransferase 2B1b (SULT2B1b) sulfates cholesterol and oxysterols. Hepatic oval cells (HOCs), thought to be progenitor cells, can be triggered in chemically injured livers. The present study focused on the role of SULT2B1b in HOC proliferation after liver injury. Our experiments revealed that the expression of SULT2B1b was increased dramatically in a chemical-induced liver injury model, mainly in HOCs. Upon challenge with a hepatotoxic diet containing 0.1 % 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), SULT2B1-/- mice presented alleviated liver injury and less HOC proliferation compared with wild-type (WT) mice, and these findings were verified by serum analysis, histopathology, immunofluorescence staining, RNA-seq, and Western blotting. HOCs derived from SULT2B1-/- mice showed lower proliferative capability than those from WT mice. SULT2B1b overexpression promoted growth of the WB-F344 hepatic oval cell line, whereas SULT2B1b knockdown inhibited growth of these cells. The IL-6/STAT3 signaling pathway also was promoted by SULT2B1b. Liquid chromatography and mass spectrometry indicated that the levels of 22-hydroxycholesterol, 25-hydroxycholesterol, and 24,25-epoxycholesterol were higher in the DDC-injured livers of SULT2B1-/- mice than in livers of WT mice. The above oxysterols are physiological ligands of liver X receptors (LXRs), and SULT2B1b suppressed oxysterol-induced LXR activation. Additional in vivo and in vitro experiments demonstrated that LXR activation could inhibit HOC proliferation and the IL-6/STAT3 signaling pathway, and these effects could be reversed by SULT2B1b. Our data indicate that upregulation of SULT2B1b might promote HOC proliferation and aggravate liver injury via the suppression of oxysterol-induced LXR activation in chemically induced mouse liver injury.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Modelos Animales de Enfermedad , Receptores X del Hígado/agonistas , Hígado/efectos de los fármacos , Oxiesteroles/farmacología , Sulfotransferasas/metabolismo , Animales , Biomarcadores/sangre , Biomarcadores/metabolismo , Carcinógenos/toxicidad , Línea Celular , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Progresión de la Enfermedad , Femenino , Hígado/metabolismo , Hígado/patología , Hígado/fisiopatología , Neoplasias Hepáticas/etiología , Receptores X del Hígado/antagonistas & inhibidores , Receptores X del Hígado/genética , Receptores X del Hígado/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Oxiesteroles/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/patología , Piridinas/toxicidad , Interferencia de ARN , Sulfotransferasas/antagonistas & inhibidores , Sulfotransferasas/química , Sulfotransferasas/genéticaRESUMEN
Intracellular lipid accumulation, inflammatory responses, and subsequent apoptosis are the major pathogenic events of metabolic disorders, including atherosclerosis and nonalcoholic fatty liver diseases. Recently, a novel regulatory oxysterol, 5-cholesten-3b, 25-diol 3-sulfate (25HC3S), has been identified, and hydroxysterol sulfotransferase 2B1b (SULT2B1b) has been elucidated as the key enzyme for its biosynthesis from 25-hydroxycholesterol (25HC) via oxysterol sulfation. The product 25HC3S and the substrate 25HC have been shown to coordinately regulate lipid metabolism, inflammatory responses, and cell proliferation in vitro and in vivo. 25HC3S decreases levels of the nuclear liver oxysterol receptor (LXR) and sterol regulatory element-binding proteins (SREBPs), inhibits SREBP processing, subsequently downregulates key enzymes in lipid biosynthesis, decreases intracellular lipid levels in hepatocytes and THP-1-derived macrophages, prevents apoptosis, and promotes cell proliferation in liver tissues. Furthermore, 25HC3S increases nuclear PPARγ and cytosolic IκBα and decreases nuclear NF-κB levels and proinflammatory cytokine expression and secretion when cells are challenged with LPS and TNFα. In contrast to 25HC3S, 25HC, a known LXR ligand, increases nuclear LXR and decreases nuclear PPARs and cytosol IκBα levels. In this review, we summarize our recent findings, including the discovery of the regulatory oxysterol sulfate, its biosynthetic pathway, and its functional mechanism. We also propose that oxysterol sulfation functions as a regulatory signaling pathway.
Asunto(s)
Ésteres del Colesterol/metabolismo , Hidroxicolesteroles/metabolismo , Inflamación/metabolismo , Sulfatasas/metabolismo , Animales , Proliferación Celular , Humanos , Metabolismo de los Lípidos , Receptores X del Hígado , Receptores Nucleares Huérfanos/fisiología , Proteínas de Unión a los Elementos Reguladores de Esteroles/fisiologíaRESUMEN
Estrogens are involved in the complex regulation of cell proliferation and apoptosis of hormone sensitive tumors including breast and endometrial cancers. Sulfation is the main pathway for estrogen metabolism, which is believed to be involved in the inactivation of estrogens in target tissues. SULT1E1 and PAPSS (PAPSS1 and PAPSS2) are responsible for the estrogen sulfation by providing catalyzing enzyme and universal sulfate donor. The present study showed the expression patterns of SULT1E1 and PAPSS in the breast and endometrial tissues by tissue array analysis and the assessment of clinical samples. The estrogen sulfation enzymes were comparatively higher in the tumorous tissues than their adjacent normal tissues. SULT1E1 overexpression inhibited the tumorigenesis in subcutaneous xenograft model. By CCK-8 assay and flow cytometry assay, overexpression of SULT1E1 and PAPSS1 by adenovirus blocked the estrogen pro-proliferating effect and promoted cell apoptosis induced by H(2)O(2) in MCF-7 cells. By real-time reverse transcription-polymerase chain reaction and western-blot assays, overexpression of SULT1E1 and PAPSS1 suppressed cell growth and triggered apoptosis by downregulating the levels of c-myc, cyclin D1 and bcl-2, meanwhile, upregulating bax expression. In conclusion, the discrepancies in expressions of SULT1E1 and PAPSS between breast and endometrial tumorous tissues and their adjacent normal tissues were prominent. Overexpression of SULT1E1 and PAPSS1 retarded MCF-7 cells growth in vivo and in vitro by arresting cell cycles and inducing apoptosis. Thus, targeting SULT1E1 and PAPSS expressions might be an important approach for estrogen-dependent cancers.
Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias Endometriales/metabolismo , Estrógenos/metabolismo , Complejos Multienzimáticos/metabolismo , Neoplasias Hormono-Dependientes/metabolismo , Sulfato Adenililtransferasa/metabolismo , Sulfotransferasas/metabolismo , Animales , Apoptosis , Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica/genética , Ciclina D1/biosíntesis , Neoplasias Endometriales/patología , Endometrio/metabolismo , Femenino , Humanos , Peróxido de Hidrógeno/farmacología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Complejos Multienzimáticos/genética , Trasplante de Neoplasias , Neoplasias Hormono-Dependientes/patología , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-myc/biosíntesis , Sulfato Adenililtransferasa/genética , Sulfotransferasas/genética , Análisis de Matrices Tisulares , Trasplante Heterólogo , Proteína X Asociada a bcl-2/biosíntesisRESUMEN
BACKGROUND: Endothelial dysfunction is a well documented evidence for the onset of atherosclerosis and other cardiovascular diseases. Lipids disorder is among the main risk factors for endothelial dysfunction in these diseases. Steroidogenic acute regulatory protein (StAR), one of the cholesterol transporters, plays an important role in the maintenance of intracellular lipid homeostasis. However, the effect of StAR on endothelial dysfunction is not well understood. Palmitic acid (PA) has been shown to decrease eNOS activity and induce inflammation, both are the causes of endothelial dysfunction, in an endothelial cell culture model. METHODS: StAR gene was introduced into primary rat aortic endothelial cells by adenovirus infection. Real-time PCR and Western blotting were performed to determine the relative genes and proteins expression level to elucidate the underlying mechanism. The free fatty acid and cholesterol quantification kits were used to detect total cellular free fatty acid and cholesterol. The levels of inflammatory factors and nitric oxide were determined by ELISA and classic Griess reagent methods respectively. RESULTS: We successfully overexpressed StAR in primary rat aortic endothelial cells. Following StAR overexpression, mRNA levels of IL-1ß, TNFα, IL6 and VCAM-1 and protein levels of IL-1ß, , TNFα and IL-6 in culture supernatant were significantly decreased, which duing to blocke NFκB nuclear translocation and activation. Moreover, StAR overexpression attenuated the PA-induced reduction of nitric oxide bioavailability by protecting the bioactivity of pAkt/peNOS/NO pathway. Furthermore, the key genes involved in lipid metabolism were greatly reduced following StAR overexpression. In order to investigate the underlying mechanism, cerulenin and lovastatin, the inhibitor of fatty acid and cholesterol synthase, were added prior to PA treatment. The results showed that both cerulenin and lovastatin had a similar effect as StAR overexpression. On the other hand, the role of StAR was inhibited when siRNA was introduced to reduce StAR expression. CONCLUSIONS: Our results showed that StAR attenuated lipid synthesis and uptake as well as PA-induced inflammation and reduction in NO bioavailability in aortic endothelial cells. StAR can ameliorate endothelial dysfunction induced by PA via reducing the intracellular lipid levels.
Asunto(s)
Aorta Torácica/metabolismo , Células Endoteliales/metabolismo , Inflamación/metabolismo , Óxido Nítrico/metabolismo , Ácido Palmítico/metabolismo , Fosfoproteínas/metabolismo , Animales , Antiinflamatorios/farmacología , Aorta Torácica/efectos de los fármacos , Aorta Torácica/inmunología , Disponibilidad Biológica , Western Blotting , Células Cultivadas , Cerulenina/farmacología , Colesterol/metabolismo , Células Endoteliales/efectos de los fármacos , Células Endoteliales/inmunología , Ensayo de Inmunoadsorción Enzimática , Inhibidores de la Síntesis de Ácidos Grasos/farmacología , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inflamación/genética , Inflamación/inmunología , Inflamación/prevención & control , Mediadores de Inflamación/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Lovastatina/farmacología , Masculino , FN-kappa B/metabolismo , Fosfoproteínas/genética , Interferencia de ARN , ARN Mensajero/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Tiempo , Transfección , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The relationship between the checkpoint kinase Chk1 and the STAT3 pathway was examined in multiple myeloma cells. Gene expression profiling of U266 cells exposed to low (nmol/L) Chk1 inhibitor [PF-477736 (PF)] concentrations revealed STAT3 pathway-related gene downregulation (e.g., BCL-XL, MCL-1, c-Myc), findings confirmed by RT-PCR. This was associated with marked inhibition of STAT3 Tyr705 (but not Ser727) phosphorylation, dimerization, nuclear localization, DNA binding, STAT3 promoter activity by chromatin immunoprecipitation assay, and downregulation of STAT-3-dependent proteins. Similar findings were obtained in other multiple myeloma cells and with alternative Chk1 inhibitors (e.g., prexasertib, CEP3891). While PF did not reduce GP130 expression or modify SOCS or PRL-3 phosphorylation, the phosphatase inhibitor pervanadate antagonized PF-mediated Tyr705 dephosphorylation. Significantly, PF attenuated Chk1-mediated STAT3 phosphorylation in in vitro assays. Surface plasmon resonance analysis suggested Chk1/STAT3 interactions and PF reduced Chk1/STAT3 co-immunoprecipitation. Chk1 CRISPR knockout or short hairpin RNA knockdown cells also displayed STAT3 inactivation and STAT3-dependent protein downregulation. Constitutively active STAT3 diminished PF-mediated STAT3 inactivation and downregulate STAT3-dependent proteins while significantly reducing PF-induced DNA damage (γH2A.X formation) and apoptosis. Exposure of cells with low basal phospho-STAT3 expression to IL6 or human stromal cell conditioned medium activated STAT3, an event attenuated by Chk1 inhibitors. PF also inactivated STAT3 in primary human CD138+ multiple myeloma cells and tumors extracted from an NSG multiple myeloma xenograft model while inhibiting tumor growth. IMPLICATIONS: These findings identify a heretofore unrecognized link between the Chk1 and STAT3 pathways and suggest that Chk1 pathway inhibitors warrant attention as novel and potent candidate STAT3 antagonists in myeloma.
Asunto(s)
Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Mieloma Múltiple , Apoptosis , Línea Celular Tumoral , ADN/metabolismo , Humanos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Factor de Transcripción STAT3/metabolismoRESUMEN
Accumulated mast cells in atherosclerotic plaques secrete a high level of tryptase that may participate in the pathogenesis of atherosclerotic disease by diverse pathways. However, the role of tryptase in the lipid metabolism of macrophages remains to be defined. In the present study, we found that the addition of tryptase into THP-1-derived macrophages increased both intracellular lipid accumulation and total cholesterol level. Tryptase promoting foam cell formation was also observed by transmission electron microscope. These effects were resisted by APC366, a selective inhibitor of mast cell tryptase. Tryptase dramatically resisted 22RHC induced activation of LXRalpha protein expression, which can be reversed by SAM-11 (a PAR-2-specific neutralizing antibody) and reduced LXRalpha, ABCG1, ABCA1 and SREBP-1c mRNA levels and ABCG1 protein level, which were all blocked by APC366. PAR-2 agonist also redeemed 22RHC stimulation to activate LXRalpha, ABCG1 protein expression, and mRNA levels of LXRalpha and its target genes in both THP-1-derived macrophages and primary human monocyte-derived macrophages. In primary macrophages that were first transfected with PAR-2 siRNA and then treated with tryptase, both the ABCG1 protein level and mRNA levels of LXRalpha and ABCG1 were higher than those in the control siRNA-treated cells. Taken together, our data clarified the PAR-2 expression of human macrophages and suggested that tryptase might promote lipid accumulation in macrophages and foam cell formation by suppressing LXRalpha activation via PAR-2/LXRalpha/LXRalpha target genes signaling pathway. This investigation sheds a new light on the role of tryptase in foam cell formation and pathogenesis of atherosclerosis.
Asunto(s)
Células Espumosas/fisiología , Macrófagos/fisiología , Receptores Nucleares Huérfanos/metabolismo , Triptasas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Células Cultivadas , Células Espumosas/citología , Humanos , Metabolismo de los Lípidos , Lipoproteínas LDL/metabolismo , Receptores X del Hígado , Macrófagos/citología , Receptores Nucleares Huérfanos/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptor PAR-2/agonistas , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Transducción de Señal/fisiología , Fracciones Subcelulares/metabolismo , Triptasas/genéticaRESUMEN
Antigen-specific immunotherapy can be limited by induced tumor immunoediting (e.g., antigen loss) or through failure to recognize antigen-negative tumor clones. Melanoma differentiation-associated gene-7/IL24 (MDA-7/IL24) has profound tumor-specific cytotoxic effects in a broad spectrum of cancers. Here we report the enhanced therapeutic impact of genetically engineering mouse tumor-reactive or antigen-specific T cells to produce human MDA-7/IL24. While mock-transduced T cells only killed antigen-expressing tumor cells, MDA-7/IL24-producing T cells destroyed both antigen-positive and negative cancer targets. MDA-7/IL24-expressing T cells were superior to their mock-engineered counterparts in suppressing mouse prostate cancer and melanoma growth as well as metastasis. This enhanced antitumor potency correlated with increased tumor infiltration and expansion of antigen-specific T cells as well as induction of a Th1-skewed immunostimulatory tumor environment. MDA-7/IL24-potentiated T-cell expansion was dependent on T-cell-intrinsic STAT3 signaling. Finally, MDA-7/IL24-modified T-cell therapy significantly inhibited progression of spontaneous prostate cancers in Hi-Myc transgenic mice. Taken together, arming T cells with tumoricidal and immune-potentiating MDA-7/IL24 confers new capabilities of eradicating antigen-negative cancer cell clones and improving T-cell expansion within tumors. This promising approach may be used to optimize cellular immunotherapy for treating heterogeneous solid cancers and provides a mechanism for inhibiting tumor escape. SIGNIFICANCE: This research describes a novel strategy to overcome the antigenic heterogeneity of solid cancers and prevent tumor escape by engineering T lymphocytes to produce a broad-spectrum tumoricidal agent.
Asunto(s)
Traslado Adoptivo/métodos , Ingeniería Celular/métodos , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Interleucinas/metabolismo , Melanoma/terapia , Neoplasias de la Próstata/terapia , Neoplasias Cutáneas/terapia , Linfocitos T/metabolismo , Animales , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Células HEK293 , Humanos , Interleucinas/genética , Masculino , Melanoma/inmunología , Melanoma/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neoplasias de la Próstata/inmunología , Neoplasias de la Próstata/patología , Neoplasias Cutáneas/inmunología , Neoplasias Cutáneas/patología , Linfocitos T/inmunología , Transfección , Microambiente Tumoral/inmunologíaRESUMEN
Interactions between the inhibitor of apoptosis protein antagonist LCL161 and the histone deacetylase inhibitor panobinostat (LBH589) were examined in human multiple myeloma (MM) cells. LCL161 and panobinostat interacted synergistically to induce apoptosis in diverse MM cell lines, including those resistant to bortezomib (PS-R). Similar interactions were observed with other histone deacetylase inhibitors (MS-275) or inhibitors of apoptosis protein antagonists (birinapant). These events were associated with downregulation of the noncanonical (but not the canonical) NF-κB pathway and activation of the extrinsic, caspase-8-related apoptotic cascade. Coexposure of MM cells to LCL161/LBH589 induced TRAF3 upregulation and led to TRAF2 and NIK downregulation, diminished expression of BCL-XL, and induction of γH2A.X. Ectopic expression of TRAF2, NIK, or BCL-XL, or short hairpin RNA TRAF3 knock-down, significantly reduced LCL161/LBH589 lethality, as did ectopic expression of dominant-negative FADD. Stromal/microenvironmental factors failed to diminish LCL161/LBH589-induced cell death. The LCL161/LBH589 regimen significantly increased cell killing in primary CD138+ cells (N = 31) and was particularly effective in diminishing the primitive progenitor cell-enriched CD138-/19+/20+/27+ population (N = 23) but was nontoxic to normal CD34+ cells. Finally, combined LCL161/LBH589 treatment significantly increased survival compared with single-agent treatment in an immunocompetent 5TGM1 murine MM model. Together, these findings argue that LCL161 interacts synergistically with LBH589 in MM cells through a process involving inactivation of the noncanonical NF-κB pathway and activation of the extrinsic apoptotic pathway, upregulation of TRAF3, and downregulation of TRAF2/BCL-XL. Notably, this regimen overcomes various forms of resistance, is active against primary MM cells, and displays significant in vivo activity. This strategy warrants further consideration in MM.
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Inhibidores de Histona Desacetilasas , Mieloma Múltiple , Animales , Caspasa 8/genética , Línea Celular Tumoral , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Ratones , Mieloma Múltiple/tratamiento farmacológico , FN-kappa BRESUMEN
OBJECTIVE: To explore the effects of down-regulated tryptase expression in mast cells on the synthesis and release of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) of vascular endothelial cells. METHODS: Tryptase-siRNA (small-interfering RNA) vector was constructed to inhibit tryptase expression in P815 cells. The medium of P815 cells treated by the tryptase-siRNA (RNAi-P815 group) or pure vector (P815 group) was collected and used to culture bEnd.3 cells. The messenger RNAs (mRNAs) of IL-6 and TNF-alpha in bEnd.3 cells and their protein levels in the medium were measured by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: IL-6 and TNF-alpha mRNAs in bEnd.3 cells cultured in RNAi-P815-conditioned medium decreased significantly compared to those in P815-conditioned medium. Consistently, IL-6 and TNF-alpha protein levels in the medium of bEnd.3 of RNAi-P815 group were lower than those of P815 group. CONCLUSION: Reduced tryptase expression significantly inhibited the synthesis and release of IL-6 and TNF-alpha in vascular endothelial cells. RNA interference targeting tryptase expression may be a new anti-inflammatory strategy for vascular diseases.
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Regulación hacia Abajo/genética , Células Endoteliales/enzimología , Células Endoteliales/metabolismo , Interleucina-6/metabolismo , ARN Interferente Pequeño/genética , Triptasas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Línea Celular , Medios de Cultivo Condicionados , Regulación Enzimológica de la Expresión Génica , Interleucina-6/genética , Ratones , ARN Mensajero/genética , Transgenes , Triptasas/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
BACKGROUND: Oxysterol sulfation plays a fundamental role in the regulation of many biological events. Its products, 25-hydroxycholesterol 3-sulfate (25HC3S) and 25-hydroxycholesterol 3, 25-disulfate (25HCDS), have been demonstrated to be potent regulators of lipid metabolism, inflammatory response, cell apoptosis, and cell survival. In the present study, we tested these products' potential to treat LPS-induced acute liver failure in a mouse model. METHODS: Acute liver failure mouse model was established by intravenous injection with LPS. The injured liver function was treated with intraperitoneal administration of 25HC, 25HC3S or 25HCDS. Serum enzymatic activities were determined in our clinic laboratory. ELISA assays were used to detect pro-inflammatory factor levels in sera. Western blot, Real-time Quantitative PCR and RT2 Profiler PCR Array analysis were used to determine levels of gene expression. RESULTS: Administration of 25HC3S/25HCDS decreased serum liver-impaired markers; suppressed secretion of pro-inflammatory factors; alleviated liver, lung, and kidney injury; and subsequently increased the survival rate in the LPS-induced mouse model. These effects resulted from the inhibition of the expression of genes involved in the pro-inflammatory response and apoptosis and the simultaneous induction of the expression of genes involved in cell survival. Compared to 25HC, 25HC3S and 25HCDS exhibited significantly stronger effects in these activities, indicating that the cholesterol metabolites play an important role in the inflammatory response, cell apoptosis, and cell survival in vivo. CONCLUSIONS: 25HC3S/25HCDS has potential to serve as novel biomedicines in the therapy of acute liver failure and acute multiple organ failure.
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Enfermedad Hepática Inducida por Sustancias y Drogas/mortalidad , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Ésteres del Colesterol/uso terapéutico , Colesterol/metabolismo , Hidroxicolesteroles/uso terapéutico , Lipopolisacáridos , Animales , Biomarcadores/sangre , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Citocinas/sangre , Regulación de la Expresión Génica/efectos de los fármacos , Hígado/patología , Pruebas de Función Hepática , Masculino , Ratones , Ratones Endogámicos C57BL , Tasa de SupervivenciaRESUMEN
Oxysterol sulfation plays an important role in regulation of lipid metabolism and inflammatory responses. In the present study, we report the discovery of a novel regulatory sulfated oxysterol in nuclei of primary rat hepatocytes after overexpression of the gene encoding mitochondrial cholesterol delivery protein (StarD1). Forty-eight hours after infection of the hepatocytes with recombinant StarD1 adenovirus, a water-soluble oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Tandem mass spectrometry analysis identified the oxysterol as 5-cholesten-3ß, 25-diol, disulfate (25HCDS), and confirmed the structure by comparing with a chemically synthesized compound. Administration of 25HCDS to human THP-1-derived macrophages or HepG2 cells significantly inhibited cholesterol synthesis and markedly decreased lipid levels in vivo in NAFLD mouse models. RT-PCR showed that 25HCDS significantly decreased SREBP-1/2 activities by suppressing expression of their responding genes, including ACC, FAS, and HMG-CoA reductase. Analysis of lipid profiles in the liver tissues showed that administration of 25HCDS significantly decreased cholesterol, free fatty acids, and triglycerides by 30, 25, and 20%, respectively. The results suggest that 25HCDS inhibits lipid biosynthesis via blocking SREBP signaling. We conclude that 25HCDS is a potent regulator of lipid metabolism and propose its biosynthetic pathway.
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Ésteres del Colesterol/análisis , Colesterol/metabolismo , Hidroxicolesteroles/análisis , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Adenoviridae/metabolismo , Animales , Células Cultivadas , Colesterol/análisis , Colesterol/biosíntesis , Ésteres del Colesterol/síntesis química , Ésteres del Colesterol/farmacología , Modelos Animales de Enfermedad , Ácido Graso Sintasas/genética , Ácido Graso Sintasas/metabolismo , Femenino , Células Hep G2 , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Hidroxicolesteroles/síntesis química , Hidroxicolesteroles/farmacología , Hidroximetilglutaril-CoA Reductasas/genética , Hidroximetilglutaril-CoA Reductasas/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Ratas , Transducción de Señal/efectos de los fármacos , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
Tryptase, the most abundant mast cell (MC) granule protein, plays an important role in atherosclerosis plaque development. To test the hypothesis that tryptase participates directly in atherosclerosis plaque haemorrhage, the gene sequence and siRNA for tryptase were cloned into a lentivirus carrier and atherosclerosis plaque haemorrhage models in ApoE-/- mice were constructed. After a cuffing-cervical artery operation, the mice were randomly divided into 6 groups. Hematoxylin and eosin(HE) staining showed that the cervical artery plaque area was much larger in the tryptase overexpression group compared to the other groups, and there was greater artery stenosis. The artery stenosis from the cuff-side in all groups was more than 90%, except the siRNA group. Tryptase promotes plaque haemorrhage distinctively because 50% of the mice in the tryptase overexpression group had plaque haemorrhage, while only 10% in the siRNA group did. The immunohistochemistry of the cervical artery plaque showed that plasminogen activator inhibitor-1 (PAI-1) expression was the lowest while tissue plasminogen activator (tPA), CD31, CD34 and VEGF was the highest in the tryptase overexpression groups. This observation was completely contrary to what was observed in the siRNA group. Tryptase promoted bEnd.3 cell growth, migration and capillary-like tube formation, which suggests that tryptase can promote microvessel angiogenesis. PAI-1 expression was inhibited, while tPA expression was increased by tryptase in bEnd.3 cells. Our in vivo and in vitro studies suggest that trypase can promote atherosclerotic plaque haemorrhage by promoting angiogenesis and regulating the balance of PAI-1 and tPA. Thus, regulating tryptase expression in MCs may provide a potential target for atherosclerosis treatment.
Asunto(s)
Apolipoproteínas E/genética , Hemorragia/enzimología , Placa Aterosclerótica/enzimología , Triptasas/fisiología , Animales , Antígenos CD34/metabolismo , Apolipoproteínas E/deficiencia , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Vectores Genéticos , Lentivirus/genética , Lípidos/sangre , Masculino , Ratones , Ratones Noqueados , Neovascularización Fisiológica , Placa Aterosclerótica/sangre , Placa Aterosclerótica/patología , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , ARN Interferente Pequeño/genética , Serpina E2/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Hydroxysteroid sulfotransferase 2B1b (SULT2B1b) is highly selective for the addition of sulfate groups to 3ß-hydroxysteroids. Although previous reports have suggested that SULT2B1b is correlated with cell proliferation of hepatocytes, the relationship between SULT2B1b and the malignant phenotype of hepatocarcinoma cells was not clear. In the present study, we found that SULT2B1 was comparatively higher in the human hepatocarcinoma tumorous tissues than their adjacent tissues. Besides, SULT2B1b overexpression promoted the growth of the mouse hepatocarcinoma cell line Hepa1-6, while Lentivirus-mediated SULT2B1b interference inhibited growth as assessed by the CCK-8 assay. Likewise, inhibition of SULT2B1b expression induced cell-cycle arrest and apoptosis in Hepa1-6 cells by upregulating the expression of FAS, downregulating the expression of cyclinB1, BCL2 and MYC in vitro and in vivo at both the transcript and protein levels. Knock-down of SULT2B1b expression significantly suppressed tumor growth in nude mouse xenografts. Moreover, proliferation rates and SULT2B1b expression were highly correlated in the human hepatocarcinoma cell lines Huh-7, Hep3B, SMMC-7721 and BEL-7402 cells. Knock-down of SULT2B1b inhibited cell growth and cyclinB1 levels in human hepatocarcinoma cells and suppressed xenograft growth in vivo. In conclusion, SULT2B1b expression promotes proliferation of hepatocellular carcinoma cells in vitro and in vivo, which may contribute to the progression of HCC.
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Carcinoma Hepatocelular/enzimología , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Hepáticas/enzimología , Sulfotransferasas/metabolismo , Análisis de Varianza , Animales , Apoptosis/fisiología , Western Blotting , Puntos de Control del Ciclo Celular/fisiología , Línea Celular Tumoral , Proliferación Celular , Cartilla de ADN/genética , Citometría de Flujo , Humanos , Ratones , Microscopía Fluorescente , Interferencia de ARN , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfotransferasas/antagonistas & inhibidoresRESUMEN
Epithelial ovarian cancer is one of the most malignant cancers in women and resistant to chemotherapy is the major obstacle for the five-year survival rate. Cisplatin is one of the effective anticancer drug used in the ovarian cancer. To find a good strategy to cure the tumors which is resistant to cisplatin, the cisplatin-resistant 3SKOV3 cells were selected from SKOV-3 ovarian cancer cells. Furthermore, the isolated mesenchymal stem cells were infused systemically to try to cure the transplanted tumor induced by 3SKOV3 cells in nude mice. The morphology and cell membrane CD44 expression were investigated by microscope and flow cytometry. The biological behaviors of resistant 3SKOV3 and its parental SKOV3 cells, including proliferation, adhesion, and cell cycle were determined by CCK8, absorbance assay and FCM methods. The transplanted tumors were set up in nude mice with 3SKOV3 cells injection. The growth rate of transplanted tumors was detected following with MSCs injection. The 3SKOV3 cells have different morphologic manifestation and expressed high level of CD44 molecule. At the same time, 3SKOV3 cells have less adhesion ability and less S-phase ratio. The isolated MSCs from bone marrow could inhibit the growth of transplanted tumor via systemic injection. The cisplatin-resistant 3SKOV3 cells have the different biological behaviors as its parental SKOV3 cells. The present study indicated that systemic MSCs have the therapeutic role on ovarian cancer. However, further investigations are in progress to elucidate the underlying mechanism.
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Proliferación Celular , Resistencia a Antineoplásicos , Trasplante de Células Madre Mesenquimatosas , Neoplasias Ováricas/terapia , Animales , Antineoplásicos , Adhesión Celular , Ciclo Celular , Línea Celular Tumoral , Cisplatino , Femenino , Humanos , Receptores de Hialuranos/metabolismo , Masculino , Ratones , Ratones Desnudos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Ratas Sprague-Dawley , Factores de Tiempo , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Estrogen sulfotransferase (SULT1E1) is a phase II drug-metabolizing enzyme known to catalyze sulfoconjugation of estrogens. 17ß-estradiol (E2) plays a pivotal role in attenuating endothelial dysfunction. E2 can be further sulfated to estradiol sulfate (E2S) using SULT1E1. To date, there are no reports of expression and function of SULT1E1 in the endothelium. We identified that SULT1E1 is highly expressed in human umbilical vein endothelial cells (HUVECs) using immunofluorescence microscopy and Western immunoblot analyses. A synthesized siRNA targeting SULT1E1 was used to successfully suppress SULT1E1 expression and inhibit estrogen sulfation in HUVECs. This led to functional depletion, as confirmed by a SULT1E1 enzyme activity assay in vitro and by an in vivo estrogen sulfation assay. Knock-down of SULT1E1 in HUVECs resulted in regulation of genes involved in inflammation and lipid metabolism. Interestingly, this regulation was attenuated by PPARγ siRNA and by exposure to the PPARγ antagonist GW9662. Compared with cell response in the absence of estrogen, the effects of SULT1E1 interference on the inflammatory response and lipid metabolism related genes in the presence of 80nM estrogen were completely opposite. When exogenous estrogen was applied, cell responses depended on the ratio of E2 to E2S, due to the activity of SULT1E1, and the different regulation of these processes. It is suggested that E2 sulfation catalyzed by SULT1E1 plays an important role in modulating endothelial cell function.
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Células Endoteliales/metabolismo , Metabolismo de los Lípidos , PPAR gamma/fisiología , Sulfotransferasas/fisiología , Estradiol/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Fase II de la Desintoxicación Metabólica , PPAR gamma/metabolismo , Interferencia de ARN , Sulfotransferasas/metabolismoRESUMEN
BACKGROUND: Epithelial ovarian cancer is one of the most malignant cancers in women because metastasis occurs in the most of patients by the time of diagnosis. Cancer cells have strong capacity to form angiogenesis or vasculogenic mimicry, which plays the major role in its malignant phenotype. Vasculogenic mimicry might contribute to the failure of the angiogenesis-targeted therapy strategies. Under the microenvironment of the tumor, hypoxia is the most common phenomena because of the vast energy and oxygen consuming. In the present study, the endothelial-like cells induced by hypoxia from SKOV-3 and ES-2 ovarian cancer cells were harvested to investigate the changes in their biological behaviors. METHODS: The endothelial-like cells from SKOV-3 and ES-2 cells were harvested by laser capture microdissection. The biological behaviors of the endothelial-like cells, including proliferation, cell cycle, apoptosis, invasion and telomerase activity were determined by MTT, FCM, Transwell chamber and TRAP-ELISA methods. HIF-1α is the most important factor for the behavior changes under hypoxic condition. Some other genes relative to biological behaviors are also changes following the changes of HIF-1α. In order to elucidate the underlying mechanisms for these changes by hypoxia, the relative genes expressions including HIF-1α, CyclinD1, Flk-1, VEGF, p53 and V-src were determined by real-time PCR. RESULTS: SKOV-3 and ES-2 cells were resistant to hypoxia by adoption of proliferation, apoptosis, differentiation and invasion. Combined with other studies, the more poorly cancer cells differentiate, the more strongly cells are resistant to hypoxia, the more possible to form vasculogenic mimicry. The changes in the expression of HIF-1α, and HIF-1α-dependent VEGF, Flk-1, Cyclin D1, and HIF-1α-independent p53 have been involved in this process. CONCLUSIONS: HIF-1α took an important role in the behavioral changes of SKOV-3 and ES-2 cells by hypoxia. At the same time, other mechanisms were also involved in this process.
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Apoptosis , Movimiento Celular , Proliferación Celular , Células Endoteliales/patología , Células Epiteliales/patología , Neoplasias Ováricas/patología , Oxígeno/metabolismo , Apoptosis/genética , Ciclo Celular , Diferenciación Celular , Hipoxia de la Célula , Línea Celular Tumoral , Movimiento Celular/genética , Forma de la Célula , Ciclina D1/genética , Células Endoteliales/metabolismo , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica , Genes src , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Rayos Láser , Microdisección/instrumentación , Invasividad Neoplásica , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Telomerasa/metabolismo , Factores de Tiempo , Proteína p53 Supresora de Tumor/genética , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
OBJECTIVE: To determine the effect of steroidogenic acute regulatory protein (StAR) overexpression on the levels of adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) in an endothelial cell line (bEnd.3). METHODS: The StAR gene was induced in bEnd.3 cells with adenovirus infection. The infection efficiency was detected by fluorescence activated cell sorter (FACS) and fluorescence microscopy. The expressions of StAR gene and protein levels were detected by real-time polymerase chain reaction (PCR) and Western blot. The gene and protein levels of ABCA1 and ABCG1 were detected by real-time PCR and Western blot after StAR overexpression. RESULTS: The result shows that StAR was successfully overexpressed in bEnd.3 cells by adenovirus infection. The mRNA and protein expressions of ABCA1 and ABCG1 were greatly increased by StAR overexpression in bEnd.3 cells. CONCLUSION: Overexpression of StAR increases ABCA1 and ABCG1 expressions in endothelial cells.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Células Endoteliales/metabolismo , Microcirculación/fisiología , Fosfoproteínas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Animales , Línea Celular , Ratones , Fosfoproteínas/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Steroidogenic acute regulatory protein (StAR) plays an important role in the maintenance of intracellular lipid homeostasis. Macrophages are the key cellular player in the pathophysiology of atherosclerosis. Imbalance of macrophage lipid homeostasis causes cellular apoptosis, which is the key process in the initiation of atherosclerosis. The present study has investigated the effects of StAR in the apoptotic process of human THP-1 derived macrophages induced by serum withdrawal or Ox-LDL. Overexpression of StAR significantly decreased the number of apoptotic macrophages by decreasing the expression of pro-apoptotic genes Caspase-3 and Bax mRNA and protein levels, as well as through increasing expression of anti-apoptotic gene Bcl-2 mRNA and protein levels in the absence and presence of Ox-LDL. The results indicate that StAR plays an important role in macrophage and foam cell apoptotic processing, which may provide a potential method for preventing atherosclerosis.