RESUMEN
The CCND1 mRNA possesses at least two distinct lengths of the 3'-untranslated region (3'UTR), with the long isoform containing multiple AU-rich elements (AREs). The tandem zinc finger (TZF) domains of human ZFP36 family members have the capacity to bind to AREs and promote mRNA degradation. Our previous study demonstrated that mutations in the TZF domain of ZFP36L1 or ZFP36L2 increased the CCND1 expression. In this study, we investigated whether ZFP36L1 and ZFP36L2 could downregulate the expression of the long 3'UTR isoform of CCND1 mRNA in human colorectal cancer (CRC) cells. Firstly, the Gene Expression Profiling Interactive Analysis 2 database indicated downregulation of ZFP36 and ZFP36L1, while E2F1 and CCND1 were upregulated in human CRC tissues compared to normal colorectal tissues. Overexpression of ZFP36L1 and/or ZFP36L2 in T-REx-293, DLD-1, and HCT116 cells led to a decrease in the total CCND1, long isoform ratio of CCND1 mRNA, and E2F1 expression. Conversely, knockdown of ZFP36L1 and ZFP36L2 in HCT116 cells resulted in an increase in total CCND1, long isoform ratio of CCND1 mRNA, and E2F1 expression. Knockdown of E2F1 decreased CCND1 expression, indicating a potential role for E2F1 in regulating CCND1 expression at the transcriptional level. These findings suggest that ZFP36L1 and ZFP36L2 play a negative role in CCND1 expression. The underlying mechanisms might involve the reduction of E2F1 transactivation at the transcriptional level and the promotion of AREs-mediated decay of the long 3'UTR isoform of CCND1 through posttranscriptional processes.
RESUMEN
The protein disulfide isomerase (PDI) family is a group of thioredoxin endoplasmic reticulum (ER)-resident enzymes and molecular chaperones that play crucial roles in the correct folding of proteins. PDIs are upregulated in multiple cancer types and are considered a novel target for cancer therapy. In this study, we found that a potent pan-PDI inhibitor, E64FC26, significantly decreased the proliferation of pancreatic ductal adenocarcinoma (PDAC) cells. As expected, E64FC26 treatment increased ER stress and the unfolded protein response (UPR), as evidenced by upregulation of glucose-regulated protein, 78-kDa (GRP78), phosphorylated (p)-PKR-like ER kinase (PERK), and p-eukaryotic initiation factor 2α (eIF2α). Persistent ER stress was found to lead to apoptosis, ferroptosis, and autophagy, all of which are dependent on lysosomal functions. First, there was little cleaved caspase-3 in E64FC26-treated cells according to Western blotting, but a higher dose of E64FC26 was needed to induce caspase activity. Then, E64FC26-induced cell death could be reversed by adding the iron chelator, deferoxamine, and the reactive oxygen species scavengers, ferrostatin-1 and N-acetylcysteine. Furthermore, the autophagosome-specific marker, light chain 3B (LC3B)-II, increased, but the autolysosome marker, sequestosome 1 (SQSTM1)/p62, was not degraded in E64FC26-treated cells. Using the FUW mCherry-LC3 plasmid and acridine orange staining, we also discovered a lower number of acidic vesicles, such as autolysosomes and mature lysosomes, in E64FC26-treated cells. Finally, E64FC26 treatment increased the cathepsin L precursor (pre-CTSL) but decreased mature CTSL expression according to Western blotting, indicating a defective lysosome. These results suggested that the PDI inhibitor, E64FC26, might initially impede proper folding of proteins, and then induce ER stress and disrupt proteostasis, subsequently leading to lysosomal defects. Due to defective lysosomes, the extents of apoptosis and ferroptosis were limited, and fusion with autophagosomes was blocked in E64FC26-treated cells. Blockade of autolysosomal formation further led to the autophagic cell death of PDAC cells.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Proteína Disulfuro Isomerasas , Proteostasis , Estrés del Retículo Endoplásmico , Apoptosis , Autofagia , Carcinoma Ductal Pancreático/tratamiento farmacológico , Neoplasias PancreáticasRESUMEN
Calcium pyrophosphate (CPP) deposition disease (CPPD) is a form of CPP crystal-induced arthritis. A high concentration of extracellular pyrophosphate (ePPi) in synovial fluid is positively correlated with the formation of CPP crystals, and ePPi can be upregulated by ankylosis human (ANKH) and ectonucleotide pyrophosphatase 1 (ENPP1) and downregulated by tissue non-specific alkaline phosphatase (TNAP). However, there is currently no drug that eliminates CPP crystals. We explored the effects of the histone deacetylase (HDAC) inhibitors (HDACis) trichostatin A (TSA) and vorinostat (SAHA) on CPP formation. Transforming growth factor (TGF)-ß1-treated human primary cultured articular chondrocytes (HC-a cells) were used to increase ePPi and CPP formation, which were determined by pyrophosphate assay and CPP crystal staining assay, respectively. Artificial substrates thymidine 5'-monophosphate p-nitrophenyl ester (p-NpTMP) and p-nitrophenyl phosphate (p-NPP) were used to estimate ENPP1 and TNAP activities, respectively. The HDACis TSA and SAHA significantly reduced mRNA and protein expressions of ANKH and ENPP1 but increased TNAP expression in a dose-dependent manner in HC-a cells. Further results demonstrated that TSA and SAHA decreased ENPP1 activity, increased TNAP activity, and limited levels of ePPi and CPP. As expected, both TSA and SAHA significantly increased the acetylation of histones 3 and 4 but failed to block Smad-2 phosphorylation induced by TGF-ß1. These results suggest that HDACis prevented the formation of CPP by regulating ANKH, ENPP1, and TNAP expressions and can possibly be developed as a potential drug to treat or prevent CPPD.
Asunto(s)
Pirofosfato de Calcio , Condrocalcinosis , Pirofosfato de Calcio/metabolismo , Condrocalcinosis/tratamiento farmacológico , Condrocalcinosis/genética , Condrocalcinosis/metabolismo , Condrocitos/metabolismo , Inhibidores de Histona Desacetilasas/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Pirofosfatasas/genética , Pirofosfatasas/metabolismoRESUMEN
BACKGROUND: With the advancement of next generation sequencing technologies (NGS), whole-genome sequencing (WGS) has been deployed to a wide range of clinical scenarios. Rapid and accurate classification of drug-resistant Mycobacterium tuberculosis (MTB) would be advantageous in reducing the amplification of additional drug resistance and disease transmission. METHODS: In this study, a long-read sequencing approach was subjected to the whole-genome sequencing of clinical MTB clones with susceptibility test profiles, including isoniazid (INH) susceptible clones (n = 10) and INH resistant clones (n = 42) isolated from clinical specimens. Non-synonymous variants within the katG or inhA gene associated with INH resistance was identified using Nanopore sequencing coupled with a corresponding analytical workflow. RESULTS: In total, 54 nucleotide variants within the katG gene and 39 variants within the inhA gene associated with INH resistance were identified. Consistency among the results of genotypic profiles, susceptibility test, and minimal inhibitory concentration, the high-INH resistance signature was estimated using the area under the receiver operating characteristic curve with the existence of Ser315Thr (AUC = 0.822) or Thr579Asn (AUC = 0.875). CONCLUSIONS: Taken together, we curated lists of coding variants associated with differential INH resistance using Nanopore sequencing, which may constitute an emerging platform for rapid and accurate identification of drug-resistant MTB clones.
Asunto(s)
Antituberculosos/farmacología , Farmacorresistencia Bacteriana/genética , Genoma Bacteriano , Isoniazida/farmacología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/efectos de los fármacos , Fenotipo , Secuenciación Completa del GenomaRESUMEN
Closely associated with visceral obesity, hepatic steatosis resulting from non-alcoholic fatty liver disease (NAFLD) exacerbates insulin resistance. Developing effective drugs to treat NAFLD is imperative. Here, we investigated the pharmacological mechanism of ugonin J (UJ) in controlling metabolic disorder and ameliorating NAFLD pathophysiology in diet-induced obese mice. The effects of UJ were assessed in 5-week-old C57BL/6 J mice fed a high-fat diet (HFD) for 12 weeks. UJ treatment averted HFD-induced body weight gain by reducing fat deposition in adipose tissues and reduced HFD-induced hyperlipidemia and hepatic inflammation. UJ also improved HFD-induced glucose tolerance and insulin resistance. Moreover, the mode of action of UJ was analyzed in palmitate (PA)-induced steatotic human HuS-E/2 hepatocytes and in hyperglycemia-simulating rat BRIN-BD11 pancreatic ß cells. In PA-induced steatotic human hepatocytes, UJ treatment promoted lipid clearance via pAMPK, pACC and CPT-1 upregulation and SREBP-1c downregulation. Interestingly, UJ upregulated Akt activity in hepatocytes and increased insulin secretion from ß cells in acute insulin secretion tests. Taken together, UJ improved adipocyte hypertrophy, hyperinsulinemia, hyperglycemia, hyperlipidemia and fat deposition in livers. UJ also reduced fatty acid accumulation by modulating key metabolic regulators. Our findings demonstrated the therapeutic potential of UJ for the treatment of NAFLD and diet-induced metabolic disorders.
Asunto(s)
Enfermedades Metabólicas/tratamiento farmacológico , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Obesidad/tratamiento farmacológico , Adipoquinas/sangre , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/patología , Animales , Línea Celular , Células Cultivadas , Dieta Alta en Grasa , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Insulina/metabolismo , Resistencia a la Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Enfermedades Metabólicas/metabolismo , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismo , Ratas , Aumento de Peso/efectos de los fármacosRESUMEN
Alternative splicing (AS) of pre-messenger (m)RNA is a pivotal mechanism in expanding proteomic diversity, which determines the functions of mammalian cells. By conducting transcriptome analyses to profile splicing events in human colorectal cancer (CRC) tissues compared to adjacent normal counterparts, we noted differential splicing profiles of serine/arginine-rich splicing factor 3 (SRSF3) and mitogen-activated protein 4 kinase 4 (MAP4K4) in cancerous tissues of CRC compared to adjacent normal tissues. In addition to SRSF3-mediated autoregulation, RNA-binding motif protein 4 (RBM4) constituted another mechanism in reprogramming the splicing profile of SRSF3. Upregulated expressions of SRSF3 in CRC cells modulated utilization of MAP4K4 exon 16 in a sequence-dependent manner. Alternatively spliced MAP4K4 variants exhibited differential effects on the phosphorylation of c-Jun N-terminal protein kinase 1 (JNK1) which subsequently modulated expression profiles of E-cadherin, N-cadherin, and vimentin, all of which are involved in the migration and invasion of CRC cells. Collectively, RBM4-SRSF3-MAP4K4 constitutes a novel mechanism for manipulating the metastasis of CRC cells through the JNK1 signaling pathway.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Neoplasias Colorrectales/genética , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Metástasis de la Neoplasia , Proteínas de Neoplasias/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-Arginina/genéticaRESUMEN
BACKGROUND: Obesity and its associated health conditions, type 2 diabetes mellitus (T2DM) and nonalcoholic fatty liver disease (NAFLD), are worldwide health problems. It has been shown that insulin resistance is associated with increased hepatic lipid and causes hepatic steatosis through a myriad of mechanisms, including inflammatory signaling. METHODS: Helminthostachys zeylanica (HZ) is used widely as a common herbal medicine to relieve fever symptoms and inflammatory diseases in Asia. In the present study, we evaluated whether HZ has therapeutic effects on obesity, NAFLD and insulin resistance. The protective effects of HZ extract were examined using free fatty acid-induced steatosis in human HuS-E/2 cells and a high-fat diet-induced NAFLD in mice. RESULTS: The major components of the HZ extract are ugonins J and K, confirmed by HPLC. Incubation of human hepatocytes, HuS-E/2 cells, with palmitate markedly increased lipid accumulation and treatment with the HZ extract significantly decreased lipid deposition and facilitated AMPK and ACC activation. After 12 weeks of a high-fat diet with HZ extract treatment, the HFD mice were protected from hyperlipidemia and hyperglycemia. HZ extract prevented body weight gain, adipose tissue expansion and adipocyte hypertrophy in the HFD mice. In addition, fat accumulation was reduced in mice livers. Moreover, the insulin sensitivity-associated index, which evaluates insulin function, was also significantly restored. CONCLUSIONS: These results suggest that HZ has a promising pharmacological effect on high-fat diet-induced obesity, hepatic steatosis and insulin resistance, which may have the potential for clinical application.
Asunto(s)
Resistencia a la Insulina , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismo , Extractos Vegetales/farmacología , Tracheophyta , Adipocitos/efectos de los fármacos , Animales , Peso Corporal/efectos de los fármacos , Línea Celular , Dieta Alta en Grasa/efectos adversos , Humanos , Metabolismo de los Lípidos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Extractos Vegetales/químicaRESUMEN
Monocyte chemotactic protein induced protein 3 (MCPIP3) belongs to the Cysâ»Cysâ»Cysâ»His (CCCH)-zinc finger protein family and contains a highly conserved CCCH-zinc finger domain and a Nedd4-BP1 YacP nuclease (NYN) domain. Previous studies showed that MCPIP3 inhibits the expression of proinflammatory genes, such as vascular cell adhesion molecule (VCAM)-1, in human endothelial cells, but the roles and functions of MCPIP3 in cancer cells are still unknown. In human colorectal cancer specimens, we found that the messenger RNA expression of MCPIP3 was significantly downregulated in cancer tissues compared to adjacent normal tissues (18/25; average fold change of 8.18). Two cell models were used to demonstrate the anti-migration activity of MCPIP3. First, Tet-on T-REx-293/HA-MCPIP3 cells were used to examine whether MCPIP3 can change epithelialâ»mesenchymal transition (EMT)-related gene expressions. Second, we used two human colorectal cancer cell lines, SW620 and HCT116, to prove the role of MCPIP3 in regulating EMT-related gene expressions. We found that overexpression of MCPIP3 inhibited cell migration according to a wound-healing assay and Transwell invasion assay and vimentin expression, and increased E-cadherin expression in these two cell lines. These results suggest that MCPIP3 might play a negative role in cell migration of human colorectal cancer cells.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Ribonucleasas/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Movimiento Celular/genética , Movimiento Celular/fisiología , Transición Epitelial-Mesenquimal/genética , Transición Epitelial-Mesenquimal/fisiología , Células HCT116 , Humanos , Ribonucleasas/genética , Vimentina/genética , Vimentina/metabolismo , Cicatrización de Heridas/genética , Cicatrización de Heridas/fisiologíaRESUMEN
An increase in mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) reportedly attenuates insulin-mediated signaling which participates in the development of brown adipose tissues (BATs). Nevertheless, the effect of MAP4K4 on brown adipogenesis remains largely uncharacterized. In this study, results of a transcriptome analysis (also referred as RNA-sequencing) showed differential expressions of MAP4K4 or SRSF3 transcripts isolated from distinct stages of embryonic BATs. The discriminative splicing profiles of MAP4K4 or SRSF3 were noted as well in brown adipocytes (BAs) with RNA-binding motif protein 4-knockout (RBM4-/-) compared to the wild-type counterparts. Moreover, the relatively high expressions of authentic SRSF3 transcripts encoding the splicing factor functioned as a novel regulator toward MAP4K4 splicing during brown adipogenesis. The presence of alternatively spliced MAP4K4 variants exerted differential effects on the phosphorylation of c-Jun N-terminal protein kinase (JNK) which was correlated with the differentiation or metabolic signature of BAs. Collectively, the RBM4-SRSF3-MAP4K4 splicing cascade constitutes a novel molecular mechanism in manipulating the development of BAs through related signaling pathways.
Asunto(s)
Adipocitos Marrones/citología , Adipogénesis , Empalme Alternativo , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Unión al ARN/genética , Factores de Empalme Serina-Arginina/genética , Adipocitos Marrones/metabolismo , Animales , Diferenciación Celular , Línea Celular , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Variación Genética , MAP Quinasa Quinasa 4/metabolismo , Ratones , Fosforilación , Análisis de Secuencia de ARN , Transducción de Señal , Quinasa de Factor Nuclear kappa BRESUMEN
Anaplastic thyroid cancer (ATC) is among the most aggressive types of malignant cancer. Epidermal growth factor (EGF) plays a crucial role in the pathogenesis of ATC, and patients with thyroid carcinoma typically exhibit increased cysteine-rich protein 61 (Cyr61). In this study, we found that EGF treatment induced cell migration, stress fiber formation, Cyr61 mRNA and protein expressions, and Cyr61 protein secretion in ATC cells. The recombinant Cyr61 protein significantly induced cell migration; however, inhibition of Cyr61 activity by a Cyr61-specific antibody abrogated EGF-induced cell migration. EGF treatment also affected epithelial-to-mesenchymal transition (EMT)-related marker protein expression, as evidenced by an increase in vimentin and a decrease in E-cadherin expression. Inhibition of Cyr61 expression by Cyr61 siRNA decreased cell migration and reversed the EMT-related marker protein expression. EGF treatment increased the phosphorylation of the extracellular signal-regulated kinase (ERK) and cAMP response element-binding protein (CREB), and finally activated Cyr61 promoter plasmid activity. Our results suggest that Cyr61 is induced by EGF through the ERK/CREB signal pathway and that it plays a crucial role in the migration and invasion of ATC cells; moreover, Cyr61 might be a therapeutic target for metastatic ATC.
Asunto(s)
Proteína 61 Rica en Cisteína/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Carcinoma Anaplásico de Tiroides/patología , Neoplasias de la Tiroides/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Proteína 61 Rica en Cisteína/genética , Transición Epitelial-Mesenquimal/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Carcinoma Anaplásico de Tiroides/genética , Carcinoma Anaplásico de Tiroides/metabolismo , Neoplasias de la Tiroides/genética , Neoplasias de la Tiroides/metabolismoRESUMEN
BACKGROUND: Glycine N-methyltransferase (GNMT) is abundantly expressed in the normal liver but is down-regulated in liver cancer tissues. GNMT knockout (Gnmt-/-) mice can spontaneously develop chronic hepatitis, fatty liver, and liver cancer. We previously demonstrated that hepatic GNMT is decreased in high-fat-diet-induced type 2 diabetes mellitus, but its contribution to metabolic syndrome is unclear. Here we show that GNMT modulates key aspects of metabolic syndrome in mice. METHODS: Eleven-week-old Gnmt-/- and wild-type (WT) mice with a C57BL/6 genetic background were used in this study. The metabolic defects of GNMT deficiency were measured by glucose and insulin tolerance tests, lipid homeostasis, gluconeogenesis, and insulin signaling. RESULTS: Gnmt-/- mice, especially females, exhibited glucose intolerance and insulin resistance. However, their body fat and lean mass, food and water intakes, and energy expenditure did not differ from those of WT mice. In addition, glucose-stimulated insulin secretion and insulin-stimulated glucagon secretion were normal in the serum and pancreatic islets of Gnmt-/- mice. Importantly, we found that GNMT deficiency increased lipogenesis and triglycerides in the liver. The elevated triglycerides disrupted the ability of insulin to induce Akt and S6 ribosomal protein phosphorylation, and then triggered insulin resistance and gluconeogenesis in female Gnmt-/- mice. CONCLUSIONS: Our data indicate that hepatic GNMT regulates lipid and glucose homeostasis, and provide insight into the development of insulin resistance through modulating the PI3K/Akt pathway.
Asunto(s)
Gluconeogénesis , Glicina N-Metiltransferasa/deficiencia , Glicina N-Metiltransferasa/genética , Insulina/metabolismo , Hígado/enzimología , Síndrome Metabólico/genética , Transducción de Señal , Animales , Femenino , Síndrome Metabólico/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
Human MCP-1-induced protein 1 (MCPIP1, also known as ZC3H12A and Regnase-1) plays important roles in negatively regulating the cellular inflammatory response. Recently, we found that as an RNase, MCPIP1 has broad-spectrum antiviral effects by targeting viral RNA. In this study, we demonstrated that MCPIP1 expression was induced by hepatitis C virus (HCV) infection in Huh7.5 hepatoma cells. MCPIP1 expression was higher in liver tissue from patients with chronic HCV infection compared with those without chronic HCV infection. Knockdown of MCPIP1 increased HCV replication and HCV-mediated expression of proinflammatory cytokines, such as TNF-α, IL-6, and MCP-1. However, overexpression of MCPIP1 significantly inhibited HCV replication and HCV-mediated expression of proinflammatory cytokines. Various mutants of functional domains of MCPIP1 showed disruption of the RNA binding and oligomerization abilities, as well as RNase activity, but not deubiquitinase activity, which impaired the inhibitory activity against HCV replication. On immunocytochemistry, MCPIP1 colocalized with HCV RNA. Use of a replication-defective HCV John Cunningham 1/AAG mutant and in vitro RNA cleavage assay demonstrated that MCPIP1 could directly degrade HCV RNA. MCPIP1 may suppress HCV replication and HCV-mediated proinflammatory responses with infection, which might contribute to the regulation of host defense against the infection and virus-induced inflammation.
Asunto(s)
Hepacivirus/fisiología , Hepatitis C Crónica/inmunología , Factores de Transcripción/fisiología , Replicación Viral , Línea Celular Tumoral , Quimiocina CCL2/biosíntesis , Células HEK293 , Hepacivirus/genética , Hepatitis C Crónica/patología , Hepatitis C Crónica/virología , Humanos , Interleucina-6/biosíntesis , Hígado/inmunología , Hígado/patología , Hígado/virología , Mutación , Estructura Terciaria de Proteína , Interferencia de ARN , ARN Interferente Pequeño , ARN Viral/metabolismo , Proteínas de Unión al ARN/genética , Ribonucleasas , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Proteasas Ubiquitina-EspecíficasRESUMEN
In chronic liver diseases, regardless of their etiology, the development of fibrosis is the first step toward the progression to cirrhosis, portal hypertension, and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the main profibrogenic cells that promote the pathogenesis of liver fibrosis, and so it is important to identify the molecules that regulate HSCs activation and liver fibrosis. Niemann-Pick type C2 (NPC2) protein plays an important role in the regulation of intracellular cholesterol homeostasis by directly binding with free cholesterol. However, the roles of NPC2 in HSCs activation and liver fibrosis have not been explored in detail. Since a high-cholesterol diet exacerbates liver fibrosis progression in both rodents and humans, we propose that the expression of NPC2 affects free cholesterol metabolism and regulates HSCs activation. In this study, we found that NPC2 is decreased in both thioacetamide- and carbon tetrachloride-induced liver fibrosis tissues. In addition, NPC2 is expressed in quiescent HSCs, but its activation status is down-regulated. Knockdown of NPC2 in HSC-T6 cells resulted in marked increases in transforming growth factor-ß1 (TGF-ß1)-induced collagen type 1 α1 (Col1a1), α-smooth muscle actin (α-SMA) expression, and Smad2 phosphorylation. In contrast, NPC2 overexpression decreased TGF-ß1-induced HSCs activation. We further demonstrated that NPC2 deficiency significantly increased the accumulation of free cholesterol in HSCs, increasing Col1a1 and α-SMA expression and activating Smad2, and leading to sensitization of HSCs to TGF-ß1 activation. In contrast, overexpression of NPC2 decreased U18666A-induced free cholesterol accumulation and inhibited the subsequent HSCs activation. In conclusion, our study has demonstrated that NPC2 plays an important role in HSCs activation by regulating the accumulation of free cholesterol. NPC2 overexpression may thus represent a new treatment strategy for liver fibrosis.
Asunto(s)
Proteínas Portadoras/metabolismo , Colesterol/metabolismo , Glicoproteínas/metabolismo , Células Estrelladas Hepáticas/metabolismo , Cirrosis Hepática/metabolismo , Animales , Western Blotting , Tetracloruro de Carbono/toxicidad , Modelos Animales de Enfermedad , Células Estrelladas Hepáticas/efectos de los fármacos , Células Estrelladas Hepáticas/patología , Humanos , Técnicas para Inmunoenzimas , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/patología , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Tioacetamida/toxicidad , Factor de Crecimiento Transformador beta1/farmacología , Proteínas de Transporte VesicularRESUMEN
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.
Asunto(s)
Proteínas Portadoras/genética , Glicoproteínas/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Sistema de Señalización de MAP Quinasas/genética , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Línea Celular , Línea Celular Tumoral , Proliferación Celular/genética , Progresión de la Enfermedad , Regulación hacia Abajo/genética , Femenino , Células HEK293 , Células Hep G2 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Tasa de Supervivencia , Proteínas de Transporte Vesicular , alfa-Fetoproteínas/genéticaRESUMEN
OBJECTIVE: Diseases associated with calcium-containing crystal deposition can lead to local bone erosion. We aimed to determine whether calcium-containing crystal-hydroxyapatite, ß-tricalcium phosphate and CPPD enhanced osteoclastogenesis and to define underlying mechanisms of action. METHODS: Osteoclastogenesis was studied by culturing murine RAW 264.7 osteoclast precursor cells with RANK ligand (RANKL)/ M-CSF and/or calcium-containing crystals, and observing the tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and TRAP activity. Resorption pit formation was used to evaluate osteoclast activity. Real-time RT-PCR analysis revealed osteoclast marker genes, including TRAP, cathepsin K and calcitonin receptor (CTR). Western blotting was used to analyse the phosphorylation levels of signal transduction molecules. RESULTS: Three kinds of calcium-containing crystal significantly enhanced RANKL/M-CSF-induced osteoclastogenesis in RAW 264.7 cells, as evidenced by the increased number of TRAP-positive multinucleated cells, TRAP activity and resorption pit formation in a dose-dependent manner. Hydroxyapatite, ß-tricalcium phosphate and CPPD treatments significantly enhanced RANKL/M-CSF-induced mRNA expression of TRAP, cathepsin K and CTR. Moreover, the three kinds of calcium-containing crystal enhanced the phosphorylation of extracellular-signal-regulated kinase and p38 in RANKL/M-CSF-treated cells. CONCLUSION: We concluded that calcium-containing crystals can promote osteoclastogenesis and bone resorption through the extracellular-signal-regulated kinase and p38 pathways. Together with synovial activation, this mechanism may be important in the pathogenesis of destructive arthropathies triggered by calcium-containing crystals.
Asunto(s)
Calcio/farmacología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/fisiología , Osteoclastos/citología , Osteogénesis/efectos de los fármacos , Ligando RANK/fisiología , Fosfatasa Ácida/fisiología , Animales , Resorción Ósea/fisiopatología , Calcio/química , Fosfatos de Calcio/farmacología , Catepsina K/fisiología , Línea Celular , Células Cultivadas , Cristalización , Durapatita/farmacología , Técnicas In Vitro , Isoenzimas/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Modelos Animales , Osteoclastos/efectos de los fármacos , Osteoclastos/fisiología , Osteogénesis/fisiología , Receptores de Calcitonina/fisiología , Fosfatasa Ácida TartratorresistenteRESUMEN
15,16-Dihydrotanshinone I (DHTS) is extracted from Salvia miltiorrhiza Bunge which is a functional food in Asia. In this study, we investigated the apoptotic effect of DHTS on the human acute myeloid leukemia (AML) type III HL-60 cell line. We found that treatment with 1.5 µg/mL DHTS increased proapoptotic Bax and Bad protein expressions and activated caspases-3, -8, and -9, thus leading to poly ADP ribose polymerase (PARP) cleavage and resulting in cell apoptosis. DHTS induced sustained c-Jun N-terminal kinase (JNK) phosphorylation and Fas ligand (FasL) expression. The anti-Fas blocking antibody reversed the DHTS-induced cell death, and the JNK-specific inhibitor, SP600125, inhibited DHTS-induced caspase-3, -8, -9, and PARP cleavage. In a xenograft nude mice model, 25 mg/kg DHTS showed a great effect in attenuating HL-60 tumor growth. Taken together, these results suggest that DHTS can induce HL-60 cell apoptosis in vitro and inhibit HL-60 cell growth in vivo; the underlying mechanisms might be mediated through activation of the JNK and FasL signal pathways.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Leucemia/tratamiento farmacológico , Fenantrenos/uso terapéutico , Salvia miltiorrhiza/química , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/aislamiento & purificación , Apoptosis/efectos de los fármacos , Proteína Ligando Fas/metabolismo , Alimentos Funcionales/análisis , Furanos , Células HL-60 , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Leucemia/metabolismo , Leucemia/patología , Masculino , Ratones Endogámicos BALB C , Ratones Desnudos , Fenantrenos/química , Fenantrenos/aislamiento & purificación , QuinonasRESUMEN
Our previous studies found that osthole markedly reduced blood glucose levels in both db/db and ob/ob mice. To improve the antidiabetic activity of osthole, a series of N-hydroxycinnamide derivatives of osthole were synthesized, and their hypoglycemia activities were examined in vitro and in vivo. Both N-hydroxycinnamide derivatives of osthole, OHC-4p and OHC-2m, had the greatest potential for activating AMPK and increasing glucose uptake by L6 skeletal muscle cells. In addition, OHC-4p and OHC-2m time- and dose-dependently increased phosphorylation levels of AMPK and p38 MAPK. The AMPK inhibitor, compound C, and the p38 MAPK inhibitor, SB203580, significantly reversed activation of AMPK and p38 MAPK, respectively, in OHC-4p- and OHC-2m-treated cells. Compound C and SB203580 also inhibited glucose uptake induced by OHC-4p and OHC-2m. Next, we found that OHC-4p and OHC-2m significantly increased glucose transporter 4 (GLUT4) translocation to plasma membranes and counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that activation of AMPK and p38 MAPK by OHC-4p and OHC-2m is associated with increased glucose uptake and GLUT4 translocation and subsequently led to amelioration of hyperglycemia. Therefore, OHC-4p and OHC-2m might have potential as antidiabetic agents for treating type 2 diabetes. Our previous studies found that osthole markedly reduced blood glucose levels in both db/db and ob/ob mice. To improve the antidiabetic activity of osthole, a series of N-hydroxycinnamide derivatives of osthole were synthesized, and their hypoglycemia activities were examined in vitro and in vivo. Both N-hydroxycinnamide derivatives of osthole, OHC-4p and OHC-2m, had the greatest potential for activating AMPK and increasing glucose uptake by L6 skeletal muscle cells. In addition, OHC-4p and OHC-2m time- and dose-dependently increased phosphorylation levels of AMPK and p38 MAPK. The AMPK inhibitor, compound C, and the p38 MAPK inhibitor, SB203580, significantly reversed activation of AMPK and p38 MAPK, respectively, in OHC-4p- and OHC-2m-treated cells. Compound C and SB203580 also inhibited glucose uptake induced by OHC-4p and OHC-2m. Next, we found that OHC-4p and OHC-2m significantly increased glucose transporter 4 (GLUT4) translocation to plasma membranes and counteracted hyperglycemia in mice with streptozotocin-induced diabetes. These results suggest that activation of AMPK and p38 MAPK by OHC-4p and OHC-2m is associated with increased glucose uptake and GLUT4 translocation and subsequently led to amelioration of hyperglycemia. Therefore, OHC-4p and OHC-2m might have potential as antidiabetic agents for treating type 2 diabetes.
Asunto(s)
Ácidos Cumáricos/administración & dosificación , Ácidos Cumáricos/síntesis química , Cumarinas/administración & dosificación , Cumarinas/química , Cumarinas/síntesis química , Diabetes Mellitus Experimental/tratamiento farmacológico , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/síntesis química , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Ácidos Cumáricos/farmacología , Cumarinas/farmacología , Diabetes Mellitus Experimental/enzimología , Transportador de Glucosa de Tipo 4/metabolismo , Hipoglucemiantes/farmacología , Músculo Esquelético/citología , Músculo Esquelético/efectos de los fármacos , Ratas , Estreptozocina , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Osthole is extracted from the Chinese herbs Cnidium monnieri and Angelica pubescens, and it was found to have antitumor activity in vitro and in vivo. A series of osthole derivatives have been synthesized, and the N-hydroxycinnamide derivatives of osthole, WJ1376-1 and WJ1398-1 were found to have the greatest potential against human colon adenocarcinoma cells. In contrast to the parental osthole, both WJ1376-1 and WJ1398-1 were found to induce multinucleation and polyploidy by microscopic observation and flow cytometry. WJ1376-1 and WJ1398-1 significantly activated ataxia telangiectasia and rad3 related (ATR) kinase, which triggered activation of the checkpoint kinase 2 (Chk2) signaling pathway and then down regulated Cdc25 phosphatase and Cdc2/cyclin B kinase activities. WJ1376-1 and WJ1398-1 also inhibited the phosphorylation of Aurora A kinase, which is associated with important processes during mitosis. The presence of a "comet" DNA fragment and phosphorylation of p53 at Ser 15 clearly indicated that DNA damage occurred with WJ1376-1 and WJ1398-1 treatment. WJ1376-1 and WJ1398-1 ultimately induced apoptosis as evidenced by the upregulation of Bad and activation of caspases-3, -7, and -9. Furthermore, WJ1376-1 and WJ1398-1 also showed a great effect in attenuating tumor growth without affecting the body weight of xenograft nude mice. Taken together, these results suggest that the toxic activities of WJ1376-1 and WJ1398-1 were dissimilar to that of the parental osthole, which can induce cell polyploidy and G2/M cell cycle arrest in colon adenocarcinoma cells and may provide a potential therapeutic target for colon cancer treatment in the future.
Asunto(s)
Apoptosis/efectos de los fármacos , Cumarinas/toxicidad , Daño del ADN/efectos de los fármacos , Ácidos Hidroxámicos/toxicidad , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Cumarinas/química , Cumarinas/uso terapéutico , Regulación hacia Abajo/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Humanos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/uso terapéutico , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Vitis thunbergii var. taiwaniana (VTT) is an indigenous Taiwanese wild grape and is used as a folk medicine in Taiwan. VTT is rich in polyphenols, especially quercetin and resveratrol derivatives, which were demonstrated to exhibit inhibitory activities against carcinogenesis and prevent some neurodegenerative diseases. (-)-Vitisin B is one of the resveratrol tetramers extracted from VTT. In this study, we investigated the mechanisms of (-)-vitisin B on the induction of apoptosis in human HL-60 promyelocytic leukemia cells. First, (-)-vitisin B significantly inhibited cell proliferation through inducing cell apoptosis. This effect appeared to occur in a time- and dose-dependent manner. Cell-cycle distribution was also examined, and we found that (-)-vitisin B significantly induced a sub-G1 population in a dose-dependent manner. In addition, (-)-vitisin B exhibited stronger inhibitory effects on cell proliferation than resveratrol. Second, (-)-vitisin B dose dependently induced apoptosis-related protein expressions, such as the cleavage form of caspase-3, caspase-8, caspase-9, poly(ADP ribose) polymerase, and the proapoptotic Bax protein. Third, (-)-vitisin B treatment also resulted in increases in c-Jun N-terminal kinase (JNK) phosphorylation and Fas ligand (FasL) expression. Moreover, the (-)-vitisin B-induced FasL expression and caspase-3 activation could be reversed by a JNK inhibitor. These results suggest that (-)-vitisin B-induced apoptosis of leukemia cells might be mediated through activation of JNK and Fas death-signal transduction.
Asunto(s)
Antineoplásicos/farmacología , Benzofuranos/farmacología , Leucemia Promielocítica Aguda/tratamiento farmacológico , Fenoles/farmacología , Vitis/química , Antineoplásicos/análisis , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis , Benzofuranos/análisis , Ciclo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/patología , Medicina Tradicional de Asia Oriental , Fenoles/análisis , Extractos Vegetales/análisis , Extractos Vegetales/farmacología , TaiwánRESUMEN
BACKGROUND: Multiple sclerosis (MS) commonly affects young adults who may be sexually active, with sexual dysfunction being a significant, but often underestimated, symptom of MS. However, no large-scaled study has investigated the association between erectile dysfunction (ED) and MS in an Asian population to date. OBJECTIVE: The objective of this study is to estimate the association between ED and a prior diagnosis of MS using a population-based dataset with a case-control design in Taiwan. METHODS: The data were sourced from National Health Insurance Research Database. We identified 38,139 patients with ED as cases and randomly selected 262,848 subjects as controls. We then used conditional logistic regression to compute the odds ratio for having previously received a diagnosis of MS between cases and controls. RESULTS: The prevalence of prior MS was 0.037% and 0.015% for cases and controls, respectively (P < 0.001). Conditional logistic regression analysis revealed that cases were 2.23 times (95% confidence interval = 1.15-4.32) more likely to have been previously diagnosed with MS than controls after adjusting for monthly income, geographic location, hypertension, diabetes, coronary heart disease, hyperlipidemia, obesity, and alcohol abuse/alcohol dependence syndromes. CONCLUSIONS: This study revealed an association between ED and prior MS even after adjusting for potential confounders.