RESUMO
BACKGROUND: Skeletal muscle atrophy is a severe condition that involves loss of muscle mass and quality. Drug intake can also cause muscle atrophy. Biguanide metformin is the first-line and most widely prescribed anti-diabetic drug for patients with type 2 diabetes. The molecular mechanism of metformin in muscle is unclear. METHODS: Myostatin expression was investigated at the protein and transcript levels after metformin administration. To investigate the pathways associated with myostatin signalling, we used real-time polymerase chain reaction, immunoblotting, luciferase assay, chromatin immunoprecipitation assay, co-immunoprecipitation, immunofluorescence, primary culture, and confocal microscopy. Serum analysis, physical performance, and immunohistochemistry were performed using our in vivo model. RESULTS: Metformin induced the expression of myostatin, a key molecule that regulates muscle volume and triggers the phosphorylation of AMPK. AMPK alpha2 knockdown in the background of metformin treatment reduced the myostatin expression of C2C12 myotubes (-49.86 ± 12.03%, P < 0.01) and resulted in increased myotube diameter compared with metformin (+46.62 ± 0.88%, P < 0.001). Metformin induced the interaction between AMPK and FoxO3a, a key transcription factor of myostatin. Metformin also altered the histone deacetylase activity in muscle cells (>3.12-fold ± 0.13, P < 0.001). The interaction between HDAC6 and FoxO3a induced after metformin treatment. Confocal microscopy revealed that metformin increased the nuclear localization of FoxO3a (>3.3-fold, P < 0.001). Chromatin immunoprecipitation revealed that metformin induced the binding of FoxO3a to the myostatin promoter. The transcript-level expression of myostatin was higher in the gastrocnemius (GC) muscles of metformin-treated wild-type (WT) (+68.9 ± 10.01%, P < 0.001) and db/db mice (+55.84 ± 6.62%, P < 0.001) than that in the GC of controls (n = 4 per group). Average fibre cross-sectional area data also showed that the metformin-treated C57BL/6J (WT) (-31.74 ± 0.75%, P < 0.001) and C57BLKS/J-db/db (-18.11 ± 0.94%, P < 0.001) mice had decreased fibre size of GC compared to the controls. The serum myoglobin level was significantly decreased in metformin-treated WT mice (-66.6 ± 9.03%, P < 0.01). CONCLUSIONS: Our results demonstrate that metformin treatment impairs muscle function through the regulation of myostatin in skeletal muscle cells via AMPK-FoxO3a-HDAC6 axis. The muscle-wasting effect of metformin is more evident in WT than in db/db mice, indicating that more complicated mechanisms may be involved in metformin-mediated muscular dysfunction.
Assuntos
Diabetes Mellitus Tipo 2 , Metformina , Animais , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Desacetilase 6 de Histona/metabolismo , Humanos , Metformina/metabolismo , Metformina/farmacologia , Metformina/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Atrofia Muscular/etiologia , Miostatina/genética , Miostatina/metabolismoRESUMO
The progression of colorectal cancer (CRC) has been well studied and understood with the development of molecular and genetic techniques. However, specific marker(s) that could be used to predict lymph node (LN) involvement, which is the most important prognostic factor for CRC, have not been identified so far. Our previous study, in which network analysis of LN(+) and LN(-) CRC gene expression was carried out with data obtained from the Cancer Genome Atlas, led to the identification of AHA1. AHA1 is a co-chaperone activator of the Hsp90 ATPase activity. However, the role of AHA1 expression in cancer cells is still unclear. To investigate how AHA1 expression regulates the cancer cell progression and/or metastasis of human CRC, the expression levels of AHA1 and Hsp90 were examined in 105 CRC tissue samples and compared with those in paired normal tissue. The RNA expression levels of AHA1 and Hsp90aa1, but not Hsp90ab, were significantly higher in cancer tissues than in adjacent paired normal tissues (p = 0.032 and p = 0.0002, respectively). In particular, AHA1, but not Hsp90aa1 and Hsp90ab, was closely associated with the TNM stage, LN stage, and tumor metastasis (p = 0.035, p = 0.012, and p = 0.0003, respectively). Moreover, the expression of AHA1 was not only higher in the CRC cell lines than in the normal colon fibroblast cell line but was also associated with the progression of these CRC cell lines. Overexpression of AHA1 in SW480 cells increased, whereas suppression of AHA1 expression in HCT116 cells reduced cell migration and invasion through the regulation of Snail, E-cadherin, pSRC, and pAKT, which are associated with EMT signaling. Taken together, our study suggests that AHA1 contributes to the metastatic advantage of human CRC.
Assuntos
Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Chaperonas Moleculares/metabolismo , Biomarcadores Tumorais , Movimento Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Linfonodos/patologia , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares/genética , Invasividade NeoplásicaRESUMO
Transdifferentiation (or activation) of hepatic stellate cells (HSCs) to myofibroblasts is a key event in liver fibrosis. Activated HSCs in the tumor microenvironment reportedly promote tumor progression. This study analyzed the effect of an inhibitor of HSC activation, retinol-binding protein-albumin domain III fusion protein (R-III), on protumorigenic functions of HSCs. Although conditioned medium collected from activated HSCs enhanced the migration, invasion, and proliferation of the hepatocellular carcinoma cell line Hepa-1c1c7, this effect was not observed in Hepa-1c1c7 cells treated with conditioned medium from R-III-exposed HSCs. In a subcutaneous tumor model, larger tumors with increased vascular density were formed in mice transplanted with Hepa-1c1c7+HSC than in mice transplanted with Hepa-1c1c7 cells alone. Intriguingly, when Hepa-1c1c7+HSC-transplanted mice were injected intravenously with R-III, a reduction in vascular density and extended tumor necrosis were observed. In an orthotopic tumor model, co-transplantation of HSCs enhanced tumor growth, angiogenesis, and regional metastasis accompanied by increased peritumoral lymphatic vessel density, which was abolished by R-III. In vitro study showed that R-III treatment affected the synthesis of pro-angiogenic and anti-angiogenic factors in activated HSCs, which might be the potential mechanism underlying the R-III effect. These findings suggest that the inhibition of HSC activation abrogates HSC-induced tumor angiogenesis and growth, which represents an attractive therapeutic strategy.
Assuntos
Carcinoma Hepatocelular/patologia , Células Estreladas do Fígado/efeitos dos fármacos , Neoplasias Hepáticas/patologia , Proteínas Recombinantes de Fusão/farmacologia , Albuminas/química , Albuminas/farmacologia , Albuminas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/terapia , Transformação Celular Neoplásica/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Feminino , Células Estreladas do Fígado/fisiologia , Neoplasias Hepáticas/irrigação sanguínea , Neoplasias Hepáticas/terapia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Patológica/genética , Neovascularização Patológica/prevenção & controle , Domínios e Motivos de Interação entre Proteínas/fisiologia , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas de Ligação ao Retinol/farmacologia , Proteínas de Ligação ao Retinol/uso terapêutico , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adenylate kinase 5 (AK5) belongs to the adenylate kinase family that catalyses reversible phosphate transfer between adenine nucleotides, and it is related to various energetic signalling mechanisms. However, the role of AK5 in colorectal cancer (CRC) has not been reported. In this study, AK5 was significantly hypermethylated in CRC compared to adjacent normal tissues (P < 0.0001) and normal tissues (P = 0.0015). Although the difference in mRNA expression was not statistically significant in all of them, the selected 49 cases of CRC tissues with AK5 hypermethylation with the cut off value of 40% showed a significant inverse correlation with mRNA expression (P = 0.0003). DNA methylation of AK5 promoter significantly decreased and AK5 expression recovered by 5-aza-2'-deoxycytidine, DNA methyltransferase inhibitor in CRC cell lines. In addition, AK5 promoter activity significantly decreased due to DNA methyltransferase, and it increased due to 5-aza. Moreover, AK5 regulated the phosphorylated AMPK and mTOR phosphorylation and inhibited the cell migration and cell invasion in CRC cell lines. Furthermore, low AK5 expression is associated with poor differentiation (P = 0.014). These results demonstrate that the AK5 promoter is frequently hypermethylated and induced methylation-mediated gene down-regulation. AK5 expression regulates AMPK/mTOR signalling and may be closely related to metastasis in colorectal adenocarcinoma.
Assuntos
Adenocarcinoma/genética , Adenilato Quinase/genética , Neoplasias Colorretais/genética , Metilação de DNA , Regulação para Baixo , Proteínas Quinases Ativadas por AMP/metabolismo , Adenocarcinoma/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Decitabina/farmacologia , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Masculino , Fosforilação , Regiões Promotoras Genéticas , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismoRESUMO
Activation of quiescent hepatic stellate cells (HSCs) to myofibroblasts plays a key role in liver fibrosis. We had previously shown that albumin and its derivative, R-III (a retinol-binding protein-albumin domain III fusion protein), inhibited HSC activation by sequestering retinoic acid (RA) and that R-III administration reduced carbon tetrachloride (CCl4)-induced liver fibrosis. In this study, we aimed to elucidate the mechanism of action of albumin downstream of RA sequestration. Nuclear factor-κB p65 was evenly distributed in the cytoplasm in activated mouse HSCs, whereas albumin expression or R-III treatment (albumin/R-III) caused the nuclear translocation of p65, probably via RA sequestration, resulting in a dramatic increase in interleukin-1beta (IL-1ß) expression. Albumin/R-III in turn induced the phosphorylation of Smad3 at the linker region, inhibiting its nuclear import in an IL-1ß-dependent manner. Consistent with the in vitro results, the level of IL-1ß mRNA expression was higher in CCl4/R-III-treated livers than in CCl4-treated livers. These findings reveal that albumin/R-III inhibits the transforming growth factor-ß-Smad3 signaling as well as the retinoic acid receptor-mediated pathway, which probably contributes to the inhibition of HSC activation, and suggest that R-III may be an anti-fibrotic drug candidate.
Assuntos
Albuminas/farmacologia , Células Estreladas do Fígado/efeitos dos fármacos , Interleucina-1beta/genética , Cirrose Hepática/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Proteína Smad3/genética , Albuminas/genética , Albuminas/metabolismo , Animais , Tetracloreto de Carbono/administração & dosagem , Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Interleucina-1beta/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacos , Cultura Primária de Células , Transporte Proteico/efeitos dos fármacos , Proteínas de Ligação ao Retinol/genética , Proteínas de Ligação ao Retinol/metabolismo , Proteínas de Ligação ao Retinol/farmacologia , Transdução de Sinais , Proteína Smad3/metabolismo , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Tretinoína/antagonistas & inibidores , Tretinoína/farmacologiaRESUMO
Korean mistletoe (Viscum album L. var. coloratum) lectin (VCA) is known as an anticancer drug. However, it is not clear whether VCA affects the self-renewal activity of mesenchymal stem cells (MSCs). Therefore, the objectives of this study were to analyze the effect of VCA on the proliferation of MSCs and expression of stemness markers. We also evaluated the usefulness of placenta-derived MSCs (PD-MSCs) as a screening tool. VCA was stably administered to MSCs, and analyzed self-renewal activities. The effect of IL-6 signaling on MSC proliferation was explored by quantitative methylation-specific PCR (qMSP) and western blot analysis. Compared with the control condition, low concentrations of VCA (10 pg/mL) induced an increase in the self-renewal activity of MSCs. Interestingly, a low concentration of VCA promoted IL-6 signaling in PD-MSCs through altered IL-6/STAT3 gene methylation. Furthermore, inhibition of IL-6 expression in PD-MSCs using an anti-IL-6 antibody caused a decrease in their self-renewal activity through IL-6/STAT3 signaling by altering IL-6/STAT3 gene methylation. These findings provide helpful data for understanding the mechanism of MSC self-renewal via VCA and show that VCA may be useful as a functional natural product for developing efficient therapies using placenta-derived stem cells.
Assuntos
Interleucina-6/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Erva-de-Passarinho/química , Lectinas de Plantas/farmacologia , Fator de Transcrição STAT3/metabolismo , Biomarcadores , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-6/genética , Placenta/citologia , Lectinas de Plantas/química , Gravidez , Fator de Transcrição STAT3/genéticaRESUMO
BACKGROUND: Chemotherapy is a standard therapeutic regimen to treat triple-negative breast cancer (TNBC); however, chemotherapy alone does not result in significant improvement and often leads to drug resistance in patients. In contrast, combination therapy has proven to be an effective strategy for TNBC treatment. Whether metformin enhances the anticancer effects of cisplatin and prevents cisplatin resistance in TNBC cells has not been reported. METHODS: Cell viability, wounding healing, and invasion assays were performed on Hs 578T and MDA-MB-231 human TNBC cell lines to demonstrate the anticancer effects of combined cisplatin and metformin treatment compared to treatment with cisplatin alone. Western blotting and immunofluorescence were used to determine the expression of RAD51 and gamma-H2AX. In an in vivo 4T1 murine breast cancer model, a synergistic anticancer effect of metformin and cisplatin was observed. RESULTS: Cisplatin combined with metformin decreased cell viability and metastatic effect more than cisplatin alone. Metformin suppressed cisplatin-mediated RAD51 upregulation by decreasing RAD51 protein stability and increasing its ubiquitination. In contrast, cisplatin increased RAD51 expression in an ERK-dependent manner. In addition, metformin also increased cisplatin-induced phosphorylation of γ-H2AX. Overexpression of RAD51 blocked the metformin-induced inhibition of cell migration and invasion, while RAD51 knockdown enhanced cisplatin activity. Moreover, the combination of metformin and cisplatin exhibited a synergistic anticancer effect in an orthotopic murine model of 4T1 breast cancer in vivo. CONCLUSIONS: Metformin enhances anticancer effect of cisplatin by downregulating RAD51 expression, which represents a novel therapeutic target in TNBC management.
Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Metformina/farmacologia , Rad51 Recombinase/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Sinergismo Farmacológico , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/patologia , Metformina/administração & dosagem , Camundongos Endogâmicos BALB C , Rad51 Recombinase/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Programmed death-ligand 1 (PD-L1) acts as an immune checkpoint inhibitor in various cancers. PD-L1 is known to be more frequently expressed in EBV (+) gastric cancer (GC). However, the mechanisms underlying the regulation of PD-L1 expression in EBV (+) GC remain unclear. We investigated the basal and inducible PD-L1 expressions in GC cells. PD-L1 expression was upregulated upon treatment with IFNγ in both EBV (-) and EBV (+) GC cells. Upon stimulation with the same concentration of IFNγ for 24 h, EBV (+) SNU-719 cells showed dramatically higher PD-L1 expression levels by activating JAK2/STAT1/IRF-1 signaling than those of EBV (-) AGS cells. PD-L1 promoter assays, chromatin immunoprecipitation, and electrophoretic mobility shift assays revealed that IFNγ-inducible PD-L1 overexpression is primarily mediated by the putative IRF-1α site of the PD-L1 promoter in EBV (+) SNU-719 cells. Moreover, EBNA1 knockdown reduced both constitutive and IFNγ-inducible PD-L1 promoter activity by decreasing the transcript and protein levels of JAK2 and subsequently STAT1/IRF-1/PD-L1 signaling. EBNA1 is suggested to be moderately enhance both constitutive and IFNγ-inducible PD-L1 expression in EBV (+) GC cells. Thus, the signaling proteins and EBNA1 that regulate PD-L1 expression are potential therapeutic targets in EBV (+) GC.
Assuntos
Interferon gama/genética , Proteínas/genética , Transdução de Sinais/genética , Neoplasias Gástricas/genética , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Infecções por Vírus Epstein-Barr/genética , Infecções por Vírus Epstein-Barr/virologia , Regulação Neoplásica da Expressão Gênica/genética , Herpesvirus Humano 4/patogenicidade , Humanos , Fator Regulador 1 de Interferon/genética , Janus Quinase 2/genética , Fator de Transcrição STAT1/genética , Neoplasias Gástricas/virologiaRESUMO
BACKGROUND: DNA hypermethylation is a key epigenetic mechanism for the silencing of many genes in cancer. Hinokitiol, a tropolone-related natural compound, is known to induce apoptosis and cell cycle arrest and has anti-inflammatory and anti-tumor activities. However, the relationship between hinokitiol and DNA methylation is not clear. The aim of our study was to explore whether hinokitiol has an inhibitory ability on the DNA methylation in colon cancer cells. RESULTS: MTT data showed that hinokitiol had higher sensitivity in colon cancer cells, HCT-116 and SW480, than in normal colon cells, CCD18Co. Hinokitiol reduced DNA methyltransferase 1 (DNMT1) and ubiquitin-like plant homeodomain and RING finger domain 1 (UHRF1) expression in HCT-116 cells. In addition, the expression of ten-eleven translocation protein 1 (TET1), a known DNA demethylation initiator, was increased by hinokitiol treatment. ELISA and FACS data showed that hinokitiol increased the 5-hydroxymethylcytosine (5hmC) level in the both colon cancer cells, but 5-methylcytosine (5mC) level was not changed. Furthermore, hinokitiol significantly restored mRNA expression of O6-methylguanine DNA methyltransferase (MGMT), carbohydrate sulfotransferase 10 (CHST10), and B-cell translocation gene 4 (BTG4) concomitant with reduction of methylation status in HCT-116 cells. CONCLUSIONS: These results indicate that hinokitiol may exert DNA demethylation by inhibiting the expression of DNMT1 and UHRF1 in colon cancer cells.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Neoplasias do Colo/genética , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Metilação de DNA/efeitos dos fármacos , Monoterpenos/farmacologia , Tropolona/análogos & derivados , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Oxigenases de Função Mista/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Tempo , Tropolona/farmacologia , Ubiquitina-Proteína LigasesRESUMO
Esophageal squamous cell carcinoma (ESCC) is a genetically complex tumor type and is a major cause of cancer-related mortality. The combination of genetics, diet, behavior, and environment plays an important role in the carcinogenesis of ESCC. To characterize the genomic aberrations of this disease, we investigated the genomic imbalances in 19 primary ESCC cases using high-resolution array comparative genomic hybridization (CGH). All cases showed either loss or gain of whole chromosomes or segments of chromosome(s) with variable genomic sizes. The copy number alterations per case affected the median 34% (~ 1,034Mb/3,000Mb) of the whole genome. Recurrent gains were 1q21.3-qter, 3q13.11-qter, 5pter-p11, 7pter-p15.3, 7p12.1-p11.2, 7q11-q11.2, 8p12-qter, 11q13.2-q13.3, 12pter-p13.31, 17q24.2, 20q11.21-qter, and 22q11.21-q11.22 whereas the recurrent losses were 3pter-p11.1, 4pter-p12, 4q28.3-q31.22, 4q31.3-q32.1, 9pter-p12, 11q22.3-qter and 13q12.11-q22.1. Amplification of 11q13 resulting in overexpression of CTTN/CCND1 was the most prominent finding, which was observed in 13 of 19 ESCC cases. These unique profiles of copy number alteration should be validated by further studies and need to be taken into consideration when developing biomarkers for early detection of ESCC.
Assuntos
Carcinoma de Células Escamosas/genética , Cromossomos Humanos Par 11/genética , Cortactina/genética , Ciclina D1/genética , Neoplasias Esofágicas/genética , Regulação Neoplásica da Expressão Gênica , Adulto , Idoso , Povo Asiático , Carcinoma de Células Escamosas/fisiopatologia , Cromossomos Humanos Par 11/metabolismo , Hibridização Genômica Comparativa , Cortactina/metabolismo , Ciclina D1/metabolismo , Neoplasias Esofágicas/fisiopatologia , Carcinoma de Células Escamosas do Esôfago , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-IdadeRESUMO
The gene MT3-MMP (also known as MMP16) encodes the membrane type 3 matrix metalloproteinase, which is a member of the matrix metalloproteinase (MMP) gene family. Several MMPs are associated with migration in colorectal cancer (CRC). However, the methylation status of the MT3-MMP promoter in CRC has not been reported. The methylation status and expression levels of MT3-MMP were investigated in primary tumor tissues and adjacent normal tissues in 105 patients with CRC, one normal colon cell line (CCD18Co), and three CRC cell lines (SW480, DLD-1, and LoVo) by quantitative methylation-specific PCR and real-time PCR. MT3-MMP was hypermethylated in 82 of 105 CRC tissues (78%), 30 of 105 adjacent normal tissues (29%), and two of 11 normal colon tissues (18%). MT3-MMP mRNA was significantly reduced in CRC compared with that in adjacent normal tissues (P < 0.05). The methylation-mediated downregulation of MT3-MMP was restored by treatment with 5-aza-2'-deoxycytidine in two CRC cell lines, and MT3-MMP promoter activity was significantly reduced by methylation. The knockdown of MT3-MMP induced cell migration, but overexpressed MT3-MMP reduced cell migration in CRC cells. These results demonstrate that the MT3-MMP promoter is frequently hypermethylated in CRC and that downregulation of MT3-MMP may be important for cell migration in CRC.
Assuntos
Adenocarcinoma/patologia , Movimento Celular/genética , Neoplasias Colorretais/patologia , Metilação de DNA/genética , Metaloproteinase 16 da Matriz/genética , Adenocarcinoma/genética , Idoso , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Decitabina , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Metaloproteinase 16 da Matriz/biossíntese , Regiões Promotoras Genéticas/genética , Interferência de RNA , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Human chorionic plate-derived mesenchymal stem cells (CP-MSCs) isolated from the placenta have been reported to demonstrate therapeutic effects in animal models of liver injury; however, the underlying epigenetic mechanism of this effect has not been elucidated. Thus, we investigated whether CP-MSCs influence epigenetic processes during regeneration of the injured liver. METHODS: CP-MSCs were engrafted into a carbon tetrachloride (CCl4)-injured rat model through direct transplantation into the liver (DTX), intrasplenic transplantation (STX), and intravenous transplantation via the tail vein (TTX). Non-transplanted (NTX) rats were maintained as sham controls. Liver tissues were analyzed after transplantation using immunohistochemistry, western blot analysis, and quantitative methylation-specific polymerase chain reaction. Proliferation and human interleukin-6 (hIL-6) enzyme-linked immunosorbent assays were performed using CCl4-treated hepatic cells that were co-cultured with CP-MSCs. RESULTS: The Ki67 labeling index, cell cyclins, albumin, IL-6, and gp130 levels were elevated in the CP-MSC transplantation groups. The concentration of hIL-6 in supernatants and the proliferation of CCl4-treated rat hepatic cells were enhanced by co-culturing with CP-MSCs (p<0.05), while the methylation of IL-6/IL-6R and STAT3 by CP-MSC transplantation decreased. CONCLUSION: These results suggest that administration of CP-MSCs promotes IL-6/STAT3 signaling by decreasing the methylation of the IL-6/SATA3 promoters and thus inducing the proliferation of hepatic cells in a CCl4-injured liver rat model. These data advance our understanding of the therapeutic mechanisms in injured livers, and can facilitate the development of cell-based therapies using placenta-derived stem cells.
RESUMO
Adenocarcinoma (AC) and squamous cell carcinoma (SCC) of non-small cell lung carcinoma (NSCLC) have different clinical presentations, morphologies, treatments, and prognoses. Recent studies suggested that fundamental genetic alterations related to carcinogenesis of each tumor type may be different. In this study, we investigated the genomic alterations of 47 primary NSCLC samples (22 ACs and 25 SCCs) as well as the corresponding normal tissue using array comparative genomic hybridization. Frequent copy number alterations (CNAs), which were identified in more than 68% of all of the cases, were evaluated in each subtype (SCC and AC), and a CNA signature was established. Among these CNAs, 37 genes from the SCCs and 15 genes from the ACs were located in a region of gain, and 4 genes from the SCCs and 13 genes from the ACs were located in a region of loss. The most frequent gain was located on 3q26-29 including the gene TP63 in SCCs and 7q11.23 and 7q36.3 in ACs. Moreover, we identified 3 genetically distinct groups (group I [16 SCC] with CNA signature of SCC; group II [7 SCC + 8 AC], which has a genetically distinctive CNA signature from SCC and AC; and group III [2 SCC + 14 AC] with CNA signature of AC) by gene clustering extracted from CNAs, which are associated with a prognosis. The present study contributed to the molecular characterization of AC and SCC of NSCLC and showed a subtype of tumor that has a unique genetic CNA signature. However, further study about the significance of these 3 distinct groups and their usefulness as a diagnostic marker of identified CNAs is necessary.
Assuntos
Adenocarcinoma/genética , Carcinoma de Células Escamosas/genética , Dosagem de Genes/genética , Neoplasias Pulmonares/genética , Adenocarcinoma/classificação , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/classificação , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Escamosas/classificação , Carcinoma de Células Escamosas/patologia , Análise por Conglomerados , Hibridização Genômica Comparativa , Feminino , Humanos , Neoplasias Pulmonares/classificação , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Análise Serial de Tecidos , Transcriptoma/genéticaRESUMO
DNA methylation is an epigenetic event that occurs frequently in colorectal cancer (CRC). Increased glucose level is a strong risk factor for CRC. Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) modulates glycogen metabolism, particularly glycogen synthesis. The aim of this study was to investigate the effect of high glucose levels on DNA methylation of PPP1R3C in CRC. PPP1R3C was significantly hypermethylated in CRC tissues (76/105, 72.38%, P <0.05) and colon cancer cell lines (P < 0.05). CRC tissues obtained from patients with high glucose levels showed that the methylation of PPP1R3C was lower than in patients who had normal levels of glucose. When DLD-1 cells were cultured under conditions of high glucose, the methylation of PPP1R3C was repressed. The expression of PPP1R3C was inversely related to methylation status. In addition, a promoter luciferase assay showed that the transcriptional activity of PPP1R3C was increased in high glucose culture conditions. The number of cells decreased when PPP1R3C was silenced in DLD-1 cells. These results suggest that PPP1R3C, a novel hypermethylated gene in CRC, may play a critical role in cancer cell growth in association with glucose levels.
Assuntos
Proteínas de Transporte/genética , Neoplasias Colorretais/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Glucose/fisiologia , Fosfoproteínas Fosfatases/genética , Idoso , Sequência de Bases , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Neoplasias Colorretais/enzimologia , Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/genética , Epigênese Genética , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fosfoproteínas Fosfatases/metabolismo , Análise de Sequência de DNARESUMO
[6]-Gingerol has been used to control diabetes and dyslipidemia; however, its metabolic role is poorly understood. In this study, [6]-gingerol increased adenosine monophosphate (AMP)-activated protein kinase (AMPK) phosphorylation in mouse skeletal muscle C2C12 cells. Stimulation of glucose uptake by [6]-gingerol was dependent on AMPKα2. Moreover, both Inhibition and knockdown of AMPKα2 blocked [6]-gingerol-induced glucose uptake. [6]-Gingerol significantly decreased the activity of protein phosphatase 2A (PP2A). Inhibition of PP2A activity with okadaic acid enhanced the phosphorylation of AMPKα2. Moreover, the interaction between AMPKα2 and PP2A was increased by [6]-gingerol, suggesting that PP2A mediates the effect of [6]-gingerol on AMPK phosphorylation. In addition, [6]-gingerol increased the phosphorylation of Akt-substrate 160 (AS160), which is a Rab GTPase-activating protein. Inhibition of AMPKα2 blocked [6]-gingerol-induced AS160 phosphorylation. [6]-gingerol increased the Rab5, and AMPKα2 knockdown blocked [6]-gingerol-induced expression of Rab5, indicating AMPK play as an upstream of Rab5. It also increased glucose transporter 4 (GLUT4) mRNA and protein expression and stimulated GLUT4 translocation. Furthermore, insulin-mediated glucose uptake and Akt phosphorylation were further potentiated by [6]-gingerol treatment. This potentiation was not observed in the presence of AMPK inhibitor compound C. In summary, our results suggest that [6]-gingerol plays an important role in glucose metabolism via the AMPKα2-mediated AS160-Rab5 pathway and through potentiation of insulin-mediated glucose regulation.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Catecóis/farmacologia , Álcoois Graxos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Linhagem Celular , Sinergismo Farmacológico , Proteínas Ativadoras de GTPase/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Camundongos , Fosforilação , Proteína Fosfatase 2/metabolismo , Ratos , Proteínas rab5 de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: The hypermethylation of Alcohol dehydrogenase iron containing 1 (ADHFE1) was recently reported to be associated with colorectal cancer (CRC) differentiation. However, the effect of alcohol on ADHFE1 hypermethylation in CRC is still unclear. METHODS: The methylation status and expression levels of ADHFE1 were investigated in primary tumor tissues and adjacent normal tissues of 73 patients with CRC, one normal colon cell line, and 4 CRC cell lines (HT-29, SW480, DLD-1, and LoVo) by quantitative methylation-specific polymerase chain reaction (QMSP) and real-time reverse transcription polymerase chain reaction (real time PCR), respectively. The effect of alcohol on the methylation status of ADHFE1 was analyzed in HT-29, SW480, DLD-1, and CCD18Co cells using QMSP, real-time PCR, immunoblot, and cell proliferation assay. RESULTS: ADHFE1 was hypermethylated in 69 of 73 CRC tissues (95%) compared to adjacent normal tissues (p<0.05). The mRNA expression of ADHFE1 was significantly reduced in CRC compared to adjacent normal tissues (p<0.05) and its expression was decreased in the alcohol consumption group (p<0.05). ADHFE1 was hypermethylated and its expression was decreased in 4 CRC cell lines compared with normal colon cell line. Alcohol induced hypermethylation of ADHFE1, decreased its expression, and stimulated cell proliferation of HT-29, SW480, and DLD-1cells. CONCLUSION: These results demonstrate that the promoter hypermethylation of ADHFE1 is frequently present in CRC and alcohol induces methylation-mediated down expression of ADHFE1 and proliferation of CRC cells.
Assuntos
Oxirredutases do Álcool/genética , Álcoois/toxicidade , Neoplasias Colorretais/genética , Metilação de DNA/efeitos dos fármacos , Proteínas Mitocondriais/genética , Oxirredutases do Álcool/biossíntese , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/induzido quimicamente , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Proteínas Mitocondriais/biossíntese , Regiões Promotoras Genéticas/efeitos dos fármacosRESUMO
Adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a cytoplasmic protein that plays a critical role in the maintenance of energy homeostasis. However, its role in the nucleus is still largely unknown. Here, we showed that AMPKα2 translocated into the nucleus during muscle differentiation. We also showed that upon treatment with 5-aminoimidazole-4-carboxy-amide-1-d-ribofuranoside (AICAR), an AMPK activator, AMPK rapidly translocated into the nucleus in rat myoblast L6 cells. On the other hand, the AMPKα2 phosphorylation-defective mutant did not translocate into the nucleus. Knockdown of AMPKα2 suppressed the differentiation-induced expression of myogenin, a differentiation marker. A physiological AMPK activator, metformin, also induced the translocation of AMPKα2 into the nucleus. Both inhibition and knockdown of AMPKα2 suppressed metformin-mediated glucose uptake. In addition, AMPKα2 was shown to directly interact with the heterogeneous nuclear ribonucleoprotein H (hnRNP H). AICAR treatment increased the phosphorylation of hnRNP H. Metformin increased the interaction between AMPKα2 and hnRNP H in the nucleus. Knockdown of hnRNP H blocked metformin-induced glucose uptake. In summary, these results demonstrate that AMPKα2 translocates into the nucleus via phosphorylation, AMPKα2 interacts with and phosphorylates hnRNP H in the nucleus, and such a protein-protein interaction modulates metformin-mediated glucose uptake.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Núcleo Celular/metabolismo , Glucose/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/metabolismo , Metformina/farmacologia , Transcitose/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Diferenciação Celular , Linhagem Celular , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/antagonistas & inibidores , Ribonucleoproteínas Nucleares Heterogêneas Grupo F-H/genética , Músculo Esquelético/citologia , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ribonucleotídeos/farmacologiaRESUMO
Rhus verniciflua STOKES (RVS) is used as an anti-cancer agent in traditional herbal medicine. However, the underlying molecular mechanism of its action is poorly understood. Here, we elucidated the mechanism of the anti-cancer mechanism of RVS in MCF-7 human breast cancer cells. We found that RVS increased the phosphorylation of AMP-activated protein kinase (AMPK) and downstream acetyl-CoA carboxylase (ACC) and suppressed cell viability in an AMPK-dependent fashion. RVS also induced an increase in reactive oxygen species (ROS) levels. RVS-induced AMPK phosphorylation was not observed in the presence of N-acetyl-cysteine (NAC), which indicated that ROS is associated with RVS-induced AMPK phosphorylation. In addition, fluorescent staining (Annexin V/propidium iodide) revealed that RVS increased the expression of Annexin V, which indicates that RVS leads to cancer-induced apoptosis. Moreover, RVS increased the phosphorylation of p53 and the expression of Bax. The inhibition of AMPK blocked RVS-induced p53 phosphorylation and Bax expression, which suggests that AMPK is involved in RVS-induced cancer apoptosis. Taken together, these results demonstrate that RVS has anti-tumor effects on MCF-7 cells through an AMPK-signaling pathway.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Acetil-CoA Carboxilase/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Casca de Planta/química , Extratos Vegetais/farmacologia , Rhus/química , Acetilcisteína/farmacologia , Anexina A5/biossíntese , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células MCF-7 , Fosforilação/efeitos dos fármacos , Extratos Vegetais/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Proteína X Associada a bcl-2/biossínteseRESUMO
BACKGROUND: Colorectal cancer (CRC) arises as a consequence of genetic events such as gene mutation and epigenetic alteration. The aim of this study was to identify new hypermethylated candidate genes and methylation-based therapeutic targets using vincristine in CRC. METHODS: We analyzed the methylation status of 27,578 CpG sites spanning more than 14,000 genes in CRC tissues compared with adjacent normal tissues and normal colon tissues using Illumina bead chip array. Twenty-one hypermethylated genes and 18 CpG island methylator phenotype markers were selected as candidate genes. The methylation status of 39 genes was validated by quantitative methylation-specific polymerase chain reaction in CRC tissues, adjacent normal tissues, normal colon cells, and three CRC cell lines. Of these, 29 hypermethylated candidate genes were investigated using the demethylating effects of 5-aza-2'-deoxycytidine (5-aza-dC) and vincristine in CRC cells. RESULTS: Thirty-two out of 39 genes were hypermethylated in CRC tissues compared with adjacent normal tissues. Vincristine induced demethylation of methylated genes in CRC cells to the same extent as 5-aza-dC. The mRNA expression of AKR1B1, CHST10, ELOVL4, FLI1, SOX5, STK33, and ZNF304 was restored by treatment with 5-aza-dC and vincristine. CONCLUSION: These results suggest that these novel hypermethylated genes AKR1B1, CHST10, ELOVL4, SOX5, STK33, and ZNF304 may be potential methylation biomarkers and therapeutic targets of vincristine in CRC.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias Colorretais/genética , Metilação de DNA , Vincristina/farmacologia , Idoso , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Ilhas de CpG , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Epigênese Genética , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Neoplásicos , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNARESUMO
BACKGROUND: Methylation-mediated inactivation of tumor-suppressor genes is a critical event during the pathogenesis of many malignancies. Vincristine is a conventional anticancer drug used to treat various types of cancers. However, few studies describe the epigenetic-based effects of vincristine. In this study, changes in the methylation of runt-related transcription factor-3 (RUNX3) were investigated in CCD18Co normal colon cells and DLD-1 colorectal adenocarcinoma cells. MATERIALS AND METHODS: CCD18Co and DLD-1 cells were treated with vincristine, and the methylation status was assessed using quantitative methylation-specific polymerase chain reaction (QMSP). Eleven normal colon tissues and 105 colorectal cancer tissues were investigated by methylation and mRNA expression of RUNX3 using QMSP and real-time reverse transcription polymerase chain reaction (real time-PCR). RESULTS: RUNX3 was demethylated after vincristine treatment in DLD-1 cells. The expression of RUNX3 mRNA was down-regulated in DLD-1 cells because of DNA hypermethylation, but was restored after vincristine treatment. In addition, hypermethylation of RUNX3 was detected in 70 out of 105 colorectal carcinomas (66.7%). RUNX3 hypermethylation was greater in colon cancer tissues than in rectal cancer tissues. The expression of RUNX3 mRNA was reduced in 68 out of 105 colorectal cancer tissues (64.8%). CONCLUSION: These results demonstrate that vincristine demethylates RUNX3 in colorectal adenocarcinoma cells, and restores its expression.