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1.
Mol Cells ; 44(3): 146-159, 2021 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-33795533

RESUMO

DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Transporte/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias do Colo do Útero/genética , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas de Transporte/metabolismo , Proliferação de Células , Regulação para Baixo , Feminino , Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Transfecção , Ubiquitina-Proteína Ligases/genética , Neoplasias do Colo do Útero/metabolismo
2.
Biochem Biophys Res Commun ; 526(4): 1061-1068, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32312517

RESUMO

Persistent infection with high-risk strains of human papillomavirus (HPV) is the primary cause of cervical cancer, the fourth most common cancer among women worldwide. Two oncoproteins encoded by the HPV genome, E6 and E7, are required for epigenetic modifications that promote cervical cancer development. We found that knockdown of HPV E6/E7 by siRNA reduced the levels of ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) but increased the levels of gelsolin (GSN) in early stage cervical cancer cells. In addition, we found that UHRF1 levels were increased and GSN levels were decreased in early stage cervical cancer compared with those in normal cervical tissues, as shown by Western blot analysis, immunohistochemistry, and analysis of the Oncomine database. Moreover, knockdown of UHRF1 resulted in increased cell death in cervical cancer cell lines. Treatment of E6/E7-transformed HaCaT (HEK001) cells and HeLa cells with the DNA-hypomethylating agent 5-aza-2'-deoxycytidine and the histone deacetylase inhibitor Trichostatin A increased GSN expression levels. UHRF1 knockdown in HEK001 cells by siRNA or the UHRF1 antagonist thymoquinone increased GSN levels, induced cell cycle arrest and apoptosis, and increased the levels of p27 and cleaved PARP. Those results indicate that upregulation of UHRF1 by HPV E6/E7 causes GSN silencing and a reduction of cell death in early stage cervical cancer, suggesting that GSN might be a useful therapeutic target in early stage cervical cancer.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Gelsolina/metabolismo , Inativação Gênica , Ubiquitina-Proteína Ligases/metabolismo , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Benzoquinonas/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/antagonistas & inibidores , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteínas E7 de Papillomavirus/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores
3.
Anim Cells Syst (Seoul) ; 23(4): 302-309, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31489252

RESUMO

Metformin is a widely used drug for the treatment of type 2 diabetes. Antidiabetic drugs are also known to influence cancer progression, as high glucose levels affect both cancer and diabetes. Metformin induces cell cycle arrest in cancer cells, but the underlying mechanism remains unclear in cervical cancer system. Here, we examined how metformin affects cell cycle arrest and apoptosis in cervical cancer cells. Western blot analysis showed that levels of O-linked N-acetylglucosamine (O-GlcNAc) and O-GlcNAc transferase (OGT) were increased in cervical cancer cells; these effects were reversed by metformin treatment. Immunoprecipitation analysis was used to examine the interplay between O-GlcNAcylation and phosphorylation in HeLa cells, revealing that metformin decreased O-GlcNAcylated AMP-activated protein kinase (AMPK) and increased levels of phospho-AMPK compared to untreated cells. These results were associated with decreased cell cycle arrest and apoptotic cell death in HeLa cells, as shown by flow cytometry. Moreover, 6-diazo-5-oxo-L-norleucine (a glutamine fructose-6-phosphate aminotransferase inhibitor) or thiamet G (an O-GlcNAcase inhibitor) decreased or increased levels of O-GlcNAcylated AMPK, and increased or decreased levels of phosphorylated AMPK, respectively, suggesting that O-GlcNAc modification affects AMPK activation. Of note, we found that metformin treatment of HeLa cells increased the levels of p21 and p27 (which are AMPK-dependent cell cycle inhibitors), leading to increased cell cycle arrest and apoptosis in HeLa cells compared to untreated cells. These findings suggest that metformin may serve as a useful antiproliferative drug in cervical cancer cells, with potential therapeutic benefit.

4.
Exp Neurobiol ; 28(2): 270-278, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31138994

RESUMO

Chronic immobilization stress (CIS) induces low levels of glutamate (Glu) and glutamine (Gln) and hypoactive glutamatergic signaling in the mouse prefrontal cortex (PFC), which is closely related to the Glu-Gln cycle. A Gln-supplemented diet ameliorates CIS-induced deleterious changes. Here, we investigated the effects of CIS and Gln supplementation on Glu-Gln cycle-related proteins to characterize the underlying mechanisms. Using the CIS-induced depression mouse model, we examined the expression of 11 proteins involved in the Glu-Gln cycle in the PFC. CIS decreased levels of glutamate transporter 1 (GLT1) and sodium-coupled neutral amino acid transporter (SNAT) 1, SANT2, SNAT3, and SNAT5. Gln supplementation did not affect the non-stressed group but significantly increased GLT1 and SNATs of the stressed group. By immunohistochemical analysis, we confirmed that SNAT1 and SNAT2 were decreased in neurons and GLT1, SNAT3, and SNAT5 were decreased in astrocytes in the medial PFC of the stressed group, but Gln-supplemented diet ameliorated these decrements. Collectively, these results suggest that CIS may cause depressive-like behaviors by decreasing Glu and Gln transportation in the PFC and that a Gln-supplemented diet could prevent the deleterious effects of CIS.

5.
Sci Rep ; 9(1): 252, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30670758

RESUMO

Glutamatergic synapses constitute a major excitatory neurotransmission system and are regulated by glutamate/glutamine (Gln) cycling between neurons and astrocytes. Gln synthetase (GS) produced by astrocytes plays an important role in maintaining the cycle. However, the significance of GS during synaptogenesis has not been clarified. GS activity and expression significantly increase from postnatal day (PD) 7 to 21, and GS is expressed prior to glial fibrillary acidic protein (GFAP) and is more abundant than GFAP throughout synaptogenesis. These observations suggest that GS plays an important role in synaptogenesis. We investigated this by inhibiting GS activity in neonatal mice and assessed the consequences in adult animals. Lower expression levels of GS and GFAP were found in the CA3 region of the hippocampus but not in the CA1 region. Moreover, synaptic puncta and glutamatergic neurotransmission were also decreased in CA3. Behaviorally, mice with inhibited GS during synaptogenesis showed spatial memory-related impairment as adults. These results suggest that postnatal GS activity is important for glutamatergic synapse development in CA3.


Assuntos
Glutamato-Amônia Ligase/metabolismo , Neurogênese/fisiologia , Memória Espacial/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Astrócitos/metabolismo , Técnicas de Observação do Comportamento , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Região CA3 Hipocampal/crescimento & desenvolvimento , Região CA3 Hipocampal/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Glutamato-Amônia Ligase/antagonistas & inibidores , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Masculino , Metionina Sulfoximina/farmacologia , Camundongos , Modelos Animais , Neurogênese/efeitos dos fármacos , Neurônios/metabolismo , Memória Espacial/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos
6.
Neuropharmacology ; 143: 143-152, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30266598

RESUMO

Emerging evidence has shown the low levels of glutamate (Glu) and glutamine (Gln) and the hypoactivity in the cortex of patients with depression. The hypoactivity is closely related with low frequency of glutamatergic signaling that is affected by the levels of Glu and Gln. Thus, we hypothesized that there might be a causality among low levels of Glu and Gln, hypoactive glutamatergic neurotransmissions, and depressive behaviors. Here, we found low Glu and Gln levels and low frequency of spontaneous excitatory postsynaptic current (sEPSC) of glutamatergic neurons in the medial prefrontal cortex (mPFC) of chronic immobilization stress (CIS)-induced depressed mice. The depressed mice also showed hypoactive Gln synthetase (GS). Inhibition of GS by methionine sulfoximine (MSO) decreased Glu and Gln levels and increased depressive behaviors with low frequency of sEPSC in the mPFC, indicating that Glu and Gln decrements cause hypoactive glutamatergic neurotransmissions and depressive behaviors. Both Glu and Gln could increase sEPSC of glutamatergic neurons in the mPFC on slice patch, but only Gln overcame MSO to increase sEPSC, suggesting that exogenous Gln would recover CIS-induced low frequency of sEPSC caused by hypoactive GS and act as an antidepressant. Expectedly, Gln supplementation showed antidepressant effects against CIS; it increased glutamatergic neurotransmissions with Glu and Gln increment in the mPFC and attenuated depressive behaviors. Moreover, selective glutamatergic activation in the mPFC by optogenetics decreased depressive behavior. In conclusion, depressive behaviors evoked by chronic stress were due to hypoactive glutamatergic neurons in the mPFC caused by low levels of Glu and Gln, and exogenous Gln can be used as an alternative antidepressant to increase glutamatergic neurotransmission.


Assuntos
Transtorno Depressivo/metabolismo , Transtorno Depressivo/terapia , Ácido Glutâmico/metabolismo , Glutamina/administração & dosagem , Glutamina/metabolismo , Córtex Pré-Frontal/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Transtorno Depressivo/patologia , Suplementos Nutricionais , Glutamato-Amônia Ligase/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/metabolismo , Neurônios/patologia , Optogenética , Córtex Pré-Frontal/patologia , Restrição Física , Estresse Psicológico/metabolismo , Estresse Psicológico/patologia , Estresse Psicológico/terapia , Transmissão Sináptica/fisiologia , Técnicas de Cultura de Tecidos
7.
Biochem Biophys Res Commun ; 503(3): 1307-1314, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30017190

RESUMO

Oxidative stress plays an important role in the development of diabetic retinopathy. Here, we examined whether α-lipoic acid (α-LA), a natural antioxidant, attenuated retinal injury in diabetic mice. The α-LA was orally administered to control mice or mice with streptozotocin-induced diabetes. We found that α-LA reduced oxidative stress, decreased and increased retinal 4-hydroxy-2-nonenal and glutathione peroxidase, respectively, and inhibited retinal cell death. Concomitantly, α-LA reversed the decreased activation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase, and increased the levels of peroxisome proliferator-activated receptor delta and sirtuin3 in diabetic mouse retinas, similar to results shown after metformin treatment of retinal pigment epithelial cells (RPE) exposed to high glucose. Moreover, α-LA lowered the levels of O-linked ß-N-acetylglucosamine transferase (OGT) and thioredoxin-interacting protein (TXNIP) in diabetic retinas that were more pronounced after metformin treatment of RPE cells. Importantly, α-LA lowered interactions between AMPK and OGT as shown by co-immunoprecipitation analyses, and this was accompanied by less cell death as measured by double immunofluorescence staining by terminal deoxynucleotide transferase-mediated dUTP nick-end labelling and OGT or TXNIP in retinal ganglion cells. Consistently, α-LA lowered the levels of cleaved poly(ADP-ribose) polymerase and pro-apoptotic marker cleaved caspase-3 in diabetic retinas. Our results indicated that α-LA reduced retinal cell death partly through AMPK activation or OGT inhibition in diabetic mice.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Retina/citologia , Retina/efeitos dos fármacos , Ácido Tióctico/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Administração Oral , Animais , Morte Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/metabolismo , Retina/metabolismo , Retina/patologia , Estreptozocina , Ácido Tióctico/administração & dosagem
8.
Oncotarget ; 9(4): 4625-4636, 2018 Jan 12.
Artigo em Inglês | MEDLINE | ID: mdl-29435130

RESUMO

O-linked N-acetylglucosamine transferase (OGT) expression is increased in various cancer types, indicating the potential importance of O-GlcNAcylation in tumorigenesis. Secretory clusterin (sCLU) is involved in cancer cell proliferation and drug resistance, and recently, liver X receptors (LXRs) and sterol response element binding protein-1 (SREBP-1) were reported to regulate sCLU transcription. Here, we found that sCLU is significantly increased in cervical cancer cell lines, which have higher expression levels of O-GlcNAc and OGT than keratinocytes. OGT knockdown decreased expression of LXRs, SREBP-1 and sCLU through hypo-O-GlcNAcylation of LXRs. Additionally, treatment with Thiamet G, O-GlcNAcase OGA inhibitor, increased expression of O-GlcNAcylation and sCLU, and high glucose increased levels of LXRs, SREBP-1 and sCLU in HeLa cells. Moreover, OGT knockdown induced G0/G1 phase cell cycle arrest and late apoptosis in cisplatin-treated HeLa cells, and decreased viability compared to OGT intact HeLa cells. Taken together, these findings suggest that OGT, O-GlcNAcylated LXRs, and SREBP-1 increase sCLU expression in cervical cancer cells, which contributes to drug resistance.

9.
Anat Cell Biol ; 51(4): 274-283, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30637162

RESUMO

Hyper-O-GlcNAcylation is a general feature of cancer which contributes to various cancer phenotypes, including cell proliferation and cell growth. Quercetin, a naturally occurring dietary flavonoid, has been reported to reduce the proliferation and growth of cancer. Several reports of the anticancer effect of quercetin have been published, but there is no study regarding its effect on O-GlcNAcylation. The aim of this study was to investigate the anticancer effect of quercetin on HeLa cells and compare this with its effect on HaCaT cells. Cell viability and cell death were determined by MTT and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling assays. O-GlcNAcylation of AMP-activated protein kinase (AMPK) was examined by succinylated wheat germ agglutinin pulldown and immunoprecipitation. Immunofluorescence staining was used to detect the immunoreactivitiy of O-linked N-acetylglucosamine transferase (OGT) and sterol regulatory element binding protein 1 (SREBP-1). Quercetin decreased cell proliferation and induced cell death, but its effect on HaCaT cells was lower than that on HeLa cells. O-GlcNAcylation level was higher in HeLa cells than in HaCaT cells. Quercetin decreased the expression of global O-GlcNAcylation and increased AMPK activation by reducing the O-GlcNAcylation of AMPK. AMPK activation due to reduced O-GlcNAcylation of AMPK was confirmed by treatment with 6-diazo-5-oxo-L-norleucine. Our results also demonstrated that quercetin regulated SREBP-1 and its transcriptional targets. Furthermore, immunofluorescence staining showed that quercetin treatment decreased the immunoreactivities of OGT and SREBP-1 in HeLa cells. Our findings demonstrate that quercetin exhibited its anticancer effect by decreasing the O-GlcNAcylation of AMPK. Further studies are needed to explore how quercetin regulates O-GlcNAcylation in cancer.

10.
Dev Reprod ; 21(3): 259-267, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29082341

RESUMO

Present study aimed to determine the effect of 'bitter melon', a popularly used fruit in Bangladesh and several other Asian countries, on high-fat-diet-induced type 2 diabetes. To investigate the effect, ethanol extract from bitter melon (BME) as a dietary supplement with mouse chow was used. BME was found to significantly attenuate the high-fat diet (HFD) -induced body weight and total fat mass. BME also effectively reduced the insulin resistance induced by the HFD. Furthermore, dietary supplementation of BME was highly effective in increasing insulin sensitivity and reducing hepatic fat and obesity. These results indicate that BME could be effective in attenuating type 2 diabetes and could therefore be a preventive measure against type 2 diabetes.

11.
J Med Food ; 20(10): 989-1001, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29040017

RESUMO

Aralia elata (Miq) Seem (AES) is a medicinal plant used in traditional Chinese and Korean medicine for the treatment of several diseases, including diabetes. This study aimed to investigate the neuroprotective effect of AES extract against high glucose-induced retinal injury in diabetic mice. AES extract (20 and 100 mg/kg body weight) was orally administered to control mice or mice with streptozotocin-induced diabetes. Protein levels of O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT), carbohydrate-responsive element-binding protein (ChREBP), sterol regulatory element-binding protein (SREBP)-1, thioredoxin-interacting protein (TXNIP), fatty acid synthase (FAS), and acetyl CoA carboxylase (ACC) were analyzed by western blotting. Colocalization of terminal deoxynucleotide transferase-mediated dUTP nicked-end labeling (TUNEL)-positive ganglion cells and OGT, ChREBP, or TXNIP were monitored using double immunofluorescence analysis. Interaction between ChREBP and OGT was assessed using coimmunoprecipitation analysis. AES extract protected the retinas from neuronal injury and decreased levels of OGT, ChREBP, TXNIP, SREBP-1, FAS, and ACC in the diabetic retinas. AES extract reduced colocalization of TUNEL-positive ganglion cells and OGT, ChREBP, or TXNIP in the diabetic retinas. Coimmunoprecipitation analysis indicated that AES extract reduced interaction between ChREBP and OGT and attenuated ganglion cell death in diabetic retinas. Moreover, the ChREBP that colocalized with OGT or the TUNEL signal was significantly decreased in diabetic mice treated with AES extract. These findings show that AES extract can alleviate OGT-, ChREBP-, TXNIP-, or SREBP-1-related retinal injury in diabetic retinopathy.


Assuntos
Aralia/química , Retinopatia Diabética/tratamento farmacológico , N-Acetilglucosaminiltransferases/metabolismo , Extratos Vegetais/administração & dosagem , Retina/enzimologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Morte Celular/efeitos dos fármacos , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Glucose/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , N-Acetilglucosaminiltransferases/genética , Retina/citologia , Retina/efeitos dos fármacos , Retina/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo
12.
Int J Ophthalmol ; 10(8): 1203-1211, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28861343

RESUMO

AIM: To investigate the role of O-GlcNAcylation of nuclear factor-kappa B (NF-κB) in retinal ganglion cell (RGC) death and analysedthe effect of Aralia elata (AE) on neurodegeneration in diabetic mice. METHODS: C57BL/6mice with streptozotocin-induced diabetes were fed daily with AE extract or control (CTL) diet at the onset of diabetes mellitus (DM). Two months after injection of streptozotocin or saline, the degree of cell death and the expression of O-GlcNAc transferase (OGT), N-acetyl-b-D-glucosaminidase (OGA), O-GlcNAcylated proteins, and O-GlcNAcylation of NF-κB were examined. RESULTS: AE did not affect the metabolic status of diabetic mice. The decrease in the inner retinal thickness (P<0.001 vs CTL, P<0.01 vs DM) and increases in RGCs with terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (P<0.001 vs CTL, P<0.0001 vs DM), glial activation, and active caspase-3 (P<0.0001 vs CTL, P<0.0001 vs DM) were blocked in diabetic retinas of AE extract-fed mice. Expression levels of protein O-GlcNAcylation and OGT were increased in diabetic retinas (P<0.0001 vs CTL), and the level of O-GlcNAcylation of the NF-κB p65 subunit was higher in diabetic retinas than in controls (P<0.0001 vs CTL). AE extract downregulated O-GlcNAcylation of NF-κB and prevented neurodegeneration induced by hyperglycemia (P<0.0001 vs DM). CONCLUSION: O-GlcNAcylation of NF-κB is concerned in neuronal degeneration and that AE prevents diabetes-induced RGC apoptosis via downregulation of NF-κB O-GlcNAcylation. Hence, O-GlcNAcylation may be a new object for the treatment of DR, and AE may have therapeutic possibility to prevent diabetes-induced neurodegeneration.

13.
Sci Rep ; 7(1): 7837, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28798347

RESUMO

Recent studies have shown that overexpression of tonicity-responsive enhancer binding protein (TonEBP) is associated with many inflammatory diseases, including diabetes mellitus, which causes neuroinflammation in the hippocampus as well as hepatic steatosis. However, the exact mechanism in diabetic neuroinflammation is unknown. We report that haploinsufficiency of TonEBP inhibits hepatic and hippocampal high-mobility group box-1 (HMGB1) expression in diabetic mice. Here, mice were fed a high-fat diet (HFD) for 16 weeks and received an intraperitoneal injection of 100 mg/kg streptozotocin (STZ) and followed by continued HFD feeding for an additional 4 weeks to induce hyperglycemia and hepatic steatosis. Compared with wild-type diabetic mice, diabetic TonEBP+/- mice showed decreased body weight, fat mass, hepatic steatosis, and macrophage infiltration. We also found that adipogenesis and HMGB1 expression in the liver and hippocampus were lower in diabetic TonEBP+/- mice compared with the wild type. Furthermore, iba-1 immunoreactivity in the hippocampus was decreased in diabetic TonEBP+/- mice compared with that in the wild type. Our findings suggest that TonEBP haploinsufficiency suppresses diabetes-associated hepatic steatosis and neuroinflammation.


Assuntos
Diabetes Mellitus Experimental/complicações , Encefalite/patologia , Fígado Gorduroso/complicações , Haploinsuficiência , Hipocampo/patologia , Fatores de Transcrição/genética , Animais , Distribuição da Gordura Corporal , Peso Corporal , Dieta Hiperlipídica , Encefalite/induzido quimicamente , Expressão Gênica , Proteína HMGB1/biossíntese , Camundongos
14.
Biochem Biophys Res Commun ; 492(3): 397-403, 2017 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-28843855

RESUMO

Retinal degeneration is an early feature of diabetic retinopathy, the major cause of blindness in the developed world. Here we investigated how the widely used antidiabetic drug metformin reduces retinal injury in diabetic mice. Metformin was orally administered to control mice or mice with streptozotocin-induced diabetes. Western blot analysis showed that levels of O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) and other related proteins such as carbohydrate-responsive element-binding protein (ChREBP) and thioredoxin-interacting protein (TXNIP) were significantly increased, and nuclear factor kappaB (NF-κB) and poly (ADP-ribose) polymerase (PARP) were activated in the diabetic retinas or retinal pigment epithelial (RPE) cells exposed to high glucose compared to controls. More importantly, RPE cells exposed to high glucose and treated with thiamet-G had higher levels of those proteins, demonstrating the role of elevated O-GlcNAcylation. Double immunofluorescence analysis revealed increased co-localization of terminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL)-positive ganglion cells and OGT, ChREBP, TXNIP, or NF-κB in diabetic retinas compared to control retinas. Co-immunoprecipitation analysis showed that interaction between OGT and ChREBP or NF-κB was increased in diabetic retinas compared to control retinas, and this was accompanied by more cell death. Notably, metformin attenuated the increases in protein levels; reduced co-localization of TUNEL-positive ganglion cells and OGT, ChREBP, TXNIP, or NF-κB; and reduced interaction between OGT and ChREBP or NF-κB. Our results indicate that OGT inhibition might be one of the mechanisms by which metformin decreases retinal cell death.


Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Experimental/patologia , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Retina/efeitos dos fármacos , Animais , Glicemia/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Células Cultivadas , Diabetes Mellitus Experimental/induzido quimicamente , Humanos , Hipoglicemiantes/administração & dosagem , Masculino , Metformina/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Retina/citologia , Retina/patologia , Estreptozocina , Aumento de Peso/efeitos dos fármacos
15.
Mol Cells ; 40(7): 476-484, 2017 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-28681591

RESUMO

C-X-C chemokine receptor 4 (CXCR4) stimulates cancer metastasis. NF-κB regulates CXCR4 expression in cancer cells, and O-GlcNAc modification of NF-κB promotes its transcriptional activity. Here, we determined whether CXCR4 expression is affected by O-GlcNAcylation of NF-κB in lung metastasis of cervical cancer. We found elevated levels of O-linked-N-actylglucosamine transferase (OGT) and O-GlcNAcylation in cervical cancer cells compared to those in non-malignant epithelial cells and detected increased expression of NF-κB p65 (p65) and CXCR4 in cervical cancer cells. Knockdown of OGT inhibited the O-GlcNAcylation of p65 and decreased CXCR4 expression levels in HeLa cells. Thiamet G treatment increased O-GlcNAcylated p65, which subsequently enhanced CXCR4 expression levels. Inhibition of O-GlcNAcylation by 6-Diazo-5-oxo-L-norleucine (DON) treatment decreased p65 activation, eventually inhibiting CXCR4 expression in HeLa cells. Lung tissues from mice engrafted with OGT-knockdown HeLa cells (shOGT) exhibited lower expression of Ki-67 and HPV E6 and E7 oncogenes compared to lung tissues from mice engrafted with control HeLa cells (shCTL). In addition, lung tissues from mice engrafted with shOGT cells exhibited lower p65 and CXCR4 immunoreactivity compared to tissues from mice engrafted with shCTL cells. Taken together, our data suggest that p65 O-GlcNAcylation promotes lung metastasis of cervical cancer cells by activating CXCR4 expression.


Assuntos
Neoplasias Pulmonares/secundário , NF-kappa B/metabolismo , Receptores CXCR4/metabolismo , Regulação para Cima , Neoplasias do Colo do Útero/patologia , Acetilglucosamina/metabolismo , Animais , Feminino , Técnicas de Silenciamento de Genes , Glicosilação , Células HeLa , Humanos , Neoplasias Pulmonares/metabolismo , Camundongos Nus , N-Acetilglucosaminiltransferases/metabolismo , Ligação Proteica , Fator de Transcrição RelA/metabolismo
16.
Curr Eye Res ; 42(7): 987-994, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28632030

RESUMO

PURPOSE: Nuclear factor-kappa B (NF-κB) has been proposed as a therapeutic target for the treatment of cataracts. The authors investigated the relationship between nuclear factor of activated T cells 5 (NFAT5) and NF-κB in ultraviolet B (UVB)-irradiated human lens epithelial (HLE) cells. METHODS: Human lens epithelial B-3 (HLE-B3) cells were exposed to UVB light at a dose of 10 mJ/cm2 and then incubated for 24 h. Cell viability was assessed by using the Cell Counting Kit-8 (CCK-8) assay. Gene expression level of NFAT5 was determined using real-time quantitative polymerase chain reaction (qPCR). Protein expression levels of NFAT5, NF-κB p65, and α-smooth muscle actin (α-SMA) and the association of NFAT5 with the NF-κB p65 subunit were measured by Western blot analysis and a co-immunoprecipitation assay, respectively. The cellular distribution of NFAT5 and NF-κB p65 was examined by triple immunofluorescence staining. RESULTS: At 24 h after UVB exposure, cell viability significantly decreased in a dose-dependent manner, and UVB light (15 and 20 mJ/cm2) significantly increased the ROS generation. UVB irradiation increased NFAT5 mRNA and protein levels and increased phosphorylation of NF-κB in HLE-B3 cells. α-SMA protein levels were increased in the irradiated cells. In addition, NFAT5 and NF-κB translocated from the cytoplasm to the nucleus, and binding between the p65 subunit and NFAT5 was increased. CONCLUSIONS: Exposure to UVB radiation induces nuclear translocation and stimulates binding between NFAT5 and NF-κB proteins in HLE-B3 cells. These interactions may form part of the biochemical mechanism of cataractogenesis in UVB-irradiated HLECs.


Assuntos
Catarata/genética , Regulação da Expressão Gênica , Cápsula do Cristalino/metabolismo , NF-kappa B/genética , Fatores de Transcrição NFATC/genética , RNA/genética , Raios Ultravioleta , Western Blotting , Catarata/patologia , Catarata/radioterapia , Sobrevivência Celular , Células Cultivadas , Imuno-Histoquímica , Imunoprecipitação , Líquido Intracelular/metabolismo , Cápsula do Cristalino/patologia , Cápsula do Cristalino/efeitos da radiação , Microscopia Confocal , NF-kappa B/biossíntese , NF-kappa B/efeitos da radiação , Fatores de Transcrição NFATC/biossíntese , Fatores de Transcrição NFATC/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
17.
Biochem Biophys Res Commun ; 483(2): 793-802, 2017 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-27845045

RESUMO

High-risk human papilloma virus (HPV) 16/18 infections are often found in lung cancer. The cellular mechanisms involved in the metastatic spread of HPV-infected cervical cancer cells remain largely elusive. High O-linked-N-acetylglucosamine (O-GlcNAc) modification has also been observed in lung cancer. In the present study, we assessed the relationship between O-GlcNAc transferase (OGT) and HPV 16/18 E6/E7, or C-X-C chemokine receptor type 4 (CXCR4), in HeLa cells and in lungs of xenografted mice. Depleting OGT with an OGT-specific shRNA significantly decreased levels of E6 and E7 oncoproteins in HeLa cells and xenograft tumors, and reduced tumor formation in vivo. Western blotting and immunofluorescence analysis showed significantly decreased expression levels of E6, E7, and HCF-1 in the lungs of xenografted mice treated with an OGT-specific shRNA compared to those treated with non-targeting shRNA. Additionally, levels of E7 or OGT co-localized with Ki-67 were significantly decreased in the lungs of xenografted mice treated with OGT-specific shRNA compared to those treated with non-targeting shRNA. Moreover, levels of CXCR4 were significantly decreased in HeLa cells and in the lungs of xenografted mice treated with OGT-specific shRNA compared to those treated with non-targeting shRNA; this may be related to reduced adhesion or invasion of circulating HPV-positive tumor cells. These findings provide novel evidence that OGT functions in metastatic spread of HPV E6/E7-positive tumor cells to the lungs through E6/E7, HCF-1 and CXCR4, suggesting OGT might be a therapeutic target for HPV-positive lung cancer.


Assuntos
Papillomavirus Humano 16/patogenicidade , Papillomavirus Humano 18/patogenicidade , Neoplasias Pulmonares/etiologia , N-Acetilglucosaminiltransferases/metabolismo , Infecções por Papillomavirus/etiologia , Animais , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Xenoenxertos , Fator C1 de Célula Hospedeira/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/virologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Nus , N-Acetilglucosaminiltransferases/antagonistas & inibidores , N-Acetilglucosaminiltransferases/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/metabolismo , Infecções por Papillomavirus/virologia , RNA Interferente Pequeno/genética , Receptores CXCR4/metabolismo , Proteínas Repressoras/metabolismo
18.
Oncotarget ; 7(28): 44596-44607, 2016 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-27331873

RESUMO

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) increases O-GlcNAc modification (O-GlcNAcylation), and transcriptional co-regulator host cell factor 1 (HCF-1) is one of OGT targets. High-risk Human Papillomaviruses (HPVs) encode E6 and E7 oncoproteins, which promote cervical cancer. Here, we tested whether O-GlcNAc modification of HCF-1 affects HPV E6 and E7 expressions and tumorigenesis of cervical cancer. We found that depleting OGT with OGT-specific shRNA significantly decreased levels of E6 and E7 oncoproteins, and cervical cancer tumorigenesis, while OGT overexpression greatly increased levels of E6 and E7 oncoproteins. Notably, OGT overexpression caused dose-dependent increases in the transcriptional activity of E6 and E7, and this activity was decreased when HCF-1 was depleted with HCF-1-specific siRNA. Moreover, OGT depletion reduced proliferation, invasion, and metastasis in cervical cancer cells. Further, high glucose enhanced the interaction between OGT and HCF-1, paralleling increased levels of E6 and E7 in cervical cancer cells. Most importantly, we found that reducing OGT in HeLa cells caused decreased tumor growth in vivo. These findings identify OGT as a novel cellular factor involved in E6 and E7 expressions and cervical cancer tumorigenesis, suggesting that targeting OGT in cervical cancer may have potential therapeutic benefit.


Assuntos
Carcinogênese/metabolismo , N-Acetilglucosaminiltransferases/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Neoplasias do Colo do Útero/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HeLa , Fator C1 de Célula Hospedeira/genética , Fator C1 de Célula Hospedeira/metabolismo , Humanos , Immunoblotting , Pessoa de Meia-Idade , N-Acetilglucosaminiltransferases/genética , Proteínas E7 de Papillomavirus/metabolismo , Interferência de RNA , Proteínas Repressoras/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia
19.
Genes Genet Syst ; 90(6): 335-42, 2016 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-26960969

RESUMO

Fetal alcohol syndrome (FAS) is a condition resulting from excessive drinking by pregnant women. Symptoms of FAS include abnormal facial features, stunted growth, intellectual deficits and attentional dysfunction. Many studies have investigated FAS, but its underlying mechanisms remain unknown. This study evaluated the relationship between alcohol exposure during the synaptogenesis period in postnatal mice and subsequent cognitive function in adult mice. We delivered two injections, separated by 2 h, of ethanol (3 g/kg, ethanol/saline, 20% v/v) to ICR mice on postnatal day 7. After 10 weeks, we conducted a behavioral test, sacrificed the animals, harvested brain tissue and analyzed hippocampal gene expression using a microarray. In ethanol-treated mice, there was a reduction in brain size and decreased neuronal cell number in the cortex, and also cognitive impairment. cDNA microarray results indicated that 1,548 genes showed a > 2-fold decrease in expression relative to control, whereas 974 genes showed a > 2-fold increase in expression relative to control. Many of these genes were related to signal transduction, synaptogenesis and cell membrane formation, which are highlighted in our findings.


Assuntos
Transtornos do Espectro Alcoólico Fetal/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/metabolismo , Álcoois/toxicidade , Animais , Feminino , Transtornos do Espectro Alcoólico Fetal/patologia , Regulação da Expressão Gênica/genética , Hipocampo/efeitos dos fármacos , Hipocampo/patologia , Humanos , Aprendizagem/efeitos dos fármacos , Camundongos , Análise em Microsséries , Gravidez , Transdução de Sinais/efeitos dos fármacos
20.
Biochem Biophys Res Commun ; 472(1): 276-80, 2016 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-26926565

RESUMO

A healthy acute stress response requires both rapid increase and rapid clearance of blood corticosteroids. We previously showed that regulators of G-protein signaling 4 (RGS4), which decreases in the paraventricular nucleus (PVN) during acute stress, forms a complex with the GABAB receptor. In the present study, we show that this decrease in RGS4 levels in the PVN during an acute stress response facilitates the return of blood corticosteroids to basal levels. Moreover, the effect of RGS4 decrease is attenuated by a GABAB receptor antagonist. These results suggest that RGS4 in the PVN regulates blood corticosteroid-related GABAB receptor signaling during the acute stress response.


Assuntos
Núcleo Hipotalâmico Paraventricular/metabolismo , Proteínas RGS/metabolismo , Receptores de GABA-B/metabolismo , Estresse Fisiológico , Animais , Corticosterona/sangue , Antagonistas de Receptores de GABA-B/farmacologia , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Compostos Organofosforados/farmacologia , Núcleo Hipotalâmico Paraventricular/efeitos dos fármacos , Proteínas RGS/antagonistas & inibidores , Proteínas RGS/genética , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacos
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