RESUMEN
α-Glucosidase inhibitors in natural products are one of the promising drugs for the treatment of type 2 diabetes. However, due to the complexity of the matrix, it is challenging to comprehensibly clarify the specific pharmacodynamic substances. In this study, a novel high-throughput inhibitor screening strategy was established based on covalent binding of α-glucosidase on chitosan-functionalized multi-walled carbon nanotubes coupled with high-resolution mass spectrometry. The synthesized MWCNTs@CS@GA@α-Glu was characterized by TEM, SEM, FTIR, Raman, and TG. Performance studies showed that the microreactor exhibited stronger thermostability and pH tolerance than that of the free one while maintaining its inherent catalytic activity. Feasibility study applying a model mixture of known α-glucosidase ligand and non-ligands indicated the selectivity and specificity of the system. By integrating ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-QTOF-MS) with ion mobility mass spectrometry (IMS), 15 ligands were obtained and tentatively identified from Tribulus terrestris L., including 8 steroidal saponins, 4 flavonoids, and 3 alkaloids. These inhibitors were further validated by in vivo experiments and molecular docking simulation.
Asunto(s)
Quitosano , Diabetes Mellitus Tipo 2 , Nanotubos de Carbono , Tribulus , alfa-Glucosidasas/metabolismo , Quitosano/química , Cromatografía Líquida de Alta Presión/métodos , Inhibidores de Glicósido Hidrolasas/farmacología , Simulación del Acoplamiento Molecular , Nanotubos de Carbono/química , Extractos Vegetales/química , Tribulus/química , Tribulus/metabolismoRESUMEN
We previously reported that all-trans-retinoic acid (ATRA) induced apoptosis in N-acetylglucosaminyltransferase V (GnT-V) repressed human hepatocarcinoma 7721 (GnT-V-AS/7721) cells via endoplasmic reticulum (ER) stress. In addition to confirming these findings, we further found that ATRA repressed the expression of betaine-homocysteine methyltransferase (BHMT) and cystathionine-beta-synthase (CBS), which are key enzymes that are involved in homocysteine metabolism, increased the level of intracellular homocysteine, and decreased the glutathione (GSH) level in GnT-V-AS/7721 cells. To investigate the effect of ATRA on homocysteine metabolism, cells were challenged with exogenous homocysteine. In GnT-V-AS/7721 cells with ATRA treatment, a significant elevation of intracellular homocysteine levels suggests that ATRA perturbs homocysteine metabolism in GnT-V-AS/7721 cells and, therefore, sensitizes the cells to homocysteine-induced ER stress. An obvious increase in the levels of GRP78/Bip protein and spliced XBP1 mRNA were observed. Furthermore, we observed that ATRA blunted the homocysteine-induced increase of GSH only in GnT-V-AS/7721 cells. These results demonstrate that ATRA intensifies ER stress and induces apoptosis in GnT-V-AS/7721 cells by disturbing homocysteine metabolism through the down-regulation of CBS and BHMT, depleting the cellular GSH and, in turn, altering the cellular redox status. In addition, we showed that ATRA did not trigger ER stress, induce apoptosis, or affect homocysteine metabolism in L02 cells, which is a cell type that is derived from normal liver tissue. These results provide support for the hypothesis that ATRA is an anticancer agent.
Asunto(s)
Antineoplásicos/farmacología , Carcinoma Hepatocelular/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Homocisteína/metabolismo , Neoplasias Hepáticas/metabolismo , N-Acetilglucosaminiltransferasas/genética , Tretinoina/farmacología , Apoptosis , Betaína-Homocisteína S-Metiltransferasa/genética , Betaína-Homocisteína S-Metiltransferasa/metabolismo , Línea Celular Tumoral , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Chaperón BiP del Retículo Endoplásmico , Humanos , N-Acetilglucosaminiltransferasas/metabolismoRESUMEN
ST13 is a cofactor of heat shock protein 70 (Hsp70). To date, all data since the discovery of ST13 in 1993 until more recent studies in 2007 have proved that ST13 is downregulated in tumors and it was proposed to be a tumor suppressor gene, but no work reported its antitumor effect and apoptotic mechanism. In the work described in this paper, ST13 was inserted into ZD55, an oncolytic adenovirus with the E1B 55-kDa gene deleted, to form ZD55-ST13, which exerts an excellent antitumor effect in vitro and in an animal model of colorectal carcinoma SW620 xenograft. ZD55-ST13 inhibited tumor cells 100-fold more than Ad-ST13 and ZD55-EGFP in vitro. However, ZD55-ST13 showed no damage of normal fibroblast MRC5 cells. In exploring the mechanism of ZD55-ST13 in tumor cell killing, we found that ZD55-ST13-infected SW620 cells formed apoptotic bodies and presented obvious apoptosis phenomena. ZD55-ST13 induced the upregulation of Hsp70, the downregulation of antiapoptotic gene Bcl-2, and the release of cytochrome c. Cytochrome c triggered apoptosis by activating caspase-9 and caspase-3, which cleave the enzyme poly(ADP-ribose) polymerase in ZD55-ST13-infected SW620 cells. In summary, overexpressed ST13 as mediated by oncolytic adenovirus could exert potent antitumor activity via the intrinsic apoptotic pathway and has the potential to become a novel therapeutic for colorectal cancer gene therapy.
Asunto(s)
Adenoviridae/genética , Apoptosis , Proteínas Portadoras/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/terapia , Terapia Genética , Mitocondrias/patología , Viroterapia Oncolítica , Proteínas Supresoras de Tumor/metabolismo , Adenoviridae/fisiología , Animales , Apoptosis/efectos de los fármacos , Proteínas Portadoras/genética , Inhibidores de Caspasas , Caspasas/metabolismo , Línea Celular Tumoral , Citocromos c/metabolismo , Regulación hacia Abajo , Femenino , Genes bcl-2/genética , Proteínas HSP70 de Choque Térmico/genética , Humanos , Ratones , Ratones Desnudos , Proteínas Supresoras de Tumor/genética , Regulación hacia ArribaRESUMEN
After transfection of alpha1,3fucosyltransferase (FucT)-VII cDNA into H7721 human hepatocarcinoma cells, the expression of alpha5, but not beta1 integrin was significantly up-regulated. This was evidenced by the increase of alpha5 integrin on cell surface as well as the increase of alpha5 mRNA and protein in the cells. However, the expressions of sialyl Lewis X (SLe(x), the product of alpha1,3FucT-VII) on both alpha5 and beta1 integrin subunits were unchanged. Concomitantly, the tyrosine autophosphorylated FAK and dephosphorylated Src (FAK and Src involve in the signal transduction of integrin alpha5beta1) were up-regulated, while the Tyr-527 phosphorylated Src was down-regulated. The above-mentioned alterations were correlated to the expressions of alpha1,3FucT-VII in different alpha1,3FucT-VII transfected H7721 cell lines. In addition, after alpha1,3FucT-VII transfection, cell adhesion to fibronectin (Fn) and chemotaxic cell migration were obviously promoted. The cell adhesion could be blocked by alpha5 integrin antibody, and cell migration was obviously attenuated by the antibodies to both alpha5 integrin and SLe(x). These findings suggest that the increased surface alpha5 integrin caused by the up-regulation of alpha5 mRNA promotes the cell adhesion to Fn, cell migratiom, and Fn-induced signaling of alpha5beta1 integrin. The up-regulation of surface SLe(x) originated from the over expression of alpha1,3FucT-VII also led to the stimulation of cell migration. This is the first time to report that alpha1,3FucT-VII can regulate the mRNA expression of integrin.
Asunto(s)
Fucosiltransferasas/metabolismo , Integrina alfa5/genética , Regulación hacia Arriba/genética , Anticuerpos/farmacología , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , ADN Complementario/genética , Citometría de Flujo , Quinasa 1 de Adhesión Focal/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Integrina alfa5/metabolismo , Integrina beta1/metabolismo , Oligosacáridos/metabolismo , Fosforilación/efectos de los fármacos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Antígeno Sialil Lewis X , Transfección , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Lithium, a therapeutic agent for bipolar disorder, can induce G2/M arrest in various cells, but the mechanism is unclear. In this article, we demonstrated that lithium arrested hepatocellular carcinoma cell SMMC-7721 at G2/M checkpoint by inducing the phosphorylation of cdc2 (Tyr-15). This effect was p53 independent and not concerned with the inhibition of glycogen synthase kinase-3 and inositol monophosphatase, two well-documented targets of lithium. Checkpoint kinase 1 (Chk1), a critical enzyme in DNA damage-induced G2/M arrest, was at least partially responsible for the lithium action. The lithium-induced phosphorylation of cdc2 and G2/M arrest was abrogated largely by SB218078, a potent Chk1 inhibitor, as well as by Chk1 siRNA or the over-expression of kinase dead Chk1. Furthermore, lithium-induced cdc25C phosphorylation in 7721 cells and in vitro kinase assay showed that the activity of Chk1 was enhanced after lithium treatment. Interestingly, the increase of Chk1 activity by lithium may be independent of ataxia telangiectasia mutated (ATM)/ATM and Rad3-related (ATR) kinase. This is because no elevated phosphorylation on Chk1 (Ser-317 and Ser-345) was observed after lithium treatment. Moreover, caffeine, a known ATM/ATR kinase inhibitor, relieved the phosphorylation of cdc2 (Tyr-15) by hydroxyurea, but not that by lithium. Our study's results revealed the role of Chk1 in lithium-induced G2/M arrest. Given that Chk1 has been proposed to be a novel tumor suppressor, we suggest that the effect of lithium on Chk1 and cell cycle is useful in tumor prevention and therapy.
Asunto(s)
Carcinoma Hepatocelular/patología , Ciclo Celular/efectos de los fármacos , Ciclina B/metabolismo , Litio/farmacología , Proteínas Quinasas/fisiología , Proteína Quinasa CDC2 , División Celular , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Ciclina B/efectos de los fármacos , Quinasas Ciclina-Dependientes , Fase G2 , Humanos , FosforilaciónRESUMEN
Two H7721 human hepatocarcinoma cell lines showing moderate and high expression of alpha1,3-fucosyltransferase (FucT)-VII cDNA were established and designated FucTVII-M and FucTVII-H, respectively. In alpha1,3-FucT-VII-transfected cells, expression of insulin receptor (InR) alpha- and beta subunits and epidermal growth factor receptor (EGFR) on the cell surface and in cells, as well as the sialyl Lewis X (SLe(x), the product of alpha1,3-FucT-VII) content of the EGFR were unchanged. However the level of SLe(x) on the InR alpha subunit (InR-alpha) was increased dramatically. Tyrosine autophosphorylation of InR-beta , but not EGFR, was elevated. Concomitantly, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), Ser/Thr phosphorylation of protein kinase B (PKB; Akt), p42/44 mitogen-activated protein kinase (MAPK), MAPK kinase (MEK), and the protein of some other signaling molecules, such as phosphoinositide-dependent kinase-1 (PDK-1), novel protein kinase (PKN), c-Raf-1 and beta-catenin were also upregulated. The activities of PKB and transcription factor TCF were concomitantly stimulated. Upregulation of InR signaling molecules and their phosphorylation was correlated with the level of SLe(x) on InR-alpha and alpha1,3-FucT-VII expression in cells. In addition, the phosphorylation intensity and difference in phosphorylation intensity between cells with different levels of alpha1,3-FucT-VII expression were attenuated significantly by the inhibitor of InR tyrosine kinase and by the mAb to SLe(x). Furthermore, insulin-induced signaling was facilitated in alpha1,3-FucT-VII-transfected cells, particularly FucTVII-H. These findings provide strong evidence that alpha1,3-FucT-VII may affect insulin signaling by upregulating the phosphorylation and expression of some signaling molecules involved in the InR-signaling pathway. These effects are likely mediated by its product, SLe(x), on the glycans of the InR. This is the first study to report that changes in the terminal structure of glycans on a surface receptor can modify cell signaling.
Asunto(s)
Fucosiltransferasas/metabolismo , Receptor de Insulina/metabolismo , Línea Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Insulina/metabolismo , Sistema de Señalización de MAP Quinasas , Oligosacáridos/metabolismo , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , Antígeno Sialil Lewis X , Transducción de Señal , Factores de Transcripción TCF/metabolismo , Transfección , Tirosina/química , beta Catenina/metabolismoRESUMEN
OBJECTIVE: Pathological myopia has a genetic background. Previous studies have mapped six loci at 18p11.31, 12q21-23, 7q36, 17q21-22, 4q22-q27 and 2q37.1 in autosomal dominant (AD) pathological myopia. The aim of the present study was to map the mutate gene associated with this disorder in Chinese population. METHODS: A family with AD pathological myopia including 12 individuals, of which 7 members were affected, consented to participate our study. Three hundred and thirty pairs of highly heterozygous microsatellite marker primers were selected for a genome-wide screening. Two-point linkage was calculated by LINKAGE package in an autosomal dominant mode with full penetrance at gene frequency of 0.0133. Multipoint LOD scores were calcu1ated by use of GENEHUNTER program. Genetic distance between marker loci examined was determined on the basis of Genethon linkage map. Haplotype analysis was performed by software of Cyrillic 2.0 based on the lowest recombination principle. RESULTS: Evidence of significant linkage was found on chromosome 15q in the family by two-point linkage analysis. The maximum LOD score was 1.76 with the markers D15S1010, D15S1007 and D15S1042 at a recombination fraction of 0.00. Multipoint linkage analysis also supported existence of linkage on this region with NPL score 5.16. Haplotype analysis refined this myopia locus to a 12 cM interval between D15S1019 and D15S146 on 15q12 - 13. No evidence of linkage was found at any known myopia loci, including AD pathological myopia loci on 18p11.31, 12q21 - 23, 7q36, 17q21 - 22, 4q22 - q27 and 2q37.1, and syndromic myopia loci on 15q15-21, 12q13.11-13.2, 6p21.3, 1q21-31, 1p21 and 21q22.3. CONCLUSIONS: Our study indicates a novel myopia locus on 15q12 - 13. There are 94 known genes locate on this region, screening for sequence of candidate genes within this region will be helpful to find the mutant gene. This study also provides additional support for genetic heterogeneity of this disorder.
Asunto(s)
Mapeo Cromosómico , Ligamiento Genético , Estudio de Asociación del Genoma Completo , Miopía Degenerativa/genética , Adolescente , Adulto , Anciano , Pueblo Asiatico/genética , Cromosomas Humanos Par 15/genética , Femenino , Genotipo , Humanos , Escala de Lod , Masculino , Persona de Mediana Edad , Linaje , Adulto JovenRESUMEN
N-acetylglucosaminyltransferase V (GnT-V) is an important tumorigenesis and metastasis-associated enzyme. To study its biofunction, the GnT-V stably suppressed cell line (GnT-V-AS/7,721) was constructed from 7,721 hepatocarcinoma cells in previous study. In this study, cDNA array gene expression profiles were compared between GnT-V-AS/7,721 and parental 7,721 cells. The data indicated that GnT-V-AS/7,721 showed a characteristic expression pattern consistent with the ER stress. The molecular mechanism of the ER stress was explored in GnT-V-AS/7,721 by the analysis on key molecules in both two unfolded protein response (UPR) pathways. For ATF6 and Ire1/XBP-1 pathway, it was evidenced by the up-regulation of BIP at mRNA and protein level, and the appearance of the spliced form of XBP-1. As for PERK/eIF2alpha pathway, the activation of ER eIF2alpha kinase PERK was observed. To confirm the results from GnT-V-AS/7,721 cells, the key molecules in the UPR were examined again in 7,721 cells interfered with the GnT-V by the specific RNAi treatment. The results were similar with those from GnT-V-AS/7721, indicating that blocking of GnT-V can specifically activate ER stress in 7,721 cells. Rate of (3)H-Man incorporation corrected with rate of (3)H-Leu incorporation in GnT-V-AS/7,721 was down-regulated greatly compared with the control, which demonstrated the deficient function of the enzyme synthesizing N-glycans after GnT-V blocking. Moreover, the faster migrating form of chaperone GRP94 associated with the underglycosylation, and the extensively changed N-glycans structures of intracellular glycoproteins were also detected in GnT-V-AS/7,721. These results supported the mechanism that blocking of GnT-V expression impaired functions of chaperones and N-glycan-synthesizing enzymes, which caused UPR in vivo.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Retículo Endoplásmico/fisiología , Regulación Neoplásica de la Expresión Génica , N-Acetilglucosaminiltransferasas/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Humanos , Proteínas Reguladoras del Hierro/metabolismo , Glicoproteínas de Membrana/metabolismo , N-Acetilglucosaminiltransferasas/fisiología , Análisis de Secuencia por Matrices de Oligonucleótidos , Polisacáridos/biosíntesis , Polisacáridos/química , Pliegue de Proteína , Interferencia de ARN , Factores de Transcripción del Factor Regulador X , Transducción de Señal , Factores de Transcripción/metabolismo , Transfección , Proteína 1 de Unión a la X-Box , eIF-2 Quinasa/metabolismoRESUMEN
Transfection of sense cDNA of N-acetylglucosaminyltransferase V (GnTV) into H7721 human hepatocellular carcinoma cells resulted in the decreased expression of surface sialyl Lewis X (SLe(x)), a sialylated fucose-containing antigen. The enzymatic mechanisms were speculated to be the concomitantly decreased expression of alpha1,3-fucosyltransferase (FucT)-III, -VI, -VII and the branching enzyme of O-glycans, core 2-beta1,6-N-acetylglucosaminyltransferase (C2GnT)-I, -II. These two glycosyltransferase families were suggested to be the key enzymes in the synthesis of SLe(x). The expression of alpha2,3-sialyltransferase (ST3)-IV, but not ST3-I, -II and -III was elevated by sense GnTV. However, it did not cause the increase of SLe(x) synthesis. Transfection of antisense GnTV into H7721 cells showed entirely opposite effects on the expression of above-mentioned SLe(x) and glycosyltransferases as the sense GnTV.
Asunto(s)
Glicosiltransferasas/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Oligosacáridos/metabolismo , Conformación de Carbohidratos , Línea Celular Tumoral , ADN sin Sentido/metabolismo , Humanos , N-Acetilglucosaminiltransferasas/genética , Antígeno Sialil Lewis X , TransfecciónRESUMEN
To explore the suppressive effects of E-cadherin on aggregation and anchorage-independent growth of human lung adenocarcinoma cells, a mammalian expression vector containing full E-cadherin cDNA was constructed and transfected into a metastatic human lung adenocarcinoma cell line A549. RT-PCR and Western blot were used to analyse expression levels of E-cadherin. Cells aggregation and anchorage-independent growth of the tumor cells before and after gene transfection were assessed respectively. The results indicated that stable transfectants showed markedly increased levels of expressed E-cadherin compared with the corresponding sham transfectants. The transfections showed more intense cellular aggregation and anchorage-independent growth.
RESUMEN
Kunming mice inoculated with hepatoma cell (H22) suspension subcutaneously at their right axilla were administered orally with antioxidants such as vitamine E, beta-carotene, glutamine, kappa-selenocarrageenan and polysaccharide-peptide of coriolus (PSP) solution. It was found that the inoculated hepatoma growth was suppressed to various extents. The two kinds of polysaccharide antioxidants improved non-specific immunity, enhanced the nitrogen monoxide (NO) content in plasma and strengthened the inhibition of hepatoma. Above antioxidants added in the culture of 7721 human hepatoma cells inhibited the cell proliferation and inducedits apoptosis. Meanwhile, the activity of glutathione peroxidase (GSH-Px) in the plasma of mice increased and the content of malondialdehyde (MDA) decreased. H(2)O(2) in low concentration improved the cancer cell proliferation and inhanced the expression of Mn-SOD c-fos and c-jun, but led to cells apoptosis or necrosis in high concentration. The mechanism of antioxidants inhibiting tumor growth and improving cancer cells apoptosis might be that, on the one hand, the antioxidants blocked the free radicals signal transduction on cancer cells proliferation, and on the other hand, they improved the release of NO through enhancing the non-specific immunity, so inhibiting the cancer cells proliferation directly.
RESUMEN
The role of alpha1,3fucosyltransferase-VII (alpha1,3 FucT-VII) in cell apoptosis was studied in human hepatocellular carcinoma H7,721 cells. After the cells were transfected with alpha1,3 FucT-VII cDNA, the expression of apoptotic protease, procaspase-3, was decreased, while the anti-apoptotic proteins, phospho-PKB and phospho-Bad were increased as compared with mock (vector) transfected cells, indicating that alpha1,3FucT-VII is a potential anti-apoptotic factor in H7,721 cells. After "alpha1,3FucT-VII" cells were irradiated by UV to induce apoptosis, the anti-apoptotic potential of alpha1,3FucT-VII became more apparent, as evidenced by the less apoptotic cell % and active cleaved caspase-3, more phospho-p38 MAPK and JNK (two anti-apoptotic signaling molecules in H7,721 cells responsible to UV stress) when compared with the "Mock" cells. In contrast, "alpha1,3FucT-VII" cells facilitated the apoptosis induced by all-trans retinoic acid (ATRA), which was verified by the greater sub-G1 (apoptotic cells) peak in flow cytometry analysis, more expressions of active caspase-3 and pro-apoptotic protein Bax, as well as less expressions of anti-apoptotic proteins, Bcl-2 and Bcl-X(L). The up regulation of alpha1,3FucT-VII mRNA and cell surface SLe(x) (alpha1,3FucT-VII product) by UV and down regulation of them by ATRA was speculated to be one of the mechanisms that alpha1,3FucT-VII decreased and increased the susceptibility of apoptosis induced by UV and ATRA respectively.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Carcinoma Hepatocelular/enzimología , Fucosiltransferasas/metabolismo , Neoplasias Hepáticas/enzimología , Tretinoina/farmacología , Rayos Ultravioleta , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/tratamiento farmacológico , Caspasa 3/metabolismo , Línea Celular Tumoral , Células Cultivadas , Fucosiltransferasas/genética , Fucosiltransferasas/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Neoplasias Hepáticas/química , Neoplasias Hepáticas/tratamiento farmacológico , Oligosacáridos/metabolismo , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Antígeno Sialil Lewis X , Transducción de Señal/efectos de los fármacos , Transducción de Señal/efectos de la radiación , Transfección , Tretinoina/químicaRESUMEN
N-Acetylglucosaminyltransferase-V (GnT-V) is a key enzyme in the processing of N-glycans during synthesis of glycoproteins. We have reported that down-regulating GnT-V could induce endoplasmic reticulum stress (ER stress) in 7721 cells, a human hepatocarcinoma cell line. In a search for mechanisms of ER stress, we found that there was a prominent decline of glucose uptake in antisense GnT-V transfectant, furthermore, a decrease of tri- or tetra-antannary sugar chain of glucose transporter 1 (GLUT1). However, distribution of GLUT1 in antisense GnT-V transfectant was not affected. Glucose deprivation has been known to activate ER stress in tumor cells. Therefore, the data presented in this study indicate that the glycosylation change and decrease of transport activity of GLUT1 may be one possible mechanism of ER stress induced by down-regulating GnT-V, and GnT-V may contribute to the regulation of glucose uptake by modifying glycosylation of GLUT1 in some tumor cells.
Asunto(s)
Retículo Endoplásmico/fisiología , Transportador de Glucosa de Tipo 1/fisiología , N-Acetilglucosaminiltransferasas/genética , Carcinoma Hepatocelular , Línea Celular Tumoral , Regulación hacia Abajo , Glucosa/deficiencia , Glucosa/metabolismo , Transportador de Glucosa de Tipo 1/biosíntesis , Glicosilación , Humanos , Neoplasias Hepáticas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We previously demonstrated that endoplasmic reticulum (ER) stress was triggered in human hepatocarcinoma 7721 cells transfected with antisense cDNA of N-acetylglucosaminyltransferase V (GnT-V-AS/7721) which were more susceptible to apoptosis induced by all-trans retinoic acid (ATRA). In the present study, we report that ATRA-induced apoptosis in GnT-V-AS/7721 cells is mediated through ER stress. We show here that ER stress is enhanced in GnT-V-AS/7721 cells with 80 microM ATRA treatment for 24 h, which is evidenced by the increase of GRP78/Bip, C/EBP-homologous protein-10 (CHOP, also known as GADD153) and spliced XBP1. Additionally, activation of caspase-12, caspase-9, and -3 was detected, and apoptosis morphology was observed in GnT-V-AS/7721 cells with ATRA treatment. These results suggest that ATRA enhances the ER stress triggered in GnT-V-AS/7721 cells, which represents a novel mechanism of ATRA to induce apoptosis. We further observed that GnT-V was significantly repressed and the structure of N-glycans was changed in GnT-V-AS/7721 cells with 80 microM ATRA treatment for 24 h, suggesting that repression of GnT-V by ATRA causes the enhanced ER stress and ER stress-mediated apoptosis in GnT-V-AS/7721 cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Tretinoina/farmacología , Secuencia de Bases , Carcinoma Hepatocelular/enzimología , Caspasas/metabolismo , Línea Celular Tumoral , Cartilla de ADN , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Chaperón BiP del Retículo Endoplásmico , Activación Enzimática , Humanos , Neoplasias Hepáticas/enzimología , N-Acetilglucosaminiltransferasas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In the present study,in order to investigate the pattern of the expression and location of oviductin using RT-PCR and Western blot,a 410bp cDNA fragment was amplified from mouse cervix tissue. Sequencing analysis and homology comparison with BLAST alignment showed that this fragment amplified from mouse cervix was homologous to the region from +310 to +714 of the mouse OGP gene in Genebank by the degree up to 100%. Western blot showed 60 KD and 30 KD components from mouse cervix same as those from mouse oviduct mucosa epithelium. Semi-quantitative RT-PCR analysis showed that expression of OGP was significantly higher at the estrous stage than that of non-estrous stage and almost no expression in the immature mouse. Those results suggest that the mRNA level of mouse cervix oviductin was related to estrous cycle,implied with estrogen-dependence. To further confirm the results,in situ hybridization histochemistry analysis showed that OGP mRNA was expressed at columnar epithelium of mouse cervix. To clarify the possible significance of oviductin expressed at the cervix, the function of oviductin on the mobility of human sperm was studied using "loss-of-function". The VAP, VCL, ALH, VSL showed no significant differences of the mobility of the human sperm in the conditioned medium blocked by the anti-CTP rOGP (rabbit Oviductin) antiserum compared with that in the conditioned medium of the normal rabbit serum. These results suggest that rOGP may not affect the mobility of human sperm in vitro.
Asunto(s)
Cuello del Útero/metabolismo , Perfilación de la Expresión Génica , Serina Endopeptidasas/genética , Animales , Animales Recién Nacidos , Western Blotting , Femenino , Humanos , Sueros Inmunes/farmacología , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos ICR , Conejos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Serina Endopeptidasas/inmunología , Serina Endopeptidasas/metabolismo , Motilidad Espermática/efectos de los fármacosRESUMEN
Alpha1,2-mannosidases, key enzymes in N-glycan processing and located both in the endoplasmic reticulum and golgi, have been targets in the development of anti-cancer therapies. Previous studies have shown its involvement in protein degradation. In this study, 1-deoxymannojirimycin, a specific inhibitor of alpha1,2-mannosidase and generating 'high mannose' type of N-glycan, was treated in human hepatocarcinoma 7721 cells and induced the endoplasmic reticulum stress. Key moleculars as XBP1 and GRP78/Bip were activated and up-regulated, which suggested the UPR pathway was activated. The cleavage of caspase-12, -9, and -3 was also detected, which implicated the ER stress was triggered and apoptosis occurred in H7721 cells. The results indicate the 'high Man' structure generated by 1-deoxymannojirimycin may constitute potential novel mechanism for ER stress and caspase-12 pathway of cell apoptosis.