RESUMO
AIM: Hepatocellular carcinoma (HCC) is the sixth most common malignancy worldwide. Liver is the largest human digestive gland with abundant Golgi apparatus involved in cell division, migration and apoptosis and others. METHODS: In the present study, Golgi apparatus of HCC and the surrounding liver tissues were isolated by sucrose density gradient centrifugation and identified by electron microscopy and enzymology methods. Using 2-D gel electrophoresis and mass spectrometry, 17 differentially expressed protein of Golgi apparatus in HCC and the surrounding liver tissue were screened and identified in the Mascot database. RESULTS: Of those differentially expressed proteins, six were upregulated and 11 were downregulated, some of them were related to the biological processes such as protein sorting, glycosylation, cell cycle regulation, transcription regulation and Golgi integrity. One protein, annexin A5, was verified to be upregulated in HCC by western blot. CONCLUSION: The differentially expressed proteins may provide new insight into HCC biology and potential diagnostic and therapeutic biomarkers.
RESUMO
CISD2 and ferroptosis participate in cancer development, but are rarely reported in ovarian cancer. This study aimed to clarify interaction between CISD2 and ferroptosis and evaluate related mechanisms. si-CISD2, wt-p53 and mut-p53 lentiviruses were transfected into SKOV-3 cells. CISD2 and p53 (wild/mutant p53) gene transcriptions were evaluated by RT-PCR. Cell viability, invasion ability, and migration capacity were determined. Expressions of ferroptosis-associated CISD2, p53, elastin, ß-catenin and levels of Gpx4 and TRF were examined. CISD2 downregulation (si-CISD2) has a significant inhibitory effect on cell activity and exerts a synergistic effect with p53. si-CISD2 and Wt-p53 markedly inhibited SKOV-3 invasion and migration capacity, compared to the downregulation control (si-NC) and overexpression control (ov-NC) group (p < 0.001). p53 expression was increased significantly in si-CISD2 treated SKOV-3, compared to si-NC treated cells (p < 0.05). si-CISD2 markedly decreased elastin and ß-catenin expression compared to the si-NC and ov-NC group (p < 0.001). si-CISD2 modulated ferroptosis-associated molecules (CDKN1A, GLS2, SAT1, SLC7A11), decreased Gpx4 and increased TRF levels in SKOV-3. si-CISD2 and Wt-p53 played an obvious synergistic role in regulating ferroptosis-associated molecules and Gpx4/TRF pathway molecules. In conclusion, CISD2 downregulation was involved in ferroptosis process of SKOV-3 cells. This effect of CISD2 was mediated by wild-type p53-associated GLS2/SAT1/SLC7A11 and Gpx4/TRF pathway.
Assuntos
Regulação para Baixo , Ferroptose , Neoplasias Ovarianas , Fosfolipídeo Hidroperóxido Glutationa Peroxidase , Transdução de Sinais , Proteína Supressora de Tumor p53 , Humanos , Ferroptose/genética , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Feminino , Regulação para Baixo/genética , Neoplasias Ovarianas/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/genética , Linhagem Celular Tumoral , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/genética , Movimento Celular/genética , Regulação Neoplásica da Expressão Gênica , Sistema y+ de Transporte de Aminoácidos/metabolismo , Sistema y+ de Transporte de Aminoácidos/genéticaRESUMO
SCOPE: The mechanisms of oleanolic acid (OA) regulating hepatic sterol regulatory element-binding protein (SREBP) 1c/stearoyl-CoA desaturase (SCD) 1 pathway to ameliorate fructose-induced hepatosteatosis are investigated. METHODS AND RESULTS: Rats treated with 10% w/v fructose solution are co-administered by OA for 5 weeks, and then sacrifice after fasting for 14 h. OA reverses the fructose-induced increase in hepatic triglyceride (TG) content and downregulates Scd1 mRNA expression. However, two upstream transcription factors, ChREBP and SREBP1c, remain at normal levels with or without fructose and/or OA. In vivo and in vitro studies using SREBP1c-/- mice and HepG2 cell models show that OA also inhibits SCD1 gene overexpression and high hepatic TG levels induced by fructose. On the other hand, in SCD1-/- mice, when the fructose diet is supplemented with high levels of oleic acid (OLA) to compensate for the deficiency of SCD1, OA inhibits hepatic SREBP1c and lipogenic gene expression and reduces hepatic OLA (C18:1) production to improve fructose and/or OLA induced liver lipid deposition. Furthermore, OA promotes PPARα and AMPK to enhance fatty acid oxidation in fructose + OLA-fed SCD1-/- mice. CONCLUSION: OA may inhibit SCD1 gene expression to ameliorate fructose-induced hepatosteatosis through SREBP1c-dependent and -independent mechanisms.
Assuntos
Ácido Oleanólico , Ratos , Camundongos , Animais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Ácido Oleanólico/farmacologia , Frutose/efeitos adversos , Frutose/metabolismo , Fígado/metabolismo , Triglicerídeos/metabolismo , Expressão Gênica , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismoRESUMO
OBJECTIVE: To compare the Golgi proteome of hepatocellular carcinoma (HCC) with that of the adjacent non-tumor tissues. METHODS: Hepatocellular carcinoma and adjacent non-tumor tissues were obtained from HCC patients. The protein expression maps in Golgi were obtained by two-dimensional gel electrophoresis (2-DE), and the differentially expressed protein spots were analyzed by PD-Quest software. Peptide mass fingerprint (PMF) of differential protein spots was obtained with MALD-TOT-MS. RESULTS: According to 2-DE maps, the average numbers of protein spots were (1153+/-49) and (1086+/-37) in hepatocellular carcinoma and the adjacent non-tumor tissues. Compared to the adjacent non-tumor tissues, 27 proteins were upregulated, and 20 proteins were downregulated in HCC Golgi. CONCLUSIONS: The Golgi proteome in HCC tissues is different from that in the adjacent non-tumor tissues, and the differential expression proteins are involved in energy metabolism, tumor metastasis, and cell cycle regulation.
Assuntos
Carcinoma Hepatocelular/metabolismo , Complexo de Golgi/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/análise , Proteoma , Anexina A5/análise , Anexina A5/metabolismo , Carcinoma Hepatocelular/patologia , Eletroforese em Gel Bidimensional/métodos , Humanos , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/patologia , Proteínas de Neoplasias/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
The Golgi complex is the central organelle of the secretory pathway and has many complicate functions. The endeavours to isolate and purify the Golgi apparatus from cultured cells will benefit further investigation of Golgi. A large number of gastric cancer cells SGC7901 were cultivated in vitro, then Golgi apparatus were isolated from the cells by differential centrifugation combined with sucrose density gradient ultra-centrifugation. Its purity was characterized biochemically by enzymatic assays, morphologically by electron microscopy (EM) and neutral red supravital staining. Finally the Golgi complex was successfully fractionated from gastric cancer cells SGC7901. The first successful isolation of Golgi apparatus from gastric cancer cells SGC7901 by using ultra-centrifugation will lead to research into the function of Golgi apparatus.
Assuntos
Complexo de Golgi/ultraestrutura , Técnicas Histológicas , Neoplasias Gástricas/ultraestrutura , Linhagem Celular Tumoral , Humanos , Neoplasias Gástricas/patologiaRESUMO
It is believed that telomerase is a terminal effector and its role is to maintain the length of telomeres. However, the main rate-limiting subunit of telomerase, human telomerase reverse transcriptase (hTERT), has been recently proved to have the ability to affect a variety of other genes such as p53 independently of the decurtation of telomere. To test whether this extra-telomeric effect could contribute to additional and immediate alterations in cellular proliferation and apoptosis, three epithelial ovarian cancer cell lines (A2780 [wild-type p53], OVCAR3 [mutant p53], and SKOV3 [p53 null]) were treated with hTERT-targeted short-hairpin RNA (shRNA), and their short-term effects were evaluated. A very fast growth inhibiting response to downregulation of hTERT was observed in all three cell lines with S-phase arrest karyotypes. But apoptosis appeared only in A2780 and OVCAR3 cells with p53 accumulation and elevated p21. In contrast, a decrease in expression of p21 was observed in SKOV3 and cell death appeared to be unaffected. These results indicated for the first time that inhibition of hTERT could induce immediate growth arrest in human ovarian carcinoma cells by blocking them in S phase, but apoptosis may only be induced via extra-telomeric effects on activation of p53 and p21.
Assuntos
Apoptose , Ciclo Celular , Proliferação de Células , Terapia Genética/métodos , Neoplasias Ovarianas/terapia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Telomerase/genética , Apoptose/genética , Ciclo Celular/genética , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Mutação , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Telomerase/metabolismo , Fatores de Tempo , Transfecção , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
Golgi reassembly and stacking protein 65 (GRASP65), which has been involved in cancer progression, is associated with tumor growth and cell apoptosis. Dihydromyricetin (DHM) has demonstrated antitumor activity in different types of human cancers. However, the pharmacological effects of DHM on ovarian cancer (OC) and the molecular mechanisms that underlie these effects are largely unknown. The present study showed that DHM reduced cell migration and invasion in a concentration- and time-dependent manner and induced cell apoptosis primarily through upregulation of Cleaved-caspase-3 and the Bax/Bcl-2 ratio in OCs. To further clarify the cancer therapeutic target, we assessed the effect of DHM on the expression of GRASP65, which is overexpressed in human ovarian cancer tissues. DHM activated caspase-3 and decreased GRASP65 expression to promote cell apoptosis, implying that downregulation of GRASP65 was related to DHM-induced cell apoptosis. Additionally, the knockdown of GRASP65 by siRNA resulted in increased apoptosis after DHM treatment, while western blot and flow cytometry analysis demonstrated that overexpression of GRASP65 attenuated DHM-mediated apoptosis. In addition, the JNK/ERK pathway may be involved in DHM-mediated caspase-3 activation and GRASP65 downregulation. Taken together, these findings provide novel evidence of the anti-cancer properties of DHM in OCs, indicating that DHM is a potential therapeutic agent for ovarian cancer through the inhibition of GRASP65 expression and the regulation of JNK/ERK pathway.
Assuntos
Antineoplásicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Flavonóis/farmacologia , Proteínas da Matriz do Complexo de Golgi/metabolismo , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Feminino , Flavonóis/uso terapêutico , Proteínas da Matriz do Complexo de Golgi/antagonistas & inibidores , Proteínas da Matriz do Complexo de Golgi/genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND: Human telomerase reverse transcriptase (hTERT) is a crucial oncogene and a key factor in cell immortalization. Apart from hematopoiesis stem cell and germ cells, normal cells and tissues almost no expression of this gene. However, the positive expression rate of hTERT in malignant tumors is as high as 80-95%. This study was designed to investigate the mechanism of knockdown of hTERT expression in the regulation of ovarian cancer carcinogenesis in nude mice transplantation model. METHODS: We identified the function of hTERT in the treatment of ovarian cancer by constructing a nude mice transplantation tumor model. Then analyzed the mechanism of hTERT in modulating ovarian cancer cell apoptosis and senescence by immunohistochemically staining. RESULTS: Our analysis identified hTERT as a novel regulator of ovarian cancer progression and is one of the most significantly upregulated genes in ovarian cancer. Down-regulation of hTERT inhibits proliferation of ovarian cancer cells by promoting apoptosis and the expression of the senescence gene p53 and p21. CONCLUSIONS: hTERT is a key oncogene in ovarian tumorigenesis and metastasis; the downregulation of hTERT significantly inhibit the proliferation of ovarian cancer and have an effect on treatment of ovarian cancer by activating cell apoptosis and senescence pathway.
RESUMO
OBJECTIVE: To investigate the effect of exogenous small interference RNA(siRNA)-mediated silencing of the cartilage homeoprotein 1 (CART1) gene on cell biological behavior in MDA-MB-231 human breast cancer cells. METHODS: We designed and chemically synthesized siRNA targeting CART1 or control, and then siRNA was transfected into MDA-MB-231 cells using RNAiMAX. The suppression effect of siRNA on CART1 expression was determined by real-time quantitative PCR and Western blotting. The effects of CART1 knockdown on MDA-MB-231 cell invasion, proliferation and cell cycle were detected by TranswellTM invasion assay, CCK-8 assay and flow cytometry, respectively. RESULTS: CART1-siRNA could significantly suppress the expression of CART1 mRNA and protein as compared with the control group. MDA-MB-231 cells transfected with CART1 siRNA had lower capacity of invasion and proliferation compared with the control group. What's more, the cell ratio increased in S phase and decreased in G2/M in the CART1 siRNA-transfected cells. CONCLUSION: Knockdown of the CART1 gene could significantly inhibit cell invasion and proliferation and induce cell cycle arrest in S phase in MDA-MB-231 human breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Proteínas de Homeodomínio/genética , Pontos de Checagem da Fase S do Ciclo Celular , Neoplasias da Mama/genética , Neoplasias da Mama/fisiopatologia , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Proteínas de Homeodomínio/metabolismo , Humanos , Invasividade Neoplásica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , TransfecçãoRESUMO
PURPOSE: The purpose of this study was to investigate the curative effect of bone-like hydroxyapatite/poly amino acid (BHA/PAA) as a carrier for poly(lactic-co-glycolic acid)-coated rifapentine microsphere (RPM) in the treatment of rabbit chronic osteomyelitis induced by Staphylococcus aureus. METHODS: RPM was prepared through an oil-in-water emulsion solvent evaporation method, and RPM was combined with BHA/PAA to obtain drug-loaded, slow-releasing materials. Twenty-six New Zealand white rabbits were induced to establish the animal model of chronic osteomyelitis. After debridement, the animals were randomly divided into three groups (n=8): the experimental group (with RPM-loaded BHA/PAA), the control group (with BHA/PAA), and the blank group. The RPM-loaded BHA/PAA was evaluated for antibacterial activity, dynamics of drug release, and osteogenic ability through in vitro and in vivo experiments. RESULTS: In vitro, RPM-loaded BHA/PAA released the antibiotics slowly, inhibiting the bacterial growth of S. aureus for up to 5 weeks. In vivo, at week 4, the bacterial colony count was significantly lower in the experimental group than in the control and blank groups (P<0.01). At week 12, the chronic osteomyelitis was cured and the bone defect was repaired in the experimental group, whereas the infection and bone defect persisted in the control and blank groups. CONCLUSION: In vitro and in vivo experiments demonstrated that RPM-loaded BHA/PAA effectively cured S. aureus-induced chronic osteomyelitis. Therefore, BHA/PAA has potential value as a slow-releasing material in clinical setting. Further investigation is needed to determine the optimal dosage for loading rifapentine.
Assuntos
Ácido Láctico/administração & dosagem , Osteomielite/tratamento farmacológico , Ácido Poliglicólico/administração & dosagem , Rifampina/análogos & derivados , Infecções Estafilocócicas/tratamento farmacológico , Aminoácidos/química , Animais , Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Doença Crônica , Preparações de Ação Retardada , Portadores de Fármacos/química , Durapatita/química , Ácido Láctico/química , Ácido Láctico/farmacologia , Microesferas , Osteomielite/microbiologia , Ácido Poliglicólico/química , Ácido Poliglicólico/farmacologia , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Coelhos , Rifampina/administração & dosagem , Rifampina/farmacologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/isolamento & purificaçãoRESUMO
AIM: To investigate the expression of three types of mucin (MUC1, MUC2, MUC5AC) and E-cadherin in human gastric carcinomas and their clinical significance. METHODS: Ninety-four gastric cancer specimens were classified according to WHO criteria and detected by immunohistochemical assay of expression of mucins and E-cadherin. RESULTS: The positive expression rates of MUC1, MUC2, MUC5AC and E-cadherin were 82% (77/94), 84% (79/94), 40% (38/94) and 56% (53/94) respectively. MUC1 expression was significantly correlated with the types of cancer (the positive rates of MUC1 in well and moderately differentiated tubular adenocarcinoma, poorly differentiated adenocarcinoma, signet-ring cell carcinoma and mucinous carcinoma were 91%, 87%, 71%, 71%, respectively, P<0.05), age of patients (the positive rates of it among the people who are younger than 40 years, between 40-60 years and over 60 year were 74%, 81%, 89%, P<0.05), lymph nodes involvement (the positive rates in the non-interfered group and the interfered group were 78%, 85%, P<0.05) and tumor size (the positive rates in the tumors with the size less than 3 cm, 3-6 cm and larger than 6 cm were 69%, 92%, 69%, P<0.05); MUC2 expression was significantly associated with types of cancers and had the strongest expression in mucinous carcinomas(the positive rates of MUC2 in well and moderately differentiated tubular adenocarcinoma, poorly differentiated adenocarcinoma, signet-ring cell carcinoma and mucinous carcinoma were 94%, 70%, 81%, 100%, P<0.05), but it had no obvious relation to age, gender, tumor location, lymph nodes involvement, depth of invasion and metastasis to extra-gastric organs (P>0.05); MUC5AC expression was not related to any of the characteristics investigated except that it had relation to gender, whereas MUC5AC showed the tendency to higher expression in less invasive lesions and lower expression in advanced stage cancers (P>0.05); No significant difference was found for E-cadherin expression. There were strong positive relationships between the expression of MUC1 and E-cadherin, MUC2 and E-cadherin, MUC1 and MUC2 (R = 0.33, R = 0.22, R = 0.32, respectively, P<0.05). According to the COX proportional hazards model, older patients, involvement of lymph nodes, different types of gastric cancer and MUC2 expression were significantly associated with poorer outcome of gastric carcinoma patients (beta = 0.08, beta = 3.94, beta = 1.33, beta = 0.75, respectively, P<0.05). CONCLUSION: MUC1 and MUC2 are good markers of different types of gastric cancer. MUC2 is especially a good marker of mucinous carcinoma. MUC1, MUC2 may interfere with the function of E-cadherin in gastric carcinomas, and have synergic effect on progression of gastric cancers.
Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/metabolismo , Caderinas/metabolismo , Mucina-1/metabolismo , Mucinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Neoplasias Gástricas/metabolismo , Adenocarcinoma/cirurgia , Adulto , Idoso , Feminino , Gastrectomia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mucina-2 , Neoplasias Gástricas/cirurgiaRESUMO
Tetrandrine (TET) is a bisbenzylisoquinoline alkaloid that is isolated from the Stephania Tetrandra. It is known to possess anti-inflammatory and immunomodulatory effects. We have shown that TET can effectively suppress the production of bacterial lipopolysaccharide (LPS)-induced inflammatory mediators, including cyclooxygenases (COXs), in macrophages. However, whether TET has an antinociceptive effect on LPS-induced hyperalgesia is unknown. In the present study, we investigated the potential antinociceptive effects of TET and the mechanisms by which it elicits its effects on LPS-induced hyperalgesia. LPS effectively evoked hyperalgesia and induced the production of PGE2 in the sera, brain tissues, and cultured astroglia. TET pretreatment attenuated all of these effects. LPS also activated inhibitor of κB (IκB) kinase ß (IKKß) and its downstream components in the IκB/nuclear factor (NF)-κB signaling pathway, including COX-2; the increase in expression levels of these components was significantly abolished by TET. Furthermore, in primary astroglia, knockdown of IKKß, but not IKKα, reversed the effects of TET on the LPS-induced increase in IκB phosphorylation, P65 phosphorylation, and COX-2. Our results suggest that TET can effectively exert antinociceptive effects on LPS-induced hyperalgesia in mice by inhibiting IKKß phosphorylation, which leads to the reduction in the production of important pain mediators, such as PGE2 and COX-2, via the IKKß/IκB/NF-κB pathway.
Assuntos
Analgésicos/uso terapêutico , Benzilisoquinolinas/uso terapêutico , Hiperalgesia/tratamento farmacológico , Quinase I-kappa B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Analgésicos/farmacologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Benzilisoquinolinas/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/metabolismo , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Quinase I-kappa B/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação/efeitos dos fármacosRESUMO
AIM: To investigate the role of P115 in the proliferation of gastric cancer cells and the mechanism involved. METHODS: The RNA and protein level of P115 and macrophage migration inhibitory factor (MIF) in gastric cancer and normal gastric tissue/cells were measured and the effect of P115 on cell proliferation was assessed. The role of P115 in cell cycle checkpoints was investigated and the related proteins and signaling pathways, such as cyclin D1, Mcm2, p53, PCNA as well as the MAPK signaling pathway were determined. The interaction between P115 and MIF and the effect of P115 on MIF secretion were examined. The data were analyzed via one-way ANOVA comparisons between groups and P < 0.05 was considered significant. RESULTS: P115 and MIF were both specifically expressed in gastric cancer tissues compared with normal gastric mucosa (both P < 0.01). The mRNA and protein levels of P115 and MIF in gastric cancer cell lines MKN-28 and BGC-823 were higher than in the human gastric epithelial cell line GES-1 (both P < 0.01). In MKN-28 and BGC-823 cell lines, P115 promoted cell proliferation and G0-G1 to S phase transition. In addition, several cell cycle-related regulators, including cyclin D1, Mcm2, PCNA, pERK1/2 and p53 were up-regulated by P115. Furthermore, the interaction between P115 and MIF was confirmed by co-immunoprecipitation assay. ELISA showed that P115 stimulated the secretion of MIF into the culture supernatant (P < 0.01) and the compensative expression of MIF in cells was observed by Western blotting. CONCLUSION: P115 promotes proliferation of gastric cancer cells through an interaction with MIF.
Assuntos
Proliferação de Células , Oxirredutases Intramoleculares/metabolismo , Fatores Inibidores da Migração de Macrófagos/metabolismo , Neoplasias Gástricas/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Proteínas da Matriz do Complexo de Golgi , Humanos , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Ligação Proteica , Interferência de RNA , RNA Mensageiro/metabolismo , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Fatores de Tempo , Transfecção , Proteínas de Transporte Vesicular/genéticaRESUMO
Maspin is a member of the serpin (serine protease inhibitor) family of protease inhibitors known to have tumor suppressor activity in diverse human cancers. However, maspin gene function and the molecular aspects in gastric carcinoma remain largely unclear. To investigate the effects of maspin on invasion of gastric carcinoma SGC7901 cell line and the underlying molecular mechanism involved in this process, we cloned short hairpin oligoes (shRNA) targeting maspin into plasmid pGenesil-1.1 eukaryotic expression vector and then transfected the recombinant plasmid pGenesil-maspin into gastric carcinoma SGC7901 cells using Lipofectamine 2000. After the maspin expression was successfully knocked down, the number of cells invading through Matrigel was obviously increased (P<0.05) in the Transwell chamber assay. By detection of reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis, respectively, we found that mRNA and protein of uPA, VEGF-C were increased significantly, and the protein level of MMP7 was also increased (P<0.05). These results suggested that maspin gene could inhibit invasion of gastric cancinoma SGC7901 cells and this inhibition maybe result from the interaction between maspin and uPA, MMP7, or VEGF-C.
Assuntos
Movimento Celular/genética , Serpinas/metabolismo , Neoplasias Gástricas/patologia , Western Blotting , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Metaloproteinase 7 da Matriz/metabolismo , Invasividade Neoplásica , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serpinas/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Transfecção , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
BACKGROUND & OBJECTIVE: MASPIN gene is closely associated with carcinogengsis and plays a key role in cell proliferation, adhesion, migration and apoptosis. This study was to construct a recombinant eukaryotic vector expressing MASPIN, and explore the effect of MASPIN overexpression on the apoptosis in human gastric carcinoma cell line SGC7901. METHODS: A eukaryotic expression vector MASIPIN/PCR2.1 was constructed and transfected into SGC7901 cells. RT-PCR and Western blot were used to detect the expression changes of MASPIN and Bax/Bcl-2. TNF-related apoptosis inducing ligand (TRAIL) (50ng/ml) was used to induce apoptosis at different time courses. DNA apoptotic ladders were determined using agarose gel electrophoresis (AGE). Cell apoptosis was measured by flow cytometry (FCM). RESULTS: Recombinant plasmid MASIPIN/PCR2.1 was successfully constructed and transfected into SGC7901 cells. The mRNA and protein levels of MASPIN were significantly higher in the MASPIN/PCR2.1 group (33.6+/-1.2, 23.4+/-1.6) than in the PCR2.1(15.0+/-1.5, 12.3+/-1.5)and the untreated group (13.7+/-2.0, 12.0+/-1.3) (P<0.05). After transfection of MASIPIN/PCR2.1, DNA apoptotic ladders appeared in SGC7901 cells and the induction of apoptosis was in a time-dependent manner. The apoptosis rates were 8.0%, 16.3% and 25.8%in the MASPIN/PCR2.1 plus TRAIL group, 3.0%, 8.2%, 14.4% in the MASPIN/PCR2.1 group, and 4.1%, 9.8%,15.9% in the TRAIL group at 12, 24, and 48 h(P<0.05). The expression levels of Bax mRNA and protein at 48 h after MASIPIN/PCR2.1 transfection were significantly higher in MASPIN/PCR2.1 plus TRAIL group(55.3+/-2.1, 75.4+/-1.3) than in the PCR2.1 group (34.3+/-1.2, 40.7+/-1.8) and the TRAIL group (43.2 +/-1.8,36.2+/-1.3)(P<0.05). The expression of Bcl-2 mRNA in the MASPIN/PCR2.1 plus TRAIL group, PCR2.1 group and TRAIL group were 28.3+/-2.5, 34.3+/-1.2, 32.8+/-2.1, respectively (P<0.05), and those of Bcl-2 protein were 17.4+/-1.5, 45.1+/-2.1, 42.8+/-1.5 in the three groups, respectively (P<0.05). CONCLUSIONS: upregulation of MASPIN/PCR2.1 can significantly enhance the sensibility of gastric cancer cell line SGC7901 to the apoptosis inducer. This maybe related to the upregulation of Bax and downregulation of Bcl-2.
Assuntos
Apoptose , Serpinas/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Vetores Genéticos , Humanos , Plasmídeos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/metabolismo , Serpinas/genética , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Transfecção , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
BACKGROUND & OBJECTIVE: Multidrug resistance-associated protein (MRP), glutathione-S-transferase-pi(GST-pi), topoisomerase IIalpha(Topo IIalpha)and lung resistance protein (LRP) play important roles in the multidrug resistance(MDR)of tumor chemotherapy. There were few reports on combined determination of the expression of MRP, GST-pi, Topo IIalpha and LRP in gastric carcinoma. This study was designed to investigate the expression and significance of MRP, GST-pi, Topo IIalpha and LRP in gastric carcinomas. METHODS: Immunohistochemistry SP method was used to determine the expression of MRP, GST-pi, Topo IIalpha, and LRP in 90 tumor samples from the patients with gastric carcinoma. Chi-square test and Fisher exact test were used to analyze the significance of the expression. RESULTS: (1)The positive expression rates of MRP, GST-pi, Topo IIalpha and LRP in gastric carcinoma were 88.9%, 91.1%, 74.4%, and 87.7%, respectively. They were all significantly higher than those in normal stomach tissues (P< 0.05). (2)The expression levels of MRP, GST-pi, and LRP in well-moderated differentiation adenocarcinoma were significantly higher than those in poor differentiation adenocarcinoma. The expression of Topo IIin well-moderated differentiation adenocarcinoma was significantly lower than that in poor differentiation adenocarcinoma. There was no difference between the expression levels of them in different degree of invasion or with lymph nodes metastasis and without lymph nodes metastasis (P > 0.05). (3) There was no correlation in any two items among the expression levels of MRP,GST-pi, topo IIalpha,and LRP. CONCLUSION: MRP, GST-pi, topo IIalpha,and LRP play important roles in the primary MDR of gastric carcinoma. The expression of them are associated with the differentiation, but are not associated with the invasion degree and lymph node metastasis.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , Glutationa Transferase/metabolismo , Isoenzimas/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Gástricas/metabolismo , Partículas de Ribonucleoproteínas em Forma de Abóbada/metabolismo , Adulto , Idoso , Resistência a Múltiplos Medicamentos , Feminino , Mucosa Gástrica/metabolismo , Expressão Gênica , Glutationa S-Transferase pi , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Gástricas/enzimologiaRESUMO
BACKGROUND & OBJECTIVE: Our previous study showed that the expression of MUC2 protein was related with the biological behavior of gastric carcinoma. The aim of the present study was to investigate the inhibitory effect in vitro of mucin gene MUC2 antisense oligodeoxynucleotide (ASODN) on its gene expression and cell proliferation on gastric cancer cells SGC7901. METHODS: Phosphorothioate MUC2 ASODN was synthesized and transfected to SGC7901 cells mediated by lipofectin. Its inhibitory effects on cell proliferation was determined by MTT method, light and electron microscopy and immunohistochemical method. RESULTS: The determination by MTT method demonstrated that MUC2 ASODN of varied concentration significantly inhibited the growth of SGC7901 cells while the control lipofectin and control N-ODN showed no such effect. The inhibitory effect was dose-dependent and time-dependent. The inhibition peaked at 48th hour after transfection, and the inhibition rate reached 55% when the MUC2 ASODN concentration was 0.5 micromol/L. After transfecting with MUC2 ASODN, SGC7901 cells showed decrease in number, volume, and karyokinesis, and increase in necroses under light microscopy. Mitochondrion swelling, increased liposomes, myelin figures, chromatin margination were found under electron microscopy. And the test by immunohistochemical method indicated that transfected MUC2 ASODN downregulated the expression levels of MUC2 protein, but upregulated the expression levels of p16 protein. CONCLUSION: MUC2 ASODN transfection could specifically inhibit SGC7901 cells proliferation.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Mucinas/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Neoplasias Gástricas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Vetores Genéticos , Humanos , Mucina-2 , Mucinas/metabolismo , Fosfatidiletanolaminas , Neoplasias Gástricas/metabolismo , TransfecçãoRESUMO
AIM:To study the distribution of arylsulfatase,beta-galactosidase and lysozyme in gastric cancer cells, and its relationship to differentiation and invasion of gastric cancer cells.METHODS: Histochemical, immunohistochemical and ruthenium red (RR) electrocytochemical technique for three types of hydrolases and proteoglycans in pericancerous matrix in 33 cases of gastric cancer were observed under light and electron microscopy.RESULTS:The expression intensities of arylsulfatase,beta-glactosidase and lysozyme in mucinous cell carcinomas were more intensive than those in well-differentiated and poorly-differentiated adenocar-cinomas (P < 0.05-0.01). The fibrous tissues smooth muscle and proteoglycans close to the cancer cells were degraded. They were found in the region far from the cancer cells. Expression of three enzymes mentioned above was low in adenocarcinoma cells, and fibrous tissues and RR granules were present and intact near the well-differentiated and poorly differentiated adenocarcinoma cells.CONCLUSION: Mucinous cell carcinoma may release various hydrolases into extra-cellular matrix, inducing degradation of pericancerous matrix and facilitating cancer cell invasion and metastasis.