RESUMEN
Programmed cell death 4 (PDCD4) is a newly identified tumor suppressor that can inhibit activator protein (AP)-1 activation and protein translation. Our previous studies indicate that lost or reduced PDCD4 expression is associated with the progression of ovarian carcinoma. However, direct evidence that PDCD4 inhibits malignant phenotype of human cancer cells is limited. In the present study, we found that PDCD4 expression in ovarian cancer cell lines (SKOV3, 3AO, and CAOV3) inhibited significantly their proliferation and cell cycle progression, and induced apoptosis. More importantly, up-regulation of PDCD4 expression decreased the colony-forming capacity of ovarian cancer cells in vitro and tumorigenic capacity in mice. These results demonstrate that PDCD4 can suppress the malignant phenotype of ovarian cancer cells, and may represent a novel therapeutic target for the treatment of ovarian cancer.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Carcinoma/patología , Neoplasias Ováricas/patología , Proteínas de Unión al ARN/metabolismo , Animales , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/fisiología , Carcinoma/genética , Ciclo Celular , Línea Celular Tumoral , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Supresores de Tumor , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Neoplasias Ováricas/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Distribución Aleatoria , Factor de Transcripción AP-1/antagonistas & inhibidores , Transfección , Carga Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Programmed cell death 4 (Pdcd4) was found to be related to apoptosis upon first discovery. It was later found to play the role of tumor suppressor gene in a variety of tumors by inhibiting transcription and translation. Recently, it has been proposed that it may play an important role in some inflammatory diseases and in the immune response. In our previous study, deficiency of Pdcd4 was found to attenuate the formation of atherosclerotic plaques. This might be because deficiency of Pdcd4 may increase IL-10 expression and lipoautophagy by macrophages and attenuate the formation of foam cells. However, the effect of Pdcd4 on the subsets of T cells in hyperlipidemic mice still remained unclear. In the present study, results showed that Pdcd4 deficiency decreased the percentage of CD8+ T cells and increased that of regulatory T cells (Tregs) under hyperlipidemic conditions both in vitro and in vivo, which may be due to the reduced expression of co-stimulatory molecules CD28 and CD137, and the enhancive expression of co-inhibitory molecules CTLA-4. These results indicated that endogenous Pdcd4 promotes immune response mediated by T cells through regulation of the co-stimulatory molecules expression, which may contribute to the development of advanced atherosclerotic plaques. The current work provides new data to understand the role of Pdcd4 in different T cell subsets under hyperlipidemic microenvironment.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/metabolismo , Hiperlipidemias/metabolismo , Proteínas de Unión al ARN/metabolismo , Subgrupos de Linfocitos T/metabolismo , Animales , Apoptosis/fisiología , Antígenos CD28/metabolismo , Linfocitos T CD8-positivos/metabolismo , Células Espumosas/metabolismo , Interleucina-10/metabolismo , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Placa Aterosclerótica/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
In this study, we investigated the in vivo role of adiponectin, an adipocytokine, on the development of atherosclerosis in rabbits mainly using adenovirus expressing adiponectin gene (Ad-APN) and intravascular ultrasonography. Serum adiponectin concentrations in rabbits after Ad-APN local transfer to abdominal aortas increased about nine times as much as those before transfer (P < 0.01), about ten times as much as the levels of endogenous adiponectin in adenovirus expressing beta-galactosidase gene (Ad-beta gal) treated rabbits (P < 0.01), and about four times as much as those in the aorta of non-injured rabbits on a normal cholesterol diet (P < 0.01). Ultrasonography revealed a significantly reduced atherosclerotic plaque area in abdominal aortas of rabbits infected through intima with Ad-APN, by 35.2% compared with the area before treatment (P < 0.01), and by 35.8% compared with that in Ad-beta gal-treated rabbits (P < 0.01). In rabbits infected through adventitia, Ad-APN treatment reduced plaque area by 28.9% as compared with the area before treatment (P < 0.01) and 25.6% compared with that in Ad-beta gal-treated rabbits (P < 0.01). Adiponectin significantly suppressed the mRNA expression of vascular cell adhesion molecule-1 (VCAM-1) by 18.5% through intima transfer (P < 0.05) and 26.9% through adventitia transfer (P < 0.01), and intercellular adhesion molecule-1 (ICAM-1) by 40.7% through intima transfer (P < 0.01), and 30.7% through adventitia transfer (P < 0.01). However, adiponectin had no effect on the expression of types I and III collagen. These results suggest that local adiponectin treatment suppresses the development of atherosclerosis in vivo in part by attenuating the expression of VCAM-1 and ICAM-1 in vascular walls.
Asunto(s)
Adiponectina/metabolismo , Aterosclerosis/terapia , Terapia Genética/métodos , Túnica Íntima/metabolismo , Adenoviridae/genética , Adiponectina/genética , Adiponectina/aislamiento & purificación , Animales , Aorta Abdominal/diagnóstico por imagen , Aterosclerosis/diagnóstico por imagen , Aterosclerosis/metabolismo , Colágeno Tipo I/análisis , Colágeno Tipo I/genética , Colágeno Tipo II/análisis , Colágeno Tipo II/genética , Dieta Aterogénica , Expresión Génica/efectos de los fármacos , Ingeniería Genética/métodos , Vectores Genéticos/administración & dosificación , Molécula 1 de Adhesión Intercelular/genética , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Modelos Animales , Pichia , Conejos , Transducción Genética/métodos , Túnica Íntima/virología , Ultrasonografía , Molécula 1 de Adhesión Celular Vascular/análisis , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
OBJECTIVE: To study the relationship between the change of regulatory T cell number in CD4+ T subset and the growth of tumor in H22 hepatocellular carcinoma-bearing mice. METHODS: Tumor-bearing mice were established by subcutaneous inoculation of H22 hepatocelluler carcinoma cells. Flow cytometry was used to detect the expression of CD4 and CD25 molecules of the T cells which came from the tumor-bearing mice. The Foxp3 gene expression was detected by RT-PCR and flow cytometry. CD4+ CD25+ T cells and CD4+ CD25- T cells were separated and purified by immuno-magnetic beads. The proliferation and suppressive function of the CD4+ CD25+ T cells coming from tumor-bearing mice was measured by [3H]-thymidines incorporation experiment in vitro, and then effect of CD4+ CD25+ T cells originated from hepatocellular carcinoma-bearing mice on tumor growth was observed in vivo. RESULTS: (1) Compared with mice of the control group, the percentage of CD4+ CD25+ T cells of CD4+ T cells in tumor-bearing mice is not only higher in draining lymph nodes (18.80% < or = 0.06%) vs. (9.50% +/- 0.03%), (P < 0.01), but also higher in non-draining lymph nodes (LN) and spleen (SP), LN: (16.28% +/- 0.02%) vs. (9.50% +/- 0.03%), P < 0.01; SP: (17.28% +/- 0.06%) vs. (11.08% +/- 0.04%), (P < 0.05). The expression of regulatory T cell specific marker Foxp3 gene was also increased. In the same tumor-bearing mice, the number of CD4+ CD25+ T cells in draining lymph node was relatively higher than the contralateral nondraining lymph node, but the difference was statistically not significant (18.8% +/- 0.06%) vs. (16.28% +/- 0.02%), (P > 0.05). (2) The CD4+ CD25+ T cells purified from tumor-bearing mice--like naturally occurring regulatory T cells--were anergic to anti-CD3 monoclonal antibody stimulation in vitro, but it could suppress CD4+ CD25- T cells proliferation. (3) The percentage of CD4+ CD25+ T cells was positively related to tumor size. It could also suppress the anti-tumor effect of CD4+ CD25- T cells in vivo. Conclusion The growth of hepatocellular carcinoma in mice can boost the amount of regulatory T cells. The amount of regulatory T cells is positively related to tumor size, indicating that attack on regulatory T cells could be used as one of modalities in cancer treatment in the future.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Neoplasias Hepáticas Experimentales/patología , Neoplasias Hepáticas/patología , Linfocitos T Reguladores/inmunología , Animales , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/metabolismo , Línea Celular Tumoral , Proliferación Celular , Femenino , Citometría de Flujo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Regulación Neoplásica de la Expresión Génica , Subunidad alfa del Receptor de Interleucina-2/inmunología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas Experimentales/inmunología , Neoplasias Hepáticas Experimentales/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/inmunología , Bazo/metabolismo , Linfocitos T Reguladores/citología , Linfocitos T Reguladores/metabolismoRESUMEN
AIM: To evaluate the inhibitory effect of antisense phosphorothioate oligonucleotide (asON) complementary to the initiator of human telomerase catalytic subunit (hTERT) on the growth of hepatoma cells. METHODS: The as-hTERT was synthesized by using a DNA synthesizer. HepG2.2.15 cells were treated with as-hTERT at the concentration of 10 micromol/L. After 72 h, these cells were obtained for detecting growth inhibition, telomerase activity using the methods of MTT, TRAP-PCR-ELISA, respectively. BALB/c(nu/nu) mice were injected HepG2.2.15 cells and a human-nude mice model was obtained. There were three groups for anti-tumor activity study. Once tumors were established, these animals in the first group were administered as-hTERT and saline. Apoptosis of tumor cells was detected by FCM. In the 2nd group, the animals were injected HepG2.2.15 cells together with as-hTERT. In the third group, the animals were given as-hTERT 24 hours postinjection of HepG2.2.15 cells. The anti-HBV effects were assayed with ELISA in vitro and in vivo. RESULTS: Growth inhibition was observed in cells treated with as-hTERT in vitro. A significant different in the value of A570-A630 was found between cells treated with as-hTERT and control (P<0.01) by MTT method. The telomerase activity of tumor cells treated with as-hTERT was reduced, the value of A450 nm was 0.42 compared to control (1.49) with TRAP-PCR-ELISA. The peak of apoptosis in tumor cells given as-hTERT was 21.12%, but not seen in saline-treated control. A prolonged period of carcinogenesis was observed in the second and third group animals. There was inhibitory effect on the expression of HBsAg and HBeAg in vivo and in vitro. CONCLUSION: As-hTERT has an anti-tumor activity, which may be useful for gene therapy of tumors.
Asunto(s)
Carcinoma Hepatocelular/patología , Neoplasias Hepáticas Experimentales/patología , Oligonucleótidos Antisentido/farmacología , Telomerasa/genética , Animales , División Celular/efectos de los fármacos , Línea Celular Tumoral , Proteínas de Unión al ADN , Humanos , Ratones , Ratones Endogámicos BALB C , Trasplante de NeoplasiasRESUMEN
AIM: To establish a mice model of hepatitis B by using HBV-transgenic mice, and to transfer HBV-specific cytotoxic T lymphocytes (CTL) induced from syngeneic BALB/c mice immunized by a eukaryotic expression vector containing HBV complete genome DNA. METHODS: HBV DNA was obtained from digested pBR322-2HBV and ligated with the vector pcDNA3. Recombinant pcDNA3-HBV was identified by restriction endonuclease assay and transfected into human hepatoma cell line HepG2 with lipofectin. ELISA was used to detect the expression of HBsAg in culture supernatant, and RT-PCR to determine the existence of HBV PreS1 mRNA. BALB/c mice were immunized with pcDNA3-HBV or pcDNA3 by intramuscular injection. ELISA was used to detect the expression of HBsAb in serum. MTT assay was used to measure non-specific or specific proliferation ability and specific killing activity of spleen lymphocytes. Lymphocytes from immunized mice were transferred into HBV-transgenic mice (2.5X10(7) per mouse). Forty-eight hours later, the level of serum protein and transaminase was detected with biochemical method, liver and kidney were sectioned and stained by HE to observe the pathological changes. RESULTS: By enzyme digestion with Eco RI, Xho I and Hind III, the recombinant pcDNA3-HBV was verified to contain a single copy of HBV genome, which was inserted in the positive direction. HepG2 cells transfected with the recombinant could stably express PreS1 mRNA and HBsAg. After immunized by pcDNA3-HBV for 4 weeks, HBsAb was detected in the serum of BALB/c mice. The potential of spleen lymphocytes for both non-specific and specific proliferation and the specific killing activity against target cells were enhanced. The transgenic mice in model group had no significant changes in the level of serum protein but had an obvious increase of ALT and AST. The liver had obvious pathological changes, while the kidney had no evident damage. CONCLUSION: A eukaryotic expression vector pcDNA3-HBV containing HBV complete genome is constructed successfully. HepG2 cells transfected with the recombinant can express PreS1 mRNA and HBsAg stably. Specific cellular immune response can be induced in mice immunized by pcDNA3-HBV. A mice model of acute hepatitis with HBV has been established.
Asunto(s)
ADN Viral , Modelos Animales de Enfermedad , Virus de la Hepatitis B/genética , Hepatitis B , Ratones Transgénicos , Linfocitos T Citotóxicos/trasplante , Animales , Línea Celular Tumoral , Genoma Viral , Hepatitis B/inmunología , Humanos , Ratones , Ratones Endogámicos BALB C , Linfocitos T Citotóxicos/inmunología , TransfecciónRESUMEN
T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been reported to participate in the pathogenesis of inflammatory diseases. However, whether Tim-3 is involved in hepatitis B virus (HBV) infection remains unknown. Here, we studied the expression and function of Tim-3 in a hydrodynamics-based mouse model of HBV infection. A significant increase of Tim-3 expression on hepatic T lymphocytes, especially on CD8+ T cells, was demonstrated in HBV model mice from day 7 to day 18. After Tim-3 knockdown by specific shRNAs, significantly increased IFN-gamma production from hepatic CD8+ T cells in HBV model mice was observed. Very interestingly, we found Tim-3 expression on CD8+ T cells was higher in HBV model mice with higher serum anti-HBs production. Moreover, Tim-3 knockdown influenced anti-HBs production in vivo. Collectively, our data suggested that Tim-3 might act as a potent regulator of antiviral T-cell responses in HBV infection.