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Six pigs underwent implantation of a portal vein infusion port by transjugular access. The technical success rate was 100% (n = 6), with no surgical complications or deaths. At 1 month after implantation, the catheter tip had moved from the splenic vein to the main portal vein, while the catheter protruded into the right ventricle through the right atrium in all cases. Hence, the infusion port system has not been used in clinical practice due to its obvious displacement after implantation. However, this study provides a new idea for future exploration of portal vein infusion pathways.
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Cateterismo Periférico/instrumentação , Veias Jugulares , Veia Porta , Dispositivos de Acesso Vascular , Animais , Cateterismo Periférico/efeitos adversos , Desenho de Equipamento , Estudos de Viabilidade , Feminino , Infusões Intravenosas , Veias Jugulares/diagnóstico por imagem , Masculino , Veia Porta/diagnóstico por imagem , Punções , Sus scrofa , Fatores de TempoRESUMO
Previous studies have shown that interleukin-24 (IL-24) has tumor-suppressing activity by multiple pathways. However, the immunogenicity moderation effect of IL-24 on malignant cells has not been explored extensively. In this study, we investigated the role of IL-24 in immunogenicity modulation of the myelogenous leukemia cells. Data show that myelogenous leukemia cells express low levels of immunogenicity molecules. Treatment with IL-24 could enhance leukemia cell immunogenicity, predominantly regulate leukemia cells to produce immune-associated cytokines, and improve the cytotoxic sensitivity of these cells to immune effector cells. IL-24 expression could retard transplanted leukemia cell tumor growth in vivo in athymic nude mice. Moreover, IL-24 had marked effects on downregulating the expression of angiogenesis-related proteins vascular endothelial growth factor, cluster of differentiation (CD) 31, CD34, collagen IV and metastasis-related factors CD147, membrane type-1 matrix metalloproteinase (MMP), and MMP-2 and MMP-9 in transplanted tumors. These findings indicated novel functions of this antitumor gene and characterized IL-24 as a promising agent for further clinical trial for hematologic malignancy immunotherapy.
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Adjuvantes Imunológicos/farmacologia , Imunomodulação , Interleucinas/uso terapêutico , Leucemia Mieloide/tratamento farmacológico , Células Mieloides/efeitos dos fármacos , Inibidores da Angiogênese/imunologia , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Linhagem Celular Tumoral , Células Matadoras Induzidas por Citocinas/imunologia , Células Matadoras Induzidas por Citocinas/metabolismo , Humanos , Interleucinas/imunologia , Interleucinas/farmacologia , Leucemia Mieloide/imunologia , Camundongos , Camundongos Nus , Células Mieloides/imunologia , Invasividade Neoplásica/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To evaluate the role of microRNA-126 (miR-126) in maintaining the integrity of the blood-retina barrier (BRB), we established a mouse model of oxygen-induced retinopathy (OIR) and measured the retinal levels of miR-126 using recombinant plasmid pCMV-MIR or pCMV-MIR-126 intravitreal injections. We also detected VCAM-1 and BCL2L11 levels. Retinal vaso-obliteration, VCAM-1 localization on retinal endothelial cells, the blood-retina vascular permeability or albumin leakage in retinas, TUNEL histology, Evans blue assays, or Western blotting for detecting albumin or tight junction levels in the retina was performed. We also detected the effect of miR-126 on the survival of Müller cells in a mouse model using vimentin fluorescence staining. Our results suggested that miR-126 may not only regulate the overexpression of VCAM-1 or BCL2L11 and lead to the reduction of retinal endothelial cell apoptosis, retinal vascular leakage, or retinal permeability in the OIR mouse model, but may also protect hypoxic retinal Müller cells via the STAT3 signaling pathway. We believe that miR-126 could also be a potential therapeutic agent to maintain the stability of the BRB in ischemic retinopathy.
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Proteína 11 Semelhante a Bcl-2/metabolismo , Barreira Hematorretiniana/fisiologia , Isquemia/metabolismo , MicroRNAs/fisiologia , Doenças Retinianas/metabolismo , Molécula 1 de Adesão de Célula Vascular/metabolismo , Animais , Animais Recém-Nascidos , Apoptose/fisiologia , Western Blotting , Permeabilidade Capilar/fisiologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Retina/citologia , Doenças Retinianas/patologia , Vasos Retinianos/metabolismo , Vasos Retinianos/patologiaRESUMO
OBJECTIVE: To evaluate the impact of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) co-activation on the prognosis of allogeneic hematopoietic stem cell transplantation (allo-HSCT) patients. METHODS: We retrospectively analyzed 330 consecutive allo-HSCT patients at First Affiliated Hospital of Soochow University from December 2011 to August 2013. CMV and EBV DNA were regularly monitored by quantitative polymerase chain reaction (PCR) from the engraftment of granuloCyte within one year after transplantation. The incidences of viremia and clinical outcomes were analyzed by χ(2) test and Kaplan-Meier analysis. RESULTS: After a median follow-up period of 16 (7-25) months, a total of 113 (34.2%) patients were identified with CMV viremia (CMV+) alone, 82 (24.8%) with EBV viremia (EBV+) alone and 32 (9.7%) with CMV and EBV co-activation (CMV/EBV+). The proportion of patients undergoing HLA mismatched transplantation and ones with acute graft-versus-host disease (aGVHD) in CMV/EBV+ group was significantly higher than CMV+ group or EBV+ group (78.1% (25/32) vs 58.5% (48/82) or 50.4% (57/113), P = 0.047,0.008; 56.3% (18/32) vs 32.9% (27/82) or 34.5% (39/113) , P = 0.022, 0.026) . The incidence of post-transplant lymphoproliferative disorder (PTLD) was similar to EBV+ group (12.5% (4/32) vs 11.0% (9/82) , P = 0.802) and so did the incidence of CMV disease when compared with CMV+ group (9.4% (3/32) vs 7.1% (8/113) , P = 0.665). The 2-year overall survival (OS) of CMV+, EBV+ and CMV/EBV+ groups was 68.7%, 61.5% and 62.4% respectively. And no significant difference existed between CMV/EBV+ and the other two groups (P = 0.598, 0.717). However, the 6-month non-relapse mortality (NRM) of CMV/EBV+ group was significantly higher than that of CMV+ or EBV+ group (18.7% vs 8.9%, P = 0.036; 18.7% vs 8.1%, P = 0.032). CONCLUSIONS: HLA mismatch transplants and aGVHD are frequent in CMV and EBV co-activation group. When compared with EBV+ or CMV+ patients, the CMV/EBV+ patients have similar incidence of PTLD or CMV disease and 2-year OS.However, the 6-month NRM is significantly higher in CMV/EBV+ group. It suggests that CMV and EBV co-activation is a risk factor for early mortality of allo-HSCT patients.
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Citomegalovirus , Transplante de Células-Tronco Hematopoéticas , Herpesvirus Humano 4 , Ativação Viral , Infecções por Citomegalovirus , Infecções por Vírus Epstein-Barr , Doença Enxerto-Hospedeiro , Humanos , Incidência , Estimativa de Kaplan-Meier , Recidiva Local de Neoplasia , Prognóstico , Estudos Retrospectivos , Fatores de Risco , Transplante Homólogo , ViremiaRESUMO
PURPOSE: The aim of this study was to develop a rating scale for the weight management of patients with congestive heart failure (CHF). METHODS: The original pool of items was created through in-depth interviews and a literature review. Scale validity was analyzed based on face validity, content validity, and structure validity. The content validity and structure validity were evaluated. The overall internal consistency reliability were assessed by using Cronbach's alpha and retest reliability test. RESULTS: A total of 190 CHF patients were enrolled but 5 refused. The original 19 items were then refined to a scale of 16 items. The final scale included four factors (weight monitoring, knowledge, confidence, and behaviours related to weight management), which accounted for 58.7% of the variance. Content validity ratio on the content validity was 0.88. The Cronbach's alpha was 0.843 and the retest reliability was 0.833. CONCLUSIONS: The Chinese CHF-related weight management scale developed has high reliability and validity. KEY WORDS: Congestive heart failure; Reliability; Scale; Validity; Weight management.
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RPB5-mediating protein (RMP) is associated with the RNA polymerase II subunit RPB5. RMP functionally counteracts the transcriptional activation of hepatitis B virus X protein that has been shown to play a role in the development of hepatocellular carcinoma (HCC). However, the effect of RMP on the growth of HCC remains unclear. In this study, we characterized the potential role of RMP in the proliferation of human HCC cells using two cell lines, SMMC-7721 and HepG2. We found that RMP expression increased when HCC cells were treated with (60)Co γ-irradiation. Cell growth and colony formation assays suggest that RMP plays an antiapoptotic role in the proliferation and growth of HCC cells. We also show that RMP depletion induced the G(2) arrest of HCC cells characterized by the decreased expression of Cdk1 and Cyclin B. Tumor formation assays further confirmed the in vivo requirement of RMP during HCC growth. In conclusion, our results demonstrate that RMP is a radiation-sensitive factor, and it may play essential roles in HCC growth by affecting the proliferation and apoptosis of HCC cells.
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Carcinoma Hepatocelular/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fase G2 , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/metabolismo , Proteínas de Neoplasias/metabolismo , RNA Polimerase II/metabolismo , Animais , Apoptose/efeitos da radiação , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Ciclina B/genética , Ciclina B/metabolismo , Raios gama , Células Hep G2 , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transplante HeterólogoRESUMO
BACKGROUND: Quinacrine (QC), an antimalarial drug, has been shown to possess anticancer effect both in vitro (cancer cell lines) and in vivo (mouse models). In the cancer cells, QC can simultaneously suppress nuclear factor-κB and activate p53 signaling, which results in the induction of the apoptosis in these cells. However, the experimental results come from a few limited cancer cell lines, and the detailed mechanisms remain unknown. OBJECTIVE: This study investigated the tumor-killing effects of QC on gastric cancer cells as well as underlying molecular pathways. METHODS: SGC-7901 cells were treated with or without QC at different concentrations for 24 hours. The effect of QC on the inhibition of SGC-7901 cell proliferation was assessed by Cell Counting Kit-8 assay. Apoptosis was detected by examining nuclear morphology and quantifying phosphatidylserine externalization. Alterations in cellular morphology were analyzed by laser scanning confocal microscopy for fluorescent analysis. Cell cycle analysis was performed by propidium iodide (PI) staining and flow cytometry. The enzyme activity changes of caspase-3 were detected by colorimetry expression method. Western blot analysis was used to detect the changes in the protein level of Bax, Bc1-2, p53, and cytochrome c in cytosol of SGC-7901 cells. RESULTS: Our results showed that QC could significantly inhibit the growth of SGC-7901 cells in a dose-dependent manner, with the IC50 mean (SD) value of 16.18 (0.64) µM, compared with nontreated controls. QC treatment (15 µM) could also induce apoptosis in SGC-7901 cells (26.30% [5.31%], compared with control group of 3.37% [0.81%]; P < 0.01), and the increasing phosphatidylserine level and the accumulation of chromatin nucleation in QC-treated cells provided further evidence. In addition, cell cycle analysis with PI staining showed that a significant S enriches, increasing from 12.00% (1.24%) (control) to 20.94% (2.40%) (QC treatment) (P < 0.01). Furthermore, increased activities of caspase-3 (increasing from 0.108 [0.019] to 0.628 [0.068]; P < 0.01) were observed in SGC-7901 cells treated with 15 µM QC. Western blot analysis showed that QC treatment significantly increased the levels of proapoptotic proteins, including cytochrome c, Bax, and p53, and decreased the levels of antiapoptotic protein Bcl-2, thus shifting the ratio of Bax/Bcl-2 in favor of apoptosis. CONCLUSIONS: Our findings suggest that QC can significantly inhibit cell growth and induce apoptosis in SGC-7901 cells, which involves p53 upregulation and caspase-3 activation pathway.
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To investigate the potential transcriptional regulation and signal pathway of a single microRNA in ischemia-induced retinal neovascularization (NV), we used oxygen-induced retinopathy (OIR) in establishing retinal NV model, and quantitative real-time reverse transcriptase PCR analyzing a microRNA (miR-126) alteration. The mice were treated with plasmid pCMV-MIR-126/liposome mixture intravitreal injection, using pCMV-MIR/liposome mixture as control. The expression levels of VEGF, IGF-2 and HIF-1α, and the level changes of total and phosphorylated p38, ERK in retina from OIR mice were determined by western blot analysis. The effects of miR-126 on retinal NV in OIR mice were identified with fluoresecin angiography and H & E staining. No effect of miR-126 intravitreal injection on retinal vessels was performed with CD31 stained retinal sections. Our results showed that miR-126 was significantly decreased in retina from OIR mice. We confirmed that restoration of miR-126 in retina overcame the high levels of VEGF, IGF-2 and HIF-1α through downregulating p38 and ERK signaling molecules in OIR model, and that miR-126 intravitreal injection reduced retinal NV in OIR model. These results suggest that miR-126 might play a potential transcriptional role in the pathogenesis in diabetic retinopathy.
Assuntos
Proteínas Angiogênicas/genética , Isquemia/tratamento farmacológico , MicroRNAs/farmacologia , Neovascularização Retiniana/tratamento farmacológico , Vasos Retinianos/efeitos dos fármacos , Proteínas Angiogênicas/metabolismo , Animais , Animais Recém-Nascidos , Biomarcadores/metabolismo , Proliferação de Células , Modelos Animais de Doenças , Regulação para Baixo , Angiofluoresceinografia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hiperóxia/metabolismo , Hiperóxia/patologia , Camundongos , Camundongos Endogâmicos C57BL , Oxigênio/toxicidade , Neovascularização Retiniana/metabolismoRESUMO
BACKGROUND: Griseofulvin, an oral nontoxic antifungal drug, has been reported to possess anticancer effect in human cancer cells, while the mechanisms are not completely understood. OBJECTIVE: The aim of this study was to investigate the cytotoxic effect of griseofulvin on K562 cells and to understand its underlying molecular pathways. METHODS: K562 cells were treated with griseofulvin at different concentrations for 24 hours, and the inhibition effect of griseofulvin on K562 cell proliferation was assessed by tetrazolium salt colorimetric assay. Apoptosis was assessed by examining nuclear morphology and quantifying phosphatidylserine externalization, and alterations in cellular morphology were analyzed by laser scanning confocal microscopy for fluorescent analysis. Flow cytometry was used in the analysis of cell cycle, mitochondrial membrane potential, and caspase pathways. RESULTS: Griseofulvin could inhibit the growth of K562 cells in a dose-dependent manner with a mean (SD) inhibitory concentration of 50% value of 15.38 (1.35) µg/mL compared with untreated controls. Apoptosis was induced in K562 cells (38.35% [2.73%]; P < 0.01) by griseofulvin with the observation of both an increase in phosphatidylserine level and accumulation of chromatin nucleation in griseofulvintreated cells. In addition, cell-cycle analysis using propidium iodide staining suggested a significant G2/M accumulation (increase from mean 17.64% [4.49%] to 48.29 [1.89%]; P < 0.01) as a result of griseofulvin treatment. Flow cytometry analysis found that griseofulvin treatment was associated with the depolarization of the mitochondrial membrane in K562 cells. Furthermore, increased activities of caspase-3 by 22.15-fold (P < 0.01) and caspase-9 by 16.73-fold (P < 0.01) were observed in K562 cells after griseofulvin treatment compared with the untreated control; a decrease of caspase-8 activity was also observed, but the change was not statistically significant. CONCLUSIONS: These findings suggest that griseofulvin inhibited growth of K562 cells and induced cell apoptosis through cell-cycle arrest and mitochondrial membrane potential decrease as well as caspase-3 and -9 activation. Further testing is needed to evaluate the potential of griseofulvin as a candidate in the chemotherapy of hematologic malignancies.
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BACKGROUND: Gene therapy is regarded as a new and promising therapeutic modality for cancers, and adenovirus is one of the most frequently used vectors. However, because of low or absent coxsackievirus and adenovirus receptor levels on the surface of many kinds of tumor cells, the efficiency of adenovirus infection of target tumor cells may be low. Meanwhile, gene therapy by a single vector carrying two or more antioncogenes can improve treatment effects and reduce side effects from vectors. In this research, we aimed to detect the antitumor effect of ING4/PTEN double tumor suppressors mediated by adenovirus modified with arginine(R)-glycine(G)-aspartate(D) (RGD) on glioma cells. METHODS: We treated U87 glioma cells with PBS, blank adenovirus or adenovirus carrying RGD, ING4, PTEN, or both ING4 and PTEN, then we detected and compared the U87 cells' growth, apoptosis, and invasion. Moreover, we established a U87 glioma transplantation tumor model to study the antitumor effect in vivo by measuring the volume and weight of tumor masses in each condition. In addition, we analyzed the transcription of related genes by fluorescent quantitative PCR and detected their expression by immunohistochemistry staining to reveal the underlying mechanisms. RESULTS: The double tumor suppressors ING4/PTEN could inhibit the growth of U87 glioma cells with a synergistic antitumor effect, and the RGD modification also acted as an antioncogene to inhibit U87 cell invasion and tumor angiogenesis. CONCLUSIONS: The ING4/PTEN double tumor suppressors mediated by adenovirus modified with RGD had a significantly synergistic antitumor effect on glioma.
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Infecções por Adenoviridae/terapia , Adenoviridae/genética , Proteínas de Ciclo Celular/antagonistas & inibidores , Glioma/terapia , Proteínas de Homeodomínio/antagonistas & inibidores , Proteínas de Membrana/antagonistas & inibidores , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Proteínas Supressoras de Tumor/antagonistas & inibidores , Aminoácidos/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Terapia Genética/métodos , Glioma/virologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas de Membrana/genética , Neovascularização Patológica , PTEN Fosfo-Hidrolase/genética , Proteínas Supressoras de Tumor/genéticaRESUMO
Elongator complex has been associated with hyperphosphorylated RNA polymerase II and is known to play critical roles in transcriptional elongation, as well as in tRNA modification and exocytosis. However, the specific mechanism of how human Elongator complex regulates cell growth and cell cycle remains unclear. To investigate the composition of human Elongator complex and its effects on cell growth, 293T cells were established that stably overexpressed Flag-Elp3 and Flag-Elp4. By using anti-Flag M2 antibody-bound resin, a core Elongator complex was purified from cells that stably overexpressed Flag-Elp3. No Elongator complex was purified from cells stably transfected with pFlagCMV4-Elp4. Interestingly, the cell growth was inhibited in 293T cells transfected with pFlagCMV4- Elp3. Flow cytometry analysis showed that most of the cells stably overexpressing Flag-Elp3 were found in G1 stage, indicating a role of the core Elongator in the G1 checkpoint for the regulation of cell cycle. We observed increased basal transcription and remarkably enhanced transcription stimulated by VP16 in 293T cells overexpressing Flag-Elp3. The transcription could also be synergistically activated by overexpressing both Elp3 and Elp4. Taken together, our results suggested that the core Elongator complex formed by Elp1, Elp2, and Elp3 was rather stable, whereas Elp4, Elp5, and Elp6 might loosely contact and work together with the core Elongator to regulate cell functions.
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Ciclo Celular/fisiologia , Histona Acetiltransferases/fisiologia , Rim/citologia , Proteínas do Tecido Nervoso/fisiologia , Proliferação de Células , Humanos , Rim/embriologia , FosforilaçãoRESUMO
OBJECTIVE: The purpose of this research was to study the influence of the regenerated silk fibroin film (SF) on cytokines expression of transfected human corneal epithelial cells (HCECs) and to investigate the possibility of constructing biomaterial complex using SF, modified by transgenic cells. METHODS: Empirical study.Ad-VEGF(165) was injected into the limbus of a rabbit's cornea to induce cornea neovascularization (CNV). CNV was evaluated by growth areas and VEGF characteristic was evaluated by immunohistochemistry. HCECs was cultivated on silk protein membrane in the cell cultivation plate. Modality of cells, activity of cell proliferation and infection efficiency of Ad-VEGF(165) were monitored to evaluate the biocompatibility of silk fibroin. The angiogenesis-related cytokines in the cell cultivation supernatant was measured using ELISA method such as vascular endothelial growth factor (VEGF), angiogenin 1 (Ang1), epidermal growth factor (EGF) and transforming growth factor-beta (TGF-beta) in the supernatant (Two-way analysis of variance). RESULTS: (1) The area of corneal neovascularization was observed to be (7.60 +/- 1.12) mm(2) at 1 week after Ad-VEGF(165) was injected and it became (12.28 +/- 2.54) mm(2) another three weeks later. Positive expression of VEGF in corneal stromal was observed by immunohistochemistry at 3 d, 1 week and 1 month after injection. (2) There was no difference noticed in amorphous, growth curve and infection efficiency of Ad-VEGF(165) between both cells culture conditions of silk protein membrane and plate cultivation. (3) After transfection, the concentration of VEGF, Ang1, EGF and TGF-beta expressions in the corneal epithelium cell cultivation supernatant with silk protein membrane as carriers was (721.67 +/- 66.97) ng/L, (1042.67 +/- 315.81) ng/L, (2421.00 +/- 0.00) ng/L, and (313.33 +/- 34.06) ng/L respectively; and the concentration of each of the aforementioned expression was (721.67 +/- 66.97) ng/L, (860.33 +/- 315.81) ng/L, (1960.33 +/- 797.90) ng/L, and (278.00 +/- 53.11) ng/L without using silk protein membrane as carriers. The increase of VEGF (F = 168.16, P < 0.0001), EGF (F = 52.76, P < 0.0001), Ang1 (F = 12.47, P = 0.001), and TGF-beta (F = 0.008, P = 0.932) in the Ad-VEGF(165) group was considered statistically significant; however, there was no evident change in the concentration of VEGF (F = 0.071, P = 0.793), EGF (F = 0.563, P = 0.465), Ang1 (F = 0.14, P = 0.714), and TGF-beta (F = 0.008, P = 0.932)expressions in the corneal epithelium cell cultivation supernatant both with or without using silk protein membrane as carriers. CONCLUSION: Same as cell HCECs culturing in the cultivation plate, through SF application, VEGF(165) destination gene could be high-level expressed in the supernatant having which the HCECs is cultured on SF, and in addition, the angiogenesis-related cytokines content of Ang1, EGF, and TGF-beta autocrine in the HCECS cultivation supernatant could be high-level expressed as well.
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Epitélio Corneano/metabolismo , Fibroínas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adenoviridae/genética , Animais , Materiais Biocompatíveis , Técnicas de Cultura de Células , Células Epiteliais/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Coelhos , Transfecção , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
As a biomaterial to be used for reparation in the case of trauma, the silk fibroin, particularly its effect on the transcription and expression of VEGF gene, is a concern. In this study, the ECV304 cell's growth shape and growth curve on the regenerated silk fibroin film were observed, and its VEGF secretion level was measured by ELISA test. It was found that the regenerated silk fibroin film did not interfere with ECV304 cell's growth and function. The L929 cell transfected with human VEGF gene grew on the regenerated silk fibroin film; the real-time quantitative RT-PCR method and ELISA test were used for detecting the transcription and expression of VEGF gene. The results showed the regenerated silk fibroin film did not interfere with the transcription and expression of VEGF gene. Therefore, the regenerated silk fibroin film is a safe biomaterial for inducing vascularization with no untoward effect on the reparation of trauma.
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Materiais Biocompatíveis , Células Endoteliais/metabolismo , Fibroínas/farmacologia , Seda , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Materiais Biocompatíveis/farmacologia , Linhagem Celular , Células Endoteliais/citologia , Humanos , Seda/farmacologia , Transcrição Gênica , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Paf1 complex was identified in yeast and characterized to function in transcription and its related events. We identified the Drosophila homological components of paf1, CDC73 and RTF1 of paf1 complex. The genes encoding Drosophila paf1, CDC73 and RTF1 were cloned and expressed. With the purified recombinant proteins of truncated components of paf1 complex, antibodies against the Drosophila paf1, CDC73 and RTF1 were generated. These antibodies have been shown to be able to detect the endogenous paf1 subunits as well as their human counterparts in the HeLa extract. On Drosophila polytene chromosomes, these antibodies have been demonstrated to locate the paf1 complex at actively transcribing sites, which co-localized with phosphorylated RNA polymerase II, indicating that paf1 complex in Drosophila is involved in transcription or the events coupling with transcription.
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Anticorpos/química , Proteínas de Drosophila/imunologia , Drosophila melanogaster , AnimaisRESUMO
The inhibitor of growth (ING) family proteins have been defined as candidate tumor suppressors. ING4 as a novel member of ING family has potential tumor-suppressive effects via multiple pathways. However, the therapeutic effect of adenovirus-mediated ING4 (Ad-ING4) gene transfer in human osteosarcoma is still unknown. In this study, we explored the in vitro and in vivo antitumor activity of Ad-ING4 in human osteosarcoma and its potential mechanism using a MG-63 human osteosarcoma cell line. We demonstrated that Ad-ING4 induced significant growth inhibition and apoptosis, upregulated the expression of P21, P27 and Bax, downregulated the Bcl-2 expression and activated Caspase-3 in MG-63 human osteosarcoma cells. Moreover, intratumoral injections of Ad-ING4 in athymic nude mice bearing MG-63 human osteosarcoma tumors significantly suppressed osteosarcoma xenografted tumor growth, increased the expression of P21, P27 and Bax, reduced the Bcl-2 and CD34 expression and microvessel density (MVD) in tumors. This retarded MG-63 osteosarcoma growth in vitro and in vivo in an athymic nude mouse model elicited by Ad-ING4 was closely associated with the increase in the expression of cell cycle-related molecules P21 and P27, decrease in the ratio of anti- to pro-apoptotic molecules Bcl-2/Bax followed by the activation of Caspase-3 leading to apoptosis via intrinsic apoptotic pathways, and the inhibition of tumor angiogenesis. Thus, our results indicate that Ad-ING4 is a potential candidate for human osteosarcoma gene therapy.
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Adenoviridae/genética , Apoptose/genética , Proteínas de Ciclo Celular/genética , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Proteínas de Homeodomínio/genética , Neovascularização Patológica/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Proteínas Supressoras de Tumor/genética , Animais , Biomarcadores , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/genética , Modelos Animais de Doenças , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Terapia Genética , Humanos , Masculino , Camundongos , Neovascularização Patológica/metabolismo , Neovascularização Patológica/terapia , Osteossarcoma/metabolismo , Osteossarcoma/terapia , Carga Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The inhibitor of growth (ING) family proteins have been defined as candidate tumor suppressors. ING4 as a novel member of ING family has potential tumor-suppressive effects via multiple pathways. However, the therapeutic effect of adenovirus-mediated ING4 (Ad-ING4) gene transfer in human osteosarcoma is still unknown. In this study, we explored the in vitro and in vivo antitumor activity of Ad-ING4 in human osteosarcoma and its potential mechanism using a MG-63 human osteosarcoma cell line. We demonstrated that Ad-ING4 induced significant growth inhibition and apoptosis, upregulated the expression of P21, P27 and Bax, downregulated the Bcl-2 expression and activated Caspase-3 in MG-63 human osteosarcoma cells. Moreover, intratumoral injections of Ad-ING4 in athymic nude mice bearing MG-63 human osteosarcoma tumors significantly suppressed osteosarcoma xenografted tumor growth, increased the expression of P21, P27 and Bax, reduced the Bcl-2 and CD34 expression and microvessel density (MVD) in tumors. This retarded MG-63 osteosarcoma growth in vitro and in vivo in an athymic nude mouse model elicited by Ad-ING4 was closely associated with the increase in the expression of cell cycle-related molecules P21 and P27, decrease in the ratio of anti- to pro-apoptotic molecules Bcl-2/Bax followed by the activation of Caspase-3 leading to apoptosis via intrinsic apoptotic pathways, and the inhibition of tumor angiogenesis. Thus, our results indicate that Ad-ING4 is a potential candidate for human osteosarcoma gene therapy.
Assuntos
Adenoviridae/genética , Apoptose/genética , Neoplasias Ósseas/terapia , Proteínas de Ciclo Celular/genética , Proteínas de Homeodomínio/genética , Neovascularização Patológica/genética , Osteossarcoma/terapia , Proteínas Supressoras de Tumor/genética , Animais , Neoplasias Ósseas/patologia , Caspase 3/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p21/biossíntese , Inibidor de Quinase Dependente de Ciclina p27/biossíntese , Ativação Enzimática/genética , Técnicas de Transferência de Genes , Genes Supressores de Tumor , Terapia Genética/métodos , Humanos , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica/terapia , Osteossarcoma/patologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Transplante Heterólogo , Proteína X Associada a bcl-2/biossínteseRESUMO
Interleukin-24 (IL-24), a member of the IL-10 cytokine gene family, causes growth suppression and apoptosis in various solid tumor cells. However, the effects of IL-24 on hematopoietic malignant cells have not been extensively explored. In this report, we constructed an RGD-engineered recombinant adenoviral vector, Ad.RGD-IL-24, and assessed its effects on human myeloid leukemia cells. Ad vector mediates gene transfer into leukemia cells with high efficiency. Ectopic over-expression of IL-24 has significant growth inhibition and differentiation inducement effects on these cells. Treatment with Ad.RGD-IL-24 could potentially induce leukemia cells' cell-cycle arrest. In addition, IL-24 expression could significantly induce apoptosis of the THP-1 cells. Ad.RGD-IL-24 had a potent effect on the up-regulation of the expression of GRP78/Bip, GADD34 and Bax, down-regulation of the expression of Bcl-2 and Mcl-1, and induced the activation of Caspase-3, which may be responsible for its apoptosis-inducing effect on THP-1 cells. Furthermore, IL-24 expression could retard transplanted leukemia cell tumor growth in vivo in athymic nude mice. These findings showed the marked antitumor activity of IL-24 and provided potential perspectives in designing therapeutic or vaccine strategies in immuno-gene therapy of myeloid leukemia.
Assuntos
Adenoviridae/genética , Interleucinas/genética , Leucemia Mieloide/terapia , Oligopeptídeos/genética , Animais , Apoptose , Ciclo Celular , Diferenciação Celular , Linhagem Celular Tumoral , Chaperona BiP do Retículo Endoplasmático , Terapia Genética , Humanos , Leucemia Mieloide/patologia , Camundongos Nus , RNA Mensageiro/metabolismo , Carga TumoralRESUMO
Interleukin-24 (IL-24)/melanoma differentiation-associated gene-7 (mda-7) is a unique cytokine-tumor suppressor that displays ubiquitous antitumor properties and tumor-specific killing activity. Oncostatin M (OSM) is the most active IL-6-type cytokine and inhibits the proliferation of various solid tumor cell lines. Multigene-based combination therapy may be an effective practice in cancer gene therapy. The therapeutic potential of a combination of IL-24 and OSM in treating cancers is still elusive. In this study, we aimed to examine the enhanced antitumor activity of adenovirus-mediated IL-24/OSM tumor suppressor gene cotransfer in human melanoma cells. We constructed an IL-24/OSM bicistronic adenovirus and assessed its combined effect on A375 human melanoma cells in vitro and in vivo by detecting and comparing apoptosis in the bicistronic antioncogene group (Ad-IL-24-OSM) and in the IL-24 or OSM single antioncogene group. We also investigated the possible mechanism underlying this effect. The bicistronic adenovirus-mediated coexpression of IL-24 and OSM induced additive growth suppression and apoptosis and an overlapping effect on the upregulation of p21, p53, Bax, and cleaved caspase-3 in vitro and in vivo. Moreover, Ad-IL-24-OSM treatment additively reduced the expression of CDK4 and cyclin D1 in A375 melanoma cells and the expression of CD34 and Cox-2 in A375 xenograft tumors in athymic nude mice. The enhanced antitumor activity elicited by Ad-IL-24-OSM was closely associated with the activation of the apoptotic pathway and the additive inhibition of tumor angiogenesis. Therefore, our results indicate that cancer gene therapy combining two or more tumor suppressors, such as IL-24 and OSM, may constitute a novel and effective therapeutic strategy for treating malignant melanoma and other cancers.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Interleucinas/genética , Melanoma/genética , Melanoma/terapia , Oncostatina M/genética , Animais , Apoptose/genética , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Interleucinas/biossíntese , Masculino , Melanoma/irrigação sanguínea , Melanoma/patologia , Camundongos , Camundongos Nus , Neovascularização Patológica/genética , Neovascularização Patológica/terapia , Oncostatina M/biossíntese , Plasmídeos/administração & dosagem , Plasmídeos/genética , Distribuição Aleatória , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Di-(2-ethylhexyl) phthalate (DEHP) has been considered as a widespread environmental persistent organic pollutant and its potential concern on human health is raised by previous studies. In particular, children are more likely to be exposed to DEHP through gastrointestinal route and consequently are more susceptible to DEHP hazards. Some reports have uncovered a positive association between DEHP exposure and an increased prevalence of allergic diseases in infants and juveniles. Allergy is a hypersensitive reaction rooted in imbalanced humoral immunity. T follicular helper cell (Tfh), an important CD4(+) Th cell subset, until recently has been identified as a key player in humoral immune response by modifying B cells functions. Tfh cells are therefore perceived as the therapeutic target of immune disorders. In the present study, focusing on the newly confirmed Tfh cells, we examined the effects of DEHP on humoral immunity and investigated the underlying mechanisms. Using ovalbumin (OVA) sensitization weanling mice model under the condition of gastrointestinal exposure to DEHP, we found that DEHP acted as an immunoadjuvant to augment OVA-specific IgE and IgG1 production, amplified germinal center formation in lymphoid nodule, as well as stimulated the expansion of CD4(+)CXCR5(+)ICOS(+)/CD4(+)CXCR5(+)PD-1(+) Tfh cells and CD19(+)CD138(+)GL7(+) plasma cells. Based on the results of immune adoptive transfusion, DEHP-related anaphylactic response was ascribed to Tfh cells hyperfunction directly. We further proved that DEHP gavage together with OVA sensitization adjuvantly promoted the synthesis of cytokines IL-21 and IL-4 and the expression of transcription factors Bcl-6 and c-Maf in Tfh cells. In conclusion, our study demonstrates that DEHP has adjuvant toxic effects on Tfh cells by synthesizing an excess of cytokines IL-21 and IL-4 via over-expression of transcription factors Bcl-6 and c-Maf, leading to an increasing secretion of allergy-related IgE and IgG1.
Assuntos
Dietilexilftalato/toxicidade , Poluentes Ambientais/toxicidade , Centro Germinativo/efeitos dos fármacos , Hipersensibilidade/etiologia , Imunidade Humoral/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Ovalbumina , Subpopulações de Linfócitos T/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Administração Oral , Transferência Adotiva , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Proliferação de Células/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Dietilexilftalato/administração & dosagem , Modelos Animais de Doenças , Poluentes Ambientais/administração & dosagem , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Hipersensibilidade/sangue , Hipersensibilidade/imunologia , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Interleucina-4/metabolismo , Interleucinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Proto-Oncogênicas c-bcl-6 , Proteínas Proto-Oncogênicas c-maf/metabolismo , Medição de Risco , Fatores de Risco , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/transplante , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo , Linfócitos T Auxiliares-Indutores/transplanteRESUMO
The smallest gene HBx of Hepatitis B virus (HBV) is recognized as an important viral oncogene (V-oncogene) in the hepatocarcinogenesis. Our previous work demonstrated that RMP is a cellular oncogene (C-oncogene) required for the proliferation of hepatocellular carcinoma (HCC) cells. Here we presented the collaboration between V-oncogene HBx and C-oncogene RMP in the development of HCC. The coexpression of HBx and RMP resulted in the cooperative effect of antiapoptosis and proliferation of HCC cells. In vivo, overexpression of RMP accelerated the growth of HBx-induced xenograft tumors in nude mice and vice versa HBx promoted the growth of RMP-driven xenograft tumors. Although HBx didn't regulate the expression of RMP, HBx and RMP interact with each other and collocalized in the cytoplasm of HCC cells. HBx and RMP collaboratively inhibited the expression of apoptotic factors and promoted the expression of antiapoptotic factors. This finding suggests that HBV may induce, or at least partially contributes to the carcinogenesis of HCC, through its V-oncoprotein HBx interacting with the C-oncoprotein RMP.