Combined Antitumor Effect of Ad-bFGF-siRNA and Ad-Vpr on the Growth of Xenograft Glioma in Nude Mouse Model
Clinical Lab, Tianjin Huan Hu Hospital, Tianjin 300060, China. Pathology & Oncology Research
(Impact Factor: 1.86).
06/2011; 17(2):237-42. DOI: 10.1007/s12253-010-9303-5
Basic fibroblast growth factor (bFGF) has been demonstrated to correlate with glioma grade and clinical outcome and has established its possible usefulness as a target for glioma therapy. Vpr has been described as an antitumor agent and displays a potent antitumor nature. Here, we try to investigate whether a combined treatment with bFGF-siRNA and Vpr gene would have a enhanced effectiveness on glioma in vitro and in vivo.After treatments with only Ad-bFGF-siRNA, only Ad-Vpr, and a combination of both, we assessed the changes in cell proliferation, cell cycle, and apoptosis in vitro by the methods of MTT, PI and FITC-AnnexinV double staining, respectively. In addition, we also evaluated the combined effect of bFGF-siRNA and Vpr gene therapy on glioma in vivo using xenograft glioma models in nude mice. Combined Ad-bFGF-siRNA and Ad-Vpr treatment was more better successful in inhibiting cell proliferation in comparison with treatments of either Ad-bFGF-siRNA or Ad-Vpr alone. Treatment of Ad-Vpr alone or a treatment of a combination of Ad-bFGF-siRNA and Ad-Vpr induced the G2/M cell cycle arrest and apoptosis; however, combined treatment was more effective than the Ad-Vpr treatment alone. Although each single treatment can slow the growth of xenograft glioma, the combined treatment with Ad-bFGF-siRNA and Ad-Vpr was better than either the Ad-bFGF-siRNA or Ad-Vpr treatment alone. Our results suggest that the combination therapy with bFGF-siRNA and Vpr gene can achieve a enhanced activity of anti-glioma, supporting the idea that the combination of these two antitumor agents could open new perspectives in glioma therapy.
Available from: Xinnu Xu
- "Western blot analysis was performed as previously described [8,9]. Briefly, the treated and untreated U251 cells were lysed in M-PER Reagent (Thermo Co, Ltd) containing the halt protease and phosphatase inhibitor cocktail. "
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ABSTRACT: Glioblastoma multiforme (GBM) carries a dismal prognosis primarily due to its aggressive proliferation in the brain regulated by complex molecular mechanisms. One promising molecular target in GBM is over-expressed basic fibroblast growth factor (bFGF), which has been correlated with growth, progression, and vascularity of human malignant gliomas. Previously, we reported significant antitumor effects of an adenovirus-vector carrying bFGF small interfering RNA (Ad-bFGF-siRNA) in glioma in vivo and in vitro. However, its mechanisms are unknown. Signal transducer and activator of transcription 3 (STAT3) is constitutively active in GBM and correlates positively with the glioma grades. In addition, as a specific transcription factor, STAT3 serves as the convergent point of various signaling pathways activated by multiple growth factors and/or cytokines. Therefore, we hypothesized that the proliferation inhibition and apoptosis induction by Ad-bFGF-siRNA may result from the interruption of STAT3 phosphorylation. In the current study, we found that in glioma cells U251, Ad-bFGF-siRNA impedes the activation of ERK1/2 and JAK2, but not Src, decreases IL-6 secretion, reduces STAT3 phosphorylation, decreases the levels of downstream molecules CyclinD1 and Bcl-xl, and ultimately results in the collapse of mitochondrial membrane potentials as well as the induction of mitochondrial-related apoptosis. Our results offer a potential mechanism for using Ad-bFGF-siRNA as a gene therapy for glioma. To our knowledge, it is the first time that the bFGF knockdown using adenovirus-mediated delivery of bFGF siRNA and its potential underlying mechanisms are reported. Therefore, this finding may open new avenues for developing novel treatments against GBM.
Available from: Alexandre Laurent
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ABSTRACT: To determine whether upregulated expression of vascular endothelial growth factor (VEGF) in VX2 cells can increase vessel density (VD) and reduce tumor necrosis.
The VX2 cell line was transfected with expression vectors containing cDNA for rabbit VEGF. Stable clones producing rabbit VEGF (VEGF-VX2) were selected. VEGF-VX2 cells (n = 5 rabbits) or nontransfected VX2 cells (controls; n = 5 rabbits) were implanted into leg muscle of 10 rabbits. The animals were sacrificed at day 21. Tumor volume, percentage of necrosis, VD, and VEGF concentration in tumor protein extract were quantified.
Overexpression of VEGF by VX2 cells augmented tumor implantation efficiency 100% and favored cyst formation. The tumor volume was significantly larger for VEGF-VX2 transfected tumors versus controls (P = .0143). Overexpression of VEGF in VX2 cells significantly increased the VD of the tumors (P = .0138). The percentage of necrosis was reduced in VEGF-VX2 tumors versus controls (19.5% vs 38.5 %; P = .002). VEGF concentration in VEGF-VX2 tumors was significantly higher than in control tumors (P = .041) and was correlated with tumor volume (ρ = .883, P = .012).
The overexpression of VEGF increased tumor growth and vascularization, favored cyst formation, and reduced tumor necrosis. This new phenotype of the VX2 tumor may offer some advantages over classic models of VX2 tumor for evaluating anticancer therapies.
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ABSTRACT: Colorectal cancer is a significant health problem, and the advanced stages of the disease have a low response rate to chemotherapy and easily acquire chemoresistance. HIV-1 viral protein R (Vpr) has been shown to possess inhibitory effects on various malignant cells in vivo and in vitro. In this study, an Ad-Vpr construct was used to infect the multidrug-resistant human colorectal cancer HCT-8/5-FU(MDR) cell line in vitro for cell viability, apoptosis, gene expression and gene activity using the MTT, flow cytometry, immunoblotting and gel shift assays, respectively. The data showed that Ad-Vpr significantly reduced HCT-8/5-FU(MDR) cell viability in a dose- and time-dependent manner. Ad-Vpr infection promoted HCT-8/5-FU(MDR) cells to undergo apoptosis and to arrest at the G2 phase of the cell cycle. The G2 cell cycle protein Cyclin B1 accumulated in the cells after Ad-Vpr infection. Furthermore, Ad-Vpr induced activation of caspase-3 and -9, but not caspase-8, in HCT-8/5-FU(MDR) cells. Ad-Vpr suppressed expression of the Bcl-xl protein, but upregulated Bax expression and cytochrome c release from the mitochondria in HCT-8/5-FU(MDR) cells. Ad-Vpr infection also resulted in a time-dependent decrease in nuclear translocation of NF-κB/p65 protein and p65 DNA-binding activity in HCT-8/5-FU(MDR) cells. The data from the current study provide mechanistic insights into understanding the molecular basis and utility of Ad-Vpr as a novel anticancer agent for multidrug resistance in human colorectal cancer.
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