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Antitumor activity of combined endostatin and thymidine kinase gene therapy in C6 glioma models.

ABSTRACT: The combination of Endostatin (ES) and Herpes Simplex Virus thymidine kinase (HSV-TK) gene therapy is known to have antitumor activity in bladder cancer. The potential effect of ES and TK therapy in glioma has not yet been investigated. In this study, pTK-internal ribosome entry site (IRES), pIRES-ES, and pTK-IRES-ES plasmids were constructed; pIRES empty vector served as the negative control. The recombinant constructs were transfected into human umbilical vein endothelial cells (HUVECs) ECV304 and C6 rat glioma cell line. Ganciclovir (GCV) was used to induce cell death in transfected C6 cells. We found that ECV304 cells expressing either ES or TK-ES showed reduced proliferation, decreased migration capacity, and increased apoptosis, as compared to untransfected cells or controls. pTK-IRES-ES/GCV or pTK-IRES/GCV significantly suppressed cell proliferation and induced cell apoptosis in C6 cells, as compared to the control. In addition, the administration of pIRES-ES, pTK-IRES/GCV, or pTK-IRES-ES/GCV therapy improved animal activity and behavior; was associated with prolonged animal survival, and a lower microvessel density (MVD) value in tumor tissues of C6 glioma rats. In comparison to others, dual gene therapy in form of pTK-IRES-ES/GCV had a significant antitumor activity against C6 glioma. These findings indicate combined TK and ES gene therapy was associated with a superior antitumor efficacy as compared to single gene therapy in C6 glioma.

SUBMITTER: Chen Y

PROVIDER: S-EPMC5055148 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Antitumor activity of combined endostatin and thymidine kinase gene therapy in C6 glioma models.

Chen Yan Y Huang Honglan H Yao Chunshan C Su Fengbo F Guan Wenming W Yan Shijun S Ni Zhaohui Z

Cancer medicine 20160701 9

The combination of Endostatin (ES) and Herpes Simplex Virus thymidine kinase (HSV-TK) gene therapy is known to have antitumor activity in bladder cancer. The potential effect of ES and TK therapy in glioma has not yet been investigated. In this study, pTK-internal ribosome entry site (IRES), pIRES-ES, and pTK-IRES-ES plasmids were constructed; pIRES empty vector served as the negative control. The recombinant constructs were transfected into human umbilical vein endothelial cells (HUVECs) ECV304 ...[more]

PMID: 27366865

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Project description:(R,R’)-4-methoxy-1-naphthylfenoterol (MNF) inhibits in vitro proliferation of several types of cancer cell lines. In this study, the in vivo antitumor effects of MNF were evaluated using rat C6 glioma cells implanted subcutaneously into the lower flank of 5 week-old NMRI/Nude female Swiss mice. Three days after the inoculation, the mice were subjected to intraperitoneal injections of saline or MNF (2mg/kg) for five days per week for16 days. Tumor volumes were measured everyday using slide calipers. Significant reductions in mean tumor volumes were observed in mice receiving MNF when compared with the saline-treated group (p<0.001, n=17-19). At the end of the study, animals were sacrificed and tumors were collected for cDNA microarray, quantitative RT-PCR and immunoblot analyses. Significant decrease in expression of genes involved in cellular proliferation, including mitotic checkpoint kinase MAD3L (Bub1b), cyclin-dependent kinase inhibitor 3 (Cdkn3) and cyclin A2 (Ccna2), as well as molecular markers for glioblastoma, such as oligodendrocyte transcription factor 1 (Olig1) and SRY-box 4 (Sox4) was observed in tumors of MNF-treated mice as compared to saline-injected controls. The efficacy of MNF against C6 glioma cancer in vivo was accompanied by marked reduction in the expression of cell cycle regulator proteins, including cyclin A and cyclin D1, in tumor extracts. This study is the first demonstration of MNF-dependent chemoprevention in a glioblastoma xenograft model in the mouse and may offer a potential mechanism for its anticancer action in vivo. Keywords: fenoterol derivative; C6 glioma; tumor xenografts; microarray analysis; Total RNA was isolated from rat C6 glioma xenografts harvested from vehicle and MNF-treated nude mice (n = 3 per group, cohort 1). This analysis was repeated in a second cohort of animals (n = 3 per group, cohort 2). Total cellular RNA was extracted using an RNeasy plus mini kit (QIAGEN, Valencia, CA), and its quality was assessed using an Agilent BioAnalyzer using RNA 6000 Nano Chips (Agilent Technologies, Santa Clara, CA). Transcriptional profiling was determined using Illumina Sentrix BeadChips (Illumina, San Diego, CA). Total RNA was used to generate biotin-labeled cRNA with the Illumina TotalPrep RNA Amplification Kit. In short, 0.5ug of total RNA was first converted into single-stranded cDNA with reverse transcriptase using an oligo-dT primer containing the T7 RNA polymerase promoter site and then copied to produce double-stranded cDNA molecules. The double-stranded cDNA was cleaned and concentrated with the supplied columns and used in an overnight in-vitro transcription reaction where single-stranded RNA (cRNA) was generated incorporating biotin-16-UTP. A total of 0.75µg of biotin-labeled cRNA was hybridized at 58 °C for 16 h to Illumina's Sentrix Rat Ref-12 Expression BeadChips. The arrays were washed, blocked and the labeled cRNA was detected by staining with streptavidin-Cy3. Hybridized arrays were scanned using an Illumina BeadStation 500X Genetic Analysis Systems scanner and the image data extracted using Illumina’s GenomeStudio software, version 1.6.1. For statistical analysis, the expression data were filtered to include only probes with a consistent signal on each chip and an Illumina detection p value < 0.02.

Project description:BackgroundDirectly targeting therapeutic suicide gene to a solid tumor is a hopeful approach for cancer gene therapy. Treatment of a solid tumor by an effective vector for a suicide gene remains a challenge. Given the lack of effective treatments, we constructed a bifidobacterial recombinant thymidine kinase (BF-rTK) -ganciclovir (GCV) targeting system (BKV) to meet this requirement and to explore antitumor mechanisms.MethodsBifidobacterium (BF) or BF-rTK was injected intratumorally with or without ganciclovir in a human colo320 intestinal xenograft tumor model. The tumor tissues were analyzed using apoptosis antibody arrays, real time PCR and western blot. The colo320 cell was analyzed by the gene silencing method. Autophagy and necroptosis were also detected in colo320 cell. Meanwhile, three human digestive system xenograft tumor models (colorectal cancer colo320, gastric cancer MKN-45 and liver cancer SSMC-7721) and a breast cancer (MDA-MB-231) model were employed to validate the universality of BF-rTK + GCV in solid tumor gene therapy. The survival rate was evaluated in three human cancer models after the BF-rTK + GCV intratumor treatment. The analysis of inflammatory markers (TNF-α) in tumor indicated that BF-rTK + GCV significantly inhibited TNF-α expression.ResultsThe results suggested that BF-rTK + GCV induced tumor apoptosis without autophagy and necroptosis occurrence. The apoptosis was transduced by multiple signaling pathways mediated by FasL and TNFR2 and mainly activated the mitochondrial control of apoptosis via Bid and Bim, which was rescued by silencing Bid or/and Bim. However, BF + GCV only induced apoptosis via Fas/FasL signal pathway accompanied with increased P53 expression. We further found that BF-rTK + GCV inhibited the expression of the inflammatory maker of TNF-α. However, BF-rTK + GCV did not result in necroptosis and autophagy.ConclusionsBF-rTK + GCV induced tumor apoptosis mediated by FasL and TNFR2 through the mitochondrial control of apoptosis via Bid and Bim without inducing necroptosis and autophagy. Furthermore, BF-rTK + GCV showed to repress the inflammation of tumor through downregulating TNF-α expression. Survival analysis results of multiple cancer models confirmed that BF-rTK + GCV system has a wide field of application in solid tumor gene therapy.

Project description:BackgroundAnti-angiogenic therapy inhibits tumor growth and is considered as a potential clinical therapy for malignant glioma. However, inevitable recurrences and unexpected tumor resistance, particularly increased invasion ability of glioma cell, were observed after anti-angiogenic treatment. The underlying mechanism remains undetermined. Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (Pyk2) are closely associated with cell migration; therefore, we investigated the possible role of these kinases in rat C6 glioma cell invasion induced by bevacizumab, a recombinant monoclonal antibody against vascular endothelial growth factor (VEGF).MethodsThe effects of bevacizumab on migration and invasion of C6 glioma cells were investigated in vitro and in vivo. The cells proliferation, migration, and invasion were determined by MTT assay, wound healing, and transwell assay, respectively. Invasive potential of glioma cells in vivo was assessed by counting vimentin-positive cells crossing the solid tumor rim by immunohistochemical staining. The total and phosphorylated protein levels of FAK and Pyk2 were detected by Western blotting.ResultsBevacizumab exposure increased migration and invasion of cultured C6 cells in a concentration-dependent manner. In addition, the continuous bevacizumab treatment also promoted tumor invasion in rat C6 intracranial glioma models. Bevacizumab treatment enhanced Pyk2 phosphorylation at Tyr402, but no effect on FAK phosphorylation at Tyr397 both in vitro and in vivo. Knockdown of Pyk2 by siRNA or inhibition of Pyk2 phosphorylation by Src kinase specific inhibitor PP1 partially inhibited bevacizumab-induced cell invasion in cultured C6 glioma cells. Furthermore, the combined administration of bevacizumab and PP1 significantly suppressed glioma cell invasion into surrounding brain tissues compared to bevacizumab treatment alone in experimental rats.ConclusionsThese results suggest that anti-VEGF treatment promotes glioma cell invasion via activation of Pyk2. Inhibition of Pyk2 phosphorylation might be a potential target to ameliorate the therapeutic efficiency of anti-VEGF treatment.

Dataset Information

Antitumor activity of combined endostatin and thymidine kinase gene therapy in C6 glioma models.

Publications

Antitumor activity of combined endostatin and thymidine kinase gene therapy in C6 glioma models.

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