Résumé
Objective: To investigate the suppression effect of mouse marrow-derived endothelial progenitor cells (EPCs) modified by cytosine deaminase (CD) gene and enzyme prodrug 5-fluorocytosine (5-Fc) on the growth of hepatocellar carcinoma in orthotopic H22 cell-transplanted mice. Methods: The EPCs were transfected by lentiviral vector carrying CD gene (plenti6.3-EGFP-CD) using Polybrene technique. The mouse hepatoma H22 cells were treated by the supernatant of EPCs carrying CD gene and 5-Fc, and then the number and morphology of the cells were observed under an inverted microscope. C57BL/6 mice bearing orthotopic transplanted H22 hepatocellar carcinoma were treated by EPCs carrying CD gene and 5-Fc. The volume of tumor in mice was monitored by magnetic resonance imaging, and the apoptosis in tumor was tested by terminal deoxynucleotidyl transferase-mediated dUTP nick and labeling (TUNEL) assay. The expression of CD protein in the transplanted tumor was identified by Western blotting. Results: The proliferation of H22 cells treated by the supernatant of EPCs carrying CD gene combined with 5-Fc was significantly reduced. Compared with the control, the tumor growth in the hepatocellular carcinoma orthotopic-transplantation mouse models treated by EPCs carrying CD gene and 5-Fc was inhibited to (47.29±5.81)% (P < 0.05), and the apoptosis index of tumor cells was markedly increased [(39.98±5.13)%]. Conclusion: The EPCs modified by CD gene combined with 5-Fc administration can effectively inhibit the proliferation of the mouse orthotopic transplanted hepatocellular carcinoma, and induce the apoptosis of tumor cells. Copyright © 2014 by TUMOR.
Résumé
Objective To evaluate the selectively killing effect of adenovirus(Ad)mediated double suicide gene driven by VEGF promoter on pancreatic cancer cell SW1990. Methods VEGFexpressing SW1990 were infected by Ad-VEGFP-CDTK and Ad-null.respectively.The infection rate was observed and the expression of CDTK was detected by RT-PCR and Western blotting.Followed by treatment with 5-FC and GCV killing effects were evaluated and bystander effects were analyzed by MTF.Pathological character of cells was observed by electron microscopy and distribution of cell cycle was detected by flow cytometry.The caspase-3 activity was detected by absorption spectrometry. Results The infection rate of the resultant recombinant Ad to SW1990 cells was not apparently different.RT-PCR and Western blotting demonstrated product of CDTK gene in SW1990 cell infected by Ad-VEGFP-CDTK.Prodrug could inhibit proliferation of SW1990 and the effect was dose-dependent.There was considerable bystander effect as observed by MTF.Apoptotic peak was also shown by flow cytometry.Morphologic features of apoptosis in SW1990 cells were displayed via electron microscopy.Cells at the G0-G1 phase was increasing and the rate at the G2-M and S phase was decreased by prodrug.The caspase-3 activity gradually rised with the increasing concentration of the prodrug. Conclusions The CDTK fusion gene system controlled by VEGF promoter has killing effect on the VEGF-expressing SW1990 cells and inducing the cell apoptosis.
Résumé
Objective: To evaluate the specific killing effect of adenovirus (Ad)-mediated double suicide gene system (CD/TK fusion genes) driven by VEGF promoter on pancreatic cancer Capan-2 cells. Methods: VEGF-positive Capan-2 cells were transfected with Ad-VEGFP-CDTK. Ad-free vector acted as negative control. The transfection efficiency was observed and the transcription of CDTK gene was detected by RT-PCR. The Capan-2 cells were treated with 5-fluorocytosine (5-FC) and ganciclovir (GCV) at different concentrations. The effects of the double suicide gene system on cell proliferation and the bystander effects were assessed by MTT assay. Then morphological changes were observed by electronic microscopy and distribution of cell cycle was determined by flow cytometry. Human pancreatic cancer Capan-2 cells were subcutaneously implanted into nude mice. The tumor inhibition rate was calculated. Results: The infection rates of the two resultant recombinant Ads in Capan-2 cells were not significantly different, and they were gradually elevated with the increase in multiple of infection (MOI) of Ads. MTT assay showed that pre-drug dose-dependently inhibited the growth of Capan-2 cells. Apparent bystander effects were also observed. Electronic microscopy demonstrated apoptotic changes of Capan-2 cells. Typical apoptotic peak was detected in double suicide gene system-treated group by flow cytometry. Cell cycle distribution analysis showed that the proportion of G0/G1 increased and the number of cells in G2/M and S phase decreased after treatment. The implanted tumor model was successfully established in nude mice. The tumor size was decreased significantly after treatment with double suicide gene system. Conclusion: The promoter of VEGF regulated double suicide gene system can specifically kill pancreatic cancer Capan-2 cells and induce apoptosis in vitro. And it significantly inhibites the growth of implanted pancreatic tumor in nude mice.