Gene therapy targeting for carcinoma regulated by E2F-1 promoter / 中华外科杂志

<p><b>OBJECTIVE</b>To study the selective effect to tumor cells mediated by a recombinant adenoviral vector carrying E2F-1 promoter.</p><p><b>METHODS</b>The AdEasy-1 adenoviral vector system was used in this experiment. Several recombinant adenovirus with tumor-targeting E2F-1 promoter were constructed and then the E2F-1 promoter gene was checked by PCR and sequencing. The two adenovirus expressing GFP gene which is regulated by E2F-1 promoter or CMV promoter were used to respectively transfect tumor cells and non-proliferating normal cells, then observed and analyzed the different results caused by different promoters. Vpr gene was cloned into the targeting recombinant adenovirus. The new adenovirus named rvAdE2F-1/vpr was used to transfect tumor cells SMMC-7721, LS174T and non-proliferating normal cells H292, L-02. The surviving rate of each group was registered; the level of E2F-1 protein expressed in normal and tumor cell lines were checked by Western Blot.</p><p><b>RESULT</b>E2F-1 promoter can regulate the downstream gene GFP selectively expressed in LS174T and its activity in LS174T was similar with CMV promoter's; Vpr gene regulated by E2F-1 promoter can suppress the proliferation of tumor cells and no toxicity to normal cells; In all of the tumor cells, a much higher level of E2F-1 was expressed compared with normal cell lines. E2F-1 promoter's activity correlated well with E2F-1 protein levels.</p><p><b>CONCLUSIONS</b>E2F-1 promoter can control a selective cell killing to cancer cells, with no effect to normal cells. The system of E2F-1 promoter is a useful method for tumor-targeting gene therapy.</p>

Application of proteomics in the research of discrepancy proteins in gastric cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To explore the discrepancy proteins in gastric cancer by proteome analysis.</p><p><b>METHODS</b>Total proteins of gastric cancer tissues and matched normal gastric epithelial tissues were separated respectively by two-dimensional polyacrylamide gel electrophoresis (2-DE). Mass spectrometry was used to test the differentially expressed proteins.</p><p><b>RESULTS</b>One thousand one hundred and forty-seven protein spots from gastric cancer tissue and 1079 spots from the normal tissue were gained. Out of 164 different protein spots, 41 were only expressed in gastric cancer tissue, 27 were unique in normal tissue, 39 were up-regulated and 57 were down-regulated in gastric cancer. Seven proteins, which were highly expressed in gastric cancer tissue, were identified.</p><p><b>CONCLUSION</b>Different protein spots between gastric cancer tissues and normal gastric epithelial tissue were gained by proteomics. The 7 discrepancy proteins were further identified. It establishes the foundation of finding specific gastric cancer proteins, which act as biomarkers for the diagnosis and prognosis of gastric cancer.</p>

Reversal of MDR1 gene-dependent multidrug resistance using short hairpin RNA expression vectors / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>RNA interference using short hairpin RNA (shRNA) can mediate sequence-specific inhibition of gene expression in mammalian cells. A vector-based approach for synthesizing shRNA has been developed recently. Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. In this study, we reversed MDR using shRNA expression vectors in a multidrug-resistant human breast cancer cell line (MCF-7/AdrR).</p><p><b>METHODS</b>The two shRNA expression vectors were constructed and introduced into MCF-7/AdrR cells. Expression of MDR1 mRNA was assessed by RT-PCR, and P-gp expression was determined by Western Blot and immunocytochemistry. Apoptosis and sensitization of the breast cancer cells to doxorubicin were quantified by flow cytometry and methyl thiazolyl tetrazolium (MTT) assays, respectively. Cellular daunorubicin accumulation was assayed by laser confocal scanning microscopy (LCSM). Statistical significance of differences in mean values was evaluated by Student's t tests. P < 0.05 was considered statistically significant.</p><p><b>RESULTS</b>In MCF-7/AdrA cells transfected with MDR1-A and MDR1-B shRNA expression vectors, RT-PCR showed that MDR1 mRNA expression was reduced by 40.9% (P < 0.05), 30.1% (P < 0.01) (transient transfection) and 37.6% (P < 0.05), 28.0% (P < 0.01) (stable transfection), respectively. Western Blot and immunocytochemistry showed that P-gp expression was significantly and specifically inhibited. Resistance against doxorubicin was decreased from 162-fold to 109-fold (P < 0.05), 54-fold (P < 0.01) (transient transfection) and to 108-fold (P < 0.05), 50-fold (P < 0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular daunorubicin accumulation. The combination of shRNA vectors and doxorubicin significantly induced apoptosis in MCF-7/AdrR cells.</p><p><b>CONCLUSIONS</b>shRNA expression vectors effectively reduce MDR expression in a sustained fashion and can restore the sensitivity of drug-resistant cancer cells to conventional chemotherapeutic agents.</p>