RESUMEN
<p><b>OBJECTIVE</b>To explore the expression of Rictor and mTOR in the colorectal cancer and their clinical significance.</p><p><b>METHODS</b>The expression levels of Rictor and mTOR in HCT116, SW480, LoVo and HCoEpiC cells were detected by indirect immunofluorescence and Western blotting. Sixty-two paraffin-embedded surgical specimens of colorectal cancer tissue and adjacent tissues were examined for Rictor expression using immunohistochemistry. The association of the expression levels of Rictor protein with the clinicopathologic features and the overall survival of the patients was analyzed.</p><p><b>RESULTS</b>The expression level of Rictor was significantly higher in colorectal cancer tissues than in the adjacent tissues (P<0.05). The expression levels of Rictor and mTOR in the colon cancer cell lines were higher than those in human normal colon epithelial cell line HCoEpiC. The expression of Rictor was correlated with Dukes stage and lymphatic metastasis of the tumors but not with other clinicopathological parameter (P>0.05). Patients with Rictor expression had a lower overall survival rate than those without Rictor expression.</p><p><b>CONCLUSION</b>Rictor overexpression is associated with the carcinogenesis and progression of colorectal cancer and can be an independent indicator for evaluating the prognosis of colorectal cancer patients.</p>
Asunto(s)
Humanos , Western Blotting , Proteínas Portadoras , Metabolismo , Línea Celular Tumoral , Neoplasias Colorrectales , Metabolismo , Progresión de la Enfermedad , Inmunohistoquímica , Metástasis Linfática , Pronóstico , Proteína Asociada al mTOR Insensible a la Rapamicina , Tasa de Supervivencia , Serina-Treonina Quinasas TOR , MetabolismoRESUMEN
<p><b>OBJECTIVE</b>To study the selective cytotoxic effect of lentivirus-mediated double suicide gene (CD/TK) against human gastric carcinoma cells SGC-7901 in vitro.</p><p><b>METHODS</b>SGC-7901 cells were infected with FGW-KDRP-CD/TK vector and the infection efficiency was observed under a fluorescence microscope. The morphological changes of the infected cells were observed by Giemsa staining. Flow cytometry (FCM) was employed for cell cycle analysis, and the expression of CD/TK was detected by RT-PCR. The infected cells were then treated with the prodrugs ganciclovir (GCV) and/or 5-fluorocytosine (5-FC) at different concentrations, and the cytotoxic effects were evaluated using MTT method.</p><p><b>RESULTS</b>The infection efficiency of the lentiviral vector in SGC-7901 cells increased with the titer of the virus, which produced no significant effect on the cancer cell morphology in vitro or on the percentages of G0-G1, G2-M and S phase cells (P>0.05). RT-PCR demonstrated the expression of CD/TK gene in SGC-7901 cells infected by FGW-KDRP-CD/TK. The infected cells were highly sensitive to the prodrugs with a dose-dependent cytotoxic effect within a specific concentration range of the drugs, whereas the non-infected cells were not sensitive to the prodrugs. Combined use of the two prodrugs produced an obviously stronger inhibitory effect than either of the them (P<0.05). When combined, GCV and 5-FC at the concentration of 0.1+40, 1+80, 10+160, and 100+320 mg/L demonstrated a synergetic effect with a CDI<1.</p><p><b>CONCLUSION</b>Lentivirus-mediated CD/TK fusion gene system can selectively kill gastric cancer cells, and the two prodrugs show a synergistic cytotoxic effect.</p>
Asunto(s)
Humanos , Adenocarcinoma , Genética , Patología , Línea Celular Tumoral , Citosina Desaminasa , Genética , Citotoxinas , Farmacología , Genes Transgénicos Suicidas , Genética , Terapia Genética , Vectores Genéticos , Genética , Lentivirus , Genética , Metabolismo , Proteínas Recombinantes de Fusión , Genética , Farmacología , Neoplasias Gástricas , Genética , Patología , Timidina Quinasa , Genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular , Genética , MetabolismoRESUMEN
<p><b>OBJECTIVE</b>To study the effect of the adenovirus containing CD/TK fusion gene controlled by the human vascular endothelial growth factor (VEGF) promoter on apoptosis of human gastric carcinoma cells SGC-7901.</p><p><b>METHODS</b>VEGF-expressing SGC-7901 cells were infected by the recombinant adenovirus Ad-VEGFP-CD/TK, and the infection efficiencies were observed with fluorescence microscopy. The toxic effect and intracellular calcium concentration induced by 5-fluorocytosine (5-FC) and ganciclovic (GCV) were determined by light microscopy, electron microscopy and flow cytometry.</p><p><b>RESULTS</b>The transfection efficiency of the recombinant adenovirus in SGC-7901 cells increased with the viral titer. At the multiplicity of infection (MOI) of 100, 5-FC and GCV could induce apoptosis of SGC-7901 cells within a given dose range in a dose- and time-dependent manner, and apoptotic changes of the cells were observed with electron microscopy. Apoptotic peak was also detected by flow cytometry. Cell cycle analysis revealed increased cell percentage in G(0)-G(1) phase and decreased percentage of cells in G(2)-M and S phases in response to treatment with the pro-drugs, which also induced marked elevation of intracellular calcium concentration in the infected cells.</p><p><b>CONCLUSIONS</b>CD/TK fusion gene system driven by VECF promoter selectively induces apoptosis of VEGF-expressing SGC-7901 cells, the action of which is probably mediated by intracellular calcium variation.</p>
Asunto(s)
Animales , Humanos , Adenoviridae , Genética , Fisiología , Apoptosis , Genética , Calcio , Metabolismo , Línea Celular Tumoral , ADN , Metabolismo , ADN Recombinante , Genética , Relación Dosis-Respuesta a Droga , Flucitosina , Farmacología , Ganciclovir , Farmacología , Genes Transgénicos Suicidas , Genética , Microscopía Electrónica , Regiones Promotoras Genéticas , Genética , Neoplasias Gástricas , Genética , Metabolismo , Patología , Virología , Factor A de Crecimiento Endotelial Vascular , GenéticaRESUMEN
<p><b>OBJECTIVE</b>To investigate the selective killing of colorectal tumor cells by lentivirus-mediated double suicide gene under the regulation of KDR promoter.</p><p><b>METHODS</b>293T packaging cells were transfected with the plasmid FGW-KDRP-CD/TK to obtain the infectious viruses. KDR-expressing LoVo cells and LS174T cells that did not produce KDR were transfected with the recombinant virus, and the transfection efficiency was evaluated by the fluorecence microscope. RT-PCR was employed to examine the expression of CDglyTK. After treatment of the cells with 5-FC and GCV, the killing effects on the two cell lines were evaluated.</p><p><b>RESULTS</b>The recombinant construct showed similar infection rate of the two cell lines. RT-PCR demonstrated that CDglyTK gene was expressed only in LoVo cells infected with FGW-KDRP-CD/TK but not in LS147T cells, and the sensitivity of the two cell lines to the prodrugs was significantly different (P<0.001). The killing effect of the double suicide gene was much stronger than that of single suicide gene administered (P<0.001).</p><p><b>CONCLUSION</b>The double suicide gene driven by KDR promoter has specific killing effect on the KDR-expressing colorectal tumor cells.</p>