Effect of p27mt gene on apoptosis of the colorectal cancer cell line Lovo

AIM： To construct p27mt recombinant adenovirus, transfect the colorectal cell line Lovo and observe the effects of p27mt on Lovo cell apoptosis and cell cycle inhibition. METHODS： We constructed recombinant adenovirus containing p27mt by homologous recombination in bacteria. The colorectal cancer cell line Lovo was infected with recombinant replication-defective adenovirus Ad- p27mt, and expression of p27mt was determined by Western blotting; the inhibitory effect of p27mt on Lovo cells was detected by cytometry. Cell cycle was determined by flow cytometry. DNA fragment analysis identified the occurrence of apoptosis. RESULTS： The recombinant adenovirus which already contained p27mt target gene was successfully constructed. When multiplicity of infection was ≥50, the infection efficiency was 100%. After transfection of Lovo cells with Ad-p27mt the cells had high p27 expression which was identified by immunoblotting assay. PI staining and flow cytometry showed that 77.96% of colorectal cancer cells were inhibited in phase G0/G1, while in the Ad-LacZ group and blank control group, 27.57% and 25.29% cells were inhibited in the same phase, respectively. DNA fragment analysis, flow cytometry and TUNEL assay demonstrated that p27mt is able to induce apoptosis in colorectal cancer cells. CONCLUSION： p27mt has an obvious blocking effect on colorectal cancer cell cycle, and most cells were inhibited in phase G0/G1. Therefore, p27mt can induce apoptosis in colorectal cells.

Impact of p27mt gene on transplantation model of human colorectal cancer in nude mice

AIM:To investigate the inhibitory and anti-metastatic effect of mutant p27 gene(p27mt) on the growth of colorectal cancer xenografts in nude mice and its underlying mechanism.METHODS:Inhibitory effect of p27mt gene on the growth of colorectal cancer xenografts was determined by measurement of tumor size before and after direct intratumoral injection of Ad-p27mt in a preestablished transplantation model of human colorectal cancer in nude mice.Cell cycle and apoptosis were detected by flow cytometry performed on single-cell suspension from an isolated tumor.Expression of MMP-9 in tumor tissue was detected by immunohistochemistry.RESULTS:The average sizes of transplantation tumors were 1.94 ± 0.67 cm3,2.75 ± 0.83 cm3 and 3.01 ± 0.76 cm3 in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.05).The average proliferation rates were 37.34% ± 1.45%,53.16% ± 3.27% and 54.48% ± 2.43%,in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.05).The average apoptosis rates were 19.79% ± 3.32%,6.38% ± 4.91% and 7.25% ± 5.20% in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.01).The average MMP-9 expression rates were 20%,75% and 66.7% in the Ad-p27mt,Ad-LacZ and control groups,respectively(P < 0.01).CONCLUSION:p27mt inhibits the growth of transplanted tumor by blocking the proliferation of cancer xenografts and by promoting apoptosis of transplantated tumor cells,as well as decrease transpl-anted tumor metastasis.