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 identif ied 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 ? ow 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.

Antitumor bioactivity of adenovirus-mediated p27mt in colorectal cancer cell line SW480

AIM: To explore the antitumor bioactivity of adenovirus- mediated mutant type p27kip1 gene in a colorectal cancer cell line SW480. METHODS: We constructed recombinant adenovirus vector expressing a mutant type p27kip1 gene (ad- p27mt), with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC), and transduced into SW480 cells. Then we detected expression of p27, Bcl-2 and Bax protein in the transductants by Western blotting, cell cycle of transductants by a digital flow cytometric system, migrating potential with Boyden Chamber and SW480 tumor cell growth inhibition in vitro and in vivo. RESULTS: We found that a recombinant adenovirus vector of expressing ad-p27mt, with mutation of Thr-187/Pro-188 (ACGCCC) to Met-187/Ile-188 (ATGATC) has potent inhibition of SW480 tumor cell growth in vitro and in vivo. Furthermore, ad-p27mt induced cell apoptosis via regulating bax and bcl-2 expressions, and G1/S arrest in SW480 cells and inhibited cell migration. CONCLUSION: ad-p27mt has a strong anti-tumor bioactivity and has the potential to develop into new therapeutic agents for colorectal cancer.