ABSTRACT
In this study, the mechanism by which Ad-p27mt inhibits the growth, invasion and metastasis of transplanted liver tumor was studied by examining the effects of Ad-27mt gene transfer on the expression of Bax, Bcl-2, VEGF and MMP-9 in the transplanted liver tumors in nude mice. The model of transplanted hepatic tumor was established in nude mice. The mice were then divided into three groups, which were injected with PBS, Ad-LacZ and Ad-p27mt and the growth of the transplanted liver tumor was observed. The expressions of P27, Bax and Bcl-2 proteins were detected by Western blotting and the expressions of VEGF and MMP-9 were immunohistochemically determined.Our result showed that the tumor size, expressions of Bax, Bcl-2 proteins, VEGF and MMP-9 were all lower than those in PBS and Ad-LacZ groups and the differences were statistically significant (P＜0.05). Our study suggested that Ad-p27mt could inhibit the growth, invasion and metastasis of hepatic cancer by lowering the expressions of VEGF and MMP-9.
ABSTRACT
Double-stranded oligomeric nucleotide encoding PEP-1 peptides was synthesized, prokaryotic expression pET15b-pep-1-p27mt recombinant constructed, E. coli BL21 (DE3)pLysS transformed and induced with IPTG to highly express fusion protein PEP-1-P27mt. Fusion protein with an N-terminal His-tag could be purified by Ni2+-resin affinity chromatography and identified by SDS-PAGE and Western blotting. Cultured EC9706 cells treated with PEP-1-P27mt revealed that PEP-1-P27mt was transduced into cells after 15 min and reached maximal intracellular concentrations in 2 h. PEP-1-P27mt of 1 μmol/L final concentration could most strongly suppress the growth. It was suggested that PEP-1 can carry P27mt across membrane, which provides a new biological protocol for using cyclin dependent kinase inhibitors p27mt in suppressing the growth of tumor cells.
ABSTRACT
Objective To assess the effects of p27mt gene transfection on the proliferation and apoptosis of human hepatocellular carcinoma cell (HCC) lines SMMC-7721. Methods A replication deficient adenovirus vector encoding p27mt (Ad-p27mt) was used and p27mt cDNA was transfected into human SMMC-7721 cell lines in vitro. The synthesis of DNA in SMMC-7721 cells was determined by using 3H-thymidine incorporation; the cell apoptosis was determined by flow cytometry, TUNEL method and DNA fragmentation analysis. Results The virus titer was 7.95?1012 cfu/ml, the transduction efficiency was 100 % when multiplicity of infection ≥50, FCM analysis revealed a sub-G1 cell peak in Ad-p27mt transduced hepatocellular carcinoma cell lines. Agarose electrophoresis showed marked ladder .The difference of apoptotic index between the Ad-p27mt group and the control group was statistically significant (58.6?4.3, vs 4.5?1.6, P
ABSTRACT
AIM: To study the inducing effect of human mutant p27 gene on apoptosis of the colorectal cancer cells. METHODS: The recombinant adenovirus Ad-p27mt was constructed to infect the colorectal cancer cell SW480. The inducing effect of Ad-p27mt on apoptosis in colorectal cancer cells was measured by flow cytometry, DNA fragment analysis and TUNEL method. RESULTS: Ad-p27mt was successfully constructed. When the multiplicity of infection (MOI) was ≥50, the infection efficiency reached 100%. After 24 h of infection, there was an apoptotic hypodiploid peak observed by flow cytometry before G_1 and there were apoptotic characteristic bands in the DNA electrophoresis. The apoptotic index detected by TUNEL method was 82.6?3.2 (Ad-p27mt group) and 5.0?3.5 (control group), respectively, the difference of which was significant (P