[Construction and antitumor effect of recombinant adenovirus vector containing heterofusion gene NT4p53(N15)Ant].
OBJECTIVE
Peptide p53(N15)Ant from the amino-terminal of p53 fused with cell penetrating peptide antennapedia (Ant) can induce rapid cell death resembling necrosis in breast cancer and pancreatic cancer, whereas it is low cytotoxic to normal cells. This study was to construct an recombinant adenovirus vector containing NT4p53(N15)Ant gene and investigate its antitumor effects.


METHODS
Adenovirus packaging system was used to construct Ad-NT4p53(N15)Ant by recombinant technique in vitro. The recombinant adenovirus was packaged in HEK-293 cells and identified by reverse transcription-polymerase chain reaction (RT-PCR). Human hepatocellular carcinoma (HCC) cell line HepG2 were infected with Ad-NT4p53(N15)Ant or parallel control recombinant adenovirus Ad-GFP. After infection of Ad-NT4p53(N15)Ant, cell morphology was observed under inverted phase contrast microscope and transmission electron microscope. The effect of Ad-NT4p53(N15)Ant on HepG2 cells was detected by MTT assay and lactate dehydrogenase (LDH) release assay.


RESULTS
NT4p53(N15)Ant significantly inhibited the proliferation of HepG2 cells. The survival rates of HepG2 cells in NT4p53(N15)Ant group were 36.67% at 48 h, 20.47% at 72 h and 17.82% at 96 h after infection, which were significantly lower than those in Ad-GFP group (P<0.05). Distinct cell membrane disruption, pore-like structures in cytoplasm, and chromatin condensation were observed in Ad-NT4p53(N15)Ant group. LDH release in supernatant of HepG2 cells were 94 U/L at 24 h, 236.3 U/L at 48 h, 267 U/L at 72 h and 313 U/L at 96 h in Ad-NT4p53(N15)Ant group, which were significantly higher than those in Ad-GFP group (P<0.05).


CONCLUSIONS
NT4p53(N15)Ant could significantly inhibit the proliferation of HepG2 cells through necrosis pathway.
Song
Li-Ping
LP
Cancer Center, The First Affiliated Hospital, Medical College, Xioan Jiaotong University, Xioan, Shaanxi, 710061, P. R. China. xaslp@126.com
Li
Yue-Ping
YP
Qiu
Shu-Dong
SD
D000256:Adenoviridae / Q000235:genetics*; D051943:Antennapedia Homeodomain Protein / Q000235:genetics*; D049109:Cell Proliferation; D002470:Cell Survival; D050939:Gene Fusion; D015316:Genetic Therapy; D005822:Genetic Vectors; D056945:Hep G2 Cells; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D009369:Neoplasms / Q000628:therapy*; D009414:Nerve Growth Factors / Q000235:genetics*; D010446:Peptide Fragments / Q000235:genetics* / Q000494:pharmacology; D010957:Plasmids; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000494:pharmacology
D051943:Antennapedia Homeodomain Protein; D009414:Nerve Growth Factors; D010446:Peptide Fragments; D016159:Tumor Suppressor Protein p53; D007770:L-Lactate Dehydrogenase; C073922:neurotrophin 4