[hTERT promoter regulated replication-selective adenovirus CNHK300 in treatment of hepatocellular carcinoma].
OBJECTIVE
To evaluate the therapeutic efficiency of replicative adenovirus CNHK300 targeted at telomerase-positive hepatocellular carcinoma.


METHODS
Human liver cancer cell line HepGII and Hep3B, human embryonic kidney cell line 293, and normal human fibroblasts of the line BJ were cultured and added with adenoviruses CNHK300, ONYX-015 (55 000 protein deleted adenovirus), or wtAd5 (wild type 5) with different multiplicity of infection (MOI) for 7 days. 293 cells were used to measure the titer of the filial generation virus from different cells. The cell survival rate was calculated by MTT method 2, 4, 6, and 8 days after. Different cells were added with CNHK300 virus and then the E1A protein in the cytoplasm was measured by western blotting. Fluorescence microscopy was used to observe the CNHK300-EGFP proliferation after the cells were cultured and added with the virus for 1.5 hours.


RESULTS
The replicative viruses CNHK300 and wtAd5 proliferated rapidly in HepGII and Hep3B cells since 24 hours after inoculation and proliferated 40625 and 65326 times respectively with a proliferation potential similar to that of the wild-type adenovirus and much higher than that of the ONYX-015 virus. CNHK300 of the MOI of 0.0002 killed half of the cancer cells, especially those of the line Hep3B, within 5 approximately 6 days, and CNHK300 virus of the MOI of 0.5 pfu/cell killed almost all the HepGII cells in the 8th day, with a killing power lower than that of the wild-type virus and higher than that of the ONYX-015 cells. The IC(50) was as low as MOI of 0.002 pfu/cell for the Hep3B cell and was as high as MOI of 100 pfu/cell for the BJ cell. CNHK300 was a less powerful killer of fibroblasts than wild-type virus. E1A expression was shown by western blotting in 293 cells and CNHK300-infected liver cancer cells, but not in the CNHK300-infected normal human fibroblasts. Fluorescence microscopy showed only isolated fluorescence-positive fibroblasts till the 10th day of infection, but obvious proliferation of CNHK300-EGFP virus since the 3rd day and fluorescence-positive cells in sheets by the 7th day, however, the fluorescent intensity was weakened since the 10th day.


CONCLUSIONS
Tumor-selective adenovirus CNHK300 replicates in telomerase-positive liver cancer cells efficiently as well as wtAd5 and causes oncolysis, but has severely attenuated proliferation and cytolysis in normal cells.
Li
Yue-Min
YM
Clinical Cancer Centre, Hospital No 307, Beijing 100039, China.
Song
San-Tai
ST
Jiang
Ze-Fei
ZF
Zhang
Qi
Q
Su
Chang-Qing
CQ
Xu
Jian-Ming
JM
Qian
Yan-Zhen
YZ
Qian
Qi-Jun
QJ
D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology; D017500:Adenovirus E1A Proteins / Q000235:genetics; D006528:Carcinoma, Hepatocellular / Q000628:therapy*; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D008113:Liver Neoplasms / Q000628:therapy*; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019098:Telomerase / Q000096:biosynthesis* / Q000235:genetics; D014779:Virus Replication
D017500:Adenovirus E1A Proteins; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D019098:Telomerase