[Treatment of hepatocellular carcinoma with a novel gene-viral therapeutic system CNHK300-murine endostatin].
OBJECTIVE
To investigate the anti-tumor effects of a novel gene-viral therapeutic system CNHK300-murine endostatin (CNHK300-mE) in hepatocellular carcinoma (HCC).


METHODS
A novel gene-viral therapeutic system named CNHK300-mE was constructed by employing the human telomerase reverse transcriptase (hTERT) promoter to drive the expression of adenovirus E1A gene and cloning the therapeutic gene murine endostatin (mE) into the adenovirus genome. Hepatocellular cells of the HepGII and Hep3B lines and normal fibroblasts of the MRC-5 line were cultured and infected with the viruses CNHK300-mE, ONYX-015, replicative adenovirus without therapeutic gene, and Ad-mE, non-replicative adenovirus with the same therapeutic gene. Ninety-six hours after the infection, tissue culture infectious dose 50 method was used to detect the titer of virus in the supernatants. MTT method was used to examine the cytolytic capability. The expression of E1A and mE were examined by Western blotting. ELISA assay was used to detect the transgene expression of mouse endostatin. Healthy nude Balb/c mice were injected with hepatic cancer cells of the SMMC 7221 line. Forty mice with tumors 5 approximately 8 mm in diameter were randomly divided into 4 groups of 20 mice: CNHK300-mE group (CNHK300-mE was injected into the tumor once every other day for 5 times), Ad-mE group (Ad-mE was injected), ONYX-015group (ONYX-015 was injected), and control group (diluent of virus was injected). 3, 7, 14, 21, and 28 days after the initial injection the size of tumor was examined. 48 hours after the finish of the whole course of treatment, the mice were killed. ELISA was used to detect the expression of mE in blood. The growth of tumor was examined by HE staining, The angiogenesis in the tumor was observed by immunohistochemistry with von Willebrand factor and The proliferation of transplanted tumor was observed by immunohistochemistry with adenovirus envelop protein hexon.


RESULTS
Ninety-six hours after the infection of the cells by CNHK300-mE virus was replicated by 6329 +/- 1830 and 25 136 +/- 6890 times in the HepGII and Hep3B cells respectively, 3296 and 12 824 times higher than in the MRC-5 cells respectively. The replication multiples of ONYX-015 virus in the HepGII and Hep3B cells were 2040 +/- 450 and 3980 +/- 740 times respectively, both significantly lower than those of CNHK300-mE virus (both P < 0.05). However, no remarkable replication of Ad-mE virus was seen in the Western blotting showed the expression of therapeutic gene mE in HepGII and Hep3B cells infected with CNHK300-mE on Ad-mE. Hep3B cells, the band of CNHK300-mE being thicker than that of Ad-mE and the band of Ad-mE being similar to that of CNHK300-mE in the MRC-5 cells. ELISA showed that the expression of mE protein in the HepGII cells infected by CNHK300-mE virus increased time-dependently during the period of 7 days after virus infection, significantly higher than the expression in the HepGII cells infected by Ad-mE virus (P < 0.05). The tumors of the CNHK300-mE virus-infected mice were significantly smaller than those of the Ad-mE and ONYX-015-infected mice (both P < 0.01). ELISA showed that the mE protein content in the blood of the CNHK300mE-infected mice was significantly higher than that of the Ad-mE group (P < 0.05). Hexon immunohistochemistry showed patchy and diffuse positive staining related to apoptosis and necrosis of tumor cells in the transplanted tumors of the CNHK300-mE virus-infected mice, however, only sporadic positive staining was seen in the Ad-mE virus-infected mice.


CONCLUSIONS
Being capable of specifically replicating in the telomerase-positive HCC cells and mediating effective expression of therapeutic gene in vitro and in vivo, the novel gene-viral therapeutic system CNHK300-mE holds potential for treatment of HCC.
Laboratory of Viral and Gene Therapy, Eastern Hepatobiliary Surgical Hospital, The Second Military Medical University, Shanghai 200438, China.
Gen-Cong
GC
Li
Ming-Ming
MM
Nie
Jia-Mei
JM
Yang
Chang-Qing
CQ
Su
Li-Chen
LC
Sun
Yan-Zhen
YZ
Qian
Jonathan
J
Sham
Guo-En
GE
Fang
Meng-Chao
MC
Wu
Qi-Jun
QJ
Qian
D000256:Adenoviridae / Q000235:genetics* / Q000502:physiology; D017500:Adenovirus E1A Proteins / Q000235:genetics; D000818:Animals; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D043169:Endostatins / Q000235:genetics* / Q000493:pharmacokinetics; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D008114:Liver Neoplasms, Experimental / Q000473:pathology / Q000628:therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D019098:Telomerase / Q000235:genetics; D014779:Virus Replication
D017500:Adenovirus E1A Proteins; D004268:DNA-Binding Proteins; D043169:Endostatins; D019098:Telomerase