Endostatin gene therapy for liver cancer by a recombinant adenovirus delivery

AIM: To investigate the expression of adenovirus-mediated human endostatin (Ad/hEndo) gene transfer and its effect on the growth of hepatocellular carcinoma (HCC) BEL-7402 xenografted tumors.METHODS: Immunohistochemistry analysis with an antiendostatin antibody was preformed to detect endostatin protein expression in HCC BEL-7402 cells infected with Ad/hEndo. MTT assay was used to investigate the effects of Ad/hEndo on proliferation of human umbilical vein endothelial cells (HUVEC). Intra-tumoral injections of lx10^9 pfu Ad/hEndo was given to treat BEL-7402 xenografted tumors in nude mice once weekly for 6 wk. Mice received injections of Ad/LacZ and DMEM were regarded as control groups. After intra-turmoral administration with Ad/hEndo, the endostatin mRNA expression in tumor tissue was analyzed by Northern blotting, and plasma endostatin levels were determined using enzyme-linked immunosorbent assay (ELISA).RESULTS: High level expression of endostatin gene was detected in the infected HCC BEL-7402 cells. Ad/hEndo significantly inhibited HUVEC cell proliferation by 57.2% at a multiplicity of infection (MOI) of 20. After 6-week treatment with Ad/hEndo, the growth of treated tumors was inhibited by 46.50% compared to the Ad/LacZcontrol group (t=-2.729, P<0.05) and by 48.56% compared to the DMEM control group (t=2.485, P<0.05). The ratio of mean tumor volume in treated animals to mean tumor volume in the control animals (T:C ratio) was less than 50% after 24 d of treatment. Endostatin mRNA in tumor tissue was clearly demonstrated as a band of approximately 1.2 kb, which was the expected size of intact and functional endostatin.Plasma endostatin levels peaked at 87.52+8.34 ng/mL at d 3 after Ad/hEndo injection, which was significantly higher than the basal level (12.23+2.54 ng/mL). By d 7,plasma levels dropped to nearly half the peak level(40.34+4.80 nglmL).CONCLUSION: Adenovirus-mediated human endostatin gene can successfully express endogenous endostatin in vitro and in vivo, and significantly inhibit the growth of BEL-7402 xenografted liver tumors in nude mice.

Quantitative detection of common deletion of mitochondrial DNA in hepatocellular carcinoma and hepatocellular nodular hyperplasia

AIM: To study the deletion of mitochondiral DNA in hepatocellular carcinoma and hepatocellular nodular hyperplasia and its significance in the development of cancer. METHODS: Deleted mtDNA (CD-mtDNA) and wild type mtDNA (WT-mtDNA) were quantitatively analyzed by using real-time PCR in 27 hepatocellular carcinomas (HCC) and corresponding noncancerous liver tissues and 27 hepatocellular nodular hyperplasiae (HNH). RESULTS: A novel CD (4 981 bp) was detected in 85% (23/27) and 83%(22/27) of HCC and HNH tumor tissues, respectively, which were significantly higher than that in paired noncancerous liver tissues (57%, 15/27) (P<0.05). The CD/WT-mtDNA ratio in HCC tumors was 0.00092 (median, interquartile range, 0.0001202-0.00105), which was significantly higher than that in paired noncancerous liver tissues (median, 0.000, quartile range, 0-0) (P=0.002, Mann-Whitney Test), and was 25 of times of that in HNH tissues (median, 0.0000374, quartile range, 0-0.0004225) (P=0.002, Mann-Whitney test). CONCLUSION: CD-mtDNA mutation plays an important role in the development and progression of HCC.