Inhibition of tumor growth in xenografted nude mice with adenovirus- mediated endostatin gene comparison with recombinant endostatin protein

摘要

Background Inhibition of tumor growth by endostatin has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in a large-scale production of the recombinant endostatin protein, rapid loss bioactivity of the protein, and the cumbersome daily administration. These limitations could be resolved by in vivo delivery and expression of the endostatin gene. In this study, we observed the effect and advantage of endostatin gene therapy mediated by a recombinant adenoviral vector (Ad/hEndo) on the growth of hepatocellular carcinoma BEL-7402 xenografted tumors, comparison with recombinant endostatin protein. Methods Hepatocellular carcinoma BEL-7402 cells were inoculated subcutaneously in the flank of Balb/c nude mice. Nine days after tumor cell inoculation, animals were given a cycle of four courses of intra-tumoral injections of Ad/hEndo of 5 x10~8 pfu (low-dose group) and 1 x10~9 pfu (high-dose group) at intervals of six days, respectively. Recombinant human endostatin protein (rhEndo) was administrated daily subcutaneously at a dose of 10 mg · kg~(-1) · d~(-1) at a site nearby the tumor for ten days. The expression of endostatin mRNA in tumor tissue was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) after Ad/hEndo injection. Dynamic changes of concentration of endostatin protein in tumor tissue were quantitated by enzyme-linked immunosorbent assay (ELISA). Results After 4 courses of treatment, the tumor growth rates of high-dose treated group with 1 x10~9 pfu of Ad/hEndo were inhibited by 42.26% compared with the Ad/LacZ control group (P = 0. 001) and by 46. 26% compared with the NIH buffer control group (P = 0.003), respectively. However, in this study, Ad/hEndo at low dose of 5 x 10~8 pfu failed to demonstrate significant inhibition of tumor growth, compared with control groups. After daily administration of recombinant human endostatin protein (rhEndo) for 9 days, the ratio of T/C (rhEndo group versus PBS group) was less than 47%. However, two days after rhEndo treatment ceased, the ratio of T/C was more than 50%. The peak of expression of endostatin mRNA in tumor tissue was at 2 or 3 days after administration intratumorally with Ad/hEndo of 1 x 10~9 pfu and gradually dropped undetectable by day 7. Dynamic analysis of endostatin concentration in tumor tissue showed that the highest level of mRNA is up at the third day after injection, and dropped to basal level three weeks later. Conclusions Endostatin gene therapy mediated by a recombinant adenoviral vector had significantly inhibited the growth of hepatocellular carcinoma BEL-7402 xenografted tumors at a high dose of 1 x 10~9 pfu compared with other groups. The analysis of dynamic expression of endostatin in vivo indicated that Ad/hEndo had acquired a high-level, relatively long-term expression in vivo and bioactivity capability.