Endostatin gene therapy for liver cancer by a recombinant adenovirus delivery

Abstract

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 anti-endostatin 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 1X10(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-tumoral 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/LacZ control 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 ng/mL).

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.

Figure 1

Immunohistochemistry analysis for endostatin pro-tein in vitro (× 100). Extensive positive staining was observed in the cytoplasm of transduced cells. This indicated that human endostatin gene mediated by recombinant adenovirus was highly expressed in BEL-7402 cells.

Figure 2

Effect of Ad/hEndo on the proliferation of HUVEC cells.

Figure 3

Effect of Ad/hEndo on growth of BEL-7402 xenografted liver tumors.

Figure 4

Northern blotting of endostatin mRNA in vivo. Lanes 1-3: RNA extracted from tumors on d 1, 4 and 8 after Ad/hEndo injection, respectively; Lane 4: RNA extracted from control tumors injected with DMEM.

Figure 5

Detection of endostatin concentration in plasma by ELISA.

Figure 6

Pathological changes of liver 1 wk after 6 wk of treatment with Ad/hEndo (HE, original magnification: × 200).