Retrovirus-mediated gene transfer of human endostatin inhibits growth of human liver carcinoma cells SMMC7721 in nude mice

Abstract

Aim: To study the effect of human endostatin mediated by retroviral gene transfer on the growth of human hepatocarcinoma cell line SMMC7721 in nude mice.

Methods: Human endostatin gene together with rat serum albumin signal peptide was transferred into human liver carcinoma SMMC7721 cells by retroviral vector pLncx to build a stable transfectant (SMMC-endo). PCR and Western blot analysis were used to verify the transfection and secretion of human endostatin gene in SMMC7721 cells. The endothelial cell proliferation assay in vitro was conducted to test the biological activity of the expressed human endostatin. The inhibitory effect of endostatin expressed by transfected SMMC7721 on the growth rates of tumor cells in vivo was observed. The mean microvessel density in the specimen was also counted.

Results: PCR amplification proved that the genome of SMMC-endo cells contained a 550bp specific fragment of endostatin gene. Western blot analysis confirmed the secretion of human endostatin gene in the conditioned medium of transfected SMMC-endo cells. The endothelial proliferation assay showed that the conditioned medium of SMMC-endo cells significantly inhibited the proliferation of human umbilical vein endothelial cells by 48 %, significantly higher than that of SMMC-pLncx (10.2 %, P<0.01). In vitro experiments revealed that only in 3 out of 5 mice tumors were formed and the mean size of flank tumors from SMMC-endo cells was 94.5 % smaller than that from the control SMMC-pLncx cells 22 days after tumor inoculation (P<0.001). The mean microvessel density in tumor samples from SMMC-endo cells was only 8.6+/-1.1, much fewer than that of 22.6+/-4.5 from SMMC-pLncx cells (P<0.01).

Conclusion: Human endostatin mediated by retroviral gene transfer can inhibit human liver carcinoma cell SMMC7721 growth in nude mice.

Figure 1

Analysis of PCR product of endostatin-transfected SMMC7721 cells by 1% agarose gel electrophoresis. 1: DNA Marker. 2: PCR product of SMMC-pLncx. 3: PCR product of SMMC-endo.

Figure 2

SDS-PAGE analysis and Western blot of endostatin expressed in supernatant of virally transduced SMMC7721cells (A) SDS-PAGE analysis; 1, supernatant of control SMMC-pLncx cells; 2, supernatant of endostatin-transfected SMMC-endo cells; 3, protein marker; (B) Western blot analysis; 1 protein marker; 2, supernatant of endostatin-transfected SMMC-endo cells.

Figure 3

Inhibition of endothelial cell proliferation by condi-tioned medium from SMMC-endo cells. Conditioned medium from SMMC-endo (2), SMMC7721 (3) and SMMC-pLncx (4) were concentrated and applied to HUVEC cells grown in 40-well plate. Three days later, cell number, as measured by absorbance(OD), was then quantified by using a colorimetric MTT assay. Bars, SD. bP<0.01, compared with conditioned medium from control SMMC-pLncx.

Figure 4

Inhibition on the growth of human liver carcinoma implanted in nude mice by human endostatin. ^bP < 0.001, SMMC-endo compared with control SMMC-pLncx.

Figure 5

Tumor sections were stained with an antibody reac-tive to CD₃₄. A: Tumor section from endostatin-transfected g1roup showed only a few positively stained vascular endothelial cells. B: Similar section of the control group showed highly vasuclarized tumor tissue. C: Microvessel density (MVD) was quantified by counting of positively stained endothelial cells from 5 fields in each tumor section. Bars, SD. ^bP < 0.01, compared with control SMMC-pLncx. × 200.