The Inhibitions Of The Tumor Cell Proliferation, Migration And Metastasis By ECRG2 In Vitro And In Vivo

Esophageal Cancer Related Gene 2 was cloned and identified in our laboratory. Previous data show that ECRG2 has similar effects with tumor suppressor gene, but the role of ECRG2 in tumor development and progress and the exact mechanism remains unclear.To gain further comprehend this issue, first, we cloned and sub-cloned the full-length ECRG2 cDNA into the pGEX-4T-l vector to produced the recombinant ECRG2 protein in E. coli, and purified ECRG2-GST fusion protein for future study by affinity chromatography on glutathione-coupled beads. Second, we generated anti-ECRG2 monoclonal antibodies (MAbs) by using c-terminal of ECRG2 peptide as immunogen; these antibodies are suitable for detecting ECRG2 by ELISA, Western blotting and Immunofluorescence microscopy. Third, we successfully constructed a recombinant plasmid pcDNA3.1-ECRG2 and transfected it into esophageal cancer cell line EC9706 by using Lipofectamin?2000. The results showed that the colony formation activity of EC9706/pcDNA3.1-ECRG2 was 18% while that of the control cell EC9706/pcDNA3.1 was 55% (P0.05). The activity of anchorage-independent proliferation of EC9706/pcDNA3.1-ECRG2 was lower than that of EC9706/pcDNA3.1 in soft agar. Fourth, we extended the examination of the role of ECRG2 by using recombinant ECRG2 and synthetic ECRG2 fragment. The EC9706 cells growth was suppressed after cell treated with these two reagents to culture medium; it confirmed the inhibition of ECRG2 in proliferation of tumor cells. When EC9706 cells were pre-incubated with these two reagents and inoculated s.c. into the flank of nude mice, the occurrence of replanted tumors was delayed, and growth of tumors was inhibited. These results suggested that ECRG2 inhibited the tumor cell proliferation in both in vitro and in vivo.The protein homologous analysis implicated that ECRG2 contain the basic structure of kazal protease inhibitor which may play a key role in the degradation of ECM components mediating tumor invasion. The structural resemblance and difference between ECRG2 and the other members of kazal family were showed that ECRG2 might have both similar and distinctive functions within cells. The endogenetic ECRG2 was not detected in the high metastasic tumor cells PG, PG cells...