The Effect Of Endostatin-P125A Mutant On Cancer Gene Therapy

Anticancer therapy targeting tumor capillary vessel, has beenattracting people's attention gradually in present years. The endostatin isan endogenous anti-angiogenic molecule with a good perspective inanti-tumor therapy. A number of research data show that the endostatinhas a significant ability to inhibit endothelial cell proliferation andmigration in vivo and in vitro. Moreover, the endostatin-P125A mutanthas been proved to have a greater capability of attaching vascularendothelial cells and a higher efficiency of inhibiting the endothelial cellsproliferation and migration recently.PURPOSE To estimate and compare the efficiency of the endothelialcell proliferation and migration between the wide type endostatin(wES)and the mutant type endostatin(mES,P125A) and provide some evidencefor the gene therapy targeted to blood vessel and mediated by thehuman-derived vector.METHODS The mES (P125A)was obtained and mutated by directPCR, and then ligated into the pcDNA3.1 vector. The pcDNA3.1/wESwas constructed to be the control. The two vectors were transfected intohepatoma cell line Bel-7402 for the detection of wES and mESexpression via ELISA assay. The stable transfected Bel-7402 cell cloneswere selected and raised. The supernatant collection of ES-containedmedia is used as the conditional media of HUVEC. The proliferation and apoptosis of HUVEC were confirmed by MTT and flow cytometry.Meanwhile, the secretary recombinant phrneo/mES with EF-1αpromoterwas constructed and electroporated to the Bel-7402. The positive cellswere selected by G418. The mES expression was detected by RT-PCRand ELISA at the levels of mRNA and protein respectively.RESULTS The full length of the mES(P125A) results in 624bpby sequencing. The ES expressions after 24h of transient transfection ofthe pcDNA3.1/wES and the pcDNA3.1/mES are 18.57±2.39 ng/ml and17.78±2.74 ng/ml respectively, which has no significant difference. Thesupernatant from the stable clones cultivation could reach a concentrationof endostatin protein at the level of 10~2ng/ml. At the level of 500ng/ml,the inhibition ratios of the wES and the mES for HUVEC proliferationare 41.4% and 64.0% respectively; and the efficiencies of inducingapoptosis are 13.0% and 32.7% respectively, both of which aresignificantly different. Twenty four stable clones that were transfectedby phrneo/mES had an 15-20ng/10~6cells/24h expression of the mES.CONCLUSION (1) The mutant ES P125A was obtainedsuccessfully. (2)The expression of transient transfection into Bel-7402has no significant difference between wES and mES (3)The Bel-7402 cellclones which highly express ES were gained successfully.(4)The mEShad greater ability of inhibiting the HUVEC proliferation and inducingthe HUVEC apoptosis than wES did in vitro.(5)The phrneo/mES could express low level of the mES after it was transfected into Bel-7402 cellline.