Adenovirus-mediated Thrombospondin 1 Gene Of Anti-angiogenesis Derivatives Metastasis And Its Inhibition Of Tumor Growth Effects Of Experimental Research

Background Recent investigations support the idea that angiogenesis is involved in the pathophysiology of both solid tumors and hematological malignancies. Within a given microenvironment, the angiogenic response is regulated by a delicate balance of angiogenesis inducers and inhibitors. Thrombospondin-1 (TSP-1) is a multifunctional extracellular glycoprotein with angiostatic properties in multiple in vitro and in vivo assays. Interestingly, there is also proangiogenic domain in this complex molecule. Development of TSP-1 as an antiangiogenic drug has been hindered by multiplicity of its functional effects, difficulties in its production and its poor pharmacokinetics. Objective To explore the effect of adenovirus-mediated delivery of an antiangiogenic fragment of human thrombospondinl (TSP1f) on K562 cell growth in nude mice. Methods TSP1f cDNA was amplified by RT-PCR from normal human peripheral blood mononuclear cells and was used as transgene to construct an adenoviral vector (ADV-TSP1f). TSPlf expression/secretion by ADV-TSP1f-infected K562 cells was demonstrated using Western blot analysis. The functional validation of adenovirus-mediated TSP-1f was determined by an in vitro human umbilical vein endothelial cell (HUVEC) proliferation inhibition assay. MTT assay was performed to determine the effect of ADV-TSPlf infection on K562 cell growth kinetics. The volume of K562 xenografts in nude mice treated with ADV-TSPlf, ADV-LacZ, or PBS intratumorally was measured every three days during 3 week long treatment. Intratumoral microvessel density (MVD) was determined by immunohistochemical staining. Results The cDNA fragments of TSP-1f (1.7 Kb) obtained by RT-PCR amplification of the RNA from normal human blood cells were sequenced using an automated sequencer and then were compared with those published in GENE BANK (GB Accession Code NM003246), where no difference was found. ADV-TSPlf stock titer after CsCl banding was 1.0×10¹¹pfu/mL. TSP-1f was expressed and secreted...