Targeting Angiogenesis and HGF Function Using an Adenoviral Vector Expressing the HGF Antagonist NK4 for Cancer Therapy

Abstract

Hepatocyte growth factor (HGF) affects tumor growth/invasion and tumor neovascularization. A proposed HGF antagonist, NK4 (an amino-terminal kringle-domain peptide of HGF), inhibits tumor growth/invasion through the competition of HGF binding to its receptor, c-Met, and acts as an angiogenesis inhibitor. To investigate the in vivo effect of NK4 gene transfer, we constructed an adenovirus vector expressing human NK4 (AdCMV.NK4). Human lung cancer cell lines (A549 and H358) infected in vitro with AdCMV.NK4 yielded NK4 protein without a change in the cell growth rate. In contrast, direct injection of AdCMV.NK4 (1 x 10(9) pfu, twice) into established subcutaneous tumors in BALB/c nu/nu mice resulted in suppression of the tumors by 64% for A549 or by 91% for H358 compared with controls (P<0.02 or P<0.01, respectively). Counting of the tumor vessels revealed suppressed vascularity by 57% in H358 tumors when using AdCMV.NK4 (P<0.0001). Furthermore, systemic NK4 delivery by intraperitoneal injection of AdCMV.NK4 effectively suppressed both angiogenesis in the Matrigel assay (86% reduction, P<0.032), subcutaneous tumor growth in vivo (by 65% for H358, P<0.001), and hematogenous lung metastases without obvious side effects. These results indicate that NK4 elicits tumor-growth suppression in vivo through its anti-angiogenic activity and anti-HGF activity and that NK4 gene transfer can be an effective tool in the treatment of cancer.

Peritumoral injection of adenovirus vector expressing NK4 combined with gemcitabine treatment suppresses growth and metastasis of human pancreatic cancer cells implanted orthotopically in nude mice and prolongs survival

“…20 In brief, the vector was generated by homologous recombination of the pJM17 plasmid 21 and shuttle plasmid vector pSV2 þ 22 containing an expression cassette with the cytomegalovirus early promoter/enhancer, followed by human NK4 cDNA 17 and a polyadenylation signal. A control vector expressing bacterial b-galactosidase (LacZ) was constructed by the same procedure with pJM17 and pCA17 containing the LacZ gene.…”

Suppression of metastasis of human pancreatic cancer to the liver by transportal injection of recombinant adenoviral NK4 in nude mice

“…20 Briefly, Ad-NK4 was generated by homologous recombination of the pJM17 plasmid 21 and the shuttle plasmid vector pSV21 22 containing an expression cassette and the cytomegalovirus early promoter/ enhancer followed by human NK4 cDNA 11 and a polyadenylation signal. A control vector with expressing the bacterial b-galactosidase enzyme (LacZ) was constructed by the same procedure with pJM17 and pCA17 containing the LacZ gene.…”

Tumor suppression effect using NK4, a molecule acting as an antagonist of HGF, on human gastric carcinomas

“…NK4 has inhibitory effect of angiogenesis driven by basic fibroblast growth factor ( bFGF ), vascular endothelial growth factor ( VEGF ), as well as HGF. 21,24,30 Considering these two-way inhibitory effects, gene therapy using NK4 seems to be an ideal potential therapy for treatment of patients with advanced gastric cancer. Basic research toward gene therapy using anti-angiogenic substances such as angiostatin and endostatin has been widely reported.…”