| Malignant tumor threatens the people's health and life. Antigiogenesis as a therapy has two significant advantages: easy accessibility to tumor sites and non-drug resistance. Most importantly, both structural and functional differences exist among blood vessels in different tissues and tumor types. So molecules that are able to effectively deliver therapeutic agents to the tumor microenvironment thus represent promising and important new tools for cancer therapy.We have obtained a specific molecule code, GX1, for gastric cancer by using phage displayed peptide library screening in vivo. Immunohistochemistry of mouse and human tissue showed that this phage peptide could bind to the endothelial cells of human gastric cancer. 99Tcm-labeled GX1 showed that it could specifically target to the gastric cancer of nude mice. These observations paved the way for the synthetic peptide's use in therapeutic approaches entailing the selective delivery of toxic agents or drugs to gastric tumor vasculature.Tumor necrosis factorα(TNFα) is a pleiotropic cytokine with antitumoral activity. TNFαexerts its effects in tumors mainly on the endothelium of the tumor-associated vasculature, with increased permeability, up-regulation of tissue factor, fibrin deposition and thrombosis, and massive destruction of the endothelial cells. Recombinant mutant human TNFα(rmhTNFα) has approved for the treatment of small cell lung cancer in China. It still used for chemical drug assistance in clinic because of high dose of usage.To further improve the therapeutic index of rmhTNFα, we fused GX1 peptide to the N-terminal of rmhTNFα, and hope that the resulting fusion protein (abbreviated to GX1-rmhTNFα) could selectively target on the gastric cancer neovesculature and alter the barrier function of endothelium when administrated systematically. The results of our studies are as follow:1.The expression and purification of GX1-rmhTNFαWe prepared expression vector pBV-220-GX1-rmhTNFαby using recombinant DNA technology. The fusion protein expressed both in solubility and deposition. The expressed protein, binding with anti-TNFαmAb specifically, is more than 7% of total bacteria protein. Then the engineering bacteria were cultured by shake flask. The solubilities were salted out by ammonium sulfate and purified by SP-Sepharose Fast Flow column and Q-sepharose Fast Flow column. The product had a purity over 95% and a specific activity of 5.65×108 IU/ml.2.The bioactivty of GX1-rmhTNFαSubsequent biological activity assay and biodistribution in vivo suggested that GX1-rmhTNFαcan reach the tumor relatively more than other organs. Therapeutic and toxic activity showed that GX1-rmhTNFαcould inhibit gasreic cancer growth and had less systemic toxicity during drug therapy.3.The mechanism of GX1-rmhTNFαImmunohistochemical analysis of GX1-rmhTNFαbiodistribution in gastric cancer showed that GX1-rmhTNFαcould bind specifically to gastric cancer vasculature after circulating for 2 hours compared with controls. Tumor perfusion assay indicated that the GX1-rmhTNFαinduced more dye into gastric cancer than rmhTNFαespecially at low dose in 2 hours. The GX1-rmhTNFαinduced cytotoxicity in HUVECs and SGC7901 cells by MTT assay, the GX1-rmhTNFαcould induce more cell apoptosis in HUVECs compared to the rmhTNFα(p < 0.05).Conclusion: The recombinant plasmid encoding GX1-rmhTNFαwas constructed successfully and the GX1-rmhTNFαprotein was produced and purified. It was showed that the GX1-rmhTNFαcould target to the gastric cancer vasculature and had lower systematical toxicity. This study has demonstrated the potential of GX1-rmhTNFαas a novel neovasculature targeting drugs for tumor treatment.
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