Synthetic Polypeptide Against CXCR4 Inhibits The Tumor Hepatic Micrometastasis In Uveal Melanoma Mouse Model

Uveal melanoma is the most common intraocular malignant tumor with very high mortality because of the hepatic metastasis. But the mechanism of the metastasis is not clear now.CXCR4 is a seven transmembrane G-coupled receptor that first drew attention as a major coreceptor of the entry of HIV. Activation of CXCR4 by SDF-1 results in activation of many downstream pathways including mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and calcium mobilization. CXCR4 is found in breast, prostate, lung cancers as well as melanoma and appears to promote metastasis of tumor cells to the distant organs. So a hypothesis comes out that the growth and metastasis of melanoma cells could be decreased by the inhibition of the CXCR4.T140 is a 14-residue peptide that possessed a high level of anti-HIV activity by mimicking SDF-1. TN14003 is a further improved compound from T140 which is far less cytotoxic and more stable in serum compared with TN140.Purpose: To evaluate the effect of synthetic polypeptide against CXCR4 in reducing the hepatic metastases in mice with uveal melanoma.Methods: B16LS9 melanoma cells line was cultured. RT-PCR,Western blot and immunocytochemistry were performed to determine the presence of CXCR4 in the melanoma cells.5×10⁵ cells/2.5μl of B16LS9 melanoma cells line were inoculated into the posterior compartment of the 24 mice's right eyes. 24 mice were divided into 3 treating groups(n=8 each): group 1, intraperitoneal injections of lmM polypeptide 3 days before inoculation of B16LS9 melanoma cells; group 2, intraperitoneal injection of lmM polypeptide at the day of inoculation of B16LS9 melanoma cells. Group 3, intraperitoneal injections of PBS at the day of inoculation of B16LS9 melanoma cells. Right eyes were enucleated at the 7^th day after the inoculation, the mice were sacrificed and livers were collected at the 28^th day. Immunohistochemical analyses (anti-CXCR4, MMP-2) were performed on the right eyes and the hepatic metastases were counted.Results: 1. CXCR4 is expressed in the mouse melanoma cell lines: RT-PCR and Western-blot showed that CXCR4 is expressed in the mouse melanoma cells. Immunofluroscent also approved the expression of the CXCR4 in same cell lines.2. Invasion of melanoma cells could be inhibited by the synthetic polypeptide in vitro: the invasive ability of the melanoma cells could be evaluated by the invasion assay which approved the effect of the synthetic polypeptide.3. Metastasis of melanoma could be decreased by the synthetic polypeptide in vivo: compare to the nontreated group, the number of hepatic micrometastasis is decreased in the group of mice which were treated with the synthetic polypeptide.Conclusions: 1. CXCR4 is expressed in the B16LS9 cells.2. TN14003 could block the invasive ability of B 16LS9 cells.3. TN14003 could decrease hepatic metastases in a mice ocular melanoma model.