Efficacy Of The Novel Small Molecular Hypoxia Inducible Factor Pathway Inhibitor KCN1 In The Control Of Micrometastases In Experimental Ocular Melanoma

Part?KCN1 effects on the expressions of HIF-1?and its downstream genes VEGF and CA9 in human and mouse melanoma cell linesObjectives To study expressions of HIF-1?, VEGF and CA9, and to evaluate the possiblity of HIF-1 pathway being an attractive therapeutic molecular target for ocular melanoma.Methods Human and mouse malignant melanoma cell lines under hypoxia condition with or without KCN1 were detected by Dual-Luciferase Reporter Assay, RT-PCR, Western Blot and ELISA for expressions of HIF-1?, VEGF and CA9, comparing their expressions in the cell lines under normoxic condition.Results Expressions of HIF-1?, VEGF and CA9 in melanoma cell lines B16LS9 and Mel270 under hypoxia condition were higher than under normoxia. With the treatment of KCN1, expressions of VEGF and CA9 were significantly repressed.Conclusions The strong upregulation of HIF-1?in malignant melanoma cell lines suggested that HIF-la might be an important factor to melanoma growth and could be an attractive therapeutic molecular target for melanoma; KCN1 can decrease the expressions of VEGF and CA9 in melanoma cells. Part?KCN1 effects on the tumor growth of primary ocular melanoma xenograft and the hepatic micrometastases and its potential mechanismsObjectives To evaluate the effect of KCN1 in reducing the size of primary ocular melanoma and the number of hepatic micrometastases in a murine model of ocular melanoma.Methods 220 mice were randomly divided into 22 groups in 3 experimental steps (dosing, timing, and longitudinal experiment) with 10 mice in each group. The posterior compartments of the right eyes of C57BL6 mice were inoculated with 5×105 B16LS9 melanoma cells. After inoculation of tumor cells, mice in KCN1 groups were intraperitoneally injected with KCN1 or an equal volume of cremophor /ethanol formulation, starting at the 1st day until sacrifice. The tumor-bearing eyes were enucleated at the 7th day to examine primary tumor growth, and the mice were sacrificed at the 28th day after inoculation to examine metastases to the liver. The histologic sections of right eyes and livers were obtained, the volumes of primary ocular tumors were evaluated using Image J, and the numbers of hepatic micrometastases were counted. Immunofluorescence were performed to evaluate the expressions of VEGF, CA9 and Ki-67 in the ocular tumor. CD-31 positive micro vascular density were examed, and serum VEGF level were detected at 1,2,3,4,5 week.Results The average size of primary ocular melanoma and hepatic micrometastases in mice treated with KCN1 were significantly reduced compared with mice with injection of control vehicles. The expressions of VEGF, CA9 and Ki-67 were suppressed by KCN1 in the tumor. Serum VEGF level and microvascular density of melanoma was reduced by KCN1. The liver showed mildly increased interstitial liquid accumulation.Conclusions This study suggests that the small molecule HIF pathway inhibitor KCN1 reduces both of the size of primary ocular melanoma and the number of hepatic micrometastases in a murine ocular melanoma model. This therapeutic response was associated with decrease of tumor proliferation and neovascularization.