The mechanism of tumor cell killing by OXI4503 was investigated by studying vascular functional and morphological changes post drug administration. SCID mice bearing MHEC5-T hemangioendothelioma were given a single dose of OXI4503 at 100 mg/kg. Tumor blood flow, measured by microsphere fluorescence, was reduced by 50% at 1 hr, and reached a maximum level 6 -24 hr post drug treatment. Tumor vascular permeability, measured by Evan's blue and hemoglobin, increased significantly from 3 hr and peaked at 18 hr. The elevated tumor vessel permeability was accompanied by an increase in vascular endothelial growth factor (VEGF) from 1 hr post drug treatment. Immunohistochemical staining for CD31 and laminin showed that tumor blood vessels were affected as early as 3 hr but more prominent from 6 hr. From 12 hr, the vessel structure was completely destroyed. Histopathological and double immunohistochemical staining showed morphological change and induction of apoptosis in endothelial cells at 1-3 hr, followed by tumor cell necrosis from 6 -72 hr. There were no statistically significant changes of Evan's blue and hemoglobin contents in liver tissue over the time course. These results suggest that OXI4503 selectively targets tumor blood vessels, and induces blood flow shutdown while it enhances tumor blood vessel permeability. The early induction of endothelial cell apoptosis leads to functional changes of tumor blood vessels and finally to the collapse of tumor vasculature, resulting in massive tumor cell necrosis. The time course of the tumor vascular response observed with OXI4503 treatment supports this drug for development as a stand alone therapy, and also lends support for the use of the drug in combination with other cancer therapies.

B16 melanoma sublines (B16-F10-BL6 and B16-F1) exhibited elevated adenosine 3',5'-cyclic monophosphate (cAMP) levels when cultured in Dulbecco's modified Eagle's medium (DMEM) in comparison to cells in RPMI-1640 medium. In parallel, cells cultured in DMEM had increased tyrosinase activity, melanization and dendrite formation, all markers of melanoma differentiation. Also, the proliferative rates of both cell lines were decreased by 80-85% when cultured in DMEM relative to cells maintained in RPMI-1640 medium. In these studies, elevated levels of the melanin precursors tyrosine (Tyr) and phenylalanine (Phe) found in DMEM were shown not to be solely responsible for the phenotypic changes observed with DMEM. Both BL6 and B16-F1 cell lines formed more experimental pulmonary tumor metastasis in syngeneic C57BL/6 mice when maintained in DMEM vs RPMI-1640 medium. Analysis of metastasis formation in nude mice with normal and depleted natural killer (NK) cell activity revealed that the enhanced lung colonizing capacity of the BL6 cells maintained in DMEM was independent of the function of T-cell or NK-cell-mediated immunity. A close association between metastatic ability of tested lines and the expression of the membrane-associated enzyme gamma-glutamyltranspeptidase (gamma-GTPase, EC 2.3.2.2) was observed. The highly metastatic BL6 cell line had 20-fold higher levels of gamma-GTPase activity than the weakly metastatic B16-F1 cell line. Both cell lines, when grown in DMEM, had elevated gamma-GTPase activity that paralleled augmentation of metastatic ability. The dramatic changes in lung-colonizing capacity of the variant B16 melanoma cells maintained in DMEM in contrast to those grown in RPMI-1640 medium may serve as a useful model in understanding certain steps involved in triggering cell differentiation as well as metastasis development.

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