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Introduction of Midkine Gene into Human Bladder Cancer Cells Enhances Their Malignant Phenotype But Increases Their Sensitivity to Antiangiogenic Therapy

Division of Urology, Department of Organ Therapeutics, Faculty of Medicine, Kobe University Graduate School of Medicine, Kobe, Japan [M. M., I. H., S. K.], and Department of Urology, Hyogo Medical Center for Adults, Akashi, Japan [H. M.]

ABSTRACT

Purpose: Midkine (MK) is a member of a family of heparin-binding growth factors, which was reported to have an important role in angiogenesis. Although MK was reported to be associated with bladder cancer progression, the functional significance of MK expression in bladder cancer progression has not been elucidated. The objectives of this study were to determine whether overexpression of MK in bladder cancer cells enhances their malignant potential and to evaluate the inhibitory effect of the antiangiogenic agent TNP-470 on the growth of MK-overexpressing bladder cancer cells in vivo.

Experimental Design: We introduced the MK gene into human bladder cancer UM-UC-3 cells that do not secrete a detectable level of MK protein and generated the MK-overexpressing cell line UM-UC-3/MK. The biological activity of secreted MK was evaluated using a human umbilical vein endothelial cell proliferation assay. To investigate the in vivo effects of MK overexpression on tumor growth, each cell line was injected s.c. and orthotopically into nude mice. To evaluate the therapeutic effects of the antiangiogenic agent, mice were given TNP-470 after s.c. injection of each cell line. The microvessel density of tumors was quantitated by immunohistochemistry of CD31.

Results: The heparin affinity-purified conditioned media of UM-UC-3/MK cells significantly enhanced human umbilical vein endothelial cell proliferation. MK expression had no effect on in vitro growth but conferred a growth advantage on both s.c. and orthotopic tumors in vivo. Furthermore, enhanced tumor growth was closely associated with increased microvessel density. Significant inhibition of tumor growth by TNP-470 treatment was observed only in UM-UC-3/MK tumors and not in control tumors.

Conclusions: We demonstrated that overexpression of the MK gene causes an increase in the angiogenic activity of cells through vascular endothelial cell growth, resulting in enhanced malignant potential of human bladder cancer cells. Moreover, the present findings suggest that TNP-470 could be used as a novel therapeutic adjunct to conventional agents for patients with advanced bladder cancer overexpressing MK.