Targeting urokinase-type plasminogen activator receptor on human glioblastoma tumors with diphtheria toxin fusion protein DTAT.

Abstract

The prognosis for patients with brain cancer is poor, and new therapies are urgently needed. Recombinant toxic proteins that specifically target tumor cells appear to be promising. Urokinase-type plasminogen activator (uPA) receptor (uPAR) is expressed on the surface of glioblastoma and some other tumor cells and endothelial cells. We synthesized a recombinant fusion protein, DTAT, which contains the catalytic portion of diphtheria toxin (DT) for cell killing fused to the noninternalizing amino-terminal (AT) fragment of uPA, and investigated its effectiveness in targeting uPAR-positive tumor cells. In vitro cytotoxicity of DTAT was measured by cell proliferation assays. For in vivo studies, athymic nude mice (four to five animals/group) bearing uPAR-expressing human glioblastoma (U118MG) cell-induced tumors were injected with DTAT or control protein. Tumor volume was assessed over time, and differences between treatments were analyzed by Student's t test. Effects of DTAT on body organ systems were evaluated in normal, tumor-free C57BL/6 mice histologically and functionally by serum enzyme tests. All statistical tests were two-sided. In vitro, DTAT was highly potent and selective in killing uPAR-expressing glioblastoma cells (U118MG, U373MG, and U87MG) and human umbilical vein endothelial cells. In vivo, compared with mice treated with control proteins, DTAT caused a statistically significant (P =.05) regression of small U118MG cell-induced tumors in all mice. Control fusion proteins that did not react with glioblastoma cells had no effect on tumor growth. DTAT given to tumor-free C57BL/6 mice had little effect on kidney, liver, heart, lung, and spleen histologies. Serum analysis in the same mice showed no elevation in blood urea nitrogen, indicating lack of effect on kidney function but a statistically significant (P =.046), albeit non-life-threatening, elevation in liver alanine aminotransferase levels. DTAT may have potential for intracranial glioblastoma therapy because of its ability to target tumor cells and tumor vasculature simultaneously and its apparent lack of systemic effects.