Induction of β-Glucuronidase Release by Cytostatic Agents in Small Tumors

Extracellular beta-glucuronidase (beta-GUS) in tumors has been investigated as a target enzyme for prodrug therapy. However, despite encouraging preclinical results, animal studies also indicate that the success of prodrug therapy might be limited by the insufficient prodrug-converting enzyme activity, especially in small tumors. We hypothesized that a single dose of a cytostatic drug might induce the release of beta-GUS in small tumors, resulting in increased levels of extracellular beta-GUS and consequently a higher efficacy of the prodrug treatment. Here we examine the extent of beta-GUS release in small C6 glioma tumors after a single treatment of doxorubicin (DOX), carmustine (BCNU) and tumor necrosis factor alpha (TNF-alpha) with positron emission tomography (PET) and the tracer 1-O-(4-(2-fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-beta-D-glucopyronuronate, [F-18]FEAnGA, which has been proven to be selective for extracellular beta-GUS. Induction of beta-GUS release was first investigated in cultured C6 glioma cells. In addition, a [F-18]FEAnGA PET study was performed in C6 tumor-bearing rats 48 h after a single treatment with different cytostatics to evaluate the extent of beta-glucuronidase release. The cleavage of [F-18]FEAnGA by beta-GUS was analyzed in tumor homogenates. The induction of tumor necrosis and leukocyte infiltration was confirmed by histochemical analysis and flow cytometry. The in vitro studies indicated that all treatments resulted in a decline of viable cells and an increase of extracellular beta-GUS activity. PET studies confirmed that beta-GUS was released in vivo and the distribution volume of the PET tracer [F-18]FEAnGA in C6 gliomas was increased significantly by 15-70%, depending on the treatment. Histochemical analysis of the tumors indicated that carmustine and TNF-alpha treatment caused a larger necrotic area with the absence of infiltrating immune cells, whereas doxorubicin induced an increase in leukocyte infiltration. These results were confirmed by flow cytometry. In conclusion, the present study demonstrates that a single dose of a cytostatic agent is able to increase the release of beta-GUS. The release in beta-GUS can be monitored by [F-18]FEAnGA PET in a noninvasive manner. This study may open the way to a two-step chemotherapy prodrug approach, in which tumors are treated with a single dose of a cytostatic drug prior to prodrug treatment.