title: Induction of β-glucuronidase release by cytostatic agents in small tumors.
abstract: Extracellular β-glucuronidase (β-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 β-GUS in small tumors, resulting in increased levels of extracellular β-GUS and consequently a higher efficacy of the prodrug treatment. Here we examine the extent of β-GUS release in small C6 glioma tumors after a single treatment of doxorubicin (DOX), carmustine (BCNU) and tumor necrosis factor α (TNF-α) with positron emission tomography (PET) and the tracer 1-O-(4-(2-fluoroethyl-carbamoyloxymethyl)-2-nitrophenyl)-O-β-d-glucopyronuronate, [(18)F]FEAnGA, which has been proven to be selective for extracellular β-GUS. Induction of β-GUS release was first investigated in cultured C6 glioma cells. In addition, a [(18)F]FEAnGA PET study was performed in C6 tumor-bearing rats 48 h after a single treatment with different cytostatics to evaluate the extent of β-glucuronidase release. The cleavage of [(18)F]FEAnGA by β-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 β-GUS activity. PET studies confirmed that β-GUS was released in vivo and the distribution volume of the PET tracer [(18)F]FEAnGA in C6 gliomas was increased significantly by 15-70%, depending on the treatment. Histochemical analysis of the tumors indicated that carmustine and TNF-α 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 β-GUS. The release in β-GUS can be monitored by [(18)F]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.
authors.affiliation: Department of Nuclear Medicine and Molecular Imaging, University Medical Center Groningen, University of Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands.
authors.forename: Inês F
authors.initials: IF
authors.lastname: Antunes
authors.forename: Hidde J
authors.initials: HJ
authors.lastname: Haisma
authors.forename: Philip H
authors.initials: PH
authors.lastname: Elsinga
authors.forename: Valentina
authors.initials: V
authors.lastname: Di Gialleonardo
authors.forename: Aren
authors.initials: A
authors.lastname: van Waarde
authors.forename: Antoon T M
authors.initials: AT
authors.lastname: Willemsen
authors.forename: Rudi A
authors.initials: RA
authors.lastname: Dierckx
authors.forename: Erik F J
authors.initials: EF
authors.lastname: de Vries
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D001932:Brain Neoplasms / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D002330:Carmustine / Q000494:pharmacology; D054697:Cytostatic Agents / Q000494:pharmacology*; D004317:Doxorubicin / Q000494:pharmacology; D005434:Flow Cytometry; D005462:Fluorine Radioisotopes; D005910:Glioma / Q000188:drug therapy / Q000201:enzymology* / Q000473:pathology; D005965:Glucuronates; D005966:Glucuronidase / Q000493:pharmacokinetics*; D007962:Leukocytes / Q000276:immunology; D008297:Male; D009336:Necrosis; D049268:Positron-Emission Tomography; D011355:Prodrugs / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: C571523:1-O-(4-(2-fluoroethylcarbamoyloxymethyl)-2-nitrophenyl)-O-glucopyronuronate; D000903:Antibiotics, Antineoplastic; D018906:Antineoplastic Agents, Alkylating; D054697:Cytostatic Agents; D005462:Fluorine Radioisotopes; D005965:Glucuronates; D011355:Prodrugs; D014409:Tumor Necrosis Factor-alpha; D004317:Doxorubicin; D005966:Glucuronidase; D002330:Carmustine