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Anthracyclines such as doxorubicin remain among the most effective agents for the treatment of solid tumors and hematological malignancies. To overcome dose-limiting side effects like cardiotoxicity, an intensive effort has been undertaken to develop promising doxorubicin prodrugs that are specifically activated at the tumor site. One approach is the application of peptide prodrugs of doxorubicin. The enzyme cathepsin B catalyzes the activation of these prodrugs, and hence, the regulation of cathepsin B by antitumor agents could influence the efficacy of peptide prodrugs using this protease. In the present investigation, the effects of doxorubicin on cathepsin B expression in the human cervix carcinoma cell line HeLa were examined. Exposure to doxorubicin induced a time- and dose-dependent up-regulation of cathepsin B expression on mRNA, protein, and activity levels. In the cathepsin B gene promoter region, a potential nuclear factor kappaB (NF-kappaB) binding site could be identified. Pretreatment of HeLa cells with specific NF-kappaB inhibitors abrogated the induction of cathepsin B expression. Doxorubicin-induced degradation of the inhibitory protein IkappaB could be prevented by pretreatment with a specific proteasome inhibitor, resulting in a significant reduction of the doxorubicin-induced cathepsin B expression. Finally, binding of NF-kappaB subunits p50 and p65 to the NF-kappaB binding site in the cathepsin B gene promoter region could be demonstrated by electrophoretic mobility shift assay. In summary, our data clearly indicate that doxorubicin induces cathepsin B expression and activity via NF-kappaB. These findings contribute to a better understanding of tumor targeting with peptide prodrugs and help to define a possible mechanism of doxorubicin toxicity in tumor cells.

HMR 1826 (N-[4-beta-Glucuronyl-3-nitrobenzyl-oxycarbonyl]doxorubicin) is a nontoxic glucuronide prodrug from which active doxorubicin is released by beta-glucuronidase. Preclinical studies aimed at dose optimization of HMR 1826, based on intratumoral pharmacokinetics, are important to design clinical studies. Using an isolated perfused human lung model, the uptake of doxorubicin into normal tissue and tumors after perfusion with 133 microg/ml (n = 6), 400 microg/ml (n = 10), and 1200 microg/ml (n = 6) HMR 1826 was compared. Extracellular tissue pH was measured, and enzyme kinetic studies were performed in vitro to investigate the effect of pH on the formation of doxorubicin. Extracellular pH was lower in tumors than in healthy tissue (6.46 +/- 0.35, n = 8 versus 7.30 +/- 0.33, n = 10; p < 0.001). In vitro, beta-glucuronidase activity was 10 times higher at pH 6.0 than at neutral pH. After perfusion with HMR 1826, there was a linear relationship between HMR 1826 concentrations in perfusate and normal lung tissue. After perfusion with 133, 400, and 1200 microg/ml HMR 1826, the final doxorubicin concentrations in normal and tumor tissue were 2.7 +/- 0.9, 11.1 +/- 5.4, and 21.8 +/- 8.4 microg/g (p < 0.05 for all comparisons), and 0.7 +/- 0.3, 8.6 +/- 2.0 microg/g (p < 0.01 versus 133 microg/g), and 8.7 +/- 4.9 microg/g, respectively. This agrees with the enzyme kinetic observations of saturation of beta-glucuronidase at 400 microg/ml HMR 1826 in the acidic environment of the tumor. Therefore, the escalation of the HMR 1826 dose most likely results in higher circulating concentrations than 400 microg/ml but does not increase the uptake of doxorubicin into tumors and, subsequently, antitumor efficacy. The isolated perfused human lung is an excellent model for preclinical investigations aimed at optimization of tissue pharmacokinetics of tumor-selective prodrugs.