Nuclear Factor- B Mediates Up-Regulation of Cathepsin B by Doxorubicin in Tumor Cells

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 timeand 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 B (NFB) binding site could be identified. Pretreatment of HeLa cells with specific NFB inhibitors abrogated the induction of cathepsin B expression. Doxorubicin-induced degradation of the inhibitory protein I B could be prevented by pretreatment with a specific proteasome inhibitor, resulting in a significant reduction of the doxorubicininduced cathepsin B expression. Finally, binding of NFB subunits p50 and p65 to the NFB 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 NFB. 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. Pharmacotherapy is the major systemic treatment of most cancer diseases. Anthracyclines such as doxorubicin are widely used in the treatment of many human solid tumors and hematological malignancies, including acute leukemias, lymphomas, Kaposi’s sarcoma, bone tumors, and stomach, breast, and ovarian cancers (Danesi et al., 2002). These cytostatic antibiotics intercalate into DNA resulting in genetic damage and cell death. Their therapeutic index is compromised by severe side effects such as irreversible cardiotoxicity, leading to heart failure (Zucchi and Danesi, 2003). One approach to prevent the dose-limiting cardiotoxicity and other adverse effects is to develop prodrugs of anticancer agents with increased selectivity for tumors by enzymatic activation in the vicinity of the tumor. Realization of such a therapeutic concept depends on the availability of an antitumoral active prodrug and a suitable enzyme that is expressed at high levels in the surrounding tumor. One example of this technique is taken from the activation of prodrugs by tumorassociated enzymes such as peptidases. For the peptide prodrug N-L-leucyl-doxorubicin (Leu-Dox), activation and release of doxorubicin by peptidases secreted from either lysosomes or cancer cells was shown, particularly by cathepsin B (Sinhababu and Thakker, 1996). Because of the instability of Leu-Dox in blood, a further candidate prodrug, Nalanyl-L-leucyl-L-alanyl-L-leucyl-doxorubicin, was developed, releasing Leu-Dox by a two-step activation mediated by pepThis work was supported by grants from the Doktor Robert Pfleger Stiftung, Bamberg, Germany, and the Deutsche Forschungsgemeinschaft (Kr 945/7-1), Bonn, Germany. ABBREVIATIONS: Leu-Dox, N-L-leucyl-doxorubicin; NFB, nuclear factor B; AMC, 7-amino-4-methylcoumarin; CA-074Me, N-[L-trans-propylcarbamoyloxirane-2-carbonyl]-L-isoleucyl-L-proline; CA-074, L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline; DMSO, dimethyl sulfoxide; CAPE, caffeic acid phenylethyl ester; E-64, trans-epoxysuccinyl-L-leucylamido-4-(4-guanidino)-butan; GAPDH, glyceraldehyde3-phosphate-dehydrogenase; HNE, 4-hydroxy-2-nonenal; Cat B, cathepsin B; dox, doxorubicin; PBS, phosphate-buffered saline; Z-AMC, 7-N-benzyloxycarbonyl-L-arginyl-L-arginylamide-4-methyl-coumarin; DTT, dithiothreitol; TBST, Tris-buffered saline/Tween 20; PCR, polymerase chain reaction; RT-PCR, reverse transcription-polymerase chain reaction; EMSA, electrophoretic mobility shift assay; FCS, fetal calf serum; MG132, N-benzyloxycarbonyl (z)-Leu-Leu-leucinal. 0026-895X/04/6505-1092–1102$20.00 MOLECULAR PHARMACOLOGY Vol. 65, No. 5 Copyright © 2004 The American Society for Pharmacology and Experimental Therapeutics 2911/1145239 Mol Pharmacol 65:1092–1102, 2004 Printed in U.S.A. 1092 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from tidases. Both prodrugs showed improved antitumor efficacy and a decreased toxicity in vivo and in vitro (Boyer and Tannock, 1993). Loadman and coworkers (1999) showed that response to the prodrug PK1, a copolymer-doxorubicin conjugate containing a peptidyl spacer, differed in tumors and was directly related to an increased lysosomal activity of cathepsin B, pointing to a pivotal role for this enzyme in prodrug-based therapy. The bioactive enzyme cathepsin B is a lysosomal endopeptidase that belongs to the cysteine protease class of the papain superfamily (Turk et al., 2000), which is ubiquitously expressed in mammalian cells. The enzyme was mainly found in the lysosomes of normal tissues and participates in intralysosomal turnover of cellular proteins and molecules assimilated from the extracellular environment as well as in the degradation of extracellular matrix components, prohormone processing (Shinagawa et al., 1990), and turnover of -amyloids in Alzheimer’s disease (Bernstein et al., 1996). Further functions of cathepsin B have been found with the development of cathepsin B-deficient mice. In this model, cathepsin B is significantly involved in the onset of pancreatitis (Halangk et al., 2000) as well as in tumor necrosis factor–mediated apoptosis of hepatocytes, hepatic inflammation, and fibrogenesis (Guicciardi et al., 2001; Canbay et al., 2003). In different cancer types, cathepsin B has been shown to be elevated, and its cellular trafficking is frequently altered in malignant cells, resulting in an increased secretion of precursor and active forms of the enzyme (Berquin et al., 1995). In addition, studies using cathepsin B antisense techniques revealed a pivotal role of cathepsin B for invasion and motility of glioblastoma (Mohanam et al., 2001) and osteosarcoma cells (Krueger et al., 1999). Variation of cathepsin B expression and activity level in different tumors and regulation of this protease could influence the therapeutic efficacy of peptide prodrugs. In HL60 cells, expression of cathepsin B was increased after treatment with differentiating agents such as phorbol esters, calcitriol, and sodium butyrate for monocytic or retinoic acids for granulocytic differentiation (Berquin et al., 1999). From these data and the lack of studies with antineoplastic agents, we investigated the effects of the anthracycline doxorubicin on cathepsin B expression using the cervix carcinoma cell line HeLa. The present study shows that doxorubicin and other anthracycline antibiotics can cause an induction of cathepsin B on mRNA, protein, and activity levels in HeLa cells by an NFB–mediated pathway. Materials and Methods Materials. The human cervix adenocarcinoma cell line HeLa was obtained from the American Type Culture Collection (Manassas, VA). Cell-culture media and serum were from Biochrom (Berlin, Germany). Purified human liver cathepsin B, the anthracyclines doxorubicin hydrochloride, daunomycin hydrochloride, and idarubicin hydrochloride, the cysteine protease inhibitor E-64, and 7-amino4-methylcoumarin (AMC) were from Sigma Chemical (Steinheim, Germany). The cathepsin B inhibitors CA-074 and CA-074Me and the cathepsin B substrate 7-N-benzoyloxycarbonyl-L-arginyl-L-arginylamid-4-methyl-coumarin were purchased from Bachem AG (Heidelberg, Germany). The NFB inhibitors caffeic acid phenylethyl ester (CAPE) and 4-hydroxy-2-nonenal (HNE) were from Alexis Biochemicals (Grünberg, Germany). The lysosomotropic fluorochrome LysoTracker Green DND-26 was from Molecular Probes (Eugene, OR). T4 polynucleotide kinase was from Roche Diagnostics (Mannheim, Germany), and [ P]ATP ( 6000 Ci/mmol) was from Amersham Biosciences (Freiburg, Germany). All materials were obtained in the highest available grade. Stock solutions of doxorubicin, daunorubicin, idarubicin, E-64, CA-074, CA-074Me, CAPE, and HNE were prepared in dimethyl sulfoxide (DMSO) (Roth, Karlsruhe, Germany), AMC was dissolved in acetone, and 7-N-benzoyloxycarbonyl-L-arginyl-L-arginylamid-4-methyl-coumarin was dissolved in 50% methanol. Stock solutions were aliquoted and stored at 20°C. Antibodies. The following antibodies were used for immunodetection, immunofluorescence, and gel shift assays: monoclonal anticathepsin B (Ab-2) (Calbiochem, Schwalbach, Germany); polyclonal anti-p50 and anti-p65 (Santa Cruz Biotechnology Inc., Heidelberg, Germany); polyclonal anti-p52 (Upstate Biotechnology, Lake Placid, NY); polyclonal anti-c-Rel (Serotec, Düsseldorf, Germany); monoclonal anti-I B (Alexis Biochemicals); monoclonal anti-glyceraldehyde-3-phosphate-dehydrogenase (Biodesign International, Kennebunk, ME), and the secondary alkaline phosphatase-conjugated swine anti-rabbit IgG and goat anti-mouse IgG (DakoCytomation, Ham-