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

Abstract

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 B (NF-B) binding site could be identified. Pretreatment of HeLa cells with specific NF-B inhibitors abrogated the induction of cathepsin B expression. Doxorubicin-induced degradation of the inhibitory protein IB could be prevented by pretreatment with a specific proteasome inhibitor, resulting in a significant reduction of the doxorubicininduced cathepsin B expression. Finally, binding of NF-B subunits p50 and p65 to the NF-B 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-B. 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.

Up‐regulation of cathepsin B expression and enhanced secretion in mitochondrial DNA‐depleted osteosarcoma cells

“…Besides the binding sites for Sp1, Ets and USF transcription factors, the 5′‐flanking region of the cathepsin B gene contains an NF‐κB binding site at position 1460–1471 bp of the promoter region (Figure 4A). It has been demonstrated that exposure of HeLa carcinoma cells to doxorubicin induced an NF‐κB‐dependent up‐regulation of cathepsin B (Bien et al, 2004). To assess NF‐κB activity in mtDNA‐depleted cells, we used a luciferase construct containing five copies of the DNA‐binding sequence of NF‐κB in its promoter region.…”

Cathepsin B Regulates Collagen Expression by Fibroblasts via Prolonging TLR2/NF‐κB Activation

“…However, conversely, CatB expression is believed to be NF- κ B-dependent as the promoter of CatB has NF- κ B binding site [43]. In almost all cell types, including fibroblasts, NF- κ B exists as a dimer of a p50 and p65 subunit that is retained in an inactive cytoplasmic complex by binding to the I κ B α .…”

Proteomic analysis of doxorubicin‐induced changes in the proteome of HepG2cells combining 2‐D DIGE and LC‐MS/MS approaches

“…Additionally, doxorubicin‐induced apoptosis involves modulation of mitochondrial structures, a process known to result in mitochondrial dysfunction, such as the leakage of cytochrome c and the opening of mitochondrial permeability transition pores 5, 12. Lysosomal proteins are also targets of doxorubicin and the drug can modify lysosomal morphology and enzyme activity 13, 14. Recently, transcriptional analysis of doxorubicin‐induced cytotoxicity was performed to gain a more global view of the effects of the drug in HepG2 cells 15.…”