Goat anti-Human Cathepsin B Polyclonal antibody, Biotin-Conjugated

RD-N, an aminomethylated derivative of riccardin D, is a lysosomotropic agent that can trigger lysosomal membrane permeabilization followed by cathepsin B (CTSB)-dependent apoptosis in prostate cancer (PCa) cells, but the underlying mechanisms remain unknown. Here we show that RD-N treatment drives CTSB translocation from the lysosomes to the nucleus where it promotes DNA damage by suppression of the breast cancer 1 protein (BRCA1). Inhibition of CTSB activity with its specific inhibitors, or by CTSB-targeting siRNA or CTSB with enzyme-negative domain attenuated activation of BRCA1 and DNA damage induced by RD-N. Conversely, CTSB overexpression resulted in inhibition of BRCA1 and sensitized PCa cells to RD-N-induced cell death. Furthermore, RD-N-induced cell death was exacerbated in BRCA1-deficient cancer cells. We also demonstrated that CTSB/BRCA1-dependent DNA damage was critical for RD-N, but not for etoposide, reinforcing the importance of CTSB/BRCA1 in RD-N-mediated cell death. In addition, RD-N synergistically increased cell sensitivity to cisplatin, and this effect was more evidenced in BRCA1-deficient cancer cells. This study reveals a novel molecular mechanism that RD-N promotes CTSB-dependent DNA damage by the suppression of BRCA1 in PCa cells, leading to the identification of a potential compound that target lysosomes for cancer treatment.

Objectives/Hypothesis. Acute otitis media, often caused by infection with Streptococcus pneumoniae, is characterized by inflammation of the middle ear mucosa. A prominent feature of the host response to bacterial infection of the middle ear mucosa is an influx of inflammatory cells that contributes to the local pool of inflammatory mediators by releasing additional inflammatory chemicals, which in turn cause further tissue injury. The objective was to identify candidate effector and signaling molecules involved in acute otitis media pathogenesis caused by S pneumoniae infection. Study Design. Male Sprague-Dawley rats were randomly assigned to 1 of 5 groups, including I control group without treatment, 2 placebo groups (12 and 48 hours) and 2 infected groups (12 and 48 hours). The rat middle ear was bilaterally inoculated with either 25 muL of tryptic soy broth (TSB group) or 25 muL of TSB containing approximately 1.24 x 10(9) cfu/mL of S pneumonias type 6A (SP group). Rats were killed at 12 and 48 hours after inoculation and the middle ear mucosa was collected. Total RNA was extracted and pooled from each group for gene expression assays. Methods. Gene expression profiles for rat middle ear mucosa at 12 and 48 hours after S pneumoniae or placebo inoculation were constructed using microarray technology (Clontech Atlas Rat 1.2 Array, 1176 cDNAs). Genes of interest were further validated by real-time polymerize chain reaction. Results. Middle ear mucosa expression of a gene cluster encoding the lysosomal cysteine proteases, cathepsins B (Ctsb), L (Ctsl), and K (Ctsk), was modified after S pneumoniae challenge. Specifically, at 12 hours, Ctsk and Ctsl messenger RNA that was abundantly expressed in the normal middle ear mucosa was decreased, whereas Ctsb transcript was induced. The changes in Ctsb and Ctsk gene expression were sustained at 48 hours. Conclusion: The constitutive expression of Ctsk and Ctsl messenger RNA in normal middle ear mucosa supports a function in the maintenance of middle ear mucosa bomeostasis, and their downregulation as an early event in acute otitis media may reflect a disruption in that function. The induction of Ctsb messenger RNA. in the infected middle ear mucosa suggests a role in early tissue injury; thus, Ctsb may represent a potential target for molecular diagnostics and/or rational intervention during the development of acute otitis media.