Injection Of Cathepsin Research Articles

Cathepsin S Evokes PAR2-Dependent Pain in Oral Squamous Cell Carcinoma Patients and Preclinical Mouse Models.

Simple SummaryOral cancer is often deadly and severely painful. Because this form of cancer pain cannot be adequately treated with current medications including opiates, new treatment approaches are needed. Cathepsin S, a lysosomal cysteine protease may play a role in oral cancer pain through a protease-activated receptor-2 (PAR2)-dependent mechanism. We undertook a series of experiments to define the role of cathepsin S in oral cancer pain. We compared cathepsin S activity in human oral cancer tumors versus patient-matched normal tissue; a human oral cancer cell line versus a benign dysplastic oral keratinocyte cell line; and in an orthotopic xenograft tongue cancer mouse model versus normal controls in mice. We localized cathepsin S in macrophages and carcinoma cells in human oral cancers. The injection of cathepsin S caused nociception in a mouse model while the injection of oral cancer cells in which the gene for cathepsin S is deleted generated less nociception. Our findings will lay the foundations for clinical trials of cathepsin S inhibitors for treating oral cancer pain.Oral squamous cell carcinoma (SCC) pain is more prevalent and severe than pain generated by any other form of cancer. We previously showed that protease-activated receptor-2 (PAR2) contributes to oral SCC pain. Cathepsin S is a lysosomal cysteine protease released during injury and disease that can activate PAR2. We report here a role for cathepsin S in PAR2-dependent cancer pain. We report that cathepsin S was more active in human oral SCC than matched normal tissue, and in an orthotopic xenograft tongue cancer model than normal tongue. The multiplex immunolocalization of cathepsin S in human oral cancers suggests that carcinoma and macrophages generate cathepsin S in the oral cancer microenvironment. After cheek or paw injection, cathepsin S evoked nociception in wild-type mice but not in mice lacking PAR2 in Nav1.8-positive neurons (Par2Nav1.8), nor in mice treated with LY3000328 or an endogenous cathepsin S inhibitor (cystatin C). The human oral SCC cell line (HSC-3) with homozygous deletion of the gene for cathepsin S (CTSS) with CRISPR/Cas9 provoked significantly less mechanical allodynia and thermal hyperalgesia, as did those treated with LY3000328, compared to the control cancer mice. Our results indicate that cathepsin S is activated in oral SCC, and that cathepsin S contributes to cancer pain through PAR2 on neurons.

Evaluation of cathepsin B activity for degrading collagen IV using a surface plasmon resonance method and circular dichroism spectroscopy

Evaluation of cathepsin B activities for degrading collagen IV and heat-denatured collagen IV (gelatin) were performed by surface plasmon resonance (SPR) and circular dichroism (CD) measurements. The optimal pH of cathepsin B activity for degrading each substrate was around 4.0. The ΔRU(15min), which is a decrease in the SPR signal at 15min after injection of cathepsin B, was smaller for collagen IV than for heat-denatured collagen IV owing to the presence of triple-helical conformation. An unstable nature of the triple-helical conformation of collagen IV at pH 4.0 was shown by the CD study. Degrading collagen IV by cathepsin B was facilitated owing to a local unwinding of the triple-helical conformation caused by proteolytic cleavage of the non-helical region. The concentration dependence of the initial velocity for degrading collagen IV by cathepsin B at pH 4.0 was biphasic, showing that cathepsin B at low concentration exhibits exopeptidase activity, while the enzyme at high concentration exhibits endopeptidase activity. The kinetic parameters for the exopeptidase activity of cathepsin B toward collagen IV and heat-treated collagen IV were evaluated and discussed in terms of the protease mechanism.

The Neutrophil Granule Protein Cathepsin G Activates Murine T Lymphocytes and Upregulates Antigen-Specific Ig Production in Mice

Cathepsin G is a neutrophil granule derived antimicrobial chymotrypsin-like enzyme. Our previous study showed that cathepsin G induces chemotactic migration of human phagocytic leukocytes and increases random migration of T lymphocytes. In this study, we investigated the capacity of cathepsin G to activate T lymphocytes and to modulate antigen-specific humoral responses in mice. We found that cathepsin G is mitogenic for and induces production of IFN-γ by murine T cells in vitro. Injection of cathepsin G in BALB/c mice immunized with keyhole limpet hemocyanin (KLH) adsorbed to aluminum hydroxide resulted in a significantly increased production of KLH-specific IgG1 and IgG2a antibodies. There was a dose-dependent increase in KLH-specific proliferation of lymphocytes from draining lymph nodes from mice treated with KLH and cathepsin G when compared with those treated with KLH alone. Subsequent analysis of IFN-γ and IL-4 release following in vitro re-stimulation of draining lymph node lymphocytes obtained from KLH-immunized mice suggested that cathepsin G augments KLH-specific Ig antibody production via activation of T cells, presumably involving both Th1 and Th2 pathways. Thus, neutrophil granule cathepsin G, in addition to its capacity to kill microbes and to enhance leukocyte motility, activates T lymphocytes and modulates humoral immunity.

Goblet cell hyperplasia in large intrapulmonary airways after intratracheal injection of cathepsin B into hamsters.

Goblet cell hyperplasia (GCH) is a frequent histologic finding in the airways of smokers. Experimental observations suggest that the process may be caused by increased proteinase activity in the airways. To investigate the possible role of cathepsin B in the development of GCH, male Syrian golden hamsters were given three intratracheal injections of bovine spleen cathepsin B or buffer (pH 5.5) at 2-day intervals. Six weeks later, we found by review of PAS-hematoxylin-stained 1-micron sections of plastic-embedded lung tissue that large intrapulmonary airways of animals given cathepsin B contained a significantly greater number of secretory cells per millimeter of airway (64.8 +/- 7.3 versus 47.5 +/- 10.3 for control animals, p less than 0.005) in association with a significant increase in the number of total cells per millimeter of airway, from 149 +/- 14 for control animals to 164 +/- 11 for cathepsin-B-treated animals (p less than 0.025). No change was observed in the number of ciliated cells (93.9 +/- 8.1/mm for control animals versus 94.8 +/- 10.3/mm for cathepsin-B-treated animals) or other cells (3.0 +/- 2.2/mm for control versus 4.3 +/- 4.1/mm for cathepsin B), indicating that selective expansion of the secretory cell population occurred. In contrast, in the main bronchi of animals given cathepsin B, no significant alterations were found in the number or percentage of secretory cells. The findings reveal that cathepsin B induces secretory cell hyperplasia in hamsters and suggest the possibility that cysteine proteinases may contribute to GCH in smokers.

Ultrastructural studies of rabbit synovitis induced by autologous IgG fragments. II. Infiltrating cells in the sublining layer.

The synovial sublining layer of rabbits with synovitis induced by intra-articular injection of cathepsin D digested autologous IgG fragments (Fab2) has been examined under the electron microscope. Twelve or more injections of autologous Fab2 led to chronic synovitis with dense mononuclear cell infiltrates containing lymphocytes, blastic cells, plasma cells and macrophages. In the infiltrates there was evidence that the lymphocytes had been activated prior to transformation into mature plasma cells. Indirect evidence suggests that T lymphocyte activation also occurred in these infiltrates. Cellular contacts between macrophages and lymphocytes or plasma cells as well as between M cells and lymphocytes were demonstrated. These contacts are tentatively interpreted as a feature of ongoing immune processes in the synovium.