• MR analysis
• SNP
• cathepsin
• causality
• endometriosis

• Humans
• Endometriosis/genetics
• Female
• Mendelian Randomization Analysis
• Cathepsins/genetics
• Polymorphism, Single Nucleotide
• Genetic Predisposition to Disease

• ECM
• antibody‐based therapy
• biologics
• cathepsins
• cell signalling
• chemotherapeutic drugs
• immunomodulation
• proteolysis
• tumour microenvironment

• Humans
• Neoplasms/drug therapy
• Neoplasms/immunology
• Neoplasms/metabolism
• Cathepsins/immunology
• Cathepsins/antagonists & inhibitors
• Cathepsins/metabolism
• Animals
• Tumor Microenvironment/drug effects

• French National Research Agency
• University of Montpellier
• Inserm Transfert
• Région Occitanie Pyrénées-Méditerranée
• Ligue Nationale contre le Cancer
• Ligue Régionale du Gard
• Ligue Régionale de l'Herault
• Ligue Régionale de la Charente Maritime

• cathepsin B
• cathepsin L
• caveolin
• human gingival fibroblast
• interleukin-6

• Animals
• Humans
• Mice
• Antineoplastic Agents/pharmacology
• Antineoplastic Agents/chemistry
• Antineoplastic Agents/chemical synthesis
• Cathepsins/antagonists & inhibitors
• Cathepsins/metabolism
• Cell Line, Tumor
• Cell Survival/drug effects
• Coordination Complexes/pharmacology
• Coordination Complexes/chemistry
• Coordination Complexes/chemical synthesis
• Green Light
• Molecular Structure
• Photochemotherapy
• Photosensitizing Agents/pharmacology
• Photosensitizing Agents/chemistry
• Photosensitizing Agents/chemical synthesis
• Ruthenium/chemistry
• Ruthenium/pharmacology

• R01 GM098285 NIGMS NIH HHS
• S10 OD028488 NIH HHS
• T32 GM142519 NIGMS NIH HHS

• Cathepsin H
• Inhibitor
• Pathophysiology
• Protease maturation
• Protease substrate

• Animals
• Mice
• Cell Adhesion
• Retinal Pigment Epithelium/metabolism
• Retinal Pigment Epithelium/pathology
• Vesicular Transport Proteins/metabolism
• Vesicular Transport Proteins/genetics
• Vesicular Transport Proteins/deficiency
• Phagosomes/metabolism
• Chediak-Higashi Syndrome/genetics
• Chediak-Higashi Syndrome/pathology
• Chediak-Higashi Syndrome/metabolism
• Peptide Hydrolases/metabolism
• Oxidative Stress
• Retinal Photoreceptor Cell Outer Segment/metabolism
• Retinal Photoreceptor Cell Outer Segment/pathology
• Disease Models, Animal
• Retina/metabolism
• Retina/pathology

• U54 AG065187 NIA NIH HHS
• R01 EY011996 NEI NIH HHS
• U54 AG054345 NIA NIH HHS
• P30 CA034196 NCI NIH HHS
• P30 EY000331 NEI NIH HHS
• R01 EY019943 NEI NIH HHS
• R01 EY027442 NEI NIH HHS
• R01 EY027860 NEI NIH HHS
• National Institutes of Health

New therapeutic targets that could improve current antitumor therapy and overcome cancer resistance are urgently needed. Promising candidates are lysosomal cysteine cathepsins, proteolytical enzymes involved in various critical steps during cancer progression. Among them, cathepsin X, which acts solely as a carboxypeptidase, has received much attention. Our results indicate that the triazole-based selective reversible inhibitor of cathepsin X named Z9 (1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-((4-isopropyl-4H-1,2,4-triazol-3-yl)thio)ethan-1-one) significantly reduces tumor progression, both in vitro in cell-based functional assays and in vivo in two independent tumor mouse models: the FVB/PyMT transgenic and MMTV-PyMT orthotopic breast cancer mouse models. One of the mechanisms by which cathepsin X contributes to cancer progression is the compensation of cathepsin-B activity loss. Our results confirm that cathepsin-B inhibition is compensated by an increase in cathepsin X activity and protein levels. Furthermore, the simultaneous inhibition of both cathepsins B and X with potent, selective, reversible inhibitors exerted a synergistic effect in impairing processes of tumor progression in in vitro cell-based assays of tumor cell migration and spheroid growth. Taken together, our data demonstrate that Z9 impairs tumor progression both in vitro and in vivo and can be used in combination with other peptidase inhibitors as an innovative approach to overcome resistance to antipeptidase therapy.

• Antitumor therapy
• Cancer
• Cathepsin B
• Cathepsin X
• Inhibitors
• Invasion

Lysosomal cysteine peptidase cathepsin B (catB) is an important tumor-promoting factor involved in tumor progression and metastasis representing a relevant target for the development of new antitumor agents. In the present study, we synthesized 11 ruthenium compounds bearing either the clinical agent nitroxoline that was previously identified as potent selective reversible inhibitor of catB activity or its derivatives. We demonstrated that organoruthenation is a viable strategy for obtaining highly effective and specific inhibitors of catB endo- and exopeptidase activity, as shown using enzyme kinetics and microscale thermophoresis. Furthermore, we showed that the novel metallodrugs by catB inhibition significantly impair processes of tumor progression in in vitro cell based functional assays at low noncytotoxic concentrations. Generally, by using metallodrugs we observed an improvement in catB inhibition, a reduction of extracellular matrix degradation and tumor cell invasion in comparison to free ligands, and a correlation with the reactivity of the monodentate halide leaving ligand.

Eleven ruthenium compounds bearing either the clinical agent nitroxoline or its potent cathepsin B (catB) inhibiting derivatives were evaluated as antimetastatic agents. We demonstrated that organoruthenation is a viable strategy for obtaining highly effective and specific inhibitors of catB activities, as shown using enzyme kinetics and microscale thermophoresis. Furthermore, we showed that the novel metallodrugs significantly impair processes of tumor progression in in vitro cell based functional assays at low noncytotoxic concentrations.

• cystatins
• cysteine cathepsins
• immune response
• myeloid-derived suppressor cells (MDSC)
• tumor-associated macrophages (TAM)

• a-subunit isoforms
• breast cancer
• cell invasion
• cell migration
• metastasis
• plasma membrane
• protein trafficking
• proton pump
• vacuolar ATPase

• Breast Neoplasms/enzymology
• Breast Neoplasms/genetics
• Breast Neoplasms/pathology
• CRISPR-Cas Systems
• Cell Line, Tumor
• Cell Membrane/enzymology
• Enzyme Inhibitors/pharmacology
• Female
• Humans
• Isoenzymes/antagonists & inhibitors
• Isoenzymes/genetics
• Isoenzymes/metabolism
• Neoplasm Invasiveness
• Neoplasm Metastasis
• RNA, Messenger/genetics
• Vacuolar Proton-Translocating ATPases/antagonists & inhibitors
• Vacuolar Proton-Translocating ATPases/genetics
• Vacuolar Proton-Translocating ATPases/metabolism

• R01 GM034478 NIGMS NIH HHS
• R37 GM034478 NIGMS NIH HHS
• HHS NIH National Institute of General Medical Sciences (NIGMS)
• Breast Cancer Alliance (BCA)
• Tufts University

Cathepsin B (CTSB) has been reported to be involved in cancer metastasis by altering extracellular matrix (ECM) remodeling and facilitating invasion. However, the contribution of CTSB to collective cell invasion in salivary adenoid cystic carcinoma (SACC) and the underlying mechanisms remain unclear. The present study demonstrated that collective cell invasion is commonly observed in SACC without a complete epithelial-mesenchymal transition signature. CTSB was found to be overexpressed in the invasive front of SACC compared to the tumor center, and was associated with a poor prognosis of patients with SACC. Subsequently, a 3D spheroid invasion assay was established in order to recapitulate the collective cell invasion of SACC and the results revealed that CTSB was only expressed in leader cells. The knockdown of CTSB by siRNA inhibited the migration and invasion of SACC-83 cells and impaired the formation of leader cells. CTSB knockdown also disrupted cytoskeletal organization, altered cell morphology and inhibited ECM remodeling by downregulating matrix metalloproteinase-9, focal adhesion kinase and Rho/ROCK function. Therefore, the present study provides evidence that CTSB may define leader cells in SACC and is required for collective cell invasion as a potential key regulator of ECM remodeling.

Lectins have been recognized as promising carrier molecules for targeted drug delivery. They specifically bind carbohydrate moieties on cell membranes and trigger cell internalization. Fungal lectin MpL (Macrolepiota procera lectin) does not provoke cancer cell cytotoxicity but is able to bind aminopeptidase N (CD13) and integrin α3β1, two glycoproteins that are overexpressed on the membrane of tumor cells. Upon binding, MpL is endocytosed in a clathrin-dependent manner and accumulates initially in the Golgi apparatus and, finally, in the lysosomes. For effective binding and internalization a functional binding site on the α-repeat is needed. To test the potential of MpL as a carrier for delivering protein drugs to cancer cells we constructed fusion proteins consisting of MpL and the cysteine peptidase inhibitors cystatin C and clitocypin. The fused proteins followed the same endocytic route as the unlinked MpL. Peptidase inhibitor-MpL fusions impaired both the intracellular degradation of extracellular matrix and the invasiveness of cancer cells. MpL is thus shown in vitro to be a lectin that can enable protein drugs to enter cancer cells, enhance their internalization and sort them to lysosomes and the Golgi apparatus.

• cancer invasion
• drug delivery
• fusion proteins
• lectin
• peptidase inhibitor

• Human cathepsin B
• Nitroxoline derivatives
• Structure-activity relationships
• Tumor invasion

• Antineoplastic Agents/chemical synthesis
• Antineoplastic Agents/chemistry
• Antineoplastic Agents/pharmacology
• Cathepsin B/antagonists & inhibitors
• Cathepsin B/metabolism
• Cell Line, Tumor
• Cell Proliferation/drug effects
• Cell Survival/drug effects
• Cysteine Proteinase Inhibitors/chemical synthesis
• Cysteine Proteinase Inhibitors/chemistry
• Cysteine Proteinase Inhibitors/pharmacology
• Dose-Response Relationship, Drug
• Drug Screening Assays, Antitumor
• Humans
• Molecular Structure
• Nitroquinolines/chemical synthesis
• Nitroquinolines/chemistry
• Nitroquinolines/pharmacology
• Structure-Activity Relationship

• Cathepsin B
• Cathepsin B inhibitors
• Collagen IV
• Surface plasmon resonance

Studies indicate secreted cathepsins are involved in metastasis. V-ATPases, which are necessary for activating intracellular cathepsins, also play a role in metastasis and are targeted to the plasma membrane of metastatic breast cancer cells. We are interested in a connection between cell surface V-ATPases, activation of secreted cathepsins and the metastatic phenotype of MDA-MB231 cells. We investigated whether V-ATPase inhibition would reduce the activity of secreted cathepsin B and cathepsin L. Using cell lysates and conditioned media, we measured cathepsin B and L activity within and outside of the cells. We found different forms of cathepsin B and L were secreted representing the pre-pro, pro and active forms of the proteases. Cathepsin B activity was higher than cathepsin L in conditioned media and in cell lysates. V-ATPase inhibition by concanamycin A decreased cathepsin B activity in conditioned media and significantly decreased cathepsin B activity in cell lysates. Cathepsin L activity showed a slight decrease in cell lysates. Changes in the activity of secreted and intracellular cathepsins following V-ATPase inhibition were supported by changes in the amounts of pro and active forms of cathepsin B in conditioned media and cathepsins B and L in cell lysates. Overall, our data shows that inactive forms of cathepsins B and L are secreted from the MB231 cells and V-ATPase activity is important for the activation of secreted cathepsin B. This indicates a connection between cell surface V-ATPases in metastatic breast cancer cells and the function of secreted cathepsin B.

• Breast cancer cells
• Cysteine cathepsins
• Protease activation
• Tumor microenvironment
• Vacuolar H+-ATPase

• Denison University

Lysosomal cysteine peptidase cathepsin B, involved in multiple processes associated with tumor progression, is validated as a target for anti-cancer therapy. Nitroxoline, a known antimicrobial agent, is a potent and selective inhibitor of cathepsin B, hence reducing tumor progression in vitro and in vivo. In order to further improve its anti-cancer properties we developed a number of derivatives using structure-based chemical synthesis. Of these, the 7-aminomethylated derivative (compound 17) exhibited significantly improved kinetic properties over nitroxoline, inhibiting cathepsin B endopeptidase activity selectively. In the present study, we have evaluated its anti-cancer properties. It was more effective than nitroxoline in reducing tumor cell invasion and migration, as determined in vitro on two-dimensional cell models and tumor spheroids, under either endpoint or real time conditions. Moreover, it exhibited improved action over nitroxoline in impairing tumor growth in vivo in LPB mouse fibrosarcoma tumors in C57Bl/6 mice. Taken together, the addition of a 2-(ethylamino)acetonitrile group to nitroxoline at position 7 significantly improves its pharmacological characteristics and its potential for use as an anti-cancer drug.

• cathepsin B
• cell migration
• inhibition
• nitroxoline derivative
• tumor invasion

Cystatin SN (CST1), a known inhibitor of cathepsin B (CatB), has important roles in tumor development. Paradoxically, CatB is a member of the cysteine cathepsin family that acts in cellular processes, such as tumor development and invasion. However, the relationship between CST1 and CatB, and their roles in tumor development are poorly understood. In this study, we observed that the knockdown of CST1 induced the activity of senescence-associated β-galactosidase, a marker of cellular senescence, and expression of senescence-associated secretory phenotype genes, including interleukin-6 and chemokine (C-C motif) ligand 20, in MDA-MB-231 and SW480 cancer cells. Furthermore, CST1 knockdown decreased extracellular CatB activity, and direct CatB inhibition, using specific inhibitors or shCatB, induced cellular senescence. Reconstitution of CST1 restored CatB activity and inhibited cellular senescence in CST1 knockdown cells. CST1 knockdown or CatB inhibition increased glycogen synthase (GS) kinase 3β phosphorylation at serine 9, resulting in the activation of GS and the induction of glycogen accumulation associated with cellular senescence. Importantly, CST1 knockdown suppressed cancer cell proliferation, soft agar colony growth and tumor growth in a xenograft model. These results indicate that CST1-mediated extracellular CatB activity enhances tumor development by preventing cellular senescence. Our findings suggest that antagonists of CST1 or inhibitors of CatB are potential anticancer agents.

• Breast cancer
• Cystatin C
• E-64
• Feedback
• Metastasis
• Pharmaceuticals
• Proteases

Hormonal-receptor positive (HRP) breast cancer patients with positive metastatic axillary lymph nodes are characterized by poor prognosis and increased mortality rate. The mechanisms by which cancer cells invade lymph nodes have not yet been fully explored. Several studies have shown that expression of IL-6 and the proteolytic enzyme cathepsin B (CTSB) was associated with breast cancer poor prognosis. In the present study, the effect of different concentrations of recombinant human IL-6 on the invasiveness capacity of HRP breast cancer cell line MCF-7 was tested using an in vitro invasion chamber assay. The impact of IL-6 on expression and activity of CTSB was also investigated. IL-6 treatment promoted the invasiveness potential of MCF-7 cells in a dose-dependent manner. Furthermore, MCF-7 cells displayed elevated CTSB expression and activity associated with loss of E-cadherin and upregulation of vimentin protein levels upon IL-6 stimulation. To validate these results in vivo, the level of expression of IL-6 and CTSB in the carcinoma tissues of HRP-breast cancer patients with positive and negative axillary metastatic lymph nodes (pLNs and nLNs) was assessed. Western blot and immunohistochemical staining data showed that expression of IL-6 and CTSB was higher in carcinoma tissues in HRP-breast cancer with pLNs than those with nLNs patients. ELISA results showed carcinoma tissues of HRP-breast cancer with pLNs exhibited significantly elevated IL-6 protein levels by approximately 2.8-fold compared with those with nLNs patients (P < 0.05). Interestingly, a significantly positive correlation between IL-6 and CTSB expression was detected in clinical samples of HRP-breast cancer patients with pLNs (r = 0.78, P < 0.01). Collectively, this study suggests that IL-6-induced CTSB may play a role in lymph node metastasis, and that may possess future therapeutic implications for HRP-breast cancer patients with pLNs. Further studies are necessary to fully identify IL-6/CTSB axis in different molecular subtypes of breast cancer.

• Cathepsin B
• Epithelial-mesenchymal transition
• Hormonal-receptor positive breast cancer
• IL-6
• Invasion
• Lymph node metastasis

Cathepsin B is a ubiquitously expressed lysosomal cysteine protease that participates in protein turnover within lysosomes. However, its protein and activity levels have been shown to be increased in cancer. Cathepsin B endopeptidase activity is involved in the degradation of extracellular matrix, a process that promotes tumor invasion, metastasis and angiogenesis. Previously, we reported an established antibiotic nitroxoline as a potent and selective inhibitor of cathepsin B. In the present study, we elucidated its anti-tumor properties in in vitro and in vivo tumor models.

Tumor and endothelial cell lines with high levels of active cathepsin B were selected for functional analysis of nitroxoline in vitro. Nitroxoline significantly reduced extracellular DQ-collagen IV degradation by all evaluated cancer cell lines using spectrofluorimetry. Nitroxoline also markedly decreased tumor cell invasion monitored in real time and reduced the invasive growth of multicellular tumor spheroids, used as a 3D in vitro model of tumor invasion. Additionally, endothelial tube formation was significantly reduced by nitroxoline in an in vitro angiogenesis assay. Finally, nitroxoline significantly abrogated tumor growth, angiogenesis and metastasis in vivo in LPB fibrosarcoma and MMTV-PyMT breast cancer mouse models. Overall, our results designate nitroxoline as a promising drug candidate for anti-cancer treatment.

• angiogenesis
• cathepsin B
• metastasis
• nitroxoline
• tumor invasion

Neuroblastoma arises from the sympathetic nervous system and accounts for 15% of childhood cancer mortality. Amplification of the oncogene N-Myc is reported to occur in more than 20% of patients. While N-Myc amplification status strongly correlates with higher tumour aggression and resistance to treatment, the role of N-Myc in the aggressive progression of the disease is poorly understood. N-Myc being a transcription factor can modulate the secretion of key proteins that may play a pivotal role in tumorigenesis. Characterising the soluble secreted proteins or secretome will aid in understanding their role in the tumour microenvironment, such as promoting cancer cell invasion and resistance to treatment. The aim of this study is to characterise the secretome of human malignant neuroblastoma SK-N-BE2 (N-Myc amplified, more aggressive) and SH-SY5Y (N-Myc non-amplified, less aggressive) cells. Conditioned media from SK-N-BE2 and SH-SY5Y cell lines were subjected to proteomics analysis. We report a catalogue of 894 proteins identified in the secretome isolated from the two neuroblastoma cell lines, SK-N-BE2 and SH-SY5Y. Functional enrichment analysis using FunRich software identified enhanced secretion of proteins implicated in cysteine peptidase activity in the aggressive N-Myc amplified SK-N-BE2 secretome compared to the less tumorigenic SH-SY5Y cells. Protein-protein interaction-based network analysis highlighted the enrichment of cathepsin and epithelial-to-mesenchymal transition sub-networks. For the first time, inhibition of cathepsins by inhibitors sensitized the resistant SK-N-BE2 cells to doxorubicin as well as decreased its migratory potential. The dataset of secretome proteins of N-Myc amplified (more aggressive) and non-amplified (less aggressive) neuroblastoma cells represent the first inventory of neuroblastoma secretome. The study also highlights the prominent role of cathepsins in the N-Myc amplified neuroblastoma pathogenesis. As N-Myc amplification correlates with aggressive neuroblastoma and chemotherapy-based treatment failure, co-treatment with cathepsin inhibitors might be a better avenue for disease management.

• N-Myc amplification
• mass spectrometry
• neuroblastoma
• proteomics
• secretome

• Copper-catalyzed azide−alkyne cycloaddition
• cysteine proteases
• human cathepsin B
• nitrile inhibitors

Large oncosomes (LO) are atypically large (1-10μm diameter) cancer-derived extracellular vesicles (EVs), originating from the shedding of membrane blebs and associated with advanced disease. We report that 25% of the proteins, identified by a quantitative proteomics analysis, are differentially represented in large and nano-sized EVs from prostate cancer cells. Proteins enriched in large EVs included enzymes involved in glucose, glutamine and amino acid metabolism, all metabolic processes relevant to cancer. Glutamine metabolism was altered in cancer cells exposed to large EVs, an effect that was not observed upon treatment with exosomes. Large EVs exhibited discrete buoyant densities in iodixanol (OptiPrep^TM) gradients. Fluorescent microscopy of large EVs revealed an appearance consistent with LO morphology, indicating that these structures can be categorized as LO. Among the proteins enriched in LO, cytokeratin 18 (CK18) was one of the most abundant (within the top 5^th percentile) and was used to develop an assay to detect LO in the circulation and tissues of mice and patients with prostate cancer. These observations indicate that LO represent a discrete EV type that may play a distinct role in tumor progression and that may be a source of cancer-specific markers.

• SILAC Proteomics
• amoeboid blebbing
• cancer metabolism
• extracellular vesicles
• tumor progression

The clioquinol–ruthenium complex [Ru(η⁶-p-cymene)(Cq)Cl] inhibits cathepsin B and reduces tumour cell invasion at non-cytotoxic concentrations, revealing a specific anti-cancer mechanism not related to a general compound-induced cytotoxicity.

Helicobacter pylori (H. pylori) infection has often no clinical signs and is one of the most common bacterial infections. All infected subjects have histology of active chronic gastritis. In some cases patients develop peptic ulcer and minority of them develop gastric cancer. Gastric cancer is multifactorial disease, thus various progressions of H. pylori infection and disease are dependent on the host genetic factors, the characteristics of the individual’s immune response, environmental factors, and different bacterial virulence factors of the individual bacterial strains. Eradication of the bacteria plays a crucial role in the treatment of these cases however antibiotic therapy does not always help. Bacteria often develop resistance to antibiotics so we recommend that not only screening for H. pylori also the strain determination should have some diagnostic value, especially in the patients who already developed gastritis. Furthermore, for such patients assessment of disease progression (atrophic or metaplastic gastritis) could be followed by polymorphism determination. Until now we cannot predict the disease based only on single polymorphism. Bacteria successfully neutralize the responses of the immune systems using different enzymes or even components of the host immune response. However, the influence of immune system and its components could represent new ways of treatments and could help to eradicate the infection.

• Helicobacter pylori
• Resistance to antibiotics
• Immune response
• Genetic factors
• Bacterial eradication

Cathepsin B is a cysteine proteinase that primarily functions as an endopeptidase within endolysosomal compartments in normal cells. However, during tumoral expansion, the regulation of cathepsin B can be altered at multiple levels, thereby resulting in its overexpression and export outside of the cell. This may suggest a possible role of cathepsin B in alterations leading to cancer progression. The aim of this study was to determine the contribution of intracellular and extracellular cathepsin B in growth, tumorigenesis, and invasion of colorectal cancer (CRC) cells. Results show that mRNA and activated levels of cathepsin B were both increased in human adenomas and in CRCs of all stages. Treatment of CRC cells with the highly selective and non‐permeant cathepsin B inhibitor Ca074 revealed that extracellular cathepsin B actively contributed to the invasiveness of human CRC cells while not essential for their growth in soft agar. Cathepsin B silencing by RNAi in human CRC cells inhibited their growth in soft agar, as well as their invasion capacity, tumoral expansion, and metastatic spread in immunodeficient mice. Higher levels of the cell cycle inhibitor p27^Kip1 were observed in cathepsin B‐deficient tumors as well as an increase in cyclin B1. Finally, cathepsin B colocalized with p27^Kip1 within the lysosomes and efficiently degraded the inhibitor. In conclusion, the present data demonstrate that cathepsin B is a significant factor in colorectal tumor development, invasion, and metastatic spreading and may, therefore, represent a potential pharmacological target for colorectal tumor therapy. © 2015 The Authors. Molecular Carcinogenesis, published by Wiley Periodicals, Inc.

• cathepsin B
• colorectal cancers
• intestinal tumorigenesis
• invasion
• p27Kip1

• 2,3-Dihydroquinazolin-4(1H)-one derivatives
• Cathepsin B
• Cathepsin H
• Endogenous proteolysis
• Non peptidyl inhibitors

4′-Phenylchalcones and their cyclised derivatives as novel inhibitors of cathepsin B, H and L, potential anticancer agents.

• Acidification
• Breast Cancer
• Invasion
• Migration
• Proton Transport
• Vacuolar ATPase

Crystalline structures activate the NLRP3 inflammasome, leading to the production of IL-1β, however, the molecular interactions responsible for NLRP3 activation are not fully understood. Cathepsin B release from the ruptured phagolysosome and potassium ion efflux have been suggested to be critical for this activation. Here, we report that Cathepsin B redistribution was not a crucial event in crystal-induced IL-1β production. Silica and monosodium urate crystal-treated macrophages with undisturbed lysosomes demonstrated strong co-localization of ASC and Caspase-1, indicative of NLRP3 inflammasome activation. Importantly, we provided evidence to suggest that macrophage cell membrane binding to immobilized crystals was sufficient to induce IL-1β release, and this activation of the NLRP3 inflammasome was inhibited by blocking potassium efflux. Therefore, this work reveals additional complexity in crystalline structure-mediated NLRP3 inflammasome regulations.

• cathepsin
• inflammation
• lysosomal proteinase
• macrophage
• reflux esophagitis

• CD spectroscopy
• Cathepsin B
• Collagen IV
• Proteolytic degradation
• Suface plasmon resonance

Cystatin SN (CST1) is one of the several salivary cystatins that form tight equimolar complexes with cysteine proteases, such as the cathepsins. High expression of CST1 is correlated with advanced pTNM stage in gastric cancer. However, the functional role of CST1 in tumorigenesis has not been elucidated. In this study, we showed that CST1 was highly expressed in colon tumor tissues, compared with nontumor regions. Increased cell proliferation and invasiveness were observed in HCT116 cell lines stably transfected with CST1 cDNA (HCT116-CST1) but not in CST3-transfected cells. We also demonstrated that CST1-overexpressing cell lines exhibited increased tumor growth as well as metastasis in a xenograft nude mouse model. Interestingly, CST1 interacted with cystatin C (CST3), a potent cathepsin B (CTSB) inhibitor, with a higher affinity than the interaction between CST3 and CTSB in the extracellular space of HCT116 cells. CTSB-mediated cellular invasiveness and proteolytic activities were strongly inhibited by CST3, but in the presence of CST1 CTSB activities recovered significantly. Furthermore, domain mapping of CST1 showed that the disulfide-bonded conformation, or conserved folding, of CST1 is important for its secretion and for the neutralization of CST3 activity. These results suggest that CST1 upregulation might be involved in colorectal tumorigenesis and acts by neutralizing the inhibition of CTSB proteolytic activity by CST3.

• Animals
• Cathepsin B/genetics
• Cathepsin B/metabolism
• Cell Membrane/metabolism
• Cell Survival
• Cells, Cultured
• Escherichia coli/immunology
• Extracellular Matrix/metabolism
• Gelatin/metabolism
• Gene Expression Regulation
• Lysosomes/metabolism
• Phagocytosis/physiology
• Phagosomes/immunology
• Phagosomes/metabolism
• Protein Transport
• Proteolysis
• Swine
• Trabecular Meshwork/cytology
• Trabecular Meshwork/immunology
• Trabecular Meshwork/metabolism

• ARRA R21EY019137-S NEI NIH HHS
• R21EY019137 NEI NIH HHS
• R21 EY019137 NEI NIH HHS
• P30EY005722 NEI NIH HHS
• R01 EY020491 NEI NIH HHS
• R01EY020491 NEI NIH HHS
• P30 EY005722 NEI NIH HHS

• sarcopenia
• cachexia
• biomarker
• skeletal muscle
• neoepitope