Immunohistochemical Evaluation of Cathepsin B, L, and S Expression in Breast Cancer Patients.

PURPOSE: Cysteine cathepsins are proteases that play a role in normal cellular physiology and neoplastic transformation. Elevated expression and enzymatic activity of cathepsins in breast cancer (BCa) indicates their potential as a target for tumor imaging. In particular cathepsin B (CTSB), L (CTSL), and S (CTSS) are used as targets for near-infrared (NIR) fluorescence imaging (FI), a technique that allows real-time intraoperative tumor visualization and resection margin assessment. Therefore, this immunohistochemical study explores CTSB, CTSL, and CTSS expression levels in a large breast cancer patient cohort, to investigate in which BCa patients the use of cathepsin-targeted NIR FI may have added value. PROCEDURES: Protein expression was analyzed in tumor tissue microarrays (TMA) of BCa patients using immunohistochemistry and quantified as a total immunostaining score (TIS), ranging from 0-12. In total, the tissues of 557 BCa patients were included in the TMA. RESULTS: CTSB, CTSL, and CTSS were successfully scored in respectively 340, 373 and 252 tumors. All tumors showed CTSB, CTSL, and/or CTSS expression to some extent (TIS > 0). CTSB, CTSL, and CTSS expression was scored as high (TIS > 6) in respectively 28%, 80%, and 18% of tumors. In 89% of the tumors scored for all three cathepsins, the expression level of one or more of these proteases was scored as high (TIS > 6). Tumors showed significantly higher cathepsin expression levels with advancing Bloom-Richardson grade (p < 0.05). Cathepsin expression was highest in estrogen receptor (ER)-negative, human epidermal growth factor receptor 2(HER2)-positive and triple-negative (TN) tumors. There was no significant difference in cathepsin expression between tumors that were treated with neoadjuvant systemic therapy and tumors that were not. CONCLUSIONS: The expression of at least one of the cysteine cathepsins B, L and S in all breast tumor tissues tested suggests that cathepsin-activatable imaging agents with broad reactivity for these three proteases will likely be effective in the vast majority of breast cancer patients, regardless of molecular subtype and treatment status. Patients with high grade ER-negative, HER2-positive, or TN tumors might show higher imaging signals.

Unveiling the Roles of Cysteine Proteinases F and W: From Structure to Pathological Implications and Therapeutic Targets.

Cysteine cathepsins F and W are members of the papain-like cysteine protease family, which have distinct structural features and functional roles in various physiological and pathological processes. This review provides a comprehensive overview of the current understanding of the structure, biological functions, and pathological implications of cathepsins F and W. Beginning with an introduction to these proteases, we delve into their structural characteristics and elucidate their unique features that dictate their enzymatic activities and substrate specificity. We also explore the intricate involvement of cathepsins F and W in malignancies, highlighting their role as potential biomarkers and therapeutic targets in cancer progression. Furthermore, we discuss the emerging roles of these enzymes in immune response modulation and neurological disorders, shedding light on their implications in autoimmune and neurodegenerative diseases. Finally, we review the landscape of inhibitors targeting these proteases, highlighting their therapeutic potential and challenges in clinical translation. This review brings together the diverse facets of cysteine cathepsins F and W, providing insights into their roles in health and disease and guiding future investigations for therapeutic advances.

Expression, Intracellular Localization, and Maturation of Cysteine Cathepsins in Renal Embryonic and Cancer Cell Lines.

Cysteine cathepsins play an important role in tumor development and metastasis. The expression of these enzymes is often increased in many types of tumor cells. Cysteine cathepsins contribute to carcinogenesis through a number of mechanisms, including proteolysis of extracellular matrix and signaling molecules on the cell surface, as well as degradation of transcription factors and disruption of signaling cascades in the cell nucleus. Distinct oncogenic functions have been reported for several members of the cysteine cathepsin family in various types of cancer, but a comparative study of all eleven cysteine cathepsins in one experimental model is still missing. In this work, we assessed and compared the expression, localization, and maturation of all eleven cysteine cathepsins in embryonic kidney cells HEK293 and kidney cancer cell lines 769-P and A-498. We found that the expression of cathepsins V, B, Z, L, and S was 3- to 9-fold higher in kidney tumor cells than in embryonic cells. We also showed that all cysteine cathepsins were present in varying amounts in the nucleus of both embryonic and tumor cells. Notably, more than half of the cathepsin Z or K and over 88% of cathepsin F were localized in tumor cell nuclei. Moreover, mature forms of cysteine cathepsins were more prevalent in tumor cells than in embryonic cells. These results can be further used to develop novel diagnostic tools and may assist in the investigation of cysteine cathepsins as potential therapeutic targets.

Investigations on Primary Cilia of Nthy-ori 3-1 Cells upon Cysteine Cathepsin Inhibition or Thyrotropin Stimulation.

In the thyroid gland, cysteine cathepsins are secreted upon thyrotropin stimulation for thyroglobulin processing, and they are present at the primary cilia of thyroid epithelial cells. Treatment with protease inhibitors resulted in the loss of cilia from rodent thyrocytes and caused redistribution of the thyroid co-regulating G protein-coupled receptor Taar1 to the endoplasmic reticulum. These findings suggest that ciliary cysteine cathepsins are important to maintain sensory and signaling properties for the proper regulation and homeostasis of thyroid follicles. Therefore, it is important to better understand how cilia structure and frequencies are maintained in human thyroid epithelial cells. Hence, we aimed to investigate the potential role of cysteine cathepsins for the maintenance of primary cilia in the normal human Nthy-ori 3-1 thyroid cell line. This was approached by determining cilia lengths and frequencies in cysteine peptidase inhibition conditions in Nthy-ori 3-1 cell cultures. Cilia lengths were shortened upon 5 h of cysteine peptidase inhibition with cell-impermeable E64. Likewise, cilia lengths and frequencies were decreased upon additional overnight treatment with the cysteine peptidase-targeting, activity-based probe DCG-04. The results suggest that cysteine cathepsin activity is required for the maintenance of the cellular protrusions not only in rodents, but also in human thyrocytes. Hence, thyrotropin stimulation was used to simulate physiological conditions that eventually lead to cathepsin-mediated thyroglobulin proteolysis, which is initiated in the thyroid follicle lumen. Immunoblotting revealed that thyrotropin stimulation conditions result in the secretion of little procathepsin L and some pro- and mature cathepsin S but no cathepsin B from the human Nthy-ori 3-1 cells. Unexpectedly, however, 24 h incubation periods with thyrotropin shortened the cilia although higher amounts of cysteine cathepsins were present in the conditioned media. These data point to the necessity of further studies to delineate which of the cysteine cathepsins plays the most prominent role in cilia shortening and/or elongation. Collectively, the results of our study provide corroboration for the hypothesis of thyroid autoregulation by local mechanisms that our group previously proposed.

Cysteine Cathepsins as Therapeutic Targets in Immune Regulation and Immune Disorders.

Cysteine cathepsins, as the most abundant proteases found in the lysosomes, play a vital role in several processes-such as protein degradation, changes in cell signaling, cell morphology, migration and proliferation, and energy metabolism. In addition to their lysosomal function, they are also secreted and may remain functional in the extracellular space. Upregulation of cathepsin expression is associated with several pathological conditions including cancer, neurodegeneration, and immune-system dysregulation. In this review, we present an overview of cysteine-cathepsin involvement and possible targeting options for mitigation of aberrant function in immune disorders such as inflammation, autoimmune diseases, and immune response in cancer.

Cysteine Cathepsins in Breast Cancer: Promising Targets for Fluorescence-Guided Surgery.

The majority of breast cancer patients is treated with breast-conserving surgery (BCS) combined with adjuvant radiation therapy. Up to 40% of patients has a tumor-positive resection margin after BCS, which necessitates re-resection or additional boost radiation. Cathepsin-targeted near-infrared fluorescence imaging during BCS could be used to detect residual cancer in the surgical cavity and guide additional resection, thereby preventing tumor-positive resection margins and associated mutilating treatments. The cysteine cathepsins are a family of proteases that play a major role in normal cellular physiology and neoplastic transformation. In breast cancer, the increased enzymatic activity and aberrant localization of many of the cysteine cathepsins drive tumor progression, proliferation, invasion, and metastasis. The upregulation of cysteine cathepsins in breast cancer cells indicates their potential as a target for intraoperative fluorescence imaging. This review provides a summary of the current knowledge on the role and expression of the most important cysteine cathepsins in breast cancer to better understand their potential as a target for fluorescence-guided surgery (FGS). In addition, it gives an overview of the cathepsin-targeted fluorescent probes that have been investigated preclinically and in breast cancer patients. The current review underscores that cysteine cathepsins are highly suitable molecular targets for FGS because of favorable expression and activity patterns in virtually all breast cancer subtypes. This is confirmed by cathepsin-targeted fluorescent probes that have been shown to facilitate in vivo breast cancer visualization and tumor resection in mouse models and breast cancer patients. These findings indicate that cathepsin-targeted FGS has potential to improve treatment outcomes in breast cancer patients.

The inhibitory effects of various ions released from S-PRG fillers on dentin protease activity.

This study investigates the effect of ions released from S-PRG fillers on host-derived enzymatic degradation of dentin collagen matrices. Dentin beams (n=80) were demineralized and distributed to eight groups following baseline dry mass and total MMP activity assessments. Each group treated with boron, fluoride, sodium, silicone, strontium, aluminium, or S-PRG eluate solutions for 5 min. Untreated beams served as control. After pre-treatment, MMP activity was reassessed, beams were incubated in complete medium for 1 week, dry mass was reassessed. Incubation media were analyzed for MMP and cathepsin-K-mediated degradation fragments. Data were analyzed with ANOVA and Tukey's test. All pretreatment groups showed significant reduction in total MMP activity (p<0.05) that was sustainable after incubation in all groups except for boron and silicone groups (p<0.05). Cathepsin-K activity did not differ between control or treatment groups. The results indicated that ions released from S-PRG fillers have the potential to partly inhibit MMP-mediated endogenous enzymatic activity.

Cysteine cathepsins: A long and winding road towards clinics.

Biomedical research often focuses on properties that differentiate between diseased and healthy tissue; one of the current focuses is elevated expression and altered localisation of proteases. Among these proteases, dysregulation of cysteine cathepsins can frequently be observed in inflammation-associated diseases, which tips the functional balance from normal physiological to pathological manifestations. Their overexpression and secretion regularly exhibit a strong correlation with the development and progression of such diseases, making them attractive pharmacological targets. But beyond their mostly detrimental role in inflammation-associated diseases, cysteine cathepsins are physiologically highly important enzymes involved in various biological processes crucial for maintaining homeostasis and responding to different stimuli. Consequently, several challenges have emerged during the efforts made to translate basic research data into clinical applications. In this review, we present both physiological and pathological roles of cysteine cathepsins and discuss the clinical potential of cysteine cathepsin-targeting strategies for disease management and diagnosis.

Inhibitory prodrug mechanism for cysteine cathepsin-targeted self-controlled drug release.

Tumour-associated macrophages (TAMs) support tumour development and have emerged as important regulators of therapeutic response to cytostatic agents. To target TAMs, we have developed a novel drug delivery approach which induces drug release as it inhibits cysteine cathepsin activity. This inhibitory prodrug (IPD) approach establishes a self-regulated system where drug release stops after all cysteine cathepsins are inhibited. This could improve the therapeutic window for drugs with severe side effects. We demonstrate and characterise this self-regulation concept with a fluorogenic IPD model. Next, we applied this IPD strategy to deliver cytotoxic drugs, as doxorubicin and monomethyl auristatin E, which are efficiently released and dose-dependently eliminate RAW264.7 macrophages. Lastly, by exploiting the increased cathepsin activity in TAM-like M2-polarised primary macrophages, we show that IPD-Dox selectively eliminates M2 over M1 macrophages. This demonstrates the potential of our IPD strategy for selective drug delivery and modulation of the tumour microenvironment.

Mussel-inspired monomer - A new selective protease inhibitor against dentine collagen degradation.

OBJECTIVES: To evaluate the inhibitory effect of a novel mussel-inspired monomer (N-(3,4-dihydroxyphenethyl)methacrylamide (DMA) on the soluble and matrix-bound proteases. METHODS: The inhibitory effect of DMA (0, 1, 5, and 10 mM) and 1 mM chlorhexidine (CHX) dissolved in 50% ethanol/water on soluble recombinant human matrix metalloproteinases (rhMMP-2, -8, and -9), as well as cysteine cathepsins (B and K) were evaluated using both fluorometric assay kits and molecular docking. The effect of CHX and DMA on matrix-bound proteases was examined by in situ zymography, and the fluorescence intensity and relative area were calculated by Image J software. All data obtained were analyzed by one-way ANOVA followed by Tukey test (α = 0.05). RESULTS: The anti-proteolytic ability of DMA increased in a dose-dependent manner except that of rhMMP-9. Inhibitory effect of 1 mM DMA against rhMMP-2, - 8, - 9, as well as cathepsin B and K was all significantly lower than 1 mM CHX (p < 0.05). The molecular docking analysis was in good agreement with the experimental results, that the binding energy of DMA was lower than CHX for all proteases. In situ zymography revealed that all DMA- and CHX-treated groups significantly inactivated the matrix-bound proteases, with a dramatic reduction of the fluorescence intensity and relative area compared with the control group (p < 0.05). SIGNIFICANCE: Under the prerequisite condition that the overall inhibitory performance on matrix-bound proteases was comparable by DMA and CHX, the more selective property of DMA could avoid inducing potential negative effects by suppressing MMP-9 when applied in dental treatment compared with CHX.

Long-term effect of curcuminoid treatment on resin-to-dentin bond strength.

Endogenous dentin proteases contribute to the degradation of collagen fibrils in the hybrid layer. Recently, inhibition of host-derived proteases by curcuminoids has shown promising results. The aim of this study was to evaluate the effect of curcuminoid treatment on the microtensile bond strength (µTBS) after 24 h or 12 months of storage. Fifty-four extracted sound human molars were flattened to mid-coronal dentin and divided into nine groups. After phosphoric acid-etching for 15 s, the dentin was experimentally treated for 60 s using 100 µM or 200 µM of curcumin, diflourobenzocurcumin, or demethoxycurcumin dissolved in 1% and 2% dimethyl sulfoxide (DMSO)/water solutions. Untreated and DMSO-treated groups served as controls. After bonding agent application, each tooth was restored with dental composite. The molars were sectioned into 0.9 × 0.9 × 6 mm beams. The µTBS testing was performed after 24 h and 12 months of storage in artificial saliva. Data were analyzed using regression analyses. Failure patterns were evaluated using scanning electron microscopy. Dentin treatment with curcuminoids did not adversely affect 24-h µTBS compared to controls. After 12 months, the µTBS of curcuminoid groups was statistically significantly higher than the controls. This study indicates the feasibility of using curcuminoids as protease inhibitors.

Primary digestive cathepsins L of Tribolium castaneum larvae: Proteomic identification, properties, comparison with human lysosomal cathepsin L.

We previously described the most highly expressed enzymes from the gut of the red flour beetle, Tribolium castaneum, as cathepsins L. In the present study, two C1 family-specific cysteine cathepsin L enzymes from the larval midgut were isolated and identified using MALDI-TOF MS analysis. The isolated T. castaneum cathepsins were characterized according to their specificity against chromogenic and fluorogenic peptide substrates, and the most efficiently hydrolyzed substrate was Z-FR-pNA with Arg in the P1 subsite. The specificity of insect digestive cathepsins was compared with human lysosomal cathepsin L, the well-studied peptidase of the C1 family cathepsins. T. castaneum digestive cathepsins efficiently hydrolyzed substrates with small and uncharged amino acid residues at P1 (Ala, Gln) more than human cathepsin L. In particular, these insect digestive cathepsins cleaved with higher efficiency the analogs of immunogenic peptides of gliadins, which contribute to autoimmune celiac disease in susceptible people, and thus insect enzymes may be useful in enzymatic treatments for this disease. A bioinformatic study supported by the proteomic analysis of the primary structures of the isolated cathepsins was used to compare tertiary models. The phylogenetic analysis of coleopteran and human cathepsins from the L subfamily indicated that insect digestive cathepsins grouped separately from lysosomal cathepsins.

[Cysteine cathepsins: structure, physiological functions and their role in carcinogenesis]. / Tsisteinovye katepsiny: stroenie, fiziologicheskie funktsii i rol' v kantserogeneze.

Cysteine cathepsins (Cts) also known as thiol proteinases belong to the superfamily of cysteine proteinases (EC 3.4.22). Cts are known as lysosomal proteases responsible for the intracellular proteins degradation. All Cts are synthesized as zymogens, activation of which occurs autocatalytically. Their activity is regulated by endogenous inhibitors. Cts can be secreted into the extracellular environment, which is of particular importance in tumor progression. Extracellular Cts not only hydrolyze extracellular matrix (ECM) proteins, but also contribute to ECM remodeling, processing and/or release of cell adhesion molecules, growth factors, cytokines and chemokines. In cancer, the expression and activity of Cts sharply increase both in cell lysosomes and in the intercellular space, which correlates with neoplastic transformation, invasion, metastasis and leads to further tumor progression. It has been shown that Cts expression depends on the cells type, therefore, their role in the tumor development differs depending on their cellular origin. The mechanism of Cts action in cancer is not limited only by their proteolytic action. The Cts influence on signal transduction pathways associated with cancer development, including the pathway involving growth factors, which is mediated through receptors tyrosine kinases (RTK) and various signaling mitogen-activated protein kinases (MAPK), has been proven. In addition, Cts are able to promote the epithelial-mesenchymal transition (EMT) by activating signal transduction pathways such as Wnt, Notch, and the pathway involving TGF-ß. So, Ctc perform specific both destructive and regulatory functions, carrying out proteolysis, both inside and outside the cell.

New Insights into the Role of Cysteine Cathepsins in Neuroinflammation.

Neuroinflammation, which is mediated by microglia and astrocytes, is associated with the progression of neurodegenerative diseases. Increasing evidence shows that activated microglia induce the expression and secretion of various lysosomal cathepsins, particularly during the early stage of neuroinflammation. This trigger signaling cascade that aggravate neurodegeneration. To date, most research on neuroinflammation has focused on the role of cysteine cathepsins, the largest cathepsin family. Cysteine cathepsins are primarily responsible for protein degradation in lysosomes; however, they also play a role in regulating a number of other important physiological and pathological processes. This review focuses on the functional roles of cysteine cathepsins in the central nervous system during neuroinflammation, with an emphasis on their roles in the polarization of microglia and neuroinflammation signaling, which in turn causes neuronal death and thus neurodegeneration.

Clickable, selective, and cell-permeable activity-based probe of human cathepsin B - Minimalistic approach for enhanced selectivity.

Human cathepsin B is a cysteine-dependent protease whose roles in both normal and diseased cellular states remain yet to be fully delineated. This is primarily due to overlapping substrate specificities and lack of unambiguously annotated physiological functions. In this work, a selective, cell-permeable, clickable and tagless small molecule cathepsin B probe, KDA-1, is developed and kinetically characterized. KDA-1 selectively targets active site Cys25 residue of cathepsin B for labeling and can detect active cellular cathepsin B in proteomes derived from live human MDA-MB-231 breast cancer cells and HEK293 cells. It is anticipated that KDA-1 probe will find suitable applications in functional proteomics involving human cathepsin B enzyme.

The Role of Cysteine Peptidases in Hematopoietic Stem Cell Differentiation and Modulation of Immune System Function.

Cysteine cathepsins are primarily involved in the degradation and recycling of proteins in endo-lysosomal compartments but are also gaining recognition as pivotal proteolytic contributors to various immune functions. Through their extracellular proteolytic activities within the hematopoietic stem cell niche, they are involved in progenitor cell mobilization and differentiation. Cysteine cathepsins, such as cathepsins L and S contribute to antigen-induced adaptive immunity through major histocompatibility complex class II antigen presentation whereas cathepsin X regulates T-cell migration. By regulating toll-like receptor signaling and cytokine secretion cysteine cathepsins activate innate immune cells and affect their functional differentiation. Cathepsins C and H are expressed in cytotoxic T lymphocytes and natural killer cells and are involved in processing of pro-granzymes into proteolytically active forms. Cytoplasmic activities of cathepsins B and L contribute to the maintenance of homeostasis of the adaptive immune response by regulating cell death of T and B lymphocytes. The expression pattern, localization, and activity of cysteine cathepsins is tightly connected to their function in immune cells. Furthermore, cysteine cathepsins together with their endogenous inhibitors, serve as mediators in the interplay between cancer and immune cells that results in immune cell anergy. The aim of the present article is to review the mechanisms of dysregulation of cysteine cathepsins and their inhibitors in relation to immune dysfunction to address new possibilities for regulation of their function.

Procathepsin V Is Secreted in a TSH Regulated Manner from Human Thyroid Epithelial Cells and Is Accessible to an Activity-Based Probe.

The significance of cysteine cathepsins for the liberation of thyroid hormones from the precursor thyroglobulin was previously shown by in vivo and in vitro studies. Cathepsin L is most important for thyroglobulin processing in mice. The present study aims at specifying the possible contribution of its closest relative, cysteine cathepsin L2/V, to thyroid function. Immunofluorescence analysis on normal human thyroid tissue revealed its predominant localization at the apical plasma membrane of thyrocytes and within the follicle lumen, indicating the secretion of cathepsin V and extracellular tasks rather than its acting within endo-lysosomes. To explore the trafficking pathways of cathepsin V in more detail, a chimeric protein consisting of human cathepsin V tagged with green fluorescent protein (GFP) was stably expressed in the Nthy-ori 3-1 thyroid epithelial cell line. Colocalization studies with compartment-specific markers and analyses of post-translational modifications revealed that the chimeric protein was sorted into the lumen of the endoplasmic reticulum and subsequently transported to the Golgi apparatus, while being N-glycosylated. Immunoblotting showed that the chimeric protein reached endo-lysosomes and it became secreted from the transduced cells. Astonishingly, thyroid stimulating hormone (TSH)-induced secretion of GFP-tagged cathepsin V occurred as the proform, suggesting that TSH upregulates its transport to the plasma membrane before it reaches endo-lysosomes for maturation. The proform of cathepsin V was found to be reactive with the activity-based probe DCG-04, suggesting that it possesses catalytic activity. We propose that TSH-stimulated secretion of procathepsin V is the default pathway in the thyroid to enable its contribution to thyroglobulin processing by extracellular means.

Significance of nuclear cathepsin V in normal thyroid epithelial and carcinoma cells.

Altered expression and/or localization of cysteine cathepsins is believed to involve in thyroid diseases including cancer. Here, we examined the localization of cathepsins B and V in human thyroid tissue sections of different pathological conditions by immunolabeling and morphometry. Cathepsin B was mostly found within endo-lysosomes as expected. In contrast, cathepsin V was detected within nuclei, predominantly in cells of cold nodules, follicular and papillary thyroid carcinoma tissue, while it was less often detected in this unusual localization in hot nodules and goiter tissue. To understand the significance of nuclear cathepsin V in thyroid cells, this study aimed to establish a cellular model of stable nuclear cathepsin V expression. As representative of a specific form lacking the signal peptide and part of the propeptide, N-terminally truncated cathepsin V fused to eGFP recapitulated the nuclear localization of endogenous cathepsin V throughout the cell cycle in Nthy-ori 3-1 cells. Interestingly, the N-terminally truncated cathepsin V-eGFP was more abundant in the nuclei during S phase. These findings suggested a possible contribution of nuclear cathepsin V forms to cell cycle progression. Indeed, we found that N-terminally truncated cathepsin V-eGFP expressing cells were more proliferative than those expressing full-length cathepsin V-eGFP or wild type controls. We conclude that a specific molecular form of cathepsin V localizes to the nucleus of thyroid epithelial and carcinoma cells, where it might involve in deregulated pathways leading to hyperproliferation. These findings highlight the necessity to better understand cathepsin trafficking in health and disease. In particular, cell type specificity of mislocalization of cysteine cathepsins, which otherwise act in a functionally redundant manner, seems to be important to understand their non-canonical roles in cell cycle progression.

Effect of protease inhibitor specificity on dentin matrix properties.

OBJECTIVE: To evaluate protease activity of dentin matrices subjected to treatment with non-specific (chlorhexidine - CHX), cysteine cathepsin specific (E-64), and cysteine cathepsin-K (CT-K) specific (Odanacatib - ODN) inhibitors. METHODS: Pulverized dentin powder obtained from human dentin disks (0.5 mm thickness) completely demineralized with 10% H3PO4 were challenged in 1 mL lactic acid (LA) (0.1M, pH 5.5) or stored in deionized water for 30 min. Aliquots of dentin powder were then immersed in 1 mL of CHX (2%), E-64 (10 µM and 20 µM) or Odanacatib (0.2 nM and 1 µM) for 30min. Degradation of dentin collagen was determined by telopeptide assays measuring the sub-product release of C-terminal cross-linked telopeptides (ICTP) and C-terminal peptide (CTX) in incubation media, which correlates with matrix metalloproteinases (MMP) and CT-K activities respectively (n = 3). The ICTP and CTX data were normalized to concentration of total protein (ICTPtp and CTXtp) in the media, measured by bicinchoninic acid assay. Dentin matrix properties were also measured by gravimetric change (n = 8) and ultimate tensile strength (UTS) (n = 10). Data were analyzed by one-way ANOVA followed by Tukey's post-hoc test and independent t-test (α = 5%). RESULTS: Telopeptide assays showed significantly lower CTXtp values after treatment with E-64 and Odanacatib. E-64 and Odanacatib at all tested concentrations significantly reduced the release of ICTPtp. Gravimetric analysis showed no significant difference between the tested inhibitors and control except for CHX after lactic acid challenge. UTS results showed significantly higher values for E-64 (20 µM) and Odanacatib (0.2 nM and 1 µM) groups in deionized water. SIGNIFICANCE: Dentin therapies targeting enzymes such as CT-K by specific inhibitors may provide superior pharmacokinetics and optimum efficacy due to precise protein binding, consequently limiting collagen degradation directly or indirectly by enzyme related pathways.

Cysteine cathepsins L and X differentially modulate interactions between myeloid-derived suppressor cells and tumor cells.

Increased proteolytic activity of cysteine cathepsins has long been known to facilitate malignant progression, and it has also been associated with tumor-promoting roles of myeloid-derived suppressor cells (MDSCs). Consequently, cysteine cathepsins have gained much attention as potential targets for cancer therapies. However, cross-talk between tumor cells and MDSCs needs to be taken into account when studying the efficacy of cathepsin inhibitors as anti-cancer agents. Here, we demonstrate the potential of the MDA-MB-231 breast cancer cell line to generate functional MDSCs from CD14+ cells of healthy human donors. During this transition to MDSCs, the overall levels of cysteine cathepsins increased, with the largest responses for cathepsins L and X. We used small-molecule inhibitors of cathepsins L and X (i.e., CLIK-148, Z9, respectively) to investigate their functional impact on tumor cells and immune cells in this co-culture system. Interactions with peripheral blood mononuclear cells reduced MDA-MB-231 cell invasion, while inhibition of cathepsin X activity by Z9 restored invasion. Inhibition of cathepsin L activity using CLIK-148 resulted in significantly increased CD8+ cytotoxicity. Of note, inhibition of cathepsins L and X in separate immune or tumor cells did not promote these functional changes. Together, our findings underlie the importance of tumor cell-immune cell interactions in the evaluation of the anti-cancer potential of cysteine cathepsin inhibitors.