High Cathepsin Research Articles

Correlation between serum cathepsin S level and severity of coronary heart disease in elderly patients

Objective To study the expression level of cathepsin S in elderly patients with different degree of coronary heart disease(CHD), and to evaluate the correlation between cathepsin S level and the severity of CHD. Methods A total of 126 elderly patients with CHD were enrolled, including 36 cases with acute myocardial infarction(group AMI), 48 cases with unstable angina(group UAP), and 42 cases with stable angina(group SAP). During the same period, 40 healthy subjects were selected as controls.Venous blood was collected immediately after admission.Arterial blood was taken through radial/femoral artery sheath in AMI patients who underwent primary percutaneous coronary intervention, and coronary arterial blood was taken in AMI patients undergoing intracoronary thrombus aspiration.SYNTAX score was assessed in patients undergoing coronary angiography.Serum levels of high sensitivity C-reactive protein(hs-CRP), matrix metalloproteinase-9(MMP-9)and cathepsin S were determined in all specimens and compared between groups. Results Serum levels of cathepsin S, MMP-9 and hs-CRP were higher in CHD patients than in the control group(P<0.05). Among CHD patients, group AMI had the highest serum levels of cathepsin S, MMP-9 and hs-CRP, followed by group UAP and group SAP(F=106.830, 197.035 and 310.442, all P=0.000). Spearman's rank correlation test suggested that cathepsin S level was positively correlated with serum levels of MMP-9(r=0.816, P=0.000)and hs-CRP(r=0.827, P=0.000). SYNTAX score was positively correlated with serum levels of cathepsin S(r=0.581, P=0.000), MMP-9(r=0.511, P=0.000), and hs-CRP(r=0.557, P=0.000). Among the 24 patients with AMI who underwent intracoronary thrombus aspiration, the levels of cathepsin S, MMP-9 and hs-CRP were higher in coronary artery blood than in peripheral artery blood(t=217.288, 3.177 and 681.479, all P=0.000). Cathepsin S and hs-CRP levels in peripheral arterial blood had positive correlations with those in coronary arterial blood respectively(r=0.962 and 0.494, P=0.000 and 0.014), but such correlation between in peripheral arterial blood versus in coronary artery blood was not found in MMP-9 levels(r=-0.188, P=0.380). Conclusions Serum cathepsin S level is higher in elderly CHD patients than in healthy people, increases along with the increased severity of CHD, and positively correlates with the degree of coronary stenosis. Key words: Coronary disease; Cathepsins

Effects of beta-hydroxy-beta-methylbutyrate supplementation on skeletal muscle in healthy and cirrhotic rats.

Beta-hydroxy-beta-methylbutyrate (HMB) is a leucine metabolite with protein anabolic effects. We examined the effects of an HMB-enriched diet in healthy rats and rats with liver cirrhosis induced by multiple doses of carbon tetrachloride (CCl4). HMB increased branched-chain amino acids (BCAAs; valine, leucine and isoleucine) in blood and BCAA and ATP in muscles of healthy animals. The effect on muscle mass and protein content was insignificant. In CCl4-treated animals alterations characteristic of liver cirrhosis were found with decreased ratio of the BCAA to aromatic amino acids in blood and lower muscle mass and ATP content when compared with controls. In CCl4-treated animals consuming HMB, we observed higher mortality, lower body weight, higher BCAA levels in blood plasma, higher ATP content in muscles, and lower ATP content and higher cathepsin B and L activities in the liver when compared with CCl4-treated animals without HMB. We conclude that (1) HMB supplementation has a positive effect on muscle mitochondrial function and enhances BCAA concentrations in healthy animals and (2) the effects of HMB on the course of liver cirrhosis in CCl4-treated rats are detrimental. Further studies examining the effects of HMB in other models of hepatic injury are needed to determine pros and cons of HMB in the treatment of subjects with liver cirrhosis.

Elevated circulating cathepsin S levels are associated with metabolic syndrome in overweight and obese individuals.

Cathepsin S is highly expressed in subcutaneous and visceral adipose tissue. Cathepsin S correlates with central obesity and contributes to the formation and progression of atherosclerosis. Here, we sought to evaluate the association of serum cathepsin S with metabolic syndrome (MS) in overweight and obese Chinese adults. We evaluated serum cathepsin S levels in a cross-sectional sample of 781 overweight and obese Chinese adults by ELISA. Glucose, insulin, lipid profile, inflammatory markers, and adipokines were also measured. Cathepsin S was significantly associated with BMI, waist circumference, waist-to-hip ratio, fasting glucose, fasting insulin, the homeostatic model assessment of insulin resistance (HOMA-IR), systolic blood pressure, C-reactive protein (CRP), triglycerides, and HDL cholesterol (all P<0.05). Plasma cathepsin S levels increased significantly (P=0.045 for trend) with increasing numbers of MS components after adjustment for potential confounders. In the highest cathepsin S quartile, the MS risk was significantly higher (odds ratio 2.30; 95% confidence interval, 1.89-2.78) than in the lowest quartile after adjustment for age, gender, alcohol consumption, smoking, education, physical activity, self-reported CVD, and family history of diabetes. This association remained strong (odds ratio 1.97; 95% confidence interval, 1.72-2.48) after controlling further for CRP, adiponectin, HOMA-IR, and BMI. Elevated circulating cathepsin S concentrations are strongly and independently associated with MS in overweight and obese Chinese adults. Prospective studies are needed to establish the role of cathepsin S in the development of MS.

A Digital Pathology-Based Shotgun-Proteomics Approach to Biomarker Discovery in Colorectal Cancer

Background: Biomarkers in colorectal cancer are scarce, especially for patients with Stage 2 disease. The aim of our study was to identify potential prognostic biomarkers from colorectal cancers using a novel combination of approaches, whereby digital pathology is coupled to shotgun proteomics followed by validation of candidates by immunohistochemistry (IHC) using digital image analysis (DIA). Methods and Results: Tissue cores were punched from formalin-fixed paraffin-embedded colorectal cancers from patients with Stage 2 and 3 disease (n = 26, each). Protein extraction and liquid chromatography-mass spectrometry (MS) followed by analysis using three different methods were performed. Fold changes were evaluated. The candidate biomarker was validated by IHC on a series of 413 colorectal cancers from surgically treated patients using a next-generation tissue microarray. DIA was performed by using a pan-cytokeratin serial alignment and quantifying staining within the tumor and normal tissue epithelium. Analysis was done in QuPath and Brightness_Max scores were used for statistical analysis and clinicopathological associations. MS identified 1947 proteins with at least two unique peptides. To reinforce the validity of the biomarker candidates, only proteins showing a significant (P < 0.05) fold-change using all three analysis methods were considered. Eight were identified, and of these, cathepsin B was selected for further validation. DIA revealed strong associations between higher cathepsin B expression and less aggressive tumor features, including tumor node metastasis stage and lymphatic vessel and venous vessel invasion (P < 0.001, all). Cathepsin B was associated with more favorable survival in univariate analysis only. Conclusions: Our results present a novel approach to biomarker discovery that includes MS and digital pathology. Cathepsin B expression analyzed by DIA within the tumor epithelial compartment was identified as a strong feature of less aggressive tumor behavior and favorable outcome, a finding that should be further investigated on a more functional level.

Morphologic and Immunohistochemical Appraisal of Primary Gastric Carcinomas

Gastric carcinoma management requires adjustments answering their genetic and morphologic heterogeneity. We aim to assess the expression and significance of a myriad of biomarkers (p53, MLH1, MSH2, PMS2, MSH6, Epstein-Barr encoding region-RNA, c-erbB2, E-cadherin, CEA, chromogranin, Ki-67, CDX2, presenilin-1, cathepsin E, MUC5AC, cyclin-dependent kinase 1) in 117 gastric carcinomas, which we have morphologically subclassified with a simple algorithm. Immunohistochemical stains were applied to 3 tissue microarrays of primary gastric carcinomas (n=117) obtained from resection specimens of untreated patients. These cases represented the morphologic subgroups that emerged from a reclassification attempt carried out according to the predominant (>50%) morphologic component they contained (adenocarcinoma, diffuse infiltrative carcinoma, mucinous carcinoma) and "mixed" carcinoma if none predominated. Cases with unusual morphology were assigned to a "special subtypes" group ("rare" tumors). Correlation of overall survival and staining patterns was carried out. Adenocarcinomas comprised 43.6% (n=51), diffuse infiltrative carcinomas 28.2% (n=33), mucinous carcinomas 6% (n=7), mixed carcinomas 6%, and "rare/other" carcinomas 16.2% (n=19) of the 117 muscle-invasive carcinoma cases. High tumor stage was associated with worse overall survival at multivariate analysis (P=0.000, log-rank). Higher cathepsin E and cyclin-dependent kinase 1 expression was associated with worse overall survival on univariate analysis (log-rank; P=0.050 and 0.001, respectively). Mismatch repair defects were seen in adenocarcinomas and "rare" tumors with MLH1 silencing. These above-mentioned points can lead to the differentiation of metabolic and phenotypic features per gastric carcinoma subtype and may help design targeted approaches.

Cathepsin L activity correlates with proteinuria in chronic kidney disease in humans.

The presence and severity of proteinuria is considered an important prognostic marker in patients with chronic kidney disease (CKD) and is associated with mortality and morbidity. Cathepsin L is highly expressed in the foot processes of podocytes in the kidney, which serves as an ultrafiltration barrier. Cathepsin L is also up-regulated in the setting of inflammation as a feature of CKD. Therefore, we postulated that proteinuria severity in CKD patients might correlate with increased serum levels of cathepsin L. In this retrospective observational study, a total of 135 patients diagnosed with CKD, 31 renal transplant patients and 48 healthy controls were included. The demographic characteristics and clinical indicators were analyzed. Serum cathepsin L activity was significantly higher in patients with CKD than in renal transplant recipients and healthy controls (P<0.01). Patients with severe proteinuria had a higher cathepsin L activity compared to those with moderate or mild proteinuria (P<0.01). Serum cathepsin L activity positively associated with age, body mass index, nitrite level, neutrophil count, high-sensitivity C-reactive protein (hs-CRP), N-terminal pro-brain natriuretic peptide, high-mobility group box-1 protein (HMGB1) and 24-h proteinuria. In the ROC analysis, the sensitivity of cathepsin L activity in diagnosis of moderate and heavy is 0.86 and the specificity is 0.73. Moreover, CKD patients with higher cathepsin L activity had a significantly higher hospital admission rate. The data also showed patients with statin administration present significantly lower cathepsin L activity (P<0.01), hs-CRP (P<0.01), HMGB1 (P<0.01) and proteinuria (P<0.01) compared to non-statin treatment group. This study revealed that serum cathepsin L activity is significantly elevated in CKD patients and its level correlates with the severity of proteinuria as well as prognosis, suggesting that serum cathepsin L may serve as a potential biomarker for CKD. Further prospective study is needed to explore its clinical implications in the future.

Cathepsin L silencing increases As2O3 toxicity in malignantly transformed pilocytic astrocytoma MPA58 cells by activating caspases 3/7

Low-grade, pilocytic astrocytomas are treated by resection, but additional therapy is necessary for those tumors with anaplastic features. Arsenic trioxide (As2O3) is emerging as an effective chemotherapeutic agent for treatment of malignant glioblastoma multiforme, where Cathepsin L silencing enables lower, less harmful As2O3 concentrations to achieve the desired cytotoxic effect. Here, we evaluated the effects of As2O3 combined with stable Cathepsin L shRNA silencing on cell viability/metabolic activity, and apoptosis in primary cultures of recurrent malignantly transformed pilocytic astrocytoma (MPA). These cells expressed high Cathepsin L levels, and when grown as monolayers and spheroids, they were more resistant to As2O3 than the U87MG glioblastoma cell line. Caspases 3/7 activity in MPA58 spheroids was not significantly affected by As2O3, possibly due to higher chemoresistance of primary biopsy tissue of less malignant astrocytoma versus the malignant U87MG cell line. However, As2O3 treatment was cytotoxic to MPA spheroids after silencing of Cathepsin L expression. While Cathepsin L silencing only slightly decreased the live/dead cell ratio in As2O3-treated MPA-si spheroids under our experimental conditions, there was an increase in As2O3-mediated apoptosis in MPA-si spheroids, as indicated by elevated caspases 3/7 activity. Therefore, Cathepsin L silencing by gene manipulation can be applied when a more aggressive approach is needed in treatment of pilocytic astrocytomas with anaplastic features.

Comparative Proteomic Analysis of Tumor Mesenchymal-Like Stem Cells Derived from High Grade versus Low Grade Gliomas.

Objective Gliomas are the most common primary brain tumors, and have been ranked as the fourth leading cause of cancer death. Tumor mesenchymal-like stem cells (tMSCs) contribute to the aggressive behavior of glial tumors by providing a favorable microenvironment for the malignant cells. The aim of our study was to identify differential proteome of tMSCs derived from low vs. high grade glioma tumors. Materials and Methods Patients with newly diagnosed low and high grade gliomas were included in this case control study. tMSCs were isolated from tumors using enzymatic digestion validated by flow cytometer analysis after sub-culturing. Differential proteomic analysis of tMSCs derived from low and high grade tumors was performed by two-dimensional gel electrophoresis and mass spectrometry. Protein spots with more than two fold differences and P values below 0.05 were considered as differentially expressed ones. Results In tMSCs isolated from low and high grade gliomas, different isoforms of mesenchymal-related proteins vimentin and transgelin were differentially expressed. Overexpressed proteins in tMSCs isolated from low grade gliomas were mitochondrial manganese-containing superoxide dismutase (Mn-SOD), 40S ribosomal protein SA, and GTP-binding nuclear protein, while in tMSCs isolated from high grade gliomas, cathepsin B, endoplasmin, ezrin, peroxiredoxin1, and pyruvate kinase (PK) were found to be significantly overexpressed. ConclusionFor the first time, we analyzed the differential proteomic profiles of tMSCs isolated from glioma tumors with different grades. It was found that molecules related to mesenchymal cells (vimentin and transglin), in addition to those related to tumor aggressiveness with potential secretory behavior (e.g. cathepsin B) were among differentially expressed proteins.

Transcription Factor EB Expression in Early Breast Cancer Relates to Lysosomal/Autophagosomal Markers and Prognosis

BackgroundDisrupting the autophagic balance to trigger autophagic death may open new strategies for cancer therapy. Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and may play a role in cancer biology and clinical behavior. MethodsThe expression of TFEB and the lysosomal cancer cell content (expression of lysosomal associated membrane protein 2a [LAMP2a] and cathepsin D) was studied in a series of 100 T1-stage breast carcinomas. Expression patterns were correlated with autophagy/hypoxia-related proteins, angiogenesis, and clinical outcome. The effect of hypoxic/acidic conditions on TFEB kinetics was studied in the MCF-7 cancer cell line. ResultsOverexpression of TFEB in cancer cell cytoplasm and the perinuclear/nuclear area was noted in 23 (23%) of 100 cases. High LAMP2a and cathepsin D expression was noted in 30 (30%) of 100 and 28 (28%) of 100 cases, respectively. TFEB expression was directly linked with LAMP2a (P < .0001, r = 0.53), cathepsin D (P = .0002, r = 0.36), light chain 3A (LC3A) (P = .02, r = 0.22), and hypoxia-inducible factor 2-alpha (HIF-2α) (P = .01, r = 0.25) expression and inversely with progesterone receptor (P = .01, r = 0.22). High vascular density was directly linked with LAMP2a (P = .05, r = 0.18) and cathepsin D (P = .005, r = 0.28). In Kaplan-Meier survival analysis, TFEB and cathepsin D expression were related to an ominous prognosis (P = .001 and P = .03, respectively). In multivariate analysis, TFEB expression sustained its independent prognostic significance (P = .05, hazard ratio 2.1). In in vitro experiments, acidity triggered overexpression of TFEB and nuclear translocation. ConclusionIntense TFEB expression and lysosomal biogenesis, evident in one fourth of early breast carcinomas, define poor prognosis. Tumor acidity is among the microenvironmental conditions that trigger TFEB overactivity. TFEB is a sound target for the development of lysosomal targeting therapies.

Role of Zinc and Lysosomal Enzymes in Type 2 Diabetes Mellitus with Periodontitis

Background: The role of zinc and lysosomal enzymes in periodontal diseases has gained attention. The incidence of periodontal disease as well as its progression can be increased in the presence of poor glycaemic control. Diabetes and periodontitis have been suggested with a vicious cycle that aggravates both conditions when present in the same patient. Even though periodontal therapy is potentially an important component of the overall management strategy for patients with diabetes, the precise mechanisms that lead to reductions in zinc and increased release of lysosomal enzymes following periodontal treatment remain, as yet, unclear. Methods: This study focuses on how serum zinc and the lysosomal enzymes, acid phosphatase, β D glucuronidase and cathepsin D are related to the subjects in control group, Type 2 diabetes mellitus with periodontitis, Type 2 diabetes mellitus without periodontitis and non-diabetes mellitus with periodontitis. Results: The expression of acid phosphatase was found to be ten times increased in T2DM with periodontitis, the levels of β D glucuronidase are nine times elevated in T2DM with periodontitis, and the highest cathepsin D enzyme activity was observed in subjects with non-diabetes with periodontitis. Conclusion: The study supports the overall hypothesis that zinc deficiency is one of the major risk factor for T2DM with periodontitis. Due to the deficiency of zinc, the lysosomal enzymes levels are elevated and the stability of lysosomal membrane gets disturbed. Keywords: Acid phosphatase, β D glucuronidase, cathepsin D, periodontitis, zinc.

Predictors of hemorrhagic transformation occurring in patients with cardioembolic ischemic stroke

Aim. To specify the role of certain clinical factors, active forms of oxygen (AFO) and Cathepsin D as predictors of hemorrhagic transformation occurring at cardioembolic ischemic stroke (CEIS).Methods. Clinical and neurological examination of 125 patients with CEIS in the acute stage was carried out. The number of leukocytes in the stage of apoptosis (АNV+-cells) and necrosis (PI+-cells), the number of leukocytes with high AFO intracellular content (AFO+-cells) and low mitochondrial membrane potential (MMP) (Mito+-cells), and Cathepsin D activity were determined.Results. Clinical and laboratory characteristics of CEIS in patients with hemorrhagic transformation (HT) were determined. Both severe stroke, and disorders of consciousness, and large stroke volume-heart size, and SBP is over than 170 mm Hg refer to clinical factors associated with HT. The number of AFO+-leukocytes and Cathepsin D activity in blood serum during the first day of the disease should be used as additional markers to predict cardioembolic ischemic stroke hemorrhagic transformation. When the count of AFO+-leukocytes is over than 40 %, along with Cathepsin D total activity, higher than 4.5, and Cathepsin D free activity, higher than 3.5 nmol of tyrosine.Conclusion. The number of leukocytes with high AFO intracellular content and Cathepsin D activity in blood serum during the first day of cardioembolic ischemic stroke can be predictors of hemorrhagic transformation progress. Determination of hemorrhagic transformation predictors should be carried out before the thrombolytic and anticoagulant therapy

Knockdown of Cathepsin L promotes radiosensitivity of glioma stem cells both in vivo and in vitro

The presence of glioma stem cells (GSCs) in tumor is relevant for glioma treatment resistance. This study assessed whether knockdown of Cathepsin L can influence GSC growth, tumor radiosensitivity, and clinical outcome. Protein levels of Cathepsin L and stem cell markers (CD133 and Nestin) were analyzed in samples from 90 gliomas of different WHO grades and 6 normal brain tissues by immunohistochemistry. Two glioma stem cell lines with overexpressed Cathepsin L were stably transfected with Cathepsin L short hairpin RNA expression vectors. The effects of Cathepsin L inhibition on radiosensitivity, self-renewal, stemness, DNA damage, and apoptosis were evaluated. In addition, an intracranial animal model and subcutaneous tumor xenografts in nude mice were used to assess tumor response to Cathepsin L inhibition in vivo. Our results proved that expressions of Cathepsin L and CD133, but not of Nestin, correlated with malignant grades of glioma tissues. GSCs with high Cathepsin L and CD133 co-expression were extraordinarily radioresistant. Cathepsin L inhibition with radiotherapy significantly reduced GSC growth, promoted apoptosis, and improved radiosensitivity. Knockdown of Cathepsin L resulted in a dramatic reduction of CD133 expression, as well as the decreased phosphorylation of DNA repair checkpoint proteins (ATM and DNA-PKcs). Furthermore, combination of Cathepsin L inhibition and radiotherapy potently blocked tumor growth and decreased blood vessel formation in vivo. Taken together, these findings suggest Cathepsin L as a promising therapeutic target for clinical therapy in GBM patients.

Pit- and trench-forming osteoclasts: a distinction that matters.

Osteoclasts (OCs) seeded on bone slices either drill round pits or dig long trenches. Whereas pits correspond to intermittent resorption, trenches correspond to continuous and faster resorption and require a distinct assembly of the resorption apparatus. It is unknown whether the distinction between pits and trenches has any biological relevance. Using OCs prepared from different blood donors, we found that female OCs achieved increased resorption mainly through pit formation, whereas male OCs did so through trench formation. Trench formation went along with high collagenolytic activity and high cathepsin K (CatK) expression, thereby allowing deeper demineralization. A specific CatK inhibitor abrogated the generation of trenches, while still allowing the generation of pits. OCs obtained from bone marrow were more prone to generate trenches than those obtained from blood. Scanning electron microscopy of bone surfaces eroded in vivo showed trenches and pits of similar size as those made by OCs in culture. We conclude that the distinction between trench- and pit-forming OCs is relevant to the differences among OCs from different skeletal sites, different individuals, including gender, and results from differences in collagenolytic power. This indicates a biological relevance and highlights the importance of discriminating between pits and trenches when assessing resorption.

ACTIVITY OF CATHEPSIN H IN THE TUMOR AND CIRCUMFERENTIAL TISSUES IN WOMEN WITH INVASIVE LOBULAR FORM OF BREAST CANCER

Breast cancer takes the first place in the structure of oncologic morbidity of female population of the planet. Potential targets in elaboration of drugs are cysteine proteinases taking part in many processes, associated with cancer progression: Hyperproliferation of tumor cells, apoptosis, tumor-induced angiogenesis, invasion and matastasis.Cathepsin H cysteine proteinase possesses both proteinase and exopeptidase activity. High cathepsin H activity has been determined in the following tumors: glioblastoma, anaplastic astrocytoma, colorectal carcinoma, in head and neck tumors in blood plasma of patients with lung cancer, melanoma.In this connection the aim of the work consisted in analysis of cathepsin H activity in the tissue of invasive lobular form of breast cancer in order to elaborate additional biochemical test for presence of a neoplasm.Samples of mammary gland without neoplasms (circumferential tissue) and samples with invasive lobular form of breast cancer at I-II stages of cancer development were analysed. The patients were informed and gave the written agreement for the use of the anatomical material for biochemical research. In the supernatant the content of protein was determined by biuret method and cathepsin Н activity was determined by the modified Bradshaw method. Cathepsin activity was expressed in mmole of leucine, formed in the process of oxytocin hydrolysis, per a mg of protein per a min at 37 °С.It was found that in case of invasive lobular form of BC, the content of protein in tumour tissue at all stages of its development was 1.6-2.6 higher than the content of protein in the circumferential tissue. It was shown that cathepsin Н activity changed depending on the stage of invasive lobular BC development. The maximal relative and specific activity of cathepsin Н in tumour tissue was determined at I-II stage of tumor development. With tumor progression relative and specific activity of cathepsin Н in tumour tissue

42 Effect of Immunosuppressive Therapies on Mucinous Pancreatic Cyst Progression

AIM: A remodelling of the pancreatic cancer tumour microenvironment and extracellular matrix (ECM) has been implicated in the prognosis of PDAC. We investigated whether the aspartic protease cathepsin D contributes to proliferation, migration and fibrogenesis of pancreatic cancer cells and its expression affects the survival of pancreatic cancer patients. MATERIALS & METHODS: Mouse pancreatic stellate cells (PSC) were isolated by Nycodenz gradient. PSC and murine pancreatic cancer PD6606 cells were used for Western blotting and immunofluorescence staining. Cathepsin D activity was inhibited with pepstatin and its expression silenced with siRNA. TGFβ1 was measured using microplate sandwich ELISA and proliferation by standard MTT assay. Tumour tissue microarrays of 404 patients from the ESPAC-3 trial (all after PDAC-resection) were stained with anti-cathepsin D (C-20) antibody and patients with the highest quartile H-score (cut-off 22.35) were compared with those in the lowest quartile using Kaplan-Meier curves, log-rank tests, and Cox proportional hazards models. All statistical tests were two-sided. RESULTS: Inhibition of cathepsin D in ductal adenocarcinoma cells resulted in significantly reduced proliferation and migration. Cathepsin D expression increased in parallel with PSC trans-differentiation to myofibroblasts. Silencing (siRNA) or inhibiting cathepsin D decreased PSC proliferation by 20-30%. There was a time-dependent activation of latent TGF β1 and cathepsin D cleaved latent TGF β1, leading to its activation. Inhibition of cathepsin D decreased activated TGF β1. In 1824 tissue microarray cores from 382 patients (94.6%) median overall patients' survival was 23.85 months (95% CI 21.0-26.2). Median survival for patients in the highest quartile cathepsin D H-score was 21.2 months (95% CI 17.3-24.8) and in the lowest quartile 27.2 months (95% CI 24.1-31.2) χLR,1DF = 4.33; P = 0.0374. DISCUSSION: In pancreatic cancer, cathepsin D expression not only regulates fibrogenesis and stromal development but also ductal adenocarcinoma cell proliferation and migration. These mechanisms make cathepsin D a driver of pancreatic cancer growth and account for the poor prognosis of patients with high cathepsin D expression in PDAC.

43 Long-Term Outcomes of IPMNs With Consensus Guidelines Criteria for Resection Undergoing Nonoperative Management

AIM: A remodelling of the pancreatic cancer tumour microenvironment and extracellular matrix (ECM) has been implicated in the prognosis of PDAC. We investigated whether the aspartic protease cathepsin D contributes to proliferation, migration and fibrogenesis of pancreatic cancer cells and its expression affects the survival of pancreatic cancer patients. MATERIALS & METHODS: Mouse pancreatic stellate cells (PSC) were isolated by Nycodenz gradient. PSC and murine pancreatic cancer PD6606 cells were used for Western blotting and immunofluorescence staining. Cathepsin D activity was inhibited with pepstatin and its expression silenced with siRNA. TGFβ1 was measured using microplate sandwich ELISA and proliferation by standard MTT assay. Tumour tissue microarrays of 404 patients from the ESPAC-3 trial (all after PDAC-resection) were stained with anti-cathepsin D (C-20) antibody and patients with the highest quartile H-score (cut-off 22.35) were compared with those in the lowest quartile using Kaplan-Meier curves, log-rank tests, and Cox proportional hazards models. All statistical tests were two-sided. RESULTS: Inhibition of cathepsin D in ductal adenocarcinoma cells resulted in significantly reduced proliferation and migration. Cathepsin D expression increased in parallel with PSC trans-differentiation to myofibroblasts. Silencing (siRNA) or inhibiting cathepsin D decreased PSC proliferation by 20-30%. There was a time-dependent activation of latent TGF β1 and cathepsin D cleaved latent TGF β1, leading to its activation. Inhibition of cathepsin D decreased activated TGF β1. In 1824 tissue microarray cores from 382 patients (94.6%) median overall patients' survival was 23.85 months (95% CI 21.0-26.2). Median survival for patients in the highest quartile cathepsin D H-score was 21.2 months (95% CI 17.3-24.8) and in the lowest quartile 27.2 months (95% CI 24.1-31.2) χLR,1DF = 4.33; P = 0.0374. DISCUSSION: In pancreatic cancer, cathepsin D expression not only regulates fibrogenesis and stromal development but also ductal adenocarcinoma cell proliferation and migration. These mechanisms make cathepsin D a driver of pancreatic cancer growth and account for the poor prognosis of patients with high cathepsin D expression in PDAC.

Cathepsins K and S: Role in Bone, Adipocytes, and Glucose Regulation

Cathepsins are a diverse group of proteases that are increasingly being recognized for their role in various disease states. The focus of this article is to review the data regarding the activity of cathepsin K and cathepsin S in adipocyte differentiation and function as well as glucose metabolism. Data from animal and human studies have shown up-regulation of cathepsin K expression in white adipose tissue of overweight/obese mice and humans. Cathepsin K appears to affect adipocyte differentiation as well as weight gain. Data from studies using cathepsin K blocking agents suggest that by blocking cathepsin K, there is reduction in adipocyte differentiation and weight gain. Cathepsin K also may play a role in glucose metabolism with higher cathepsin K levels being associated with insulin resistance. Cathepsin S is also overexpressed in adipose tissue of overweight/obese subjects. Both murine and human models have been studied to further investigate its role in adipocytes and obesity. Cathepsin S appears to be involved in glucose dysregulation as well as pathogenesis of diabetes mellitus. Further studies are necessary to better characterize the role of cathepsin K and S and to examine whether there is potential for targeted therapy for prevention and treatment of obesity as well as diabetes mellitus.

Abstract IA18: A brain metastasis-promoting role for cathepsin S identified from analysis of tumor- and stroma-supplied proteolytic networks

Abstract Cancer cells in an aggressive primary tumor are adept at exploiting their local tissue microenvironment. In contrast, when metastatic cells leave these favorable surroundings, they must possess or acquire traits that will allow them to survive and colonize foreign, potentially hostile tissue environments. The obstacles that metastasizing tumor cells encounter vary from organ to organ, and are highly influenced by non-cancerous stromal cells of the tumor microenvironment. For example, the blood-brain barrier, composed of endothelial cells, astrocytes and pericytes, presents a far more formidable structure for tumor cells to penetrate, compared to the fenestrated capillaries in the bone marrow. While the primary tumor microenvironment has emerged as an important regulator of cancer progression, it is less well understood how different tissue environments influence metastatic processes. We used a dual species-specific microarray platform to uncover tumor-stroma interactions that modulate organ tropism of brain, bone and lung metastasis in animal models of cancer. Among the differentially regulated tumor- and stroma-specific genes, we identified cathepsin S as a novel regulator of breast-to-brain metastasis. In breast cancer patients, high cathepsin S expression at the primary site correlated with decreased brain metastasis-free survival. Both macrophages and tumor cells produce cathepsin S, and only the combined depletion significantly reduced brain metastasis in experimental models in vivo. We show that cathepsin S specifically mediates blood-brain barrier transmigration via proteolytic processing of the junctional adhesion molecule (JAM)-B. Pharmacological inhibition of cathepsin S significantly reduced experimental brain metastasis, supporting its consideration as a therapeutic target for this disease. Citation Format: Lisa Sevenich, Robert Bowman, Steve Mason, Daniela Quail, Franck Rapaport, Benelita Elie, Edi Brogi, Priscilla Brastianos, William Hahn, Leslie Holsinger, Joan Massagué, Christina Leslie, Johanna A. Joyce. A brain metastasis-promoting role for cathepsin S identified from analysis of tumor- and stroma-supplied proteolytic networks. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr IA18. doi:10.1158/1538-7445.CHTME14-IA18

OXIDATIVE STRESS IN HUMAN THYROID GLAND UNDER IODINE DEFICIENCY NODULAR GOITER: FROM HARMLESSNESS TO HAZARD DEPENDING ON COPPER AND IODINE SUBCELLULAR DISTRIBUTION

&lt;p&gt;&lt;strong&gt;Background.&lt;/strong&gt; Thyroid disorders are the second most common endocrinopathies found in humans and animals. Determination of their key molecular markers presents a special interest.&lt;br /&gt;&lt;strong&gt;Objective.&lt;/strong&gt; We studied iodine and copper accumulation in nodular, paranodular and contralateral (not affected tissue by node) tissues of human thyroid gland in relation to the level of metal-binding proteins, potential antioxidants, and oxidative changes in tissue for this goal. Lower level of organificated iodine and higher level and mass fraction of inorganic iodine and copper in the nodular and paranodular tissue versus contralateral part of thyroid gland was established.&lt;br /&gt;&lt;strong&gt;Results.&lt;/strong&gt; The level of both metal-binding and apo-form of metallothioneins was higher. Content of reduced glutathione was lower in node-affected tissue compared to the contralateral part. Signs of oxidative stress (higher activity of superoxide dismutase, catalase, glutathione-transferase and level of oxyradicals) and cytotoxicity (higher cathepsin D activity, higher level of DNA strand breaks and glycolysis activation) in affected tissue were observed. The range of indice variability in paranodular tissue was smaller than in nodule compared to the parenchyma of contralateral part.&lt;br /&gt;&lt;strong&gt;Conclusions.&lt;/strong&gt; Excess of copper unbound to metallothionein in goitrous-changed tissue and high level of inorganic iodine could be the reason for elevated DNA fragmentation and increased lysosomal membrane permeability and activation of antioxidant defense. The main criterions of goiter formation were represented by low level of organificated iodine and high level of DNA damage in thyroid gland.&lt;/p&gt;&lt;p&gt;&lt;strong&gt;KEY WORDS:&lt;/strong&gt; iodine deficiency nodular colloidal goiter, iodine, copper, metallothioneins, oxidative stress, cytotoxicity&lt;/p&gt;

Impact of the Enhanced Permeability and Retention (EPR) Effect and Cathepsins Levels on the Activity of Polymer-Drug Conjugates

Polymer-drug conjugates have demonstrated clinical potential in the context of anticancer therapy. However, such promising results have, to date, failed to translate into a marketed product. Polymer-drug conjugates rely on two factors for activity: (i) the presence of a defective vasculature, for passive accumulation of this technology into the tumour tissue (enhanced permeability and retention (EPR) effect) and (ii) the presence of a specific trigger at the tumour site, for selective drug release (e.g., the enzyme cathepsin B). Here, we retrospectively analyse literature data to investigate which tumour types have proved more responsive to polymer-drug conjugates and to determine correlations between the magnitude of the EPR effect and/or expression of cathepsin B. Lung, breast and ovarian cancers showed the highest response rate (30%, 47% and 41%, respectively for cathepsin-activated conjugates and 31%, 43%, 40%, across all conjugates). An analysis of literature data on cathepsin content in various tumour types showed that these tumour types had high cathepsin content (up to 3835 ng/mg for lung cancer), although marked heterogeneity was observed across different studies. In addition, these tumour types were also reported as having a high EPR effect. Our results suggest that a pre-screening of patient population could bring a more marked clinical benefit.