Downregulation Of Cathepsin Research Articles

Down regulation of Cathepsin W is associated with poor prognosis in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is associated with a very poor prognosis. Therefore, there has been a focus on identifying new biomarkers for its early diagnosis and the prediction of patient survival. Genome-wide RNA and microRNA sequencing, bioinformatics and Machine Learning approaches to identify differentially expressed genes (DEGs), followed by validation in an additional cohort of PDAC patients has been undertaken. To identify DEGs, genome RNA sequencing and clinical data from pancreatic cancer patients were extracted from The Cancer Genome Atlas Database (TCGA). We used Kaplan–Meier analysis of survival curves was used to assess prognostic biomarkers. Ensemble learning, Random Forest (RF), Max Voting, Adaboost, Gradient boosting machines (GBM), and Extreme Gradient Boosting (XGB) techniques were used, and Gradient boosting machines (GBM) were selected with 100% accuracy for analysis. Moreover, protein–protein interaction (PPI), molecular pathways, concomitant expression of DEGs, and correlations between DEGs and clinical data were analyzed. We have evaluated candidate genes, miRNAs, and a combination of these obtained from machine learning algorithms and survival analysis. The results of Machine learning identified 23 genes with negative regulation, five genes with positive regulation, seven microRNAs with negative regulation, and 20 microRNAs with positive regulation in PDAC. Key genes BMF, FRMD4A, ADAP2, PPP1R17, and CACNG3 had the highest coefficient in the advanced stages of the disease. In addition, the survival analysis showed decreased expression of hsa.miR.642a, hsa.mir.363, CD22, BTNL9, and CTSW and overexpression of hsa.miR.153.1, hsa.miR.539, hsa.miR.412 reduced survival rate. CTSW was identified as a novel genetic marker and this was validated using RT-PCR. Machine learning algorithms may be used to Identify key dysregulated genes/miRNAs involved in the disease pathogenesis can be used to detect patients in earlier stages. Our data also demonstrated the prognostic and diagnostic value of CTSW in PDAC.

Narciclasine suppresses oral cancer metastasis by modulating cathepsin B and extracellular signal–related kinase pathways

Oral cancer is a malignancy with unfavorable prognosis due to its high rates of recurrence and lymph node metastasis. Narciclasine is extracted from Narcissus species (Amaryllidaceae), which have antitumor and anti-inflammatory properties. However, the antitumor properties of narciclasine toward oral cancer remain unclear. The present study explored the antimetastatic effects of narciclasine in oral cancer as well as the underlying molecular mechanisms. We treated three oral cancer cell lines with noncytotoxic concentrations of narciclasine and discovered a dose-dependent antimetastatic effect. Mitogen-activated protein kinase (MAPK) pathways, including extracellular signal–related kinase (ERK), p38, and c-Jun N-terminal kinase (JNK), were regulated by narciclasine. We further discovered the ERK pathway to directly affect narciclasine-induced metastasis inhibition by combining treatment with narciclasine and ERK inhibitor. Furthermore, downregulation of cathepsin B (CTSB) in the SAS and SCC-47 cell lines revealed the critical role of CTSB in the antimetastatic effect of narciclasine. Our findings indicate that narciclasine inhibits oral cancer metastasis by regulating the ERK pathway and CTSB. This study provides evidence of the mechanism of narciclasine-induced inhibition oral cancer metastasis and suggests potential targets for use in oral cancer treatment.

Cathepsin D as biomarker in cerebrospinal fluid of nusinersen-treated patients with spinal muscular atrophy.

The therapeutic landscape of spinal muscular atrophy (SMA) has changed dramatically during the past 4years, but treatment responses differ remarkably between individuals, and therapeutic decision-making remains challenging, underlining the persistent need for validated biomarkers. We applied untargeted proteomic analyses to determine biomarkers in cerebrospinal fluid (CSF) samples of SMA patients under treatment with nusinersen. Identified candidate proteins were validated in CSF samples of SMA patients by Western blot and enzyme-linked immunosorbent assay. Furthermore, levels of peripheral neurofilament heavy and light chain were determined. Untargeted proteomic analysis of CSF samples of three SMA type 1 patients revealed the lysosomal protease cathepsin D as a candidate biomarker. Subsequent validation analysis in a larger cohort of 31 pediatric SMA patients (type 1, n=12; type 2, n=9; type 3, n=6; presymptomatically treated, n=4; age = 0-16years) revealed a significant decline of cathepsin D levels in SMA patients aged ≥2months at the start of treatment. Although evident in all older age categories, this decline was only significant in the group of patients who showed a positive motor response. Moreover, downregulation of cathepsin D was evident in muscle biopsies of SMA patients. We identified a decline of cathepsin D levels in CSF samples of SMA patients under nusinersen treatment that was more pronounced in the group of "treatment responders" than in "nonresponders." We believe that our results indicate a suitability of cathepsin D levels as a possible biomarker in SMA also in older patients, in combination with analysis of peripheral neurofilament light chain in adolescents or alone in adult patients.

Knockdown of CD44 inhibits proliferation, migration and invasion of osteosarcoma cells accompanied by downregulation of cathepsin S

BackgroundOsteosarcoma (OS) is a malignant bone tumour of mesenchymal origin. These tumours are characterised by rich vascularisation, therefore promoting rapid proliferation and facilitating metastasis. CD44 has been reported to be involved in OS, but its role and molecular mechanisms in the pathogenesis of the disease are not fully determined.MethodsIn this study, we investigated the antitumor effect of CD44 on the development of OS and further explored the molecular mechanisms. The expression of CD44, cathepsin S and MMP-9 was detected by Western blot (WB) and reverse transcription-polymerase chain reaction (RT-qPCR) in different cell lines (MG63, U2OS OS and hFOB 1.19). To elucidate the role of CD44 in OS, MG63 and U2OS cells were treated with small interference RNA (siRNA) to knock down CD44, and the knockdown efficiency was validated with GFP and RT-qPCR. Furthermore, cell proliferation was assayed using Cell Counting Kit‑8 (CCK-8) and colony formation assays, and cell migration and invasion were assayed by transwell and wound-healing assays.ResultsWe found that CD44 expression in the MG63 and U2OS OS cell lines was markedly increased compared to that of the human osteoblast hFOB 1.19 cell line. Knockdown of CD44 inhibited proliferation, migration and invasion of MG63 and U2OS cells. Cathepsin S expression in the MG63 and U2OS OS cell lines was increased compared to that of the human osteoblast hFOB 1.19 cell line. When CD44 was knocked down, its expression level went down.ConclusionTaken together, our data reinforced the evidence that CD44 knockdown inhibited cell proliferation, migration and invasion of OS cells accompanied by altered expression of cathepsin S. These findings offer new clues for OS development and progression, suggesting CD44 as a potential therapeutic target for OS.

Cathepsin L involved in the freezing resistance of murine normal hatching embryos and dormant embryos

The cryopreservation of mammalian embryos is an important technology in embryo engineering. The discovery and application of the embryo's own high freezing resistance factors are the main methods to improve the utilization of mammalian embryos in cryopreservation. Cathepsin L gene expression in the frozen and thawed dormant embryos displayed a significant difference from those normal hatched ones. The aim of the present study was to dig out the potential role of Cathepsin L in anti-freezing capacity of murine blastocysts by investigating the location and expression of Cathepsin L in frozen and thawed both activated and dormant hatching blastocysts. Different concentrations of Cathepsin L recombinant protein and E-64d were then respectively added into the embryo cryoprotectant and pre-cryo culture medium. Our results found that down-regulation of Cathepsin L improves the freezing resistance of murine normal hatching embryos by reducing apoptosis. Cathepsin L inhibitors can be used to improve the efficiency of cryopreservation and recovery of blastocysts in vitro. Our study provides a theoretical basis for the further development and application of Cathepsin L.

Downregulation of cathepsin C alleviates endothelial cell dysfunction by suppressing p38 MAPK/NF-κB pathway in preeclampsia

ABSTRACT Endothelial cell dysfunction is an essential pathophysiological feature of preeclampsia (PE). It has been reported that cathepsin C is upregulated in the maternal vascular endothelium of PE patients. The excessive activation of p38 MAPK leads to various diseases, including PE. NF-κB pathway can promote uteroplacental dysfunction, endothelial stress and development of PE. Moreover, it has been verified that cathepsin C can activate p38 MAPK/NF-κB pathway. In the present work, hypoxia/reoxygenation (H/R) injury model of HUVECs was established to discuss the biological functions of cathepsin C in endothelial cell dysfunction and to elucidate the underlying molecular mechanism. The correlation between cathepsin C and p38 MAPK/NF-κB pathway in H/R-stimulated HUVECs as well as the effects of cathepsin C and p38 MAPK/NF-κB pathway on viability, apoptosis, invasion, in vitro angiogenesis of HUVECs and oxidative stress were assessed. The results revealed that H/R injury elevated cathepsin C expression and activated p38 MAPK/NF-κB pathway in HUVECs and cathepsin C knockdown inhibited the activity of p38 MAPK/NF-κB pathway in H/R-stimulated HUVECs. Downregulation of cathepsin C improved viability, inhibited apoptosis and enhanced invasion of H/R-stimulated HUVECs. In addition, downregulation of cathepsin C alleviated oxidative stress and induced stronger HUVEC angiogenesis in vitro. Furthermore, the protective effects of cathepsin C knockdown against endothelial cell dysfunction were reversed by p38 MAPK activator anisomycin. In other words, downregulation of cathepsin C could improve HUVEC viability and enhance anti-apoptotic capacity, anti-oxidative capability, invasive ability, as well as angiogenic potential of H/R-stimulated HUVECs by repressing p38 MAPK/NF-κB pathway.

Downregulation of Cathepsin B Reduces Proliferation and Inflammatory Response and Facilitates Differentiation in Human HaCaT Keratinocytes, Ameliorating IL-17A and SAA-Induced Psoriasis-Like Lesion.

Psoriasis is a common inflammatory dermatology disease. Strongly expressed serum amyloid A (SAA) promotes psoriasis exacerbation through inducing IL-17 secretion. What's more, SAA can stimulate the release of cathepsin B. The current work was performed to demonstrate the specific effects of cathepsin B silencing on inflammatory response, proliferation, and differentiation of IL-17A and SAA-induced keratinocytes and to report the precise role of cathepsin B in psoriasis-like lesion. HaCaT keratinocytes received treatment with IL-17A (0, 10, 50, 100 ng/ml) or SAA (0, 1, 5, 10, 20 μg/ml) for 24 h to establish psoriasis-like keratinocytes model. HaCaT keratinocytes were transfected with small interfering RNA (siRNA)-cathepsin B for the functional experiments. Cathepsin B mRNA and protein levels were separately assessed by performing RT-qPCR and Western blot analysis. Then, CCK-8 for detection of cell proliferative capacity and Western blot assay for detection of Ki67 and PCNA expression were adopted to evaluate the influence of silenced cathepsin B on proliferation of IL-17A/SAA-induced HaCaT keratinocytes. Furthermore, IL-6, IL-1β, TNF-α, and p-NF-κB p65 were detected to assess the effects of cathepsin B knockdown on inflammatory response in IL-17A/SAA-induced HaCaT keratinocytes. In addition, assessment of KRT10, FLG, and LOR levels were applied to analyze the function of cathepsin B silencing on differentiation of IL-17A/SAA-induced HaCaT keratinocytes. Cathepsin B expression is distinctly elevated in IL-17A/SAA-induced HaCaT keratinocytes. IL-17A or SAA treatment enhanced proliferation, promoted the release of inflammatory factors, and arrested differentiation in HaCaT keratinocytes. Furthermore, downregulation of cathepsin B reduced proliferation, suppressed inflammatory response, and boosted differentiation in IL-17A/SAA-induced HaCaT keratinocytes. To sum up, cathepsin B silencing rescued excessive proliferation and inflammatory response and scarce differentiation in HaCaT keratinocytes induced by IL-17A and SAA. These findings prompted that cathepsin B might be a promising therapeutic target for psoriasis-like lesion, which helps to develop an anti-psoriatic agent.

MMP-3 plays a major role in calcium pantothenate-promoted wound healing after fractional ablative laser treatment

Ablative fractional laser treatment leads to a loss of matrix metalloproteinase-3 (MMP-3) expression; therefore, in the present in vitro study, we addressed the role of MMP-3 and its regulation by calcium pantothenate in wound healing processes at the molecular level. Utilizing confocal laser microscopy, we investigated MMP-3 protein expression in fractional ablative CO2 laser-irradiated skin models. In addition, we established full-thickness 3D skin models using fibroblasts and keratinocytes with a MMP-3 knockdown that were irradiated with a fractional ablative Er:YAG laser to set superficial injuries with standardized dimensions and minimal thermal damage to the surrounding tissue. We revealed an upregulation of MMP-3 protein expression in laser-irradiated skin models receiving aftercare treatment with calcium pantothenate. Skin models with MMP-3 knockdown exhibited a slower wound closure after laser treatment compared to controls. Gene expression profiling detected an MMP-3 knockdown-dependent upregulation of cytokines and chemokines (e.g. IL-36B, CXCL17, IL-37, CXCL5), antimicrobial peptides (e.g., S100A7, S100A12), epidermal crosslinking enzymes (TGM5), and differentiation markers (e.g., LOR, KRT1, FLG2). We also detected a downregulation of cathepsin V and MMP-10, both of which play a prominent role in wound healing processes. After fractional ablative laser injury, an aftercare treatment with calcium pantothenate accelerated wound closure in MMP-3 expressing models faster than in MMP-3 knockdown models. Our data substantiate a major role of MMP-3 in wound healing processes after ablative laser treatments. For the first time, we could show that calcium pantothenate exerts its wound healing-promoting effects at least partly via MMP-3.

Inhibition of Cathepsin S Induces Mitochondrial Apoptosis in Glioblastoma Cell Lines Through Mitochondrial Stress and Autophagosome Accumulation

Cathepsin S (CTSS), a lysosomal cysteine protease, is overexpressed in various cancers, including glioblastoma (GB). A high level of CTSS is associated with tumor progression and poor outcome in GB. However, the underlying mechanisms of its role in the biological characteristics of G5B remain to be elucidated. Here, we uncovered a potential role of CTSS in the lysosomes and mitochondria of GB cells (GBCs). Downregulation of CTSS in GBCs could increase the expression of autophagy-related proteins; however, there was no significant change in p62, suggesting autophagy blockade. Moreover, inhibition of CTSS increased the expression of mitochondrial calcium uniporter (MCU) and enhanced mitochondrial Ca2+ uptake ability, causing mitochondrial Ca2+ overload, the generation of copious reactive oxygen species (ROS) and eventual mitochondrial apoptosis. Additionally, elevated damage to mitochondria exacerbated the burden of autophagy. Finally, we found that silence of MCU could alleviate the inhibition of CTSS-induced autophagosome accumulation and mitochondrial stress. Collectively, these results demonstrate that CTSS plays an important role in the process of autophagic flux and mitochondrial functions in GBCs.

Integrative analyses of SARS-CoV-2 genomes from different geographical locations reveal unique features potentially consequential to host-virus interaction, pathogenesis and clues for novel therapies

We have performed an integrative analysis of SARS-CoV-2 genome sequences from different countries. Apart from mutational analysis, we have predicted host antiviral miRNAs targeting virus genes, PTMs in the virus proteins and antiviral peptides. A comparison of the analyses with other coronavirus genomes has been performed, wherever possible. Our analysis confirms unique features in the SARS-CoV-2 genomes absent in other evolutionarily related coronavirus family genomes, which presumably confer unique infection, transmission and virulence capabilities to the virus. For understanding the crucial factors involved in host-virus interactions, we have performed Bioinformatics aided analysis integrated with experimental data related to other corona viruses. We have identified 42 conserved miRNAs that can potentially target SARS-CoV-2 genomes. Interestingly, out of these, 3 are previously reported to exhibit antiviral activity against other respiratory viruses. Gene expression analysis of known host antiviral factors reveals significant over-expression of IFITM3 and down regulation of cathepsins during SARS-CoV-2 infection, suggesting its active role in pathogenesis and delayed immune response. We also predicted antiviral peptides which can be used in designing peptide based drugs against SARS-CoV-2. Our analysis explores the functional impact of the virus mutations on its proteins and interaction of its genes with host antiviral mechanisms.

Gallic acid Reduces Experimental Colitis in Rats by Downregulation of Cathepsin and Oxidative Stress

Gallic acid Reduces Experimental Colitis in Rats by Downregulation of Cathepsin and Oxidative Stress

Deficiency of Selected Cathepsins Does Not Affect the Inhibitory Action of ECTV on Immune Properties of Dendritic Cells

ABSTRACTEctromelia virus (ECTV), an orthopoxvirus, undergoes productive replication in conventional dendritic cells (cDCs), resulting in the inhibition of their innate and adaptive immune functions. ECTV replication rate in cDCs is increased due to downregulation of the expression of cathepsins – cystein proteases that orchestrate several steps during DC maturation. Therefore, this study was aimed to determine if downregulation of cathepsins, such as B, L or S, disrupts cDC capacity to induce activating signals in T cells or whether infection of cDCs with ECTV further weakens their functions as antigen-presenting cells. Our results showed that cDCs treated with siRNA against cathepsin B, L and S synthesize similar amounts of pro-inflammatory cytokines and exhibit comparable ability to mature and stimulate alloreactive CD4+ T cells, as untreated wild type (WT) cells. Moreover, ECTV inhibitory effect on cDC innate and adaptive immune functions, observed especially after LPS treatment, was comparable in both cathepsin-silenced and WT cells. Taken together, the absence of cathepsins B, L and S has minimal, if any, impact on the inhibitory effect of ECTV on cDC immune functions. We assume that the virus-mediated inhibition of cathepsin expression in cDCs represents more a survival mechanism than an immune evasion strategy.

A novel derivative of artemisinin inhibits cell proliferation and metastasis via down-regulation of cathepsin K in breast cancer

Breast cancer is one of the main diagnosis cancers annually worldwide. It is difficult to thorough cure due to drug resistance and the high possibility of metastasis. SM934 is a novel water-soluble artemisinin analog, and has been reported to have a promising therapeutic effect on multiple autoimmune diseases. In this study, SM934 was combined with Testosterone, which is related to prostate cancer, and the reaction product was SM934-Testosterone. We aimed to explore whether SM934, Testosterone and SM934-Testosterone could inhibit tumorigenesis and metastasis of breast cancer cells. Moreover, the mechanism also remains to be clarified. The results of our study showed that among the three compounds, only SM934-Testosterone treatment could lead to the suppression of cell proliferation and metastasis with IC50 = 30.66 ± 2.13 μM at 24 h in MDA-MB-231 and IC50 = 31.11 ± 1.79 μM at 24 h in SK-BR-3, where apoptosis was induced. But SM934-Testosterone showed little effects on breast cancer in vivo due to its poor water-solubility. Furthermore, computational target prediction and experimental validation demonstrated that Cathepsin K was the target of SM934-Testosterone. SM934-Testosterone inhibited the expression of Cathepsin K in breast cancer cells. Then, down-regulation of Cathepsin K in cancer cells by transfected with Cathepsin K shRNA inhibited cell proliferation and metastasis of breast cancer cells. Moreover, pathway enrichment was performed to understand the mechanism of action that Cathepsin K could adjust apoptosis regulator Bcl-X, and knockdown of Cathepsin K by SM934-Testosterone resulted in the reduction of Bcl-xL, which has been reported to be related to the proliferation and metastasis of cells. Collectively, SM934-Testosterone inhibited proliferation and metastasis ability of breast cancer cells via inhibiting the expression of Cathepsin K followed by the inhibition of Bcl-xL.

Down-regulation of cathepsin S and matrix metalloproteinase-9 via Src, a non-receptor tyrosine kinase, suppresses triple-negative breast cancer growth and metastasis

Triple-negative breast cancer (TNBC) is a highly metastatic breast cancer with poor prognosis. In the present study, we demonstrated that Src, a non-receptor tyrosine kinase, might provide an effective therapeutic strategy to overcome TNBC invasion and metastasis, which are mediated via the synergistic action of the lysosomal enzyme cathepsin S (CTSS) and gelatinase MMP-9. Knock-down of MMP-9 and CTSS using siRNAs resulted in a synergistic suppression of MDA-MB-231 cell invasion, which was similarly observed with pharmacological inhibitors. During the screening of new drug candidates that suppress both CTSS and MMP-9, BJ-2302, a novel 7-azaindolin-2-one derivative, was discovered. Src, an upstream activator of both pathways (PI3K/Akt and Ras/Raf/ERK) responsible for the expression of CTSS and MMP-9, was identified as a high-affinity target of BJ-2302 (IC90: 3.23 µM) through a Src kinase assay and a drug affinity responsive target stability (DARTS) assay. BJ-2302 effectively suppressed MDA-MB-231 cell invasion (Matrigel invasion assay) and metastasis (chorioallantoic membrane assay xenografted with MDA-MB-231-luc2-tdTomato cancer cells). Unlike Z-FL-COCHO (potent CTSS inhibitor), BJ-2302 did not induce any cytotoxicity in MCF-10A normal breast epithelial cells. Additionally, BJ-2302 (1 mg/kg) strongly suppressed TNBC cell proliferation in vitro and tumor growth in a xenograft mouse tumor model. The anti-metastatic and anti-tumor effects of BJ-2302 were superior to those of Z-FL-COCHO (1 mg/kg) or batimastat (30 mg/kg), a pan-MMP inhibitor. In summary, inhibition of Src kinase suppressed TNBC tumor growth and metastasis, and Src inhibitors such as BJ-2302 may constitute a novel therapeutic tool to treat breast cancer that expresses high levels of CTSS and MMP-9.

Trifluoperazine, a novel autophagy inhibitor, increases radiosensitivity in glioblastoma by impairing homologous recombination

BackgroundResistance to adjuvant radiotherapy is a major cause of treatment failure in patients with glioblastoma (GBM). Autophagy inhibitors have been shown to enhance the efficacy of radiotherapy for certain solid tumors. However, current inhibitors do not penetrate the blood-brain-barrier (BBB). Here, we assessed the radiosensitivity effects of the antipsychotic drug trifluoperazine (TFP) on GBM in vitro and in vivo.MethodsU251 and U87 GBM cell lines as well as GBM cells from a primary human biopsy (P3), were used in vitro and in vivo to evaluate the efficacy of TFP treatment. Viability and cytotoxicity was evaluated by CCK-8 and clonogenic formation assays. Molecular studies using immunohistochemistry, western blots, immunofluorescence and qPCR were used to gain mechanistic insight into the biological activity of TFP. Preclinical therapeutic efficacy was evaluated in orthotopic xenograft mouse models.ResultsIC50 values of U251, U87 and P3 cells treated with TFP were 16, 15 and 15.5 μM, respectively. TFP increased the expression of LC3B-II and p62, indicating a potential disruption of autophagy flux. These results were further substantiated by a decreased Lysotracker Red uptake, indicating impaired acidification of the lysosomes. We show that TFP and radiation had an additive effect when combined. This effect was in part due to impaired TFP-induced homologous recombination. Mechanistically we show that down-regulation of cathepsin L might explain the radiosensitivity effect of TFP. Finally, combining TFP and radiation resulted in a significant antitumor effect in orthotopic GBM xenograft models.ConclusionsThis study provides a strong rationale for further clinical studies exploring the combination therapy of TFP and radiation to treat GBM patients.

Salidroside improves pulmonary fibrosis by down-regulation of cathepsin B and NF-κBp65 in rats

To investigate the mechanism of salidroside in improvement of pulmonary fibrosis in rats. Methods: SD rats were subjected to 6 groups: a blank group, a model group, a pirfenidone group, a high-dose salidroside group, a middle-dose salidroside group, and a low-dose salidroside group. The contents of ALB, ALP, LDH, PC-III and COL4, and the expression levels of cathepsin B (CB) and NF-κBp65 in the 6 groups were analyzed. Results: Lung coefficient and oxygen partial pressure in the blank group were lower than those in the model group (P<0.05). Compared with the model group, lung coefficients in the pirfenidone group, the high-dose salidroside group, the middle-dose salidroside group, and the low-dose salidroside group were decreased, while oxygen partial pressures were statistically increased (P<0.05). The contents of ALB, ALP and LDH in the model group were statistically increased (P<0.05) in a dose-dependent manner. Compared with the blank group, the contents of GSH, HYP, PC-III and COL4, and the expression levels of CB and NF-κBp65 in the model group were significantly increased (P<0.05), which were attenuated by alidroside in a dose-dependent manner (P<0.05). Conclusion: Salidroside can improve pulmonary fibrosis in rats, and the mechanism may be related to reduce the expression of CB and NF-κBp65.

Inhibition of Cathepsin S Induces Mitochondrial ROS That Sensitizes TRAIL-Mediated Apoptosis Through p53-Mediated Downregulation of Bcl-2 and c-FLIP.

Cathepsin S is highly expressed in various cancer cells, and it has protumoral effects, including promotion of migration, invasion, and neovascularization. In this study, we show that inhibition of cathepsin S could sensitize cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. An inhibitor of cathepsin S (Z-FL-COCHO; ZFL) markedly induced apoptosis in human renal cancer cells treated with TRAIL. In contrast, combined treatment with ZFL and TRAIL had no effect on normal cells. ZFL downregulated Bcl-2 expression at the transcriptional level in a p53-dependent manner, and overexpression of Bcl-2 also markedly blocked apoptosis induced by combined treatment with ZFL and TRAIL. In addition, ZFL induced downregulation of c-FLIP, and overexpression of c-FLIP blocked the apoptosis induced by ZFL plus TRAIL. Moreover, ZFL increased the expression of Cbl, an E3 ligase of c-FLIP, in a p53-dependent manner, and knockdown of Cbl markedly prevented c-FLIP downregulation and the apoptosis induced by ZFL plus TRAIL. Interestingly, ZFL induced p53 expression via production of mitochondrial reactive oxygen species (ROS). We also demonstrated that downregulation of cathepsin S by small interfering RNA sensitized TRAIL-mediated apoptosis in Caki cells. These results reveal the importance of cathepsin S on resistance against TRAIL, and inhibition of cathepsin S activity plays a crucial role in TRAIL-mediated cell death of cancer cells. Our results indicated that inhibition of cathepsin S stimulates TRAIL-induced apoptosis through downregulation of Bcl-2 and Cbl-mediated c-FLIP by ROS-mediated p53 expression. Antioxid. Redox Signal. 27, 215-233.

Mycobacterium bovis BCG Interferes with miR-3619-5p Control of Cathepsin S in the Process of Autophagy.

Main survival mechanism of pathogenic mycobacteria is to escape inimical phagolysosomal environment inside the macrophages. Many efforts have been made to unravel the molecular mechanisms behind this process. However, little is known about the involvement of microRNAs (miRNAs) in the regulation of phagolysosomal biosynthesis and maturation. Based on a bottom up approach, we searched for miRNAs that were involved in phagolysosomal processing events in the course of mycobacterial infection of macrophages. After infecting THP-1 derived macrophages with viable and heat killed Mycobacterium bovis BCG (BCG), early time points were identified after co-localization studies of the phagosomal marker protein LAMP1 and BCG. Differences in LAMP1 localization on the phagosomes of both groups were observed at 30 min and 4 h. After in silico based pre-selection of miRNAs, expression analysis at the identified time points revealed down-regulation of three miRNAs: miR-3619-5p, miR-637, and miR-324-3p. Consequently, most likely targets were predicted that were supposed to be mutually regulated by these three studied miRNAs. The lysosomal cysteine protease Cathepsin S (CTSS) and Rab11 family-interacting protein 4 (RAB11FIP4) were up-regulated and were considered to be connected to lysosomal trafficking and autophagy. Interaction studies verified the regulation of CTSS by miR-3619-5p. Down-regulation of CTSS by ectopic miR-3619-5p as well as its specific knockdown by siRNA affected the process of autophagy in THP-1 derived macrophages.

Hepatic metallothioneins in molecular responses to cobalt, zinc, and their nanoscale polymeric composites in frog Rana ridibunda

Despite numerous studies suggesting a dramatic decline of amphibians, the biochemical mechanisms of adaptation in these animals to polluted environment are poorly studied. The aim of this study was to elucidate the ability to release cobalt (Co) and zinc (Zn) from their nanoscale complexes (NCs) derived from the polymeric substance of N-vinylpyrrolidone (PS) in the liver of amphibian (Rana ridibunda). Frog males were subjected to 14days exposure to waterborne Co2+ (50μg/L), Zn2+ (100μg/L), as well as corresponding concentrations of Co-NC, Zn-NC or PS. Main attention was paid to MT's interrelations with indices of stress and toxicity. Only Co2+ and Zn2+ caused elevation of the correspondent metal in MTs. Co2+ caused down-regulation of cathepsin D activity, while Zn2+, Zn-NC and the PS up-regulated this activity. Zn2+ provoked 1.6 times increase of metal-bounded form of the MT (MT-Me), while all other exposures caused the elevation of the ratio of MT total protein concentration (MT-SH) and concentrations of the MT-Me and/or immunoreactive (MTi) form (up to ~10 times) accompanied by a decrease in the levels of oxyradicals. The increased DNA fragmentation and down-regulation of caspase-3 activity in relation to the redox state of glutathione and/or lactate/pyruvate were shown at all exposures. These data indicate the vulnerability of the redox state of cellular thiols and inability to release Co and Zn from NCs in frog's liver.

Downregulation of cathepsin L suppresses cancer invasion and migration by inhibiting transforming growth factor‑β‑mediated epithelial‑mesenchymal transition.

Cathepsin L, a lysosomal acid cysteine protease, was found to be overexpressed in several types of human carcinomas. However, its functional roles in tumor progression and the underlying mechanisms remain largely unclear. In the present study, we investigated a novel functional aspect of cathepsinL in regulating transforming growthfactor‑β (TGF‑β)‑induced epithelial‑mesenchymal transition (EMT) in A549 and MCF‑7 cells and examined its possible mechanisms. We found that TGF‑β‑induced cell morphologic changes of EMT were associated with the increased protein level of cathepsin L in A549 and MCF‑7 cells, suggesting that cathepsin L may be involved in the regulation of EMT. Furthermore, we showed that silencing of cathepsinL blocked TGF‑β‑induced cell migration, invasion and actin remodeling and inhibited TGF‑β‑mediated EMT. We also demonstrated that the mechanism of how cathepsin L knockdown regulates EMT may be explained by the suppression of EMT‑inducing molecules, such as Snail, which is associated with the phosphatidylinositol 3‑kinase (PI3K)‑AKT and Wnt signaling pathways. Moreover, we proved that cathepsinL knockdown in A549 cells significantly inhibited xenograft tumor growth and EMT invivo. The results showed a new mechanism to determine cathepsinL involvement in the regulation of cancer invasion and migration. These results showed that cathepsin L knockdown is important in regulating EMT and suggest that cathepsin L may be utilized as a new target for enhancing the efficacy of chemotherapeutics against epithelial cancer.