Expression Levels Of Cathepsin Research Articles

Targeting Caspases 3/6 and Cathepsins L/B May Decrease Laminopathy-Induced Apoptosis in Alzheimer’s Disease

Background: Laminopathy is a pathological manifestation observed in Alzheimer’s disease (AD), leading to neuronal apoptosis. Objective: Our objective was to assess inhibitors of enzymes involved in laminopathy. Methods: The mRNA expression of the cathepsins L and B, caspases 3 and 6, lamins b1 and b2, granzymes A and B, and lamins A and C were extracted and analyzed from GSE5281 and GSE28146 datasets. A total of 145 ligands were selected for molecular docking. Subsequently, 10 ns and 100 ns atomistic molecular dynamics (MD) and Martini 3 were performed with NAMD for two selected ligands (PubChem id: 608841 and ChEMBL id: 550872). Results: The mRNA expression level highlighted caspase 6 and lamin A/C upregulation in the hippocampus of the AD samples, in contrast to cathepsin B, lamin b2, and caspase 3. Moreover, there was a strong correlation between the expression level of cathepsin B, lamin A/C, and caspase 6 in the AD group. The MD results suggested molecule with ChEMBL id of 550872 had higher free binding energy, while in longer simulation the molecule with PubChem id of 608841 was suggested to be more stable in complex with the receptor. Conclusions: Our findings suggest that lamins A/C, cathepsins B/L, caspase 6, and lamin B2 are associated with laminopathy as potential factors contributing to apoptosis in AD. We propose that simultaneous inhibition of caspases 6 and cathepsins L may decrease the rate of apoptosis triggered by lamin degradation. Nevertheless, further studies are required to confirm these observations due to the lack of in vivo findings.

The E3 ubiquitin ligase TRIM31 attenuates NLRP3 inflammasome activation in Helicobacter pylori-associated gastritis by regulating ROS and autophagy

BackgroundThe NLRP3 inflammasome activation is the molecular basis of Helicobacter pylori (Hp)-associated gastritis. Tripartite motif (TRIM) 31 is involved in diverse pathological events. However, whether TRIM31 plays a role in the activation of NLRP3 inflammasome in Hp infection is not clarified.MethodsA mouse model of chronic Hp infection was established, and the gastric tissues were subjected to the polymerase chain reaction, western blotting, histopathological analysis, and RNA sequencing. The mitochondrial membrane potential and ROS in the human gastric epithelium GES-1 cells with or without Hp infection were measured by flow cytometry. GES-1 cells with or without TRIM31 knockdown were transfected with mCherry-EGFP-LC3 adenovirus. After rapamycin and bafilomycin A1 stimulation, autophagy flux in the above primed GES-1 cells was assessed by laser confocal microscope. Lysosomal acidification and expression levels of cathepsin B and cathepsin D in GES-1 cells with Hp infection were measured.ResultsNLRP3 inflammasome was activated in the gastric tissues of mice with chronic Hp infection in vivo and the GES-1 cells with Hp infection in vitro. TRIM31 was downregulated in Hp infection. TRIM31 negatively regulated the NLRP3 inflammasome activation. Enhanced ROS, impaired autophagy flux, and decreased expression of lysosomal cathepsin B and cathepsin D were observed in TRIM31-deficient GES-1 cells with Hp infection. In turn, inhibition of ROS led to the decreased expression of NLRP3 inflammasome.ConclusionsTogether, our data identified that TRIM31 negatively regulated the activation of NLRP3 inflammasome in Hp-associated gastritis by affecting ROS and autophagy of gastric epithelial cells.BaPkEPhQjKRgDMqnjfdJ-VVideo abstract

Unravelling the suitability of Branchinecta gaini as a potential biomonitor of contaminants of emerging concern in the Antarctic Peninsula region

AbstractThe occurrence and impact of contaminants of emerging concerns (CECs) have been investigated in Antarctica much less than in other parts of the world. Although legacy anthropogenic pollutants can reach Antarctica via long-range transport, CECs mainly originate from local sources. Here, we investigated the ability of a freshwater crustacean, the Antarctic fairy shrimp Branchinecta gaini, to cope with nanoscale titanium dioxide (n-TiO2), a widely used pigment in consumer products (e.g. paintings), including those for personal care (e.g. sunscreens). An in vivo acute short-term exposure study (9 h, n-TiO2 concentration range 50–200 μg ml-1) was performed and the expression levels of several genes involved in stress response were evaluated. No effect on the expression of heat-shock protein chaperone genes was found, with the exception of Hsp70a, which was significantly upregulated at 200 μg ml-1 n-TiO2. Similarly, cytochrome P450 was upregulated at 100 and 200 μg ml-1 n-TiO2, while the expression levels of cathepsin L and of antioxidant genes such as superoxide dismutase and glutathione peroxidase were significantly reduced with increasing concentrations of n-TiO2. This study shows for the first time the responsiveness and sensitivity of an Antarctic freshwater crustacean to n-TiO2 exposure and supports its suitability as a biomonitor of CECs in Antarctica.

Oncotype DX 21-gene test has a low recurrence score in both pure and mixed mucinous breast carcinoma.

The Oncotype DX 21-gene test can be used to predict chemotherapy efficacy in patients with estrogen receptor (ER)-positive and HER2-negative breast cancer; however, the data on the 21-gene recurrence score (RS) for mucinous breast carcinoma (MBC) are limited. The present study aimed to evaluate the distribution pattern and clinical value of the 21-gene RS in patients with MBC. A total of 38 pure MBC (PMBC) and 11 mixed MBC (MMBC) cases were retrospectively analyzed, and a total of 29 ER-positive and HER2-negative MBCs underwent the Oncotype DX 21-gene test. There were no statistically significant differences between the PMBCs and MMBCs in age, tumor size and molecular subtype; however, patients with MMBC showed a significantly higher incidence rate of nodal metastases compared with that in patients with PMBC (72.7 vs. 16.2%, respectively). Following surgery, 87.8 and 59.2% of the enrolled patients received endocrine therapy and chemotherapy, respectively. With a median follow-up of 65.6 months, the 5-year disease-free survival and overall survival rates were 97.0 and 100.0%, respectively. The 21-gene test revealed that the proportions of patients with MBC categorized into low (RS <18), intermediate (RS ≥18-30) and high (RS ≥30) risk groups were 51.7, 44.8 and 3.5%, respectively, and there was no statistically significant difference between the PMBC and MMBC cases. Notably, among the genes in the 21-gene RS testing, the expression levels of cathepsin V, progesterone receptor (PR) and CD68 were significantly higher in the PMBC group compared with that in the MMBC group. In conclusion, the current study demonstrated that patients with MBC had a favorable prognosis, and both PMBC and MMBC cases had a low- and intermediate-risk RS, which suggests that a considerable proportion of patients may be able to avoid chemotherapy. In addition, the high expression level of PR, based on the 21-gene test in PMBCs, indicated that they may have a more favorable response to endocrine therapy than MMBCs.

Synergistic inhibition effects of tea polyphenols as adjuvant of oxytetracycline on Vibrio parahaemolyticus and enhancement of Vibriosis resistance of Exopalaemon carinicauda

Antibiotics worldwide were abused to kill all kinds of pathogens in shrimp industry, which resulted in the emergence of many drug-resistant strains, such as Vibrio parahaemolyticus. Natural plant extracts (NPEs) as adjuvant of antibiotics offer an effective alternative to minimize the problems. We evaluated the antimicrobial activities of different combinations of eight NPEs and eight antibiotics allowed to be used in aquaculture and found tea polyphenols (TP) was the optimum adjuvant of oxytetracycline against V. parahaemolyticus. The smallest FIC index of 0.14 was obtained with the combination of 1/64 MIC TP and 1/8 MIC oxytetracycline. The combination of TP and oxytetracycline could improve the survival rate to 53.3 ± 2.74% of Exopalaemon carinicauda in vitro, higher than that in the TP or oxytetracycline group significantly. The activities of digestive enzymes (pepsin and amylase) and immune enzyme including lysozyme, alkaline phosphatase (AKP), the expression levels of cathepsin D (CatD) and Toll receptor (Toll) of E. carinicauda were up-regulated significantly. The changes of immune parameters thereby promoted the growth and improved the immunity of E. carinicauda, which led to the increase in resistance against V. parahaemolyticus. The results showed that the synergistic inhibition mechanisms of TP and antibiotics may be involved in the immune defence process and the increased abilities of digestion. The combination could reduce the use of oxytetracycline when the better therapeutic effect was achieved.

Effects of Sudden Drop in Salinity on Osmotic Pressure Regulation and Antioxidant Defense Mechanism of Scapharca subcrenata.

Salinity is an important ecological factor that impacts the growth and survival of aquatic organisms. The salinity of seawater in coastal and estuarine areas is often subject to dynamic changes because of seasonal rainfall and continental runoff. Thus, the current study investigated the effects of sudden changes in salinity on the survival rate and osmotic pressure regulation mechanisms of bottom-sowing seedlings of the economically important ark shell, Scapharca subcrenata. By simulating the sudden changes that occur in seawater salinity after rainstorms, the results showed that the osmotic pressure of the hemolymph and Na+, K+, Ca2+, and Cl– concentrations first decreased and then increased. When the salinity decreased from 30 to 14‰, hemoglobin, soluble total protein, taurine, and total free amino acid gradually increased; maximum levels of hemoglobin, soluble total protein, and taurine occurred once the salinity increased to 22‰ at 96 h. After 96 h, the total free amino acid content increased until 144 h. The reactive oxygen species (ROS) content and total antioxidant capacity (T-AOC) peaked at 96 h, whereas the expression levels of Mn-superoxide dismutase (MnSOD) and catalase (CAT) increased earlier, indicating that, with continuous ROS generation, antioxidant defense mechanisms were activated to avoid oxidative damage. Expression levels of cathepsin C (CTSC), cathepsin D (CTSD), heat shock protein 20 (HSP20), and heat shock protein 70 (HSP70) were significantly higher than in the control group at 48 h (salinity level 14‰); the expression levels of HSP20, heat shock protein 90 (HSP90), MnSOD, and glutathione peroxidase (GPx) remained high, indicating that they were still required for osmotic pressure regulation to maintain the dynamic balance between the generation and removal of ROS as the salinity level increased. These results not only add to our basic understanding of the aquatic ecology of S. subcrenata, but also provide a theoretical ground for improving the survival rate of bottom-sowing, propagation, and release of S. subcrenata seedlings.

Distinctive alteration in the expression of autophagy genes in Drosophila models of amyloidopathy and tauopathy

Background Alzheimer’s disease (AD) is one the most common types of dementia. Plaques of amyloid beta and neurofibrillary tangles of tau are two major hallmarks of AD. Metabolism of these two proteins, in part, depends on autophagy pathways. Autophagy dysfunction and protein aggregation in AD may be involved in a vicious circle. The aim of this study was to investigate whether tau or amyloid beta 42 (Aβ42) could affect expression of autophagy genes, and whether they exert their effects in the same way or not. Methods Expression levels of some autophagy genes, Hook, Atg6, Atg8, and Cathepsin D, were measured using quantitative PCR in transgenic Drosophila melanogaster expressing either Aβ42 or Tau R406W. Results We found that Hook mRNA levels were downregulated in Aβ42-expressing flies both 5 and 25 days old, while they were increased in 25-day-old flies expressing Tau R406W. Both Atg6 and Atg8 were upregulated at day 5 and then downregulated in 25-day-old flies expressing either Aβ42 or Tau R406W. Cathepsin D expression levels were significantly increased in 5-day-old flies expressing Tau R406W, while there was no significant change in the expression levels of this gene in 5-day-old flies expressing Aβ42. Expression levels of Cathepsin D were significantly decreased in 25-day-old transgenic flies expressing Tau R406W or Aβ42. Conclusion We conclude that both Aβ42 and Tau R406W may affect autophagy through dysregulation of autophagy genes. Interestingly, it seems that these pathological proteins exert their toxic effects on autophagy through different pathways and independently.

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

Cathepsin S regulates renal fibrosis in mouse models of mild and severe hydronephrosis.

As a member of the cysteine protease family, cathepsin S (CTSS) serves an important role in diseases such as cancer, arthritis and atherosclerosis. Nevertheless, its role in renal fibrosis is unknown. In the present study, the effects of CTSS on renal fibrosis in mild (group M) and severe (group S) hydronephrosis were studied by reverse transcription-quantitative PCR (RT-qPCR), western blot analysis (WB), Masson's trichrome staining and immunohistochemical staining in mouse models. The effects of CTSS on extracellular matrix (ECM) deposition and epithelial-mesenchymal transition (EMT) and the potential mechanisms were further studied by RT-qPCR and WB in transforming growth factor (TGF-β1)-stimulated TCMK-1 cells. Compared with group N (no hydronephrosis), the expression levels of CTSS in the M and S groups were significantly higher, and a significant increase in ECM deposition was observed in the S group. In addition, compared with group N, the expression levels of TGF-β1, α-smooth muscle actin (α-SMA), SMAD2, SMAD3, phosphorylated (p)SMAD2 and pSMAD3 in groups M and S were significantly higher, whereas the expression of E-cadherin was significantly lower. Inhibition of CTSS expression increased the expression levels of TGF-β1, α-SMA, fibronectin, collagen-I, SMAD2, SMAD3, pSMAD2 and pSMAD3, whereas E-cadherin expression decreased. A significant increase in CTSS was observed in the TGF-β1-stimulated TCMK-1 cell line. ECM deposition and EMT were also intensified. The opposite outcomes occurred after intervention with small interfering RNA targeting CTSS. In conclusion, CTSS affected EMT and the deposition of ECM. CTSS may mediate the regulation of fibrosis by the TGF-β/SMAD signaling pathway. CTSS may serve an important role in the treatment of renal fibrosis.

Transcriptomic and Quantitative Proteomic Analyses Provide Insights Into the Phagocytic Killing of Hemocytes in the Oyster Crassostrea gigas

As invertebrates lack an adaptive immune system, they depend to a large extent on their innate immune system to recognize and clear invading pathogens. Although phagocytes play pivotal roles in invertebrate innate immunity, the molecular mechanisms underlying this killing remain unclear. Cells of this type from the Pacific oyster Crassostrea gigas were classified efficiently in this study via fluorescence-activated cell sorting (FACS) based on their phagocytosis of FITC-labeled latex beads. Transcriptomic and quantitative proteomic analyses revealed a series of differentially expressed genes (DEGs) and proteins present in phagocytes; of the 352 significantly high expressed proteins identified here within the phagocyte proteome, 262 corresponding genes were similarly high expressed in the transcriptome, while 140 of 205 significantly low expressed proteins within the proteome were transcriptionally low expressed. A pathway crosstalk network analysis of these significantly high expressed proteins revealed that phagocytes were highly activated in a number of antimicrobial-related biological processes, including oxidation–reduction and lysosomal proteolysis processes. A number of DEGs, including oxidase, lysosomal protease, and immune receptors, were also validated in this study using quantitative PCR, while seven lysosomal cysteine proteases, referred to as cathepsin Ls, were significantly high expressed in phagocytes. Results show that the expression level of cathepsin L protein in phagocytes [mean fluorescence intensity (MFI): 327 ± 51] was significantly higher (p < 0.01) than that in non-phagocytic hemocytes (MFI: 83 ± 26), while the cathepsin L protein was colocalized with the phagocytosed Vibrio splendidus in oyster hemocytes during this process. The results of this study collectively suggest that oyster phagocytes possess both potent oxidative killing and microbial disintegration capacities; these findings provide important insights into hemocyte phagocytic killing as a component of C. gigas innate immunity.

EUS-FNA cytological material from pancreatic lesions: the expression of cathepsins and its predictive value of malignancy.

To assess the expression of cathepsins in pancreatic samples obtained by endoscopic ultrasonography and fine needle aspiration (EUS-FNA) and to investigate their relationship with the staging of the pancreatic ductal adenocarcinoma (PDAC). We prospectively included patients with solid pancreatic masses, in which EUS-FNA were performed. Cathepsins B, L, S and H expression was determined in FNA samples. Seventeen FNA were performed. All cytological material was from PDAC. Expression of cathepsins was predominantly low (B 65%, L 23%, S 76%, and H 41%). We found no correlation between the expression levels and the extension of the neoplasm. Expression of cathepsins in the cytological material of PDAC is diverse but still poor to be useful in the pre-operative diagnosis. There is no correlation between the expression levels of cathepsins and the extension of the PDAC.

Cathepsin K modulates invasion, migration and adhesion of oral squamous cell carcinomas invitro.

Cathepsin K was initially discovered as an osteoclast-specific cysteine proteinase, but the enzyme is also expressed in various cancers including oral squamous cell carcinomas. This study aimed to clarify the function of cathepsin K in oral squamous cell carcinomas. Expression levels of cathepsin K were examined in six types of cell carcinomas. Carcinomas overexpressing cathepsin K were constructed. Effects of cathepsin K overexpression and treatment with odanacatib, a specific cathepsin K inhibitor, on cell invasion, migration and adhesion were analysed. Different levels of cathepsin K were expressed in carcinomas. Cathepsin K was predominantly localised in lysosomes. Cathepsin K overexpression impaired the proliferation of carcinomas. Invasion analysis showed that cathepsin K overexpression enhanced invasion and migration of carcinomas, whereas inhibition of cathepsin K by odanacatib caused the opposite effects in carcinomas. Cathepsin K overexpression also increased cell adhesion and slightly increased surface expression of the adhesion receptor CD29/integrin β1 . The enhanced invasion of carcinomas resulting from cathepsin K overexpression is probably due to the increased cell migration and adhesion. Thus, cathepsin K is implicated not only in protein degradation but also in invasion, migration and adhesion of oral squamous cell carcinomas.

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.

Cyr61 and YB-1 are novel interacting partners of uPAR and elevate the malignancy of triple-negative breast cancer.

The triple-negative breast cancer (TNBC) is a very aggressive tumor type often occurring in young women and is associated with a bad prognosis for the patients. TNBC lacks established targets for breast cancer therapy, such as the estrogen receptor (ER), progesterone receptor (PR) and the human epidermal growth factor receptor 2 (HER2). Therefore, novel therapeutic targets and strategies are needed for an improved treatment of this breast cancer subtype. TNBC and respective cell lines often overexpress proteins of the urokinase plasminogen activator system (uPAS) including uPA, its receptor uPAR and inhibitor PAI-1, which together with co-factors contribute to the malignancy of TNBC. Here, two novel interacting partners of uPAR, the cysteine-rich angiogenic inducer 61 (Cyr61) and the Y-box-binding protein 1 (YB-1) were identified and their differential expression demonstrated in TNBC cells as well as in tumors. In the TNBC cohort, both interactors significantly correlated with expression levels of cathepsin B, c-Met and the tumor grade. In addition, expression levels of Cyr61 significantly correlated with cathepsin D (p=0.03), insulin receptor (p≤0.001), insulin-like growth factor receptor 1 (IGF1R, p=0.015) and also with YB-1 (p=0.0004) levels. The interactions of uPAR with Cyr61 significantly correlated with expression levels of tumor-promoting biomarkers including plasminogen (p=0.0014), cathepsin B (p=0.032), c-Met (p=0.0192) as well as with the tumor grade (p=0.02). In multivariate survival analysis, YB-1 showed independent prognostic value (p=0.01). As the novel interacting partners, also together with uPAR, contribute to tumor progression and metastasis, both may be potential therapeutic targets in breast cancer.

Comparisons of orthodontic root resorption under heavy and jiggling reciprocating forces during experimental tooth movement in a rat model.

ObjectiveRoot mobility due to reciprocating movement of the tooth (jiggling) may exacerbate orthodontic root resorption (ORR). "Jiggling" describes mesiodistal or buccolingual movement of the roots of the teeth during orthodontic treatment. In the present study, buccolingual movement is described as "jiggling." We aimed to investigate the relationship between ORR and jiggling and to test for positive cell expression in odontoclasts in resorbed roots during experimental tooth movement (jiggling) in vivo.MethodsMale Wistar rats were divided into control, heavy force (HF), optimal force (OF), and jiggling force (JF) groups. The expression levels of cathepsin K, matrix metalloproteinase (MMP)-9 protein, interleukin (IL)-6, cytokine-induced neutrophil chemoattractant 1 (CINC-1; an IL-8-related protein in rodents), receptor activator of nuclear factor κB ligand (RANKL), and osteoprotegerin protein in the dental root were determined using immunohistochemistry.ResultsOn day 21, a greater number of root resorption lacunae, which contained multinucleated odontoclasts, were observed in the palatal roots of rats in the JF group than in rats from other groups. Furthermore, there was a significant increase in the numbers of cathepsin K-positive and MMP-9-positive odontoclasts in the JF group on day 21. Immunoreactivities for IL-6, CINC-1, and RANKL were stronger in resorbed roots exposed to jiggling than in the other groups on day 21. Negative reactivity was observed in the controls.ConclusionsThese results suggest that jiggling may induce ORR via inflammatory cytokine production during orthodontic tooth movement, and that jiggling may be a risk factor for ORR.

Expression profile of cathepsins indicates the potential of cathepsins B and D as prognostic factors in breast cancer patients.

Breast cancer is one of the most prevalent types of cancer in women and contributes to 32% of all female cancer cases. Cathepsins, a family of proteins, are known to have a critical role in human cancers. However, previous studies on the systematic analysis of the role of cathepsin family members in breast cancer are limited. The aim of the present study was to identify biological markers to predict prognosis and treatment response of breast cancer patients, as well as to elucidate novel therapeutic targets. The present study analyzed the expression of six members of cathepsin family, including cathepsins B, G, D, K, L and V in 188 breast cancer tissue specimens using immunohistochemistry. The data showed that all members of the tested cathepsin families featured cytoplasmic staining. Notably, expression of cathepsin L was associated with advanced tumor stages, while cathepsins B and K expression levels were associated with positive estrogen receptor expression; in addition, cathepsin K expression was also demonstrated to be associated with progesterone receptor expression. Cathepsins V and D expression levels were found to be associated with breast cancer metastasis, while the expression levels of cathepsins B and D were associated with poor disease-free survival in breast cancer patients. In addition, univariate analysis demonstrated that breast cancer metastasis to the bone and the expression of cathepsin B protein were associated with poor disease-free survival. In conclusion, the results of the present study indicated that the altered expression of cathepsins, in particular cathepsins B and D, contributed to the progression of breast cancer and poor disease-free survival in breast cancer patients.

Lack of vitamin D receptor causes stress-induced premature senescence in vascular smooth muscle cells through enhanced local angiotensin-II signals

ObjectivesThe inhibition of the renal renin-angiotensin system by the active form of vitamin D contributes to the cardiovascular health benefits of a normal vitamin D status. Local production of angiotensin-II in the vascular wall is a potent mediator of oxidative stress, prompting premature senescence. Herein, our objective was to examine the impact of defective vitamin D signalling on local angiotensin-II levels and arterial health. MethodsPrimary cultures of aortic vascular smooth muscle cells (VSMC) from wild-type and vitamin D receptor-knockout (VDRKO) mice were used for the assessment of cell growth, angiotensin-II and superoxide anion production and expression levels of cathepsin D, angiotensin-II type 1 receptor and p57Kip2. The in vitro findings were confirmed histologically in aortas from wild-type and VDRKO mice. ResultsVSMC from VDRKO mice produced more angiotensin-II in culture, and elicited higher levels of cathepsin D, an enzyme with renin-like activity, and angiotensin-II type 1 receptor, than wild-type mice. Accordingly, VDRKO VSMC showed higher intracellular superoxide anion production, which could be suppressed by cathepsin D, angiotensin-II type 1 receptor or NADPH oxidase antagonists. VDRKO cells presented higher levels of p57Kip2, impaired proliferation and premature senescence, all of them blunted upon inhibition of angiotensin-II signalling. In vivo studies confirmed higher levels of cathepsin D, angiotensin-II type 1 receptor and p57Kip2 in aortas from VDRKO mice. ConclusionThe beneficial effects of active vitamin D in vascular health could be a result of the attenuation of local production of angiotensin-II and downstream free radicals, thus preventing the premature senescence of VSMC.

Inhibitory effect of cathepsin B-RNAi-lentivirus on mouse retinal neovascularization

Background Retinal neovascularization disease is a group of threatening-vision eye diseases.Researches showed that cathepsin B is involved in angiogenesis.Exploring a drug which inhibit retinal blood vessels will provide the basis for the molecular mechanism of these diseases.Objective This study was to investigate the inhibitory role of cathepsin B-RNAi-lentivirus on retinal angiogenesis.Methods Sixty 7-day-old C57BL/6J mice were raised together with maternal mice in the closed box with the oxygen concentration of （75-2）% for 5 days to establish the retinal angiogenesis mouse models.The mice were then taken into the normal air environment for continuous raise and were randomized into 3 groups.NC-GFP-LV of 1 μl and the equal volume of cathepsin B-RNAi-lentivirus was intravitreously injected respectively in 40 eyes in the control group and the gene treatment group,and no drug was administered in the 40 eyes of the model group.The mice were sacrificed and retinas were obtained.Expression of cathepsin B protein in the retina was detected by Western blot assay （cathepsin B/β-actin）.Real-time PCR was used to detect and compare the expression level of cathepsin B mRNA （2△△Ct）.FITC-dextran was used to perform heart infusion for the retinal stretched preparation 5 days after intravitreously injection.Retinal neovascularization was examined by fluorescent angiography.Results The expression level （2-△△Ct） of cathepsin B mRNA was 0.74 ±0.12 in the gene treatment group,showing a significant decline in comparison with 1.66±0.17 and 1.58±0.29 in the model group and control group （q--0.746,1.588,P〈 0.01）.The expression level of cathepsin B protein （cathepsin B/β-actin） in the retina was 0.64±0.06,0.93±0.09 and 0.96±0.09 respectively in the gene treatment group,model group and control group,indicating a significant reduce in the gene treatment group （q =0.637,0.894,P〈0.01）.Distorted vessels were seen in the mice retinas of the model group with more branches and vascular anastomosis,and fluorescine leakage was exhibited under the fluorescence microscope.However,the vessels were regular with less branches and angiogenesis.Conclusions Cathepsin B-RNAi-lentivirus can effectively inhibit oxygen-induced retinal angiogenesis in mouse. Key words: Lentivirus; Cathepsin B; Angiogenesis

Assessment of the Roles of Cathepsins B, H and L in the Progression of Colorectal Cancer

Cysteine cathepsins are important regulators and signaling molecules of an unimaginable number of biological processes, while they concurrently play an essential role in cancer progression, invasion and metastasis. The purposes of our study were to: a) compare the expression levels of cathepsins B, H and L in the supernatants of colon cancer tissues from 74 patients versus the corresponding enzymic expressions of supernatants in the adjacent normal colorectal tissues; and b) correlate our results to the grade of the malignancy by using an enzyme-linked immunosorbent assay (ELISA). The findings indicated that cathepsins B, H and L of all malignant tissues exhibited significantly higher expression levels than their corresponding controls. Furthermore, cathepsin B expression levels doubled in all tumor samples and this increase remained quite steady with tumor stage advancement, in contrast to cathepsin H expression which rose significantly as malignancy progressed. Specifically, cathepsin H concentration was higher than the corresponding control: 155% in B1 stage and 204.44% in D stage. Among the three investigated proteases, cathepsin L has shown the highest increase, which in D stage stood 261.03% higher than the corresponding control. The results at hand suggested that cysteine protease H and L expression levels could be of critical value in the diagnosis and progression of colon cancer.

Screen and validation of differentially expressing genes related to silicotic pulmonary fibrosis in rats

To screen the differentially expressing genes between silicotic lung tissue and normal lung tissue, to identify the differentially expressing genes of matrix metalloproteinase-12 (MMP-12) and Cathepsin E and to explore the roles of those genes in silicosis development. Thirty male SD rats were divided randomly into two groups: control group (6 rats) and exposure group (24 rats) which was exposed to SiO2 by intra-tracheal perfusion. On the 30 th, 60 th and 90 th days after exposure, 8 rats in model group and 2 rats in control group were executed and the lung tissues were obtained. The morphologic changes of lung tissues were observed with HE staining and VG staining under a light microscope. The gene microarrays were used to identify differentially expressing genes of lung tissues in rats exposed to SiO2 for 60 days. Two significantly up-regulated genes, MMP-12 and Cathepsin E, were validated using RT-PCR, immunohistochemistry and Western Blot assay. A total of 338 differentially expressing genes were identified from the 26 962 genes between silicotic rats and normal rats, including 267 up-regulated genes and 71 down-regulated genes. The results of RT-PCR showed that in the lung tissues of exposure group on the 30 th, 60 th and 90 th days, the mRNA expression levels of MMP-12 were 4.306, 5.338, 6.713 times higher than those in the control group, the mRNA expression levels of Cathepsin E were 1.434, 2.974, 3.889 times higher than those in the control group, respectively. The results of immunohistochemical showed that in the lung tissues of exposure group on the 30th, 60th and 90th days, the mRNA expression levels of MMP-12 were 1.435, 1.746, 2.069 times higher than those in the control group, the mRNA expression levels of Cathepsin E were 1.372, 1.663, 2.103 times higher than those in the control group, respectively. The results of immunohistochemical showed that in the lung tissues of exposure group on the 30th, 60th and 90th days, the expression levels of MMP-12 protein were 1.214, 1.531, 1.959 times higher than those in the control group, the expression levels of Cathepsin E protein were 1.262, 1.828, 1.907 times higher than those in the control group, respectively. Compared with the control group, the mRNA and protein expression levels of MMP-12 and Cathepsin E in lung tissues of exposure group were significantly up-regulated (P < 0.05). The differentially expressing genes in rat lung tissues screened by gene chip were validated, which suggested that a complex gene regulatory network may be contributed to occurrence of silicosis. MMP-12 and Cathepsin E genes may be involved in the development of silicotic pulmonary fibrosis by degrading the basement membrane of alveolar wall and participating in the immune response.