New multienzymatic complex formed between human cathepsin D and snake venom phospholipase A2

Background Cathepsin D (CatD) is a lysosomal proteolytic enzyme expressed in almost all tissues and organs. This protease is a multifunctional enzyme responsible for essential biological processes such as cell cycle regulation, differentiation, migration, tissue remodeling, neuronal growth, ovulation, and apoptosis. The overexpression and hypersecretion of CatD have been correlated with cancer aggressiveness and tumor progression, stimulating cancer cell proliferation, fibroblast growth, and angiogenesis. In addition, some studies report its participation in neurodegenerative diseases and inflammatory processes. In this regard, the search for new inhibitors from natural products could be an alternative against the harmful effects of this enzyme. Methods An investigation was carried out to analyze CatD interaction with snake venom toxins in an attempt to find inhibitory molecules. Interestingly, human CatD shows the ability to bind strongly to snake venom phospholipases A2 (svPLA2), forming a stable muti-enzymatic complex that maintains the catalytic activity of both CatD and PLA2. In addition, this complex remains active even under exposure to the specific inhibitor pepstatin A. Furthermore, the complex formation between CatD and svPLA2 was evidenced by surface plasmon resonance (SPR), two-dimensional electrophoresis, enzymatic assays, and extensive molecular docking and dynamics techniques. Conclusion The present study suggests the versatility of human CatD and svPLA2, showing that these enzymes can form a fully functional new enzymatic complex.

Cathepsin D knockdown regulates biological behaviors of granulosa cells and affects litter size traits in goats / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Cathepsin D (CTSD), the major lysosomal aspartic protease that is widely expressed in different tissues, potentially regulates the biological behaviors of various cells. Follicular granulosa cells are responsive to the increase of ovulation number, hence indirectly influencing litter size. However, the mechanism underlying the effect of CTSD on the behaviors of goat granulosa cells has not been fully elucidated. This study used immunohistochemistry to analyze CTSD localization in goat ovarian tissues. Moreover, western blotting was applied to examine the differential expression of CTSD in the ovarian tissues of monotocous and polytocous goats. Subsequently, the effects of CTSD knockdown on cell proliferation, apoptosis, cell cycle, and the expression of candidate genes of the prolific traits, including bone morphogenetic protein receptor IB (

Lysosomal Hydrolase Cathepsin D Non-proteolytically Modulates Dendritic Morphology in Drosophila / 神经科学通报·英文版

The main lysosomal protease cathepsin D (cathD) is essential for maintaining tissue homeostasis via its degradative function, and its loss leads to ceroid accumulation in the mammalian nervous system, which results in progressive neurodegeneration. Increasing evidence implies non-proteolytic roles of cathD in regulating various biological processes such as apoptosis, cell proliferation, and migration. Along these lines, we here showed that cathD is required for modulating dendritic architecture in the nervous system independent of its traditional degradative function. Upon cathD depletion, class I and class III arborization (da) neurons in Drosophila larvae exhibited aberrant dendritic morphology, including over-branching, aberrant turning, and elongation defects. Re-introduction of wild-type cathD or its proteolytically-inactive mutant dramatically abolished these morphological defects. Moreover, cathD knockdown also led to dendritic defects in the adult mushroom bodies, suggesting that cathD-mediated processes are required in both the peripheral and central nervous systems. Taken together, our results demonstrate a critical role of cathD in shaping dendritic architecture independent of its proteolytic function.

Pepstatin Pr show anti-fibrosis effect related to YAP-TGFβ-Smad pathway / 药学学报

Liver fibrosis is a tissue repair compensatory response to liver injury caused by various chronic factors, ultimately leading to liver cirrhosis, liver failure and even hepatocellular carcinoma. Abnormal activation of hepatic stellate cells is the cellular basis of liver fibrosis development. Pepstatin Pr, the derivative of pepstatin A, was isolated from Streptomyces sp. CPCC 202950. Our purpose was to investigate the anti-fibrotic activity of pepstatin Pr and explore its molecular mechanism. Hepatic stellate cell LX-2 was stimulated by TGFβ1 and sub- sequently treated with pepstatin Pr. Its cytotoxicity was detected by sulforhodamine B (SRB) assay. The expression of COL1A1, α-SMA and cathepsin D, signaling proteins TGFβ, Smad and YAP/TAZ were detected by Western blot or real-time PCR. The results showed that pepstatin Pr was not cytotoxic to LX-2 cells. And pepstatin Pr significantly reduced the mRNA and protein expression of COL1A1 and α-SMA, which are important liver fibrosis markers. Pepstatin Pr also repressed the protein expression level of cathepsin D, TGFβ1, YAP/TAZ, the phospholation level of Smad2, and YAP nuclear translocation. In conclusion, pepstatin Pr exhibits anti-fibrotic effects in TGFβ1-stimulaed LX-2 cells by mediating YAP-TGFβ-Smad pathway.

Partial Characterization of Two Cathepsin D Family Aspartic Peptidases of Clonorchis sinensis

Cathepsin D (CatD, EC 3.4.23.5) is a member belonging to the subfamily of aspartic endopeptidases, which are classified into the MEROPS clan AA, family A1. Helminth parasites express a large set of different peptidases that play pivotal roles in parasite biology and pathophysiology. However, CatD is less well known than the other classes of peptidases in terms of biochemical properties and biological functions. In this study, we identified 2 novel CatDs (CsCatD1 and CsCatD2) of Clonorchis sinensis and partially characterized their properties. Both CsCatDs represent typical enzymes sharing amino acid residues and motifs that are tightly conserved in the CatD superfamily of proteins. Both CsCatDs showed similar patterns of expression in different developmental stages of C. sinensis, but CsCatD2 was also expressed in metacercariae. CsCatD2 was mainly expressed in the intestines and eggs of C. sinensis. Sera obtained from rats experimentally infected with C. sinensis reacted with recombinant CsCatD2 beginning 2 weeks after infection and the antibody titers were gradually increased by maturation of the parasite. Structural analysis of CsCatD2 revealed a bilobed enzyme structure consisting of 2 antiparallel β-sheet domains packed against each other forming a homodimeric structure. These results suggested a plausible biological role of CsCatD2 in the nutrition and reproduction of parasite and its potential utility as a serodiagnostic antigen in clonorchiasis.

The role of lysosomes in manganese-induced toxicity in SK-N-SH cells / 中华劳动卫生职业病杂志

Objective@#To investigate the role of lysosomes in manganese-induced toxicity in human neuroblastoma SK-N-SH cells.@*Methods@#SK-N-SH cells were treated with MnCl2 at doses of 0.062 5, 0.125, 0.25, 0.5, 1.0, 2.0 and 4.0 mmol/L for 24 h, and the cell viability was detected by MTT assay. Cells were treated with MnCl2 at doses of 0.125, 0.25, 0.5 and 1.0mmol/L for 24 h, and lysosomes labeled with lysotracker red were observed by laser confocal microscopy, the expression levels of LAMP1 and CTSD were detected by western blot, and CTSD activity was detected by Cathepsin D Activity Fluorometric Assay Kit.@*Results@#Compared with the control group, the survival rates of SK-N-SH cells were decreased significantly in the 0.5-4.0 mmol/L MnCl2 treatment groups (P<0.01) , the relative fluorescence intensities of 0.5 and 1.0 mmol/L MnCl2 treatment groups were increased (P<0.01) . Compared with the control group, the 0.125-0.5 mmol/L MnCl2 treatment groups had significant increase in the the expression of LAMP1 (P<0.01) . Compared with the control group, the expression of m-CTSD was significantly increased at the does of 0.125-0.25 mmol/L MnCl2, while it was decreased at the does of 1.0 mmol/L (P<0.01) . Otherwise, it wasn’t observed significant difference of the activity of CTSD between different MnCl2 treatment groups.@*Conclusion@#MnCl2 could cause cytotoxicity in SK-N-SH cells. Lysosomes may play a normal function at low doses of manganese, but they may be damaged at high doses of manganese. As an organelle that can degradate substrates in autophagy, lysosomes participate in the neurotoxic mechanism of manganese.

Evaluation of serum cathepsin D concentrations in coronary artery disease

Background Coronary artery disease (CAD) cannot be sufficiently explained by the presence of traditional risk factors. Cathepsin D has been proposed to serve as a surrogate marker of atherosclerosis but its alterations in CAD patients have not been studied. Objective To evaluate serum cathepsin D concentrations in relation to the presence and severity of CAD. Materials and methods A total of 104 subjects were recruited; 71 patients with suspected CAD and 33 healthy subjects. Thirty-four patients had >50% coronary stenosis of at least one artery (CAD+); the remaining 37 patients had <50% stenosis (CAD−) based on angiography. CAD+ patients were sub-divided into three sub-groups with single (SVD; n = 15), double (2VD; n = 9), and triple vessel (3VD; n = 10) disease. Serum soluble cathepsin D concentrations were determined using an enzyme-linked immunosorbent assay (ELISA). Results Serum cathepsin D concentrations were significantly higher in the CAD+ compared with healthy control (p = 0.016) but not CAD− group (p = 0.098). Within the CAD+ group, patients with 3VD had significantly higher serum cathepsin D concentrations compared with the SVD group (p = 0.025), and also compared with the CAD− (p = 0.011) and SVD (p = 0.001) groups. No significant associations were found between serum cathepsin D concentrations and potential confounders including age, sex, blood pressure, smoking history and dyslipidemia. Conclusion Serum cathepsin D concentrations may be associated with the presence of CAD.

Ganglioneuroblastoma - A Case Report

Neuroblastomas are among the most important tumors of extra cranial origin in pediatric patients. They arise from emyonal cells involved in the development of the sympathetic nervous system, whose differentiation has been arrested [1, 2]. This article describes an atypical presentation of ganglioneuroblastoma in a 52 year old male patient. X– Ray left hand in AP and Lateral views showed it as a soft tissue swelling. Excision was done under digital block, and excised specimen was sent to department of pathology for histo pathological examination. HPE showed it as gangioneuroblastoma and pathologists advised to get immunohistochemistry for cathepsin D to be done.

Expression of EGFR, HSP90A and Cathepsin D in nasopharyngeal carcinoma and their relationship with tumor invasion and metastasis / 重庆医学

Objective To investigate the expressions of epidermal growth factor receptor (EGFR),heat shock protein 90A (HSP90A) and cathepsin D in nasopharyngeal carcinoma and their relationship with tumor invasion and metastasis.Methods From January 2014 to December 2015,58 patients with nasopharyngeal carcinoma (group A) in the First Affiliated Hospital of University of South China were selected,and the positive expression rates of EGFR,HSP90A and cathepsin D in biopsy specimens of nasopharyngeal carcinoma were detected by immunohistochemistry method.The positive expression rates of EGFR,HSP90A and cathepsin D in biopsy chronic rhinitis tissues of 30 patients with chronic rhinitis (group B) were also detected.T staging,N staging and lymphatic vessel density (LVD) of patients in group A were analyzed.In group A,the T staging,N staging and LVD of between patients with positive expression of EGFR,HSP90A and eathepsin D and those with negative expression of these indicators were compared,and the correlations of the expression of EGFR,HSP90A and cathepsin D to T staging,N staging and LVD were analyzed.Results The positive expression rates of EGFR,HSP90A and cathepsin D in group A were 86.21％,82.76％ and 87.93％ respectively,which were higher than 30.00％,26.67％ and 33.33％ in group B (P＜0.05).In group A,the percentages of patients on T3-T4 and N2-N3 stages and LVD of patients with positive expression of EGFR,HSP90A and cathepsin D protein respectively were higher than those of patients with negative expression of corresponding protein (P ＜ 0.05).Spearman correlation analysis showed that the positive expression rates of EGFR,HSP90A and cathepsin D in nasopharyngeal carcinoma were positively correlated with the T staging,N staging and LVD (EGFR:r=0.844,0.795,0.785;HSP90A:r=0.862,0.825,0.822;eathepsin D:r=0.842,0.815,0.863,P＜0.05).Conclusion EGFR,HSP90A and cathepsin D expression in nasopharyngeal carcinoma are closely related to invasion and metastasis of tumor,which may be used as reference indexes for the evaluation of tumor invasion and metastasis in nasopharyngeal carcinoma.

Regulatory role of cathepsin D in degradation of advanced glycation end products by human dermal fibroblasts / 中华皮肤科杂志

Objective To investigate the regulatory role of cathepsin D (CatD) in the degradation of intracellular advanced glycation end products (AGEs) endocytosed by human dermal fibroblasts (HDFs).Methods Cultured HDFs were treated with 1 μnol/L CA074Me (an inhibitor of CatB and CatL),75 μmol/L pepstatin A (an inhibitor of CatD) and 1 μmol/L MG-132 (an inhibitor of20S proteasome) separately for 4 hours,and then cell counting kit 8 (CCKS) assay and fluorometric assay were performed to determine the cellular viability and protease activity,respectively.The cells in the CA074Me group,pepstatin A group and MG-132 group were additionally treated with AGE-bovine serum albumin (BSA) for 8 hours,and the cells in the blank control group were treated with phosphate-buffered saline (PBS) alone.After 8-hour cultivation,the cells in the above groups were subsequently reincubated with fresh culture medium containing the corresponding inhibitors for 24 hours.Then,flow cytometry was performed to assess the mean fluorescence intensity of intracellular AGE-BSA at different time points.Some other HDFs were treated with 37.5,75 and 150 μmol/L pepstatin A and PBS separately for 4 hours,and then the cells in the 4 groups were treated with 200 mg/L AGE-BSA for 8 hours,followed by the removal of AGE-BSA from the medium and the treatment with 37.5,75 and 150 μmol/L pepstatin A and PBS respectively.Enzyme-linked immunosorbent assay (ELISA) was conducted to measure the mean concentration of intracellular AGE-BSA at different time points,and the degradation rate of AGE was calculated.Some HDFs were divided into 3 groups:blank control group receiving no treatment,NC group transfected with an empty vector,and CatD group transfected with a CatD-overexpressing lentiviral vector.Fluorescence microscopy was conducted to estimate the transfection efficiency.Reverse transcription-PCR,Western blot analysis and fluorometric assay were performed to determine the mRNA and protein expression,and activity of CatD respectively.Then,the cells in the above 3 groups were incubated with AGE-BSA for 8 hours,followed by the removal of AGE-BSA from the medium and the treatment with fresh culture medium.The detection methods were same as the above experiment,and the degradation rate was calculated.Results The cellular proliferative activity in the 1-μmol/L CA074Me group,75-μmnol/L pepstatin A group and 1-μ mol/L MG-132 group was more than 90％,and there was no significant difference between the 3 groups and the control group (100％,F =1.525,P ＞ 0.05).Twenty-four hours after the removal of AGE-BSA from the medium,the fluorescence intensities of intracellular AGE-BSA in the CA074Me + AGE-BSA group (275.00 ± 10.15) and MG-132 + AGE-BSA group (259.00 ± 11.14) significantly decreased compared with those at the 8-hour time point (295.00 ± 6.56 and 285.67±8.74 respectively;paired t test,t =4.778,6.154 respectively,both P ＜ 0.05),while no significant difference was observed in the fluorescence intensities of intracellular AGE-BSA in the pepstatin A + AGE-BSA group between the 8-hour time point and 32-hour time point (P ＞ 0.05).The degradation rates of intracellular AGE-BSA within 24 hours in the 37.5,75 and 150 μmol/L pepstatin A groups were 9.64％ ± 1.27％,5.62％ ± 0.47％ and 3.21％ ± 0.73％ respectively;there were significant differences among the 3 groups (F =45.876,P ＜ 0.05),and the degradation rate significantly decreased along with the increase of pepstatin A concentration (P ＜ 0.05).Fluorescence microscopy showed no fluorescent cells in the blank control group,while the NC group and CatD group both showed a high proportion (＞ 80％) of fluorescent cells.The mRNA and protein expression as well as the activity of CatD were significantly higher in the CatD group than in the blank control group and NC group (all P ＜ 0.05).The CatD + AGE-BSA group showed a significantly higher degradation rate of intracellular AGE-BSA within 24 hours compared with the AGE-BSA group and NC + AGE-BSA group (both P ＜ 0.05).Conclusion CatD can promote the degradation of intracellular AGE-BSA endocytosed by HDFs.

Correlation analysis between the expression of cathepsin D and the accumulation of advanced glycation end products in skin tissues / 中华皮肤科杂志

Objective To determine the expression of cathepsin D and advanced glycation end products (AGEs)in skin tissues from patients of different ages or skin tissues with different degrees of sun exposure,to evaluate their correlation,and to preliminarily investigate the role of cathepsin D in the degradation and accumulation of AGEs in photoaged skin.Methods Skin tissues were collected from sunexposed and sun-protected body sites in patients aged 15-20 years,35-40 years,55-60 years or 75-80 years.These skin tissues were divided into 8 groups according to age of patients and degrees of sun exposure,and there were 6 specimens in each group.Immunohistochemical and immunofluorescent methods were used to measure the expression of cathepsin D and AGEs in the skin tissues.Statistical analysis was carried out by factorial design analysis of variance,Wilcoxon rank sum test and Kruskal-Wallis rank sum test for analyzing associations of the expression of cathepsin D and AGEs with age and sun exposure,as well as by Pearson correlation analysis for assessing the correlation between cathepsin D expression and AGEs expression.Results Immunohistochemical study showed that the expression of cathepsin D markedly decreased along with the increase of age,but the accumulation of AGEs gradually increased along with the increase of age.In the same age group,the cathepsin D expression was lower in the sun-exposed skin tissues than in the sun-protected skin tissues,while the accumulation of AGEs was more in the sun-exposed skin tissues than in the sun-protected skin tissues.Factorial design analysis of variance showed that sun exposure could decrease the expression of cathepsin D (F =58.70,P ＜ 0.001),but increase the accumulation of AGEs (F =158.18,P ＜ 0.001).Moreover,the increase of age could lead to decreased expression of cathepsin D (F =79.49,P ＜ 0.001),and increased expression of AGEs (F =106.06,P ＜0.001).Compared with the sun-protected skin tissues,the sun-exposed skin tissues in all the age groups showed significantly lower absorbance value of cathepsin D (35-40 years:0.020 ± 0.005 vs.0.032 ± 0.005;55-60 years:0.012 ± 0.004 vs.0.026 ± 0.002;75-80 years:0.002 ± 0.001 vs.0.013 ± 0.004;all P ＜0.001),but higher absorbance value of AGEs (35-40 years:0.030 ± 0.008 vs.0.010 ± 0.003;55-60years:0.066 ± 0.010 vs.0.021 ± 0.004;75-80 years:0.085 ± 0.015 vs.0.035 ± 0.009;all P ＜ 0.001)except the age group of 15-20 years.No matter whether the skin tissues were sun-exposed or sunprotected,there were significant differences in the expression of cathepsin D and AGEs among different age groups (all P ＜ 0.001).The results of double immunofluorescence staining were similar to those of immunohistochemical study.Pearson correlation analysis showed that the expression of cathepsin D in the sun-exposed skin tissues was highly negatively correlated with the accumulation of AGEs (r =-0.915,P ＜0.05),while they were moderately negatively correlated in the sun-protected skin tissues (r =-0.730,P ＜0.05).Conclusions Along with the increase of age,the expression of cathepsin D in skin tissues decreased,but the expression of AGEs increased.In the sun-protected skin tissues,the expression of cathepsin D was moderately negatively correlated with the expression of AGEs,while they were highly negatively correlated in the sun-exposed skin tissues,suggesting that cathepsin D may play an important role in the degradation and accumulation of AGEs in photoaged skin.

Correlation of the expression of HIF-1α and CTSD with vasculogenic mimicry and its clinicopathological features in esophageal squamous cell carcinoma / 临床与实验病理学杂志

Purpose To investigate the expression of vasculogenic mimicry (VM) and hypoxia inducible factor-1 α (HIF-1α),cathepsin D (CTSD) in esophageal squamous cell carcinoma (ESCC) and its relationship with clinicopathological parameters.Methods 120 specimens of primary ESCC and their clinical data were collected.Dual staining of CD34 and PAS were performed to validate the existence of VM in ESCC.Immunohistochemical of EnVision two-step method was used to detect the expression of VM-related markers vascular endothelial cadherin (VE-cadherin) and vascular endothelial grow factor (VEGF),HIF-1α and CTSD in 120 cases of ESCC.The relationship between HIF-1α,CTSD and clinical pathological parameters were analyzed.Results The positive rate of HIF-1α,CTSD was correlated with infilatrating,lymph node metastasis,clinical stage and cancer embolus.VM existed in 25 of 120 cases.The expression of HIF-1α and CTSD in cases with VM was obviously higher than that without VM,the expression of HIF-1 α and CTSD was positively correlated with VM in ESCC.The expression of CTSD in case with HIF-1 α was obviously lower than that without HIF-1α,the expression of CTSD was positively correlated with HIF-1α in ESCC.The expression of VE-cadherin and VEGF in cases with CTSD was obviously higher than that without CTSD,the level of VE-cadherin and VEGF expression was positively correlated with CTSD in ESCC.Conclusion CTSD may promote VM formation in ESCC via HIF-1α.High expression of HIF-1α and CTSD indicate poor prognosis of ESCC patients.

Effects of advanced glycation end products on the expressions and activity of cathepsin D in ultraviolet A- irradiated human dermal fibroblasts / 中华皮肤科杂志

Objective To investigate the effects of advanced glycation end products(AGE)on the expressions and activity of cathepsin D(CatD)in ultraviolet A(UVA)?irradiated human dermal fibroblasts. Methods Human dermal fibroblasts were isolated and harvested from the circumcised foreskin of children, and subjected to a primary culture. CCK?8 assay was performed to screen non?cytotoxic concentrations of AGE?bovine serum albumin (BSA). Some fibroblasts were incubated with 50, 100 and 300 mg/L AGE?BSA separately for 24 hours, with untreated cells as the control group. Then, reverse transcription(RT)?PCR, Western?blot analysis and a fluorimetric assay were performed to measure the mRNA and protein expressions as well as activity of CatD, respectively. Some fibroblasts were classified into six groups: control group receiving no treatment, AGE?BSA group and BSA group treated with the highest non?cytotoxic concentration of AGE?BSA and the same concentration of BSA respectively for 24 hours, UVA group irradiated by 10 J/cm2 UVA, UVA?AGE?BSA group and UVA?BSA group treated with AGE?BSA and BSA at the above non?cytotoxic concentration respectively for 24 hours both before and after UVA radiation at 10 J/cm2. After the treatments, RT?PCR, Western?blot analysis and a fluorimetric assay were conducted to detect mRNA and protein expressions and activity of CatD respectively. Results AGE?BSA of 50- 200 mg/L exhibited no obvious influence on cellular proliferation of fibroblasts. The fibroblasts incubated with AGE?BSA of 50, 100 and 200 mg/L showed a significant increase in the mRNA expression(0.267 ± 0.007, 0.348 ± 0.007, and 0.418 ± 0.006 respectively), protein expression (1.403 ± 0.181, 2.233 ± 0.090 and 2.477 ± 0.111 respectively), and activity(1.760 ± 0.080, 2.330 ± 0.060 and 2.890 ± 0.080 respectively)of CatD compared with the control group(mRNA:0.161 ± 0.006;protein:0.903 ± 0.200;activity:1.100 ± 0.090, all P < 0.05). AGE?BSA increased CatD expressions and activity in a dose?dependent manner. The mRNA and protein expressions as well as activity of CatD were significantly higher in the UVA group than in the control group (mRNA expression: 0.480 ± 0.005 vs. 0.155 ± 0.005; protein expression: 2.583 ± 0.199 vs. 0.920 ± 0.235;activity:2.970 ± 0.110 vs. 1.110 ± 0.040, all P<0.05), but significantly lower in the UVA?AGE?BSA group than in the UVA group(mRNA expression:0.394 ± 0.008 vs. 0.480 ± 0.005;protein expression:2.070 ± 0.125 vs. 2.583 ± 0.199;activity: 2.560 ± 0.060 vs. 2.970 ± 0.110, all P < 0.05). Conclusion AGEs could increase CatD expressions and activity in human dermal fibroblasts not receiving UVA irradiation, but inhibit their increase in UVA?induced human dermal fibroblasts.

Performance of transfection with a complex plasmid encoding green fluorescent protein tagged cathepsin D in researches on chronic photodamaged fibroblasts / 中华皮肤科杂志

Objective To evaluate the performance of transfection with a complex plasmid encoding green fluorescent protein tagged CatD (GFP-CatD)in researches on chronic photodamaged fibroblasts. Methods Human dermal fibroblasts (HSFs)were irradiated with ultraviolet A (UVA)at 25 J/cm2 once a day for 21 consecutive days to establish a chronic photodamaged cell model. A plasmid encoding GFP-CatD was constructed and transfected into some chronic photodamaged fibroblasts (experimental group). The photodamaged HSFs receiving no treatment served as the blank control group, and those transfected with the negative plasmid encoding GFP only as the negative control group. After additional culture, fluorescence microscopy and Western-blot analysis were performed to observe and measure the expression of GFP-CatD in HSFs respectively, flow cytometry and methyl thiazolyl tetrazolium (MTT)assay to evaluate the apoptosis and proliferation of chronic photodamaged fibroblasts respectively. Results Fluorescence microscopy showed the expression of GFP-CatD in cytoplasm of chronic photodamaged fibroblasts at 96 hours after transfection with the GFP-CatD-encoding plasmid. Western-blot analysis revealed that the expression of CatD in the experimental group was 1.28 times that in the blank control group. There were no significant differences in the apoptosis rate(4.29% ± 1.30%vs. 3.03% ± 1.70% , P > 0.05)or proliferative rate (45.20% ± 4.70% vs. 43.60 ± 3.90% , P > 0.05)between the experimental group and blank control group. Conclusion CatD could be traced in chronic photodamaged fibroblasts with no changes in biological activity or cell cycle after transfection with the GFP-CatD-encoding complex plasmid.

Activity of cathepsin D and alpha-1 antitrypsin in patients with hip or knee osteoarthritis / 中南大学学报(医学版)

Objective: To assess the activity of cathepsin D (CAT-D) and alpha-1 antitrypsin (AAT) in blood in patients with hip or knee osteoarthritis, and to explore whether these two enzymes could be served as serum biomarkers for cartilage degeneration. Methods: hTe activity of CAT-D and AAT in blood serum of 44 women and 26 men with hip or knee osteoarthritis was determined by the method of ELISA before total joint replacement and on the 10th day atfer the surgery. One hundred healthy volunteers were chosen as the control. All datawere analyzed by using SPSS19.0 sotfware. Results: Compared with the controls, the activity of CAT-D in patients with osteoarthritis was decreased by 25% (P0.05), but it was increased by 80% after the surgery than that in the control group (P0.05). hTe gender, hypertension, diabetes and age did not affect the activities of the 2 enzymes (P>0.05). Conclusion: AAT might be a possible inflammatory indicator in the osteoarthritis. CAT-D and AAT enzymes are not affected by gender, age, hypertension and diabetes, etc, and they might be served as potential biomarkers for cartilage degradation.

Proteomic fingerprinting of N-linked glycoproteins involved in hepatocellular carcinoma / 解剖学报

Objective To identify differentially expressed N-linked glycoproteins between hepatocellular carcinoma ( HCC) and adjacent non-tumorous liver tissues .Methods N-linked glycoproteome was extracted by multi-lectin affinity chromatography comprising concanavalin A (ConA), lentil lectin (LCH), and snowdrop lectin (GNA) and subsequently subjected to two-dimensional electrophoresis ( 2DE ) and mass spectrometry ( MS ) for identification of differential glycoproteins between 10 pairs of HCC and adjacent non-cancer tissue .Western blotting was used to verify different expression of human liver carboxylesterase 1 (hCE1), haptoglobin (HP)and cathepsin D (CD).Invasion potential in vitro was examined after si-RNA mediated CD gene scilencing .Results LC-ESI-MS/MS identified a total of 28 differentially expressed glycoproteins (14 up-regulation and 14 down-regulated).Western blotting detected consistent down-regulation of hCE1 and HP, and up-regulation of pro-cathepsin D (pCD) in HCC.Up-regulation of ConA-binding CD (ConA-CD), however , was verified in HCC only after ConA-CD enrichment by ConA chromatography .Down-regulation of CD expression mediated by CD-siRNA markedly inhibited the in vitro invasive potential of SNU449 and SNU473.Conclusion Dysregulation of HP , hCE1 expression and alteration of glycans linked to CD may play crucial roles in pathogenesis of HCC.

The Expression of Multiple Proteins as Prognostic Factors in Colorectal Cancer: Cathepsin D, p53, COX-2, Epidermal Growth Factor Receptor, C-erbB-2, and Ki-67

BACKGROUND/AIMS: A single gene mutation alone cannot explain the poor prognosis of colorectal cancer. This study aimed to establish a correlation between the expression of six proteins and the prognosis of colorectal cancer patients. METHODS: Tissue samples were collected from 266 patients who underwent surgery for colorectal cancer at our institution from January 2006 to December 2007. The expression of six proteins were determined using immunohistochemical staining of specimens. RESULTS: Cathepsin D, p53, COX-2, epidermal growth factor receptor, c-erbB-2, and Ki-67 expression were detected in 38.7%, 60.9%, 37.6%, 35.7%, 30.1%, and 74.4% of the samples, respectively. The expression of cathepsin D was significantly correlated with reduced cancer-free survival (p=0.036) and colorectal cancer-specific survival (p=0.003), but the other expression levels were not. In a multivariate analysis, cathepsin D expression was found to be an independent prognostic factor for poorer colorectal cancer-specific survival (hazard ratio, 8.55; 95% confidence interval, 1.07 to 68.49). Furthermore, patients with tumors expressing four or more of the proteins had a significantly decreased cancer-free survival rate (p=0.006) and colorectal cancer-specific survival rate (p=0.002). CONCLUSIONS: Patients with cathepsin D positivity had a poorer outcome than patients who were cathepsin D-negative. Thus, cathepsin D may provide an indicator for appropriate intensive follow-up and adjuvant chemotherapy.

Proteomic Analysis between U87MG and U343MG-A Cell Lines: Searching for Candidate Proteins for Glioma Invasion

BACKGROUND: To investigate the molecular basis for invasion of malignant gliomas, proteomic analysis approach was carried out using two human glioma cell lines, U87MG and U343MG-A that demonstrate different motility and invasiveness in in vitro experiments. METHODS: High-resolution two-dimensional gel electrophoresis and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry analysis were performed. RESULTS: Nine distinct protein spots that were recognized with significant alteration between the two cell lines. Five of these protein spots were up-regulated in U87MG and four were up-regulated in U343MG-A. CONCLUSION: Among these proteins, cathepsin D was shown to be one of the important proteins which are related with glioma invasion. However, further studies are necessary to reveal the exact role and mechanism of cathepsin D in glioma invasion.

Progress of cathepsin D in cancer / 国际外科学杂志

Tumor cell invasion and metastasis are associated with the proteolytic activity of various types of proteinases.Among them,cathepsin D,which is a lysosomal proteinase,has received more attention recently.Various studies have shown that the lysosomal aspartic protease cathepsin D is over-expressed and hyper-secreted by numerous cancer cell lines.Indeed it plays an essential role in the multiple steps of tumor progression,in stimulating cancer cell proliferation,tumor invasion and metastasis,fibroblast outgrowth and angiogenesis,as well as in inhibiting tumor apoptosis.In addition,CD is also a key mediator of induced- apoptosis.The aim of this article is to review the current knowledge on Cathepsin D action in cancer progression and metastasis,as well as its dual function in apoptosis.

Relative prognostic significance of the cathepsin D and c-erbB-2 protein in breast cancer / 肿瘤研究与临床

Objective To investigate the expression of c-erbB-2 and cathepsin D (cath-D) in primary breast carcinoma, and to analyze its relationship to the patients' clinicopathological status and its prognostic significance. MethodsThe immnohistochemical test were used to detect the expression of c-erbB-2 and cath-D in 128 patients with primary breast carcinoma. All data were statistically analyzed.ResultsThe positive expression rate of c-erbB-2 and cath-D was 36.5 % and 40.9 %, relatively. Cath-D was correlated with tumor size (x2 =21.0, P ＜0.0001) and lymph node status (35.29 % vs 63.83 %, P ＜0.0001). The Kaplan-Meier curve and log rank test indicated that the positive c-erbB-2 expression was a significant prognostic factor for patients disease free survival (P＜0.01). Conclusionc-erbB-2 and cath-D show a clinicopathological invasive characteristic. They are prognostic factors for patients with primary breast carcinoma, and also can be prognostic indicator for patients.