Effect of vitamin E and human placenta cysteine peptidase inhibitor on expression of cathepsins B and L in implanted hepatoma Morris 5123 tumor model in Wistar rats

AIM: To examine the effectiveness of human placental inhibitors, by injecting vitamin E to rats with transplanted Morris-5123 hepatoma, on the expression of cathepsins B and L in tumor, liver, lung and blood sera after transplantation of Morris 5123 hepatoma. METHODS: Animals were divided into 10 groups receiving three different concentrations of vitamin E and inhibitors along or in combination and compared with negative control (healthy rats) and positive control (tumor rats). Effectiveness of treatment was evaluated with regard to survival time, tumor response and determination of the activities of proteolytic enzymes and their inhibitors using flurogenic substrates. RESULTS: Cathepsins B and L activities were elevated by 16-fold in comparison with negative control tissues, and their endogenous inhibitor activity decreased by 1.2-fold before treatment. In several cases, tumors completely disappeared following vitamin E plus human placental cyteine protease inhibitor (CPI) compared with controls. The number of complete tumor responses was higher when 20 m/kg vitamin E plus 400 μg of CPI was used, i.e. 7/10 rats survived more than two mo. Cathepsins B and L were expressed significantly in tumor, liver, lung tissues and sera in parallel to the increasing of the endogenous inhibitor activity compared with the controls after treatment(P<0.0001) CONCLUSION: The data indicate formation of metastasis significantly reduced in treated rats, which might provide a therapeutic basis for anti-cancer therapy.

Icariin plus curcumol enhances autophagy through the mTOR pathway and promotes cathepsin B-mediated pyroptosis of prostate cancer cells

Objective:To examine the effect of icariin plus curcumol on prostate cancer cells PC3 and elucidate the underlying mechanisms.Methods:We employed the Cell Counting Kit 8 assay and colony formation assay to assess cell viability and proliferation.Autophagy expression was analyzed using monodansylcadaverine staining.Immunofluorescence and Western blot analyses were used to evaluate protein expressions related to autophagy,pyroptosis,and the mTOR pathway.Cellular damage was examined using the lactate dehydrogenase assay.Moreover,cathepsin B and NLRP3 were detected by co-immunoprecipitation.Results:Icariin plus curcumol led to a decrease in PC3 cell proliferation and an enhancement of autophagy.The levels of LC3-Ⅱ/LC3-Ⅰand beclin-1 were increased,while the levels of p62 and mTOR were decreased after treatment with icariin plus curcumol.These changes were reversed upon overexpression of mTOR.Furthermore,3-methyladenine resulted in a decrease in inflammatory cytokines,pyroptosis-related protein levels,and lactate dehydrogenase concentration,compared to the icariin plus curcumol group.Inhibiting cathepsin B reversed the regulatory effects of icariin plus curcumol.Conclusions:Icariin plus curcumol demonstrates great potential as a therapeutic agent for castration-resistant prostate cancer by enhancing autophagy via the mTOR pathway and promoting pyroptosis mediated by cathepsin B.These findings provide valuable insights into the molecular mechanisms underlying the therapeutic potential of icariin and curcumol for prostate cancer treatment.

大鼠脑外伤后溶酶体酶Cathepsin-B和D的表达

目的研究大鼠脑外伤后溶酶体酶cathepsin-B和-D是否被激活及其不同时段表达变化,阐述其与凋亡执行因子caspase-3表达的关系,并探讨对脑外伤诊断及形成时间的意义。方法采用自由落体打击法建立脑外伤动物模型,并对模型及对照样本进行免疫荧光、双标和激光共聚焦检测,结果用SPSS10.0软件处理。结果脑外伤后1hcathepsin-B表达即增加,4～8d达高峰,脑外伤后32d仍处于高表达水平;cathepsin-D的表达于脑外伤后12h增加,4～8d达高峰,32d的表达仍然高于12h的表达水平。脑外伤初期,cathepsin-B和-D阳性细胞与caspase-3阳性细胞重叠较少,脑外伤后6h开始增加,32d仍然有很多阳性细胞重叠。结论脑外伤后cathepsin-B和-D被激活,其激活在脑外伤早期可能抑制细胞凋亡执行因子caspase-3的激活,之后(6h后)则与caspase-3起协同作用,共同促进细胞死亡;cathepsin-B和-D表达的时程变化对于脑外伤的法医学诊断和中晚期的时间推断有参考意义。

Autophagy Plays a Potential Role in the Process of Sea Cucumber Body Wall “Melting” Induced by UV Irradiation

The changes of tissue appearances and structures in the process of UV-induced ＂melting＂ for sea cucumber （Stichopus japonicus） body wall were studied. And the localization and determination of acid phosphatase （ACP）, Cathepsin B and Cathepsin L activities were also investigated. The results show that the connective tissue was damaged with many hollows emerging and the regular collagen bundles were broken apart into irregular fragments. Margination of condensed chromatin at the nuclear membrane was observed. Both Golgi＇s body and endoplasmic reticulure swelled, curled, and eventually double- or multi-lamellar vesicles were formed. A number of autophagic vesicles distributed in all through the whole cytoplasm. ACP becomes more active after UV irradiation. The activities of cathepsin B and cathepsin L increased in UV-treated sea cucumbers and both achieved their maximum under certain conditions, It indicates that autophagy plays a potential role in the ＂melting＂ process for sea cucumber body wall induced by UV irradiation.

Evaluation of serum catnepsin B and D in relation to diniGopatnological staging of colorectal cancer

AIM: Proteolytic degradation of the extracellular matrix facilitates cancer invasion and promotes metastasis. The study aims at evaluation of preoperative and postoperative serum cathepsins B and D levels in correlation with selected anatomoclinical features of colorectal cancer.METHODS: Blood samples were collected from 63colorectal cancer patients before curative operation of the tumor 10 d later. Blood that was obtained from 20healthy volunteers, served as a control. The activity of cathepsin B was measured with Bz-DL-arginine-pNA as a substrate at pH 6.0, while cathepsin D activity was determined with urea-denatured hemoglobin (pH 4.0).RESULTS: The preoperative and postoperative activities of cathepsin B were significantly (P＜0.00001) lower in serum of colorectal cancer patients than in control group.However, postoperative values of this protease were significantly increased in comparison with preoperative ones (P = 0.031). Activity of cathepsin D appeared to be significantly higher in colorectal cancer sera (P＜0.00001)compared with controls. No statistically significant differences between preoperative and postoperative activity of cathepsin D were noted (P = 0.09). We revealed a strong linkage of cathepsins' levels with lymph node status and pT stage of colorectal cancer.CONCLUSION: Blood serum activities of cathepsin B and D depend on the time of sampling, tumor size and lymph node involvement. Significantly, increased activity of cathepsin D could indicate a malignant condition of the large intestine. In our work, the serum postoperative decrease of cathepsin B activity appears as an obvious concomitant of local lymph node metastasis-the wellknown clinicopathological feature of poor prognosis.

CATHEPSIN B EXPRESSION AND ITS RELATIONSHIP WITH MICROVESSEL DENSITY AND BIOLOGICAL BEHAVIOUR OF COLORECTAL CARCINOMA

Objective: To investigate cathepsin B(CB) expression in colorectal carcinoma and its relationship with microvessel density (MVD) and biological behavior. Methods: CB and MVD were detected by immunohistochemistry in 47 cases of colorectal carcinoma. Results: The expression of CB in mucinous colorectal carcinoma was significantly higher than that in no-mucinous colorectal carcinoma. There was significant difference (P<0.05). The MVD in group with positive CB was stronger than that in group with negative CB. There was also significant difference (P<0.05). Conclusion: The results suggest that CB expression has correlation with MVD, invasion and metastasis in colorectal carcinoma, especially in mucinous colorectal carcinoma.

Role of cathepsin B-mediated apoptosis in fulminant hepatic failure in mice

AIM: To investigate the pathogenic role of cathepsin B and the protective effect of a cathepsin B inhibitor (CA074Me) in fulminant hepatic failure in mice. METHODS: LPS/D-Gal N was injected into mice of the model group to induce fulminant hepatic failure; the protected group was administered CA-074me for 30 min before LPS/D-Gal N treatment; the normal group was given isochoric physiologic saline. Liver tissue histopathology was determined with HE at 2, 4, 6 and 8 h after Lps/D-Gal injection. Hepatocyte apoptosis was examined by TUNEL method. The expression of cathepsin B in liver tissues was investigated by immunohistochemistry, Western blot and RT-PCR. RESULTS: Compared with the normal group, massive typical hepatocyte apoptosis occurred in the model group; the number of apoptotic cells reached a maximum 6 h after injection. The apoptosis index (AI) in the protected group was clearly reduced (30.4 ± 2.8 vs 18.1 ± 2.0, P < 0.01 ). Cathepsin B activity was markedly increased in drug-treated mice compared with the normal group (P < 0.01). Incubation with LPS/D-Gal N at selected time points resulted in a timedependent increase in cathepsin B activity, and reached a maximum by 8 h. The expression of cathepsin B was significantly decreased in the protected group (P < 0.01). CONCLUSION: Cathepsin B plays an essential role in the pathogenesis of fulminant hepatic failure, and the cathepsin B inhibitor CA-074me can attenuate apoptosis and liver injury.

Hepatoprotective effects of cathepsin B inhibitor on acute hepatic failure induced by lipopolysaccharide/D-galactosamine in mice

BACKGROUND:Increasing evidence suggests that the inactivation of cathepsin B attenuates hepatocyte apoptosis and liver damage.This study aimed to investigate the protective effects of a cathepsin B inhibitor(CA-074me) on lipopolysaccharide(LPS)/D-galactosamine(D-GalN)-induced acute hepatic failure(AHF) in mice.METHODS:Mice were intraperitoneally injected with a combination of LPS/D-GalN to induce AHF with or without CA-074me pretreatment.The cumulative survival rates were calculated 48 hours after the induction of AHF.As well as changes in biochemical indicators and liver histology,hepatocyte apoptosis was assessed using a TUNEL method.Serum tumor necrosis factor-α(TNF-α) production,caspase-3,caspase-8,and caspase-9 activity was evaluated.Cytosolic cytochrome c and Bcl-2 expression were measured by Western blotting.RESULTS:The marked elevation in serum aminotransferase activity and prothrombin time found in LPS/D-GalN-treated mice was significantly improved by pretreatment with CA074me.The efficacy of CA-074me was also confirmed by histological analysis and TUNEL assay.The survival rate significantly improved in LPS/D-GalN-induced mice given CA-074me compared with untreated mice.LPS/D-GalNinduced caspase-3 and caspase-9 activation was remarkably suppressed by CA-074me.However,the increased levels of serum TNF-α and elevated caspase-8 activity in AHF mice were not significantly reduced by CA-074me.Moreover,CA074me sharply reduced the increased expression of cytosolic cytochrome c and markedly augmented Bcl-2 expression.CONCLUSION:These results suggest that CA-074me has a protective effect in acute hepatic failure induced by LPS/D-GalN.

The role of autophagic and lysosomal pathways in ischemic brain injury

Autophagy is involved in neural cell death after cerebral ischemia. Our previous studies showed that rapamycin-induced autophagy decreased the rate of apoptosis, but the rate of apoptosis was in- creased after the autophagy inhibitor, 3-methyladenine, was used. In this study, a suture-occluded method was performed to generate a rat model of brain ischemia. Under a transmission electron microscope, autophagic bodies and autophagy lysosomes were markedly accumulated in neurons at 4 hours post brain ischemic injury, with their numbers gradually reducing over time. Western blotting demonstrated that protein levels of light chain 3-11 and cathepsin B were significantly in- creased within 4 hours of ischemic injury, but these levels were not persistently upregulated over time. Confocal microscopy showed that autophagy was mainly found in neurons with positive light chain 3 signal. Injection of rapamycin via tail vein promoted the occurrence of autophagy in rat brain tissue after cerebral ischemia and elevated light chain 3 and cathepsin B expression. However, in- jection of 3-methyladenine significantly diminished light chain 3-11 and cathepsin B expression. Results verified that autophagic and lysosomal activity is increased in ischemic neurons. Abnormal components in cells can be eliminated through upregulating cell autophagy or inhibiting autophagy after ischemic brain injury, resulting in a dynamic balance of substances in cells. Moreover, drugs that interfere with autophagy may be potential therapies for the treatment of brain injury.

Putative roles of cathepsin B in Alzheimer's disease pathology: the good, the bad, and the ugly in one?

Alzheimer＇s disease（AD）is a fatal progressive neurodegenerative disorder characterized by loss in memory,cognition,and executive function and activities of daily living.AD pathogenesis has been shown to involve loss of neurons and synapses,cholinergic deficits,amyloid-beta protein（Aβ）deposition,tau protein hyperphosphorylation, and neuroinflammation.

Relation of Cystatin C and Cathepsin B Expression to the Pathological Grade and Invasion of Human Gliomas

OBJECTIVE To explore the relation of cystatin C and cathepsin B expression to the pathological grade and invasion of human gliomas. METHODS A immunohistochemical method was used to detect the expression of cystatin C and cathepsin B in 57 glioma samples. RESULTS The expression of cystatin C in high-grade （Grade Ⅲ-Ⅳ ）gliomas was significantly weaker than that in low-grade（Grade Ⅰ-Ⅱ, P=0.0001）. On the other hand, the expression of cathepsin B in high-grade gliomas was significantly stronger than that in low-grade （P=0.0001）. Cystatin C expression correlated inversely with cathepsin B expression in gliomas （P=0.01）. CONCLUSION Cystatin C and cathepsin B expression is related to the pathological grade and invasion of gliomas. Combining detection of cystatin C and cathepsin B expressions might provide significant information for clinical assessment of maglignant phenotypes and invasion of gliomas.

八子补肾胶囊对衰老小鼠骨质量的保护作用及其对SIRT6/NF-κB/cathepsin K通路的影响

目的探讨八子补肾胶囊(BZBS)对D-半乳糖(D-gal)和亚硝酸钠(NaNO2)诱导的衰老小鼠的骨保护作用及可能的作用机制。方法ICR雄性小鼠连续腹腔注射D-gal和NaNO2三个月造成衰老小鼠模型,并于造模第25天开始用BZBS干预,连续干预65 d。然后收集股骨,分别用HE染色评价骨微结构的变化,三点弯曲实验评估骨生物力学性能,Micro-CT扫描观察骨组织形态计量学参数,红外光谱法分析骨材料特性,免疫组织化学染色法测定骨组织中的SIRT6、NF-κB和cathepsin K蛋白表达水平。结果BZBS能明显抑制D-gal和NaNO2诱导的衰老小鼠的骨形态破坏,提高骨组织矿物质含量。此外,BZBS能上调衰老小鼠血清中的T-AOC、SOD、GSH、GSH/GSSG水平,降低MDA水平,提高骨组织中SIRT6的表达,抑制NF-κB乙酰化,降低cathepsin K的表达。结论BZBS能提高衰老小鼠的骨质量,这种作用可能与调节氧化还原平衡进而调控SIRT6/NF-κB/cathepsin K信号通路有关。

Catechu Inhibiting Cathepsin B Activity and Motility of HT1080 Cells

We reported that Catechu extract has a significant inhibitory effect on cathepsin B activity. The IC50 value for the Catechu extract against cathepsin B was 7.6 μg/mL. In addition, we showed that HT1080 human fibrosarcoma cells express cathepsin B and Catechu modulate the invasion and motility of these cells. These data may provide molecular mechanisms for the therapeutic effects of Catechu.

Anti-Cancer Effects of Novel Doxorubicin Prodrug PDOX in MCF-7 Breast Cancer Cells

Ac-Phe-Lys-PABC-DOX（PDOX） is a smart doxorubicin（DOX） prodrug designed to decrease toxicities while maintaining the potent anticancer effects of DOX. This study was aimed at elucidating the effectiveness and toxicities of DOX and PDOX in patient-derived MCF-7 breast cancer cells in vitro. The MCF-7 cells were exposed to both PDOX and DOX, and cytotoxicities, cell cycle and P53/P21 signaling alterations were studied. Abundant cathepsin B was found in the MCF-7 cells, and treatment with PDOX and DOX triggered dose- and time-dependent cytotoxicity and resulted in a significant reduction in cell viability. The IC50 of PDOX and DOX was 3.91 and 0.94 μmol/L, respectively. Both PDOX and DOX caused an up-regulation of the P53/P21-related signal pathway, and PDOX significantly increased expression of P53 and caspase 3, and arrested the cell cycle at the G1/G2 phase. As compared with DOX, PDOX reduced toxicities, and it may have different action mechanisms on breast cancer cells.

S53G SNP in Cathepsin B: Can Be Used as a Molecular Biomarker to Breast Cancer?

Cathepsin B (CTS B) is a proteolytic enzyme that participates in several important biological processes. However, when it is altered can be involved in development of breast cancer, a disease with high incidence and mortality rate among women. Many studies have shown a correlation between high CTS B expression and breast tumor. Furthermore, it has been shown that the SNP rs1803250 (S53G) leads to structural and functional changes in protein that can make it pathogenic. The present study aimed to evaluate a possible association of SNP rs1803250 (S53G) with breast cancer. For this, real-time PCR was performed on a sample collected in the State of Pará, with 127 patients and 122 controls. The SNP frequency in this region was 0.12, according to a research project in progress that aims to identify Amerindians molecular alterations. This indicates that the SNP is found in region with a distribution close to worldwide frequency of 0.09. Our results showed that the SNP was in Hardy-Weinberg Equilibrium in collected sample, but C variant allelic frequency was 0.08 in both patients and control groups, which is extremely similar to global average. Moreover, homozygotes CC was not found in the sample and SNP genotypes frequency in patients and control groups was not significantly different. In addition, statistical analysis showed that the SNP did not have to correlate with tumor subtypes nor with tumor staging. Therefore, according to the analyzed sample, the SNP rs1803250 has no association with breast cancer and it cannot be considered a molecular biomarker for breast cancer.

天麻干预脑缺血后星形胶质细胞损伤的作用机制研究

目的探讨天麻对脑缺血后星形胶质细胞损伤是否具有直接保护作用及Cathepsin B-Caspase信号通路与天麻保护细胞的作用有无关联。方法建立动物缺血性脑中风模型,缺血再灌注28 d,随机分为假手术组、模型组、天麻低剂量(50 mg/kg)组、天麻中剂量(100 mg/kg)组、天麻高剂量(150 mg/kg)组,天麻各剂量组每天给予不同剂量的天麻,假手术组给予同体积的溶剂,28 d后观察各组大鼠行为学、神经症状以及脑梗死体积。通过培养原代大鼠大脑皮层星形胶质细胞,然后进行氧糖剥夺再灌注(OGD/R),建立体外的细胞模型,缺氧缺糖再灌注导致的星形胶质细胞损伤,使用乳酸脱氢酶(LDH)试剂盒检测,采用蛋白免疫印迹法(Western Blot)检测星胶标志蛋白胶质纤维酸性蛋白(GFAP)及凋亡蛋白Cathepsin B、Caspase-3表达。结果天麻低、中、高剂量组脑梗死体积较模型组明显缩小(P<0.01)。Western Blot检测结果显示:缺血再灌注后,大鼠的皮层缺血区星形胶质细胞标志蛋白GFAP表达下调(P<0.01),通路相关蛋白Cathepsin B、Caspase-3表达上调(P<0.01)。给予天麻干预之后,与模型组相比,天麻各剂量组GFAP表达升高,且缺血区Cathepsin B、Caspase-3表达明显降低(P<0.05或P<0.01)。LDH检测结果显示:随着天麻(0.1μmol/L、1μmol/L、10μmol/L)给药剂量的增加,星形胶质细胞得以不同程度的保护(P<0.05或P<0.01)。体外Western Blot检测结果显示:天麻(0.1μmol/L、1μmol/L、10μmol/L)可有效地降低OGD/R处理的星形胶质细胞中或Cathepsin B、Caspase-3的表达(P<0.05或P<0.01)。结论天麻可通过Cathepsin B-caspase的细胞凋亡途径进而对缺血再灌注引起的细胞损伤发挥保护作用。

Autophagy activation aggravates neuronal injury in the hippocampus of vascular dementia rats

It remains unclear whether autophagy affects hippocampal neuronal injury in vascular dementia. In the present study, we investigated the effects of autophagy blockade on hippocampal neuro- nal injury in a rat model of vascular dementia. In model rats, hippocampal CA1 neurons were severely damaged, and expression of the autophagy-related proteins beclin-1, cathepsin B and microtubule-associated protein 1 light chain 3 was elevated compared with that in sham-operated animals. These responses were suppressed in animals that received a single intraperitoneal injection of wortmannin, an autophagy inhibitor, prior to model establishment. The present results confirm that autophagy and autophagy-related proteins are involved in the pathological changes of vascular dementia, and that inhibition of autophagy has neuroprotective effects.

特异性坏死性凋亡抑制剂-1(Nec-1)减轻脊髓损伤模型大鼠胶质瘢痕的形成

目的观察特异性坏死性凋亡抑制剂-1(Nec-1)对脊髓损伤模型大鼠胶质瘢痕形成的影响及其机制。方法将大鼠随机分为假手术组、模型组(坠落法)及受体相互作用蛋白激酶1(RIPK1)抑制剂Nec-1组(在大鼠侧脑室注射10μmol/L Nec-110μL)。BBB评分法测定大鼠后肢运动功能,免疫荧光检测胶质瘢痕形成,Western blot检测组织蛋白酶(cathepsin)B、caspase-3、胶质纤维酸性蛋白(GFAP)和vimentin表达。结果术后第14天,与假手术组相比,模型组和Nec-1组BBB评分值均显著降低(P<0.05),与模型组相比,Nec-1组BBB评分值显著升高(P<0.05)。术后第14天,假手术组未见NF-200荧光,模型组及Nec-1组均可见NF-200荧光,与模型组相比,Nec-1组NF-200荧光标记强度显著降低(P<0.05)。术后第7天及14天,与假手术组相比,模型组、Nec-1组cathepsin B、caspase-3、GFAP、vimentin蛋白水平显著升高(P<0.05),与模型组相比,Nec-1组cathepsin B、caspase-3、GFAP和vimentin蛋白水平显著降低(P<0.05)。结论Nec-1可能通过调控cathepsinB和caspase表达,减轻大鼠脊髓损伤后胶质瘢痕形成,促进神经功能恢复。

Diagnostic usefulness of selected proteases and acute phase factors in patients with colorectal adenocarcinoma

BACKGROUND Uncontrolled growth and loss of control over basic metabolic functions,leading to invasive proliferation and metastases,are the salient traits of malignant tumors in general and colorectal cancer in particular.Invasion and metastases hinder effective tumor treatment.While surgical techniques and radiotherapy can be used to remove tumor focus,only chemotherapy can eliminate dispersed neoplastic cells.However,the efficacy of the latter method is limited in the advanced stages of the disease.Therefore,recognition of the mechanisms involved in neoplastic cell spreading is indispensable for developing effective therapies.AIM To use a number of biomarkers involved in cancer progression and identify a panel that could be used for effective early diagnosis.METHODS We recruited 185 patients with colorectal adenocarcinoma(98 men,87 women with median age 63).Thirty-five healthy controls were sex and age-matched.Dukes’staging was as follows:A=22,B=52,C=72,D=39.We analyzed patients'blood serum before surgery.We determined:(1)Cathepsin B(CB)with Barrett's method(fluorogenic substrate);(2)Leukocytic elastase(LE)in a complex with alpha 1 trypsin inhibitor(AAT)using the immunoenzymatic MERCK test;(3)Total sialic acid(TSA)with the colorimetric periodate-resorcinol method;(4)Lipid-bound sialic acid(LASA)with the colorimetric Taut's method;and(5)The antitrypsin activity(ATA)employing the colorimetric test.RESULTS In patients,the values of the five biochemical parameters were as follows:CB=16.1±8.8 mU/L,LE=875±598μg/L,TSA=99±31 mg%,LASA=0.68±0.33 mg%,and ATA=3211±1504 U/mL.Except for LASA,they were significantly greater than those of controls:CB=11.4±6.5 mU/L,LE=379±187μg/L,TSA=71.4±15.1 mg%,LASA=0.69±0.28 mg%,and ATA=2016±690 U/mL.For CB and LASA,the differences between the four Dukes’stages and controls were not statistically significant.The inter-stage differences for CB and LASA were also absent.The receiver operating characteristic(ROC)analysis revealed the potential diagnostic value of CB,TSA,and ATA.The area under ROC,sensitivity,and specificity for these three parameters were:0.85,72%,90%;0.75,66%,77%;and 0.77,63%,84%,respectively.The sensitivity and specificity for the threeparameter panel CB-TSA-ATA were equal to 88.2%and 100%,respectively.CONCLUSION The increased value of CB,TSA,and ATA parameters are associated with tumor biology,invasion,and metastasis of colorectal cancer.The presented evidence suggests the potential value of the CB-TSA-ATA biochemical marker panel in early diagnostics.