Determination of cysteine peptidases-like activity and their inhibitors in the serum of patients with ovarian cancer treated by conventional chemotherapy and vitamin E

Enzymatic activity of cysteine peptidases (cathepsins B and L) --associated with carcinogenesis is controlled by their specific inhibitors. The study was objected to the effects enhanced by taxol and cisplatin in patients pretreated with the vitamin E, by determining the levels of cathepsins B and L in sera of patients with ovarian cancer. The activity of cysteine peptidase (CP) and their inhibitors (CPI) in serum from patients with ovarian cancer and noncancerous patients were measured by using fluorogenic substrate before and after the routine anticancer chemotherapy, and a complementary combination of chemotherapy with vitamin E. The cat B and L activities were significantly higher in patient sera with ovarian cancer than non-cancerous patients (0.0001 pounds sterling). The results shows that, inhibitory activity of CPI and complex form were significantly decreased from 4.6 mEU/mg protein in a group of non-cancerous patients to 0.7 mEU/mg protein in a group of patients with ovarian cancer (p < or = 0.0001). Supplementation with vitamin E after a cycle of therapy with toxic drugs caused a decrease of the cysteine peptidases activities, that is 2.8-fold in patients to whom 40 0mg of vitamin E per day was given in comparison with control, and 6-fold after the third course. The CPI and DCPI complex increased 3-fold and 2.3 fold respectively, as compared to a group of patients were vitamin E was not administered. We observed that vitamin E administered to the patients with ovarian cancer in periods between anticancer drugs therapy courses decreases the cysteine peptidases activity and increases the enzyme-inhibitor complexes level     

Variant Cathepsin L Activity from Gastric Cancer Tissue

Cathepsin L activity was partially purified by S-Sepharose FF chromatography, concanavalin-A Sepharose chromatography, phenyl-Superose column chromatography, Mono S column chromatography, and TSK G3000SWXL column chromatography from gastric cancer tissue. The optimal pH of cathepsin L from gastric cancer tissue was 7.4, and the activity was retained even at alkaline pH. Heat stability tests showed that cathepsin L from gastric cancer tissue was heat stable; that is, 65% activity was retained after incubation at 56°C for 60 min. The molecular weight of cathepsin L from gastric cancer tissue was estimated as 115 kD by gel filtration or 110 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme showed a different affinity for wheat germ agglutinin-Sepharose than cathepsin L from gastric normal mucosa. These results suggest that cathepsin L from gastric cancer tissue may play an important role in gastric cancer invasion through the destruction of the surrounding extracellular matrix by its proteolytic activity.     

Inhibition of proteinase-like peptidase activities in serum and tissue from breast cancer patients

The inhibitory profiles of several proteinase-like peptidases active on synthetic peptide (MCA) substrates, present in sera and 100,000g supernatants of malignant tissue from patients with breast cancer, have been studied using a series of known inhibitors including epoxysuccinyl peptides (E-64, Ep-475), Z-Phe-Phe-diazomethane, PMSF, iodoacetamide, 1-10-O-phenanthroline, leupeptin, aprotinin, elastatinal and alpha 2-macroglobulin. While in general the inhibition profiles confirmed reported substrate specificities some anomalies were observed. In particular, the serum activities on two cathepsin B substrates were unaffected by specific cysteine proteinase inhibitors and in breast tissue only 20-37% of activity towards these two substrates was apparently due to the presence of endopeptidases. However, the potent inhibition of other proteinase-like activities by the epoxysuccinyl peptides and leupeptin, or similar inhibitors, may be useful agents in the study of methods of combating tumour spread.     

miRNA-340和cyclin D1在胃癌组织中的表达及临床意义

目的:探讨胃癌组织中miRNA-340（miR-340）和cyclin D1的表达水平,并分析二者与胃癌患者临床病理参数的关系。方法:选取2019年12月至2020年06月期间我院病理科胃癌手术切除组织及对应癌旁正常胃黏膜组织(距离肿瘤边缘≥5 cm)共60例。采用qRT-PCR检测组织中miR-340的表达水平,采用免疫组化染色检测cyclin D1蛋白表达水平,并分析二者与胃癌患者临床病理参数的关系。结果:胃癌组织中miR-340的表达水平（2.38±0.51）明显低于癌旁正常黏膜组织（2.70±0.54）,差异具有统计学意义（P＜0.05）;胃癌组织中cyclin D1蛋白的表达水平（48.33％）明显高于癌旁正常黏膜组织（16.67％）,差异具有统计学意义（P＜0.05）。胃癌组织中miR-340表达与cyclin D1蛋白表达呈负相关（r=-0.367,P＜0.05）。胃癌组织中miR-340的表达与肿瘤直径、肿瘤的分化程度、浸润深度、临床分期有关（P＜0.05）,cyclin D1蛋白的表达与肿瘤的分化程度、浸润深度、临床分期、神经侵犯及淋巴结转移有关（P＜0.05）。结论:胃癌组织中miR-340和cyclin D1蛋白表达异常,且与不良预后相关,二者联合有望成为胃癌诊疗的潜在生物标志物。     

Enhanced production of matrix metalloproteinases and activation of matrix metalloproteinase 2 (gelatinase A) in human gastric carcinomas

We examined the production and tissue localization of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in gastric carcinoma tissues. MMP-I (tissue collagenase), MMP-9 (gelatinase B) and TIMP-2 were immunolocalized in carcinoma cells and MMP-2 (gelatinase A) on tumor cell membranes, whereas no or little immunostaining for MMP-3 (stromelysin-1) and TIMP-I was seen in carcinoma cells. Stromal cells in carcinoma tissue were also positively stained for these MMPs and TIMPs. MMP-2 immunostaining was observed exclusively on advanced gastric carcinoma cells and correlated with vascular invasion by tumor cells. Sandwich enzyme immunoassays revealed enhanced production of MMP-1, MMP-2, MMP-3, MMP-9 and TIMP-I by carcinoma tissues. Gelatinolytic activities were significantly higher in carcinoma samples than in normal controls. Using gelatin zymography, active forms of MMP-2 and MMP-9 were more frequently detected in carcinoma tissue, and the activation rate of the zymogen of MMP-2 (proMMP-2), but not that of proMMP-9, correlated well with degree of local invasion and lymphatic permeation. Our data indicate an enhanced production of 4 MMPs in gastric carcinoma tissue and suggest that activation of pro-MMP-2 may be a key step for spreading of gastric carcinoma cells. © 1996 Wiley-Liss, Inc.     

Effects of combined in vivo treatment of transplantable solid mammary carcinoma in wistar rats using vitamin E and cysteine peptidase inhibitors from human placenta

Cysteine cathepsin B and its endogenous inhibitor play an important role in tumor progression. Increase in cathepsin B expression and reduced levels of its inhibitors were associated with tumor malignancy in breast cancer. The objective of this study was to investigate the effects of a new therapy combining vitamin E and placental inhibitor on the level of endogenous protease inhibitor in sera and tumor tissues with mammary cancer. The inhibitor was used in doses of 100 and 200 micrograms per animal for 8 days. Vitamin E was added after the last treatment with inhibitor and was injected daily in doses of 10 and 20 mg per animal for one mouth. The size and survival time of treated animals as well as cathepsin B and the inhibitor activity in tumor and sera before and after treatment in comparison with the control groups were determined. The activity of cathepsin B significantly decreased both in tumor tissues and in sera (P < or = 0.0001). Cathepsin B activity in tumor tissue homogenates and in sera decreased two-fold and three-fold, respectively, after the animals were treated with vitamin E at a dose of 20 mg, and decreased five-fold and 15-fold, respectively, when treated with vitamin E plus inhibitor in comparison with untreated animals. Endogenous inhibitor activity increased six-fold and 12-fold in the sera and tissue homogenates, respectively, after the animals were treated with 200 micrograms of cysteine protease inhibitor plus 20 mg of vitamin E, in comparison with untreated animals. The total cure responses were higher in eight of 10 rats, as compared with untreated animals. The combination of placental inhibitor and vitamin E resulted in a significant reduction in breast metastasis and might provide a therapeutic basis for anti-metastasis therapy.     

Post-Translational Regulation of Cathepsin B, but not of Other Cysteine Cathepsins, Contributes to Increased Glioblastoma Cell Invasiveness In Vitro

Cells that migrate away from a central tumour into brain tissue are responsible for inefficient glioblastoma treatment. This migratory behaviour depends partially on lysosomal cysteine cathepsins. Reportedly, the expression of cathepsins B, L and S gradually increases in the progression from benign astrocytoma to the malignant glioblastoma, although their specific roles in glioma progression have not been revealed. The aim of this study was to clarify their specific contribution to glioblastoma cell invasion. The differences between the matrix invading cells and non-invading core cells from spheroids derived from glioblastoma cell culture and from glioblastoma patients’ biopsies, and embedded in type I collagen, have been studied at the mRNA, protein and cathepsin activity levels. Analyses of the two types of cells showed that the three cathepsins were up-regulated post-translationally, their specific activities increasing in the invading cells. The cystatin levels were also differentially altered, resulting in higher ratio of cathepsins B and L to stefin B in the invading cells. However, using specific synthetic inhibitors and silencing strategies revealed that only cathepsin B activity was involved in the invasion of glioblastoma cells, confirming previous notion of cathepsin B as tumour invasiveness biomarker. Our data support the concept of specific roles of cysteine cathepsins in cancer progression. Finally the study points out on the complexity of protease regulation and the need to include functional proteomics in the systems biology approaches to understand the processes associated with glioma invasion and progression.     

胃癌组织Trx-1、FN1表达及其与临床病理特征和患者生存的关系

目的探讨胃癌组织硫氧还蛋白-1(Trx-1)、纤维连接蛋白1(FN1)表达及其与临床病理特征和患者生存的关系。方法收集接受胃癌根治术切除标本105例,并切取距离肿瘤边缘5 cm以上的癌旁组织作为对照,通过免疫组化检测胃癌及癌旁组织中Trx-1、FN1的表达情况,分析Trx-1、FN1表达与临床病理特征及生存的关系。结果胃癌组织中Trx-1、FN1均呈高水平表达(P<0.05),癌旁组织中Trx-1、FN1呈低表达(P<0.05);Trx-1表达与胃癌组织分化、浸润深度有关(P<0.05),FN1表达与胃癌组织分化、浸润深度、淋巴结转移有关(P<0.05);随访105例中,3年总生存率为58.10%,Trx-1高表达组3年生存率为45.00%,低于低表达组的75.56%(P<0.05);FN1高表达组3年生存率为46.88%,低于低表达组的75.61%(P<0.05)。结论Trx-1、FN1在胃癌组织中均呈高表达,与胃癌的侵袭、发展相关,可能参与了肿瘤恶化的调控,并影响患者生存情况。     

Role of cysteine endopeptidases in cancerogenesis

Role of cysteine endopeptidases in cancerogenesis steps: neoplastic transformation, invasion and metastasis is reviewed and discussed. Positive correlation between tumor invasiveness, as well as its metastatic potential and secretion of cysteine endopeptidase (particularly cathepsins B and L) has been documented well in literature. Based on our recent results we postulate that serum endopeptidase-like activity could be used as a marker of cancer aggressiveness in diagnostic procedures in oncology. We also propose that the cysteine endopeptidase inhibitor levels (total, active and latent) could be useful factors for recognising the activation of the organism self-defence mechanisms against cancer. In addition, our idea of use of urinary cysteine peptidase inhibitors (UCPI) as potential anticancer agents is presented and discussed.     

Enrichment of collagen and gelatin degrading activities in the plasma membranes of human cancer cells

Interactions between connective tissue substrates and proteinases localized to the surface of cancer cells are implicated in cancer invasion. In this report we have compared the enrichment of collagen and gelatin degrading activities and cysteine proteinase(s) in well-characterized (enzyme markers and electron microscopy) subcellular membrane fractions isolated from human small cell lung cancer lines (NCI-H69 and NCI-H82) and the RWP-1 pancreatic cancer line. With each cell line collagenolytic, gelatinolytic, and cysteine proteinase activities were enriched 5- to 128-fold in the plasma membrane fractions with differences noted between microvilli versus smooth membrane profiles. Incubation of tumor plasma membranes with methyl-3H-labeled collagen resulted in extensive degradation of the gamma, beta, alpha 1, and alpha 2 chains, suggesting the combined action of metalloproteinases. Treatment of tumor plasma membranes with the chaotropic agent, 2 M KCl, did not diminish membrane collagen- or gelatin-degrading activity, but extensively leached out the cysteine proteinase, suggesting that the latter enzyme is not an integral membrane protein. Enzyme inhibitors specific for metalloproteinases and cysteine proteinase were used to corroborate enzymatic classification. In conclusion, we have demonstrated variations in the localization of proteinases in the plasma membrane domains of different human cancer cells.     

Immunostained cathepsins B and L correlate with depth of invasion and different metastatic pathways in early stage gastric carcinoma

BACKGROUND: With the recent development of minimal treatment for early stage gastric carcinoma, identifying specific indicators of the metastatic potential of primary tumors has become more important. Cathepsin B and cathepsin L, both lysosomal cysteine proteases, degrade the extracellular matrix during tumor progression. Although many studies have shown their relation to human cancer progression, little is known about their roles in the early stage. The clinicopathologic significance of cathepsins was therefore studied in early stage gastric carcinoma. METHODS: Expression of both cathepsins was studied immunohistochemically in 51 tissue specimens from gastric carcinomas that invaded the submucosal layer or muscularis propria. The relation between their expression and clinicopathologic factors was analyzed. RESULTS: Both cathepsins were expressed at higher levels in tumors that invaded the muscularis propria than in those within the submucosa (P < 0.05). In addition, tumors with lymphatic invasion showed higher cathepsin B expression than those without it (P < 0.05), whereas tumors with venous invasion showed higher cathepsin L expression than those without it (P < 0.05). No other clinicopathologic factors correlated with expression of either cathepsin. CONCLUSIONS: Tumors with overexpression of cathepsins have powerful potential for invasiveness in the early stage of gastric carcinoma. Moreover, the authors hypothesize that cathepsins may be one of the determinants of the metastatic route. To the authors' knowledge, this is the first report on specific proteases concerning the mode of metastasis, and the results of this study suggest that therapeutic strategies for early stage gastric carcinoma might need to be changed according to the status of cathepsins.     

基质金属蛋白酶-2和组织蛋白酶D与胃癌生物学行为及腹膜转移的关系

目的:探讨基质金属蛋白酶-2(matrixmetalloproteinase-2,MMP-2)和组织蛋白酶D(cathepsinD,CD)在胃癌组织中的表达与胃癌生物学行为、腹膜转移的关系。方法:应用免疫组织化学方法检测55例胃癌MMP-2和CD表达情况。其中45例行腹腔脱落细胞学检查。结果:MMP-2和CD与浸润深度、胃浆膜分型、腹腔脱落癌细胞和临床分期密切相关(P<0.01);CD与淋巴结转移显著相关(P<0.01),MMP-2与淋巴结转移无明显关系,但淋巴结转移阳性者MMP-2表达率高于淋巴结转移阴性者。结论:MMP-2和CD高表达胃癌具有更强的侵袭、淋巴结转移能力及腹膜转移倾向。MMP-2和CD能较好的反映胃癌的恶性生物学行为,有助于判断预后。结转移显著相关(P<0.01),MMP-2与淋巴结转移无明显关系,但淋巴结转移阳性者MMP-2表达率高于淋巴结转移阴性者。结论:MMP-2和CD高表达胃癌具有更强的侵袭、淋巴结转移能力及腹膜转移倾向。MMP-2和CD能较好的反映胃癌的恶性生物学行为,有助于判断预后。     

INPP4B mRNA 及蛋白在胃癌患者外周血中的表达及意义

目的：探讨 INPP4B mRNA 及蛋白在胃癌患者外周血中的表达水平,并分析其表达水平与临床病理参数（TNM分期、淋巴结转移和浸润深度）的关系。方法：应用实时荧光定量 PCR（QRT －PCR）及酶联免疫吸附实验（ELISA）测定50例胃癌患者及30例健康志愿者外周血 INPP4B mRNA 及蛋白的表达水平。结果：胃癌患者外周血中 INPP4B mRNA 比健康志愿者外周血中 INPP4B mRNA 的表达水平显著下降（P ＜0．05）,IN-PP4B 蛋白在胃癌患者外周血中的浓度[（845．118±293．560）ng／L],显著低于健康志愿者外周血中的浓度[（2089．204±603．829）ng／L]（P ＜0．05）。INPP4B mRNA 及蛋白的表达水平在胃癌外周血中与 TNM分期、淋巴结转移和浸润深度相关（P ＜0．05）。结论：外周血中 INPP4B mRNA 及蛋白的表达与胃癌关系密切,可作为反映胃癌发生、发展过程的有效分子指标。     

Clinical features of cystatin A expression in patients with pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy known, with an extremely poor prognosis due to the lack of an efficient diagnostic scheme and no radical treatment option, except surgery. Therefore, understanding the pathophysiology of, and finding a novel biomarker to detect, PDAC should be prioritized. We observed an increase in mRNA expression of the cysteine protease inhibitor cystatin A (CSTA) in CD4⁺ T cells in peripheral blood cells of nine patients with PDAC, compared with the expression in seven healthy volunteers. Moreover, we confirmed significantly higher CSTA mRNA expression in a larger cohort of 41 patients with PDAC compared with that in 20 healthy volunteers. Correspondingly, the serum CSTA concentrations in 36 patients with PDAC were higher than those in 37 healthy volunteers, and this increase was correlated with PDAC clinical stage. Furthermore, the expression of CSTA and cathepsin B, which is a lysosomal cysteine protease inhibited by CSTA, was observed in tumor tissues and tumor‐infiltrating immune cells in 20 surgically resected PDAC tissues by immunohistochemical staining. Expression of CSTA was detected in some tumor tissues and many tumor‐infiltrating immune cells. Cathepsin B expression was also observed in most tumor tissues and tumor‐infiltrating immune cells. In conclusion, CSTA and its substrate cathepsin B are involved in PDAC‐related inflammation. The increment of CSTA expression in peripheral blood of patients with PDAC may have a potential role as a PDAC immunopathologic biomarker.     

长链非编码RNA HOTAIR在胃癌中的表达及临床意义

目的:探讨长链非编码RNA HOTAIR在胃癌及癌旁组织中的表达及其与临床病理特征之间的关系。方法:利用实时定量PCR法检测55例胃癌组织及其配对癌旁正常组织中长链非编码RNA HOTAIR的表达情况,并分析其与胃癌临床病理学特征之间的联系。结果:实时定量PCR法检测HOTAIR在胃癌组织中的相对表达量为0.0232±0.0073,其配对癌旁正常组织相对表达量为0.0071±0.0017,差异具有统计学意义（P＜0.05）。长链非编码RNA HOTAIR的表达与胃癌患者肿瘤淋巴结转移、远处转移、TNM分期显著相关（P＜0.05）,与年龄、性别、分化程度等无关（P＞0.05）。结论:长链非编码RNA HOTAIR在胃癌组织中表达显著高于癌旁组织,与胃癌转移、TNM分期等病理因素相关,可能在胃癌的侵袭、转移过程中起重要作用。     

The expression of lysosomal proteinases and their inhibitors in breast cancer: Possible relationship to prognosis of the disease

Proteolytic enzymes have been proposed as new biological prognostic indicators to facilitate decisions about treatment of breast cancer patients following surgery. We reported earlier that the activities of cysteine proteinases (CP), cathepsin (Cat) B and cathepsin (Cat) L and the expression of stefin A might be associated with breast tumor progression and prognosis. Here, the protein concentrations of Cats D, B and L and stefin A have been measured in a series of 60 matched pairs of breast tumours and control adjacent tissues, using ELIS As developed in our laboratory. Median tumor concentrations of Cat D (47 pm/mg), Cat B (222 ng/mg) and Cat L (88 ng/mg) were significantly (p<0.0005) increased by 7 fold, 27 fold and 6 fold, respectively. Much greater increases in the activities of Cat B (63 fold) and of Cat L (274 fold) were found, indicating enhanced activation of cysteine proteinases in tumors, due either to proteolytic activation of proCat B and proCat L and/or to a decrease in specific endogenous cystatins. However, the 1.6-fold decreased (p<0.0001) levels of inhibition by cystatins could not be entirely responsible for more than 100-fold increased ratio of CP:cystatins activity. Moreover, stefin A was either increased or decreased in tumor samples, resulting in a 1.4-fold median increase in tumors. Comparing the biological parameters with the established histo-pathological prognosticators, we found that the increased protein concentration of Cat B was associated with lymph node involvement (p<0.009) and higher stage (p<0.003), and both Cat B and Cat L activities were more increased in high grade tumours (p<0.05). Survival analysis revealed that stefin A was the most significant prognostic factor for disease-free (p<0.008) and overall survival (p<0.02), followed by increased Cat B activity and protein concentration. Cat L was of borderline significance while Cat D was not significant for prognosis. We conclude that enhanced activation of CP, due partially to an imbalance between cysteine proteinases and inhibitors is linked to the progression of breast cancer. Larger sample size is needed to confirm the prognostic significance of stefin A, Cat B and Cat L.     

基质金属蛋白酶-7和E-钙黏附素在胃癌组织中的表达及其相关性

目的探讨基质金属蛋白酶．7（MMP．7）、E-钙黏附素（E—CD）在胃癌组织中的表达及其与胃癌侵袭、转移的相关性。方法采用原位杂交法,检测78例胃癌组织中MMP-7mRNA和E—CDmRNA的表达,观察二者与浸润深度和淋巴结转移之间的关系。结果胃癌组织中MMP-7mRNA和E-CDmRNA的阳性表达与浸润深度和淋巴结转移有关。MMP-7mRNA阳性表达的胃癌组织中E—CDmRNA的阳性表达率[9．09％（6／66）[低于MMP-7mRNA阴性表达的胃癌组织中E—CDmRNA的阳性表达率[66．70％（8／12）1,二者呈负相关（r=-0．269,P〈0．05）。结论MMP-7mRNA和E—CDmRNA的表达与胃癌的浸润、转移密切相关,且二者的表达呈负相关。检测MMP-7mRNA和E—CDmRNA在胃癌患者胃镜活检组织中的表达有助于预后判断。