以腺相关病毒为载体的小干扰RNA对肝星状细胞中金属蛋白酶组织抑制因子的抑制作用

目的 观察以腺相关病毒（AAV）为载体含有针对大鼠金属蛋白酶组织抑制因子（TIMP）-1具有较强抑制作用的小干扰RNA（siRNA）感染大鼠星状细胞系HSC-T6后TIMP-1的表达抑制作用。方法 针对大鼠TIMP-1mRNA基因序列挑选一段22bp片段，在体外构建为短发夹siRNA（short hairpin siRNA，shRNA）表达载体后，将其包装为重组AAV并感染大鼠肝星状细胞系HSC-T6后，于感染后30d及90d应用荧光定量PCR方法及Westem blot方法分别检测TIMP-1 mRNA及蛋白质表达情况，同时通过PCR技术以感染后细胞的基因组DNA为模板扩增外源基因验证其长效表达。结果 经PCR、酶切及序列测定证实含有siRNA-TIMP-1基因的重组AAV载体质粒已成功克隆。将重组质粒包装成病毒后感染HSC-T6细胞，与对照组细胞相比，感染后30d及90d细胞TIMP-1 mRNA水平明显降低（P〈0．01），感染后30d TIMP-1蛋白表达水平较对照组细胞相比下降约60％，而感染后90d，TIMP-1蛋白表达几乎下降90％。PCR结果显示在重组病毒感染后90d细胞基因组DNA中仍可扩增出外源基因，证实外源基因可长期表达。结论 重组病毒rAAV／siRNA-TIMP-1／neo可长期有效地抑制TIMP-1基因的表达。     

Serum tissue inhibitor of metalloproteinase 1 (TIMP-1) and vascular endothelial growth factor A (VEGF-A) are associated with prognosis in esophageal cancer patients

PurposeThe matrix metalloproteinases, tissue inhibitors of metalloproteinases and angiogenesis contribute to growth and spread of cancer. We investigated the correlation between pretreatment serum levels of tissue inhibitor of metalloproteinase 1 (TIMP-1) and vascular endothelial growth factor A (VEGF-A), and clinicopathologic features and survival in patients with esophageal cancer (EC).Material/MethodsSerum TIMP-1 and VEGF-A were measured by enzyme-linked immunosorbent assay (ELISA) in 89 patients with EC, and 30 healthy controls.ResultsSerum TIMP-1 and VEGF-A levels were significantly higher in patients with esophageal carcinoma than in the control group (p=0.001 and p<0.001, respectively). High levels of TIMP-1 were associated with histological type (p<0.001), tumor depth (p<0.001), stage (p<0.001) and lymph node metastases (p=0.001). Subgroup analysis showed that tumor size (p<0.001), tumor depth (p<0.001), stage (p<0.001), lymph node metastases (p=0.002), distant metastases (p=0.009) and resectability (p=0.003), were correlated with an elevated level of VEGF-A. Patients with elevated levels of TIMP-1 and VEGF-A had a significantly lower overall survival (p=0.02 and p=0.048, respectively), and disease-free survival (TIMP-1, p<0.001).ConclusionHigh serum levels of TIMP-1 and VEGF-A were found to be associated with tumor progression and unfavorable prognosis in patients with EC.     

Cytoplasmic Pin1 expression is correlated with poor prognosis in colorectal cancer

Objective

The aim of this study was to determine the clinicopathological significance and prognostic role of Pin1 expression and subcellular localization in colorectal cancer (CRC).

Thrombospondin-1 (TSP-1) up-regulates tissue inhibitor of metalloproteinase-1 (TIMP-1) production in human tumor cells: Exploring the functional significance in tumor cell invasion

Thrombospondin-1 (TSP-1), a matrix-bound adhesive glycoprotein, has been shown to modulate tumor progression. We previously demonstrated that TSP-1 up-regulates matrix metalloproteinases MMP-2 and MMP-9. Our studies suggested that the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) is a key determinant in tumor cell invasion. We now report that TSP-1 up-regulates TIMP-1 expression in both human breast and prostate cancer cell lines. The effect of TSP-1 on TIMP-1 expression was examined in human breast adenocarcinoma cell lines (MDA-MB-231) and human prostate cancer cell lines (PC3-NI and PC3-ML) treated with exogenous TSP-1. TIMP-1 expression was also examined in TSP-1 stably transfected breast cancer cell line (MDA-MB-435). Northern and western blot analysis revealed TIMP-1 mRNA and TIMP-1 protein expression increased with increasing concentrations of TSP-1. This effect was inhibited by antibodies against the type I repeat domain of TSP-1 further suggesting that TSP-1 mediates TIMP-1 secretion. Inhibition of TSP-1 induced TIMP-1 levels increased tumor cell invasion. We conclude that TSP-1 is involved in influencing the critical balance between MMPs and their inhibitors, maintaining the controlled degradation of the extracellular matrix needed to support metastasis and our results may provide an explanation for the divergent activities reported for TSP-1 in tumor progression.     

The expression of BIRC5 is correlated with loss of specific chromosomal regions in breast carcinomas

Expression of BIRC5 (survivin), a member of the inhibitor of apoptosis protein (IAP) family, is elevated in fetal tissues and in various human cancers. Mechanisms up-regulating BIRC5 in cancer are poorly understood. Here, we show that overexpression of BIRC5 induces a high proliferation level in MCF-7 breast tumor cells. In a population of 191 breast carcinomas, BIRC5 expression is not affected by BIRC5 promoter polymorphism at -31, or BIRC5 gene copy number. However, a significant correlation was found between expression of demethylase (dMTase) and expression of BIRC5. In addition, among 13 chromosomal regions tested for allelic loss [loss of heterozygosity (LOH)], two regions close to D3S1478 and D6S264 were related to BIRC5 expression. In tumors with LOH at D3S1478 and/or D6S264, BIRC5 expression was significantly increased. These regions have been suggested to harbor tumor suppressor genes and/or common fragile sites that may play a role in increasing genetic instability. These results suggest that genes located near D3S1478 and D6S264 might work by inhibiting, directly or indirectly, BIRC5 expression and thus their loss leads to its up-regulation. In addition, BIRC5 expression may induce breast tumor proliferation by promoting genetic instability. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

Increased expression of tissue inhibitor of metalloproteinases type 1 (TIMP-1) in a more tumourigenic colon cancer cell line

Genetic changes occurring in the late stages of colonic tumour progression have received much less attention than those occurring in the early stages. As described in the accompanying paper, SW480 and SW620 cell lines provide a useful model for studying the advanced stages of progression for colon cancer. Comparison of the two cell lines by differential display reveals that SW620 cells express lower levels of the CC3 tumour suppressor gene and also lower levels of the tissue inhibitor of metalloproteinases-3 (TIMP-3) gene. Northern blot analysis for TIMP-3 confirms this finding and shows a similar difference in the expression of TIMP-2, which seems logical since TIMPs inhibit enzymes that play a role in tumour invasion. For this reason, it was surprising to find that TIMP-1 messenger RNA expression is markedly increased in SW620 cells. Consistent with this finding, western blot analysis shows a ten-fold increase in TIMP-1 protein secretion by SW620 cells. It is noteworthy that high TIMP-1 expression is associated with poor prognosis in colorectal cancer. This association between TIMP-1 expression and tumour progression may be related to additional growth factor-like effects described for TIMP-1 in some systems.     

Infiltrative capacity of T leukemia cell lines: A distinct functional property coupled to expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinases-1 (TIMP-1)

Infiltrative capacity was found to distinguish separate T leukemia cell lines. Of seven T-cell lines four exhibited capacity to infiltrate Matrigel. Analysis of infiltration was performed at the single-cell level throughout the Matrigel using a depth meter. Further, we examined differences in migration capacity and metalloproteinase production between infiltrating and non-infiltrating T-cell lines. The capacity to infiltrate was not directly correlated to the capacity to adhere to the Matrigel or to migrate on/to extracellular matrix components. It is concluded that infiltration capacity does not simply reflect capacity to migrate but represents a distinct functional property. The production of metalloproteinases and their inhibitors by the separate T-cell lines was analyzed using rt PCR, biosynthetic labelling, zymography, immunoprecipitation and ELISA. All T-cell lines with capacity to infiltrate produced matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) while non-infiltrating cell lines did not express MMP-9. Expression of MMP-1, 2, 3, 10, 14 and 17 showed no correlation to capacity to infiltrate. Analysis of infiltration in the presence of a metalloprotease inhibitor showed an increased number of cells within the gel. This enhancement of infiltration suggests that the function of MMPs and/or their inhibitors in lymphocyte infiltration is more complex than previously thought. This revised version was published online in July 2006 with corrections to the Cover Date.     

氧化型胆固醇下调血管平滑肌细胞金属蛋白酶组织抑制剂基因表达

目的:研究氧化型胆固醇对血管平滑肌细胞MMP-9及TIMP-1表达的影响。方法:离体培养兔主动脉血管平滑肌细胞,分别用胆固醇、Triol与25-OH负载细胞,狭缝杂交测定TIMP-1mRNA表达,细胞免疫化学测定MMP-9与TIMP-1蛋白表达。结果:Triol与25-OH(1 mg/L,24 h)抑制TIMP-1 mRNA及蛋白表达,对MMP-9蛋白表达无影响。结论:氧化型胆固醇可以下调血管平滑肌细胞TIMP-1基因表达。     

Coordinate regulation of matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 expression in human vascular smooth muscle cells

The expression of matrix metalloproteinases (MMPs) and their inhibitors (TIMPs) by human vascular smooth muscle cells (SMC) was monitored as a function of the phenotypic modulation in vitro. Cell phenotype was manipulated by varying serum concentration and cell density. Synthetic phenotype was characterized by a minimum expression of the contractile proteins and a maximal proliferation rate. Contractile phenotype was quiescent and expressed a maximal level of contractile proteins. Synthetic cells expressed the highest levels of both MMP-1 and TIMP-1 and displayed maximal collagenolytic activity. No significant change was detected in MMP-2 expression or catalytic activity. Enzyme immunoassays revealed that MMP-1 expression fell by 77+/-2.4-95+/-0.5%, and that of TIMP-1 by 34+/-0.5-59+/-1.9%, as the cells acquired a contractile phenotype. The level of the MMP-1/TIMP-1 complex was similarly reduced by 78+/-2.9-85+/-1.6%. These data demonstrate that the expression of MMP-1 and TIMP-1 are coordinately regulated with SMC phenotype.     

TC-1 (C8orf4) expression is correlated with differentiation in ovarian carcinomas and might distinguish metastatic ovarian from metastatic colorectal carcinomas

Thyroid cancer 1 (TC-1, C8orf4) is involved in the development of many cancers. In this study, we investigated the correlation between the expression of TC-1 and the clinicopathological characteristics of ovarian and colorectal adenocarcinomas. We also explored the possible use of TC-1 as a marker to distinguish between metastatic tumors of the ovary and colorectum. We used immunohistochemistry to examine the expression level of TC-1 in 100 ovarian and 100 colorectal adenocarcinomas and 25 metastatic carcinomas with the ovary or colorectum as primary site. TC-1 was expressed in all ovarian carcinoma samples. The high expression rate of TC-1 was 84 % in ovarian carcinomas, which was much higher than that observed in colorectal adenocarcinomas (35 %, P?<?0.001). High expression of TC-1 significantly correlated with poor differentiation of ovarian carcinomas (P?=?0.013). To explore the value of TC-1 in distinguishing metastatic ovarian cancers from colorectal cancers, we found the area under the receiver operator characteristic curve of TC-1 to be 0.819 (95 % confidence interval, 0.760–0.878; P?<?0.001). Furthermore, TC-1 was highly expressed in 100 % of nine metastatic ovarian cancers, but only in 31 % of 16 metastatic colorectal cancers. The higher expression of TC-1 in ovarian compared to colorectal adenocarcinomas suggests its potential use as a marker, to distinguish between metastatic ovarian and colorectal adenocarcinomas.     

High expression of tissue inhibitor of metalloproteinase-2 in serous ovarian carcinomas and the role of this expression in ovarian tumorigenesis

Tissue inhibitors of metalloproteinases (TIMPs) play key roles in maintaining homeostasis of the extracellular matrix by controlling matrix metalloproteinases (MMPs). In addition to their role in regulating MMPs, TIMPs have also been shown to have pluripotential effects on cell growth, apoptosis, and differentiation. The aim of this study was to evaluate TIMP-2 level in serous ovarian tumor tissues and to understand further the role of TIMP-2 protein in ovarian tumorigenesis. The expression of TIMP-2 was assessed by immunohistochemistry in a total of 57 ovarian specimens, including 5 normal ovaries, 12 benign serous cystadenomas, 20 serous borderline tumors, and 20 serous carcinomas. In addition, we transfected a TIMP-2 plasmid into the gynecologic cancer cell lines SKOV-3, 2774, and HeLa and then assayed cell growth, apoptosis, and MMP-2 activation. We found that TIMP-2 immunostaining was significantly more frequent in serous carcinomas, mainly in tumor epithelium, compared with cells of the other tissues studied. Tissue inhibitor of metalloproteinase-2 overexpression in ovarian cancer cells did not mediate proapoptosis, inhibited cisplatin-induced apoptosis, and induced MMP-2 expression. These findings suggest that TIMP-2 may function to favor tumor growth in serous ovarian tumorigenesis. Additional research is now needed to elucidate further the role of TIMP-2 in the biologic behavior of ovarian serous tumors.     

基质金属蛋白酶-9及其组织抑制物-1在人胎盘的表达及意义

目的研究基质金属蛋白酶-9(matrix metalloproteinases,MMP-9)及其组织抑制物-1(tissue inhibitor of metalloproteinases,TIMP-1)在不同孕期胎盘中的表达及与滋养层细胞侵蚀、子宫-胎儿血管系统建立的关系.方法应用原位杂交法检测56例胎盘(早孕16 例、中孕20 例、晚孕20例)中MMP-9及TIMP-1mRNA的表达.结果 MMP-9及TIMP-1mRNA主要表达于滋养细胞、绒毛间质血管壁及蜕膜组织中;MMP-9mRNA的表达早、中孕组明显强于晚孕组,TIMP-1mRNA的表达晚孕组明显强于早、中孕组,差异均有极显著性(P<0.01).结论 MMP-9及TIMP-1协同表达可能在滋养层细胞侵蚀、孕卵着床、血管重建过程中发挥一定的作用.     

Matrix metalloproteinase-1 is associated with poor prognosis in oesophageal cancer

The matrix metalloproteinases (MMPs) are a family of closely related proteolytic enzymes which are involved in the degradation of different components of the extracellular matrix. There is increasing evidence to indicate that individual MMPs have an important role in tumour invasion and tumour spread. Monoclonal antibodies specific for MMP-1, MMP-2, or MMP-9 have been produced, using as immunogens peptides selected from the amino acid sequences of individual MMPs. The presence of MMP-1, MMP-2, and MMP-9 in oesophageal cancer was investigated by immunohistochemistry on formalin-fixed, wax-embedded sections of oesophageal cancers. The relationship of individual MMPs to prognosis and survival was determined. MMP-1 was present in 24 per cent of oesophageal cancers, while MMP-2 and MMP-9 were present in 78 and 70 per cent of tumours, respectively. The presence of MMP-1 was associated with a particularly poor prognosis (log rank test 8·46, P<0·004) and was an independent prognostic factor (P=0·02). The identification of individual MMPs in oesophageal cancer provides a rational basis for use in the treatment of oesophageal cancer of MMP inhibitors which are currently undergoing clinical trial. © 1998 John Wiley & Sons, Ltd.