Involvement of the mitogen-activated protein kinase kinase 2 in the induction of cell dissociation in pancreatic cancer

In our previous investigation, mitogen-activated protein kinase kinase 2 (MEK2) was detected as a factor which was correlated to the potential of invasion-metastasis. In this study, the immunocytochemical, immunohistochemical and mRNA expressions of MEK2 were examined in pancreatic cancer cell lines and tissue samples, respectively. Constitutive expressions of MEK2 and phosphorylated MEK (p-MEK) were observed in PC-1.0 and ASPC-1 cells, which exhibited a growth pattern of single cells, whereas the relevant expressions were quite faint in PC-1 cells and CAPAN-2 cells, which exhibited a growth pattern of island-like clonies. Simultaneous inductions of MEK2 expressions and cell dissociation were observed after the treatment with a conditioned medium (CM) of PC-1.0 cells. The expression of MEK2 and p-MEK were reduced and the cell aggregation was found in PC-1.0 and ASPC-1 cells after U0126 (a MEK inhibitor) treatment. In vivo, both the MEK2 and p-MEK overexpressed in human pancreatic cancer tissues and p-MEK was found to be more strongly expressed in the invasive front than that in the center of tumor (P<0.05). MEK2 is closely related to pancreatic cancer cell dissociation. MEK2 activation is probably involved in the first step of the cascade in the invasion-metastasis of pancreatic cancer.     

Prostaglandin E2 enhances pancreatic cancer invasiveness through an Ets-1-dependent induction of matrix metalloproteinase-2

Accumulating evidence suggests an important role for cyclooxygenase-2 (COX-2) in the pathogenesis of a wide range of malignancies. Here we tested the hypothesis that the COX-2 product prostaglandin E(2) (PGE(2)) increases cellular invasive potential by inducing matrix metalloproteinase-2 (MMP-2) expression and activity through an extracellular signal-regulated kinase (ERK)/Ets-1-dependent mechanism in pancreatic cancer. PANC-1 and MIAPaCa-2 pancreatic cancer cells were treated with PGE(2) or rofecoxib, a selective COX-2 inhibitor. MMP-2 expression and activity were assayed using Western blot analysis and zymography, respectively. MMP-2 promoter activity was analyzed with a luciferase-based assay. Ets-1 activity was analyzed using gel shift assay. Ets-1 expression was specifically silenced using RNA interference. Cellular invasive and migratory potentials were determined using a Boyden chamber assay with or without Matrigel, respectively. Exogenous PGE(2) induced MMP-2 expression and activity and increased ERK1/2 phosphorylation, Ets-1 binding activity, and MMP-2 promoter activity. PGE(2) also increased cellular migratory and invasive potentials. The mitogen-activated protein kinase kinase inhibitor PD98059 and Ets-1 silencing each abolished PGE(2)-induced increases in MMP-2 expression. PD98059 and Ets-1 silencing each abrogated the effect of PGE(2) on cellular invasive potential but not on cellular migratory potential. Rofecoxib suppressed MMP-2 expression and activity, Ets-1 binding activity, MMP-2 promoter activity, and cellular migratory and invasive potentials. These results suggest that PGE(2) mediates pancreatic cancer cellular invasiveness through an ERK/Ets-1-dependent induction of MMP-2 expression and activity. They also suggest that COX-2 inhibition may represent a strategy to inhibit invasive potential in pancreatic cancer.     

Claudin-23在胰腺癌细胞解离中的表达和定位变化

目的:探讨claudin-23表达和定位变化与胰腺癌细胞解离的关系。方法:利用RT-PCR、Western blot及免疫细胞化学方法观察胰腺癌细胞解离过程中claudin-23 mRNA和蛋白表达及其定位变化。结果:claudin-23 mRNA及蛋白在解离型高转移株胰腺癌细胞PC-1.0中低表达,而在非解离型低转移株胰腺癌细胞PC-1中高表达,表达MEK1-shRNA的PC-1.0亚细胞克隆中claudin-23 mRNA及蛋白表达增加,而在表达MEK2-shRNA的PC-1.0亚细胞克隆中表达变化不明显,但claudin-23蛋白定位发生明显变化。此外,表达MEK2-shRNA的PC-1.0亚细胞克隆呈岛样细胞克隆方式生长。与此相反,培养液上清中添加细胞解离因子后,claudin-23 mRNA和蛋白表达明显减少。结论:Claudin-23表达和定位变化与胰腺癌细胞解离状态密切相关,可能的分子机制为通过MEK信号转导通路调节claudin-23的表达和定位变化,从而调控胰腺癌的细胞解离。     

Gli1 contributes to the invasiveness of pancreatic cancer through matrix metalloproteinase-9 activation

The hedgehog (Hh) signaling pathway has been reported to be associated with the growth of pancreatic cancer, but its role in the invasive phenotype is poorly understood. Therefore, we investigated the role of the Hh pathway in pancreatic cancer cell invasiveness using a Matrigel invasion assay. Blockade of the Hh pathway by cyclopamine inhibited pancreatic cancer cell invasion in association with a decreased expression of matrix metalloproteinase (MMP)-9. By contrast, activation of the Hh pathway by the addition of exogenous Sonic hedgehog increased cell invasion and MMP-9 expression. Stable transfection of pancreatic cancer cells with Gli1 increased their invasiveness, which was associated with activation of MMP-9. We also showed that inhibition of MMP-9 by small interfering RNA blocked the increased invasiveness of Gli1-transfected cells. Furthermore, inhibition of Gli1 by small interfering RNA suppressed the invasiveness and MMP-9 expression of pancreatic cancer cells. Taken together, these findings suggest that members of the Hh pathway, especially Gli1, play an important role in the invasiveness of pancreatic cancer cells through the regulation of MMP-9 expression. ( Cancer Sci 2008; 99: 1377–1384)     

Claudin-23在胰腺癌细胞解离中的表达和定位变化

目的：探讨claudin-23表达和定位变化与胰腺癌细胞解离的关系。方法：利用RT—PCR、Western blot及免疫细胞化学方法观察胰腺癌细胞解离过程中claudin-23mRNA和蛋白表达及其定位变化。结果：claudin-23mRNA及蛋白在解离型高转移株胰腺癌细胞PC-1．0中低表达,而在非解离型低转移株胰腺癌细胞PC-1中高表达,表达MEK1-shRNA的PC-1．0亚细胞克隆中claudin-23mRNA及蛋白表达增加,而在表达MEK2-shRNA的PC-1．0亚细胞克隆中表达变化不明显,但claudin-23蛋白定位发生明显变化。此外,表达MEK2-shRNA的PC-1．0亚细胞克隆呈岛样细胞克隆方式生长。与此相反,培养液上清中添加细胞解离因子后,claudin-23mRNA和蛋白表达明显减少。结论：Claudin-23表达和定位变化与胰腺癌细胞解离状态密切相关,可能的分子机制为通过MEK信号转导通路调节claudin-23的表达和定位变化,从而调控胰腺癌的细胞解离。     

Prognostic value of matrix metalloproteinase-7 expression in patients with non-small cell lung cancer

The prognostic value of matrix metalloproteinase-7 (MMP-7) for survival of patients with non-small cell lung cancer (NSCLC) remains controversial. We performed a meta-analysis of the literatures to clarify its impact. Trials were selected for meta-analysis if they provided an independent assessment of MMP-7 in NSCLC and reported the analysis of survival data based on MMP-7 status. Pooled hazard ratio (HR) with 95 % confidence interval (95 % CI) was used to evaluate the associations between MMP-7 expression and survival of NSCLC patients. Heterogeneity and publication bias were also assessed. Seven studies involving 1,446 patients were identified. The combined HR for all studies was 1.28 (95 % CI 0.86–1.91; P?=?0.22). Subgroup analysis revealed that MMP-7 overexpression had a favorable impact on survival in Caucasians (HR?=?0.74; 95 % CI 0.55–0.99; P?=?0.043) but showed a poor survival prognosis in Asians (HR?=?1.74; 95 % CI 1.05–2.88, P?=?0.031). Its effect also appeared significant when the analysis was restricted to Asian patients with squamous cell cancer (HR?=?3.42; 95 % CI 1.92–6.11, P?=?0.000) and adenocarcinoma (HR?=?2.1; 95 % CI 1.34–3.29, P?=?0.001). Our meta-analysis suggests that there are ethnic differences in the clinical significance of MMP-7 expression for patients with NSCLC.     

Analysis of invasion-metastasis mechanism in pancreatic cancer: involvement of tight junction transmembrane protein occludin and MEK/ERK signal transduction pathway in cancer cell dissociation

Mitogen-activated protein kinase kinase 2 (MEK2) was detected as an invasion-metastasis related factor between highly invasive (PC-1.0) and weakly invasive (PC-1) pancreatic cancer cell lines in our previous study. On the other hand, tight junction (TJ) was found to be correlated with carcino-genesis and tumor development. In this study, the expressions and correlation of TJ transmembrane protein occludin and MEK/extracellular signal-regulated kinase (ERK) signaling pathway were analyzed to clarify the regulatory mechanism of cell dissociation in pancreatic cancer cells. Two hamster (PC-1.0 and PC-1) and human (AsPC-1 and CAPAN-2) pancreatic cancer cell lines were analyzed immunocytochemically with anti-occludin, phosphorylated MEK1/2 (p-MEK1/2), phosphorylated ERK1/2 (p-ERK1/2) antibodies. MEK1/2 inhibitor U0126 significantly induced the expression of occludin at the cell-cell junction and substantially suppressed the p-MEK1/2 and p-ERK1/2 expressions in PC-1.0 and AsPC-1 cells. In contrast, dissociation factor (DF) treatment obviously disrupted the occludin expressions at the sites of cell-cell junction and markedly induced the p-MEK1/2 and p-ERK1/2 expressions in PC-1 and CAPAN-2 cells. In addition, occludin expressions at cell-cell junction were restored and p-MEK1/2 and p-ERK1/2 expressions were suppressed by subsequent U0126-treatment in DF treated PC-1 and CAPAN-2 cells. Correspondingly, light microscopic images showed that DF induced the dissociation of cell island-like colonies in PC-1 and CAPAN-2 cells, and U0126-treatment induced cell aggregation in these pancreatic cancer cells. Occludin is involved in the cell dissociation in pancreatic cancer cells. Moreover, MEK/ERK signaling pathway probably regulates the cell dissociation status of pancreatic cancer through influencing the intracellular localization and expression of occludin.     

ＭＭＰ-７和ｕＰＡＲ在非小细胞肺癌的表达及其意义

目的：检测ＭＭＰ-１、ＭＭＰ-３、ＭＭＰ-７和尿激酶纤溶酶原激活物受体（ｕＰＡＲ）在非小细胞肺癌（ＮＳＣＬＣ）的表达水平,分析其表达水平与肺癌侵袭转移以及相关临床病理特征之间的关系。方法：免疫组织化学方法检测６６例ＮＳＣＬＣ和１０例正常肺组织中ＭＭＰ-１、ＭＭＰ-３、ＭＭＰ-７和ｕＰＡＲ的蛋白表达水平。结果：免疫组织化学检测显示ＮＳＣＬＣ组织ＭＭＰ-１和ＭＭＰ-３表达率较低,而ＭＭＰ-７和ｕＰＡＲ表达率较高,分别达到５５％和６１％,明显高于正常肺组织。ＭＭＰ-７的表达与ＮＳＣＬＣ淋巴结转移、分化程度以及预后密切相关;ｕＰＡＲ的表达与ＮＳＣＬＣ预后密切相关,与相关临床病理因素无关;ＭＭＰ-７和ｕＰＡＲ的表达呈正相关。结论：ＭＭＰ-７和ｕＰＡＲ的表达可作为ＮＳＣＬＣ患者预后评价的指标,可能成为抗肿瘤治疗的靶点。     

胰腺癌MMP-2和TIMP-2表达与预后的关系

目的:探讨胰腺癌组织基质质属蛋白酶-2(MMP-2)和基质金属蛋白酶抑制剂-2(TIMP-2)表达与肿瘤侵袭转移和预后的关系.方法:通过免疫组化方法检测35例胰腺癌病理标本和10正常胰腺标本MMP-2和TIMP-2的半定量表达情况及其与肿瘤临床病理参数和预后的关系,生存分析采用Kaplan-meier方法.结果:胰腺癌MMP-2和TIMP-2表达阳性率明显高于正常对照组,与淋巴结转移和临床分级密切相关,但与胰腺癌的分化程度无关,Kaplan-meier分析发现MMP-2和TIMP-2高表达患者的生存时间明显低于MMP 2和TIMP 2低表达患者(P<0.001).结论:胰腺癌MMP-2和TIMP-2表达与肿瘤侵袭转移和预后密切相关,有助于确定术后治疗方案.     

Tenascin-C stimulates glioma cell invasion through matrix metalloproteinase-12

The capacity of glioma cells to invade extensively within the central nervous system is a major cause of the high morbidity rate of primary malignant brain tumors. Glioma cell invasion involves the attachment of tumor cells to extracellular matrix (ECM), degradation of ECM components, and subsequent penetration into adjacent brain structures. These processes are accomplished in part by matrix metalloproteinases (MMP) within a three-dimensional milieu of the brain parenchyma. As the majority of studies have used a two-dimensional monolayer culture system, we have used a three-dimensional matrix of collagen type I gel to address glioma-secreted proteases, ECM, and invasiveness of glioma cells. We show that in a three-dimensional collagen type I matrix, the presence of tenascin-C, commonly elevated in high-grade gliomas, increased the invasiveness of glioma cells. The tenascin-C-mediated invasiveness was blocked by metalloproteinase inhibitors, but this did not involve the gelatinases (MMP-2 and MMP-9) commonly implicated in two-dimensional glioma growth. A thorough analysis of 21 MMPs and six members of a disintegrin and metalloproteinase domain showed that MMP-12 was increased in gliomas by tenascin-C in three-dimensional matrix. Furthermore, examinations of resected specimens revealed high MMP-12 levels in the high-grade glioblastoma multiforme tumors. Finally, a function-blocking antibody as well as small interfering RNA to MMP-12 attenuated the tenascin-C-stimulated glioma invasion. These results identify a new factor, MMP-12, in regulating glioma invasiveness through interaction with tenascin-C.     

Expression and prognostic value of matrix metalloproteinase-7 in colorectal cancer

The purpose of this study was to evaluate expression and prognostic value of matrix metalloproteinase-7 (MMP-7) in colorectal cancer (CRC) patients. CRC tissues and corresponding distal normal mucosa tissues of 118 CRC patients were assessed by immunohistochemistry. Correlations between MMP-7 expression, patients' clinic pathological features, and overall survival rate were analyzed. We found that positive expression of MMP- 7 in CRC tissues was significantly higher than that in distal normal mucosa (61.0% vs. 39.8%, p=0.001). Poor histological differentiation, advanced clinical stage and lymph node metastasis were significantly correlated with MMP-7 expression in CRC. The overall survival rate was significantly higher in the MMP-7 negative group than the positive group (Log-rank test=9.957, p=0.002). MMP-7 appeared as a significant independent prognostic factor through multivariate survival analysis. Collectively, we found MMP-7 expression to be correlated with progression and metastasis of CRC and thus could be used as a predictive marker of prognosis in CRC patients.     

基质金属蛋白酶-7在正常子宫内膜和子宫内膜癌组织中的表达

目的：研究MMP-7在正常子宫内膜和子宫内膜癌组织中的表达及其与临床病理特征之间的关系。方法：采用免疫组化和RT-PCR方法检测76例正常子宫内膜组织和41例子宫内膜癌组织中MMP-7蛋白及MMP-7mRNA表达水平。正常子宫内膜组织按28天月经周期分为增殖早期（EPn=10）、增殖中期（MPn=11）、增殖晚期（LPn=13）和分泌早期（Esn=11）、分泌中期（MSn=12）、分泌晚期（LSn=10）以及月经期（Mn=9）。统计方法采用秩和检验。结果：MMP-7蛋白主要定位于子宫内膜腺上皮细胞。MMP-7高表达于分泌晚期和月经期，低表达于分泌早、中期（u=2．13P=0．032；u=4．178P=0．0001）；子宫内膜癌组织中MMP-7表达随着肿瘤分化程度的降低、肌层浸润程度的加深和淋巴结的转移而逐渐升高。结论：正常月经周期中，MMP-7可能与子宫内膜的剥脱和重建有关；子宫内膜癌组织中，MMP-7参与肿瘤细胞的侵袭和转移。     

Matrix metalloproteinase-7 and matrix metalloproteinase-9 are associated with unfavourable prognosis in superficial oesophageal cancer

If oesophageal carcinoma is detected in the superficial stage, the prognosis is better than for advanced oesophageal carcinoma. But the factors which predict the prognosis and treatment policy remain unclear. Matrix metalloproteinase-7 (MMP-7) and matrix metalloproteinase-9 (MMP-9) have been reported to have close associations with tumour invasion and metastasis. In this study, we retrospectively studied the relations between MMP-7 and MMP-9 expression in immunohistochemistry, clinicopathologic factors, and prognosis in 55 superficial oesophageal carcinomas. MMP-7 and MMP-9 expression occurred in 23.6% and 47.3% of the patients, respectively. MMP-7 expression was significantly correlated with the presence of nodal metastasis (P=0.004). MMP-9 expression was significantly correlated with the depth of tumour invasion (P=0.004), lymphatic permeation (P=0.001), nodal metastasis (P=0.049), and pathologic differentiation grade (P=0.003). By the log-rank test, MMP-7 expression and MMP-9 expression on the invasive front were related to the prognosis. In multivariate analysis, MMP-9 expression on the invasive front was an independent prognostic indicator. The combined expression of MMP-7 and MMP-9 may be a good marker for the degree of malignancy of oesophageal cancer and for the presence of lymphatic metastasis.     

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。     

Survivin和MMP-7在结肠癌组织中的表达及临床意义

背景与目的：临床发现同一分期的结肠癌患者实行同一方案治疗预后不同,寻找能单独或结合临床病理分期更准确地判断患者预后的指标非常必要。本研究选择有争论的增殖转移相关蛋白生存素（survivin）、基质金属蛋白酶7（matrix metalloproteinase-7,MMP-7）,研究它们与结肠癌患者临床病理参数及预后的关系,为结肠癌的分子病理分期提供理论基础。方法：选取1995年1月至2003年5月在中山大学肿瘤防治中心住院手术的620例有完整随访资料的结肠癌患者临床资料及病理蜡块,应用组织芯片及免疫组化技术检测survivin、MMP-7表达,研究其与结肠癌患者临床病理参数及预后的关系。结果：本组病例survivin表达正常肠黏膜阳性率为0,而癌组织阳性率为41．0％．两者差异有统计学意义（P〈0．001）。MMP-7表达正常肠黏膜阳性率为40．9％,而癌组织阳性率为88．8％,两者差异有统计学意义（P〈0．05）。x^2检验分析结果显示survivin的表达各TNM分期之间差异有统计学意义（P〈0．05）,分期越晚阳性率越高。MMP-7的表达在患者的年龄、性别、肿瘤部位、肿瘤最大径、肿瘤分化程度、肿瘤大体、组织类型、TNM分期之间差异均无统计学意义（P〉0．05）。Cox单因素及多因素分析结果显示MMP-7阳性、survivin阳性是结肠癌患者肿瘤死亡的独立危险因素。结论：MMP-7、survivin均与结肠癌的发生相关,是影响结肠癌患者肿瘤死亡的独立危险因素。     

Prognostic impact of matrix metalloproteinase-9 in operable non-small cell lung cancer

We assessed the clinical impact of MMP-9 expression on long-term survival in patients with operable non-small cell lung cancer (NSCLC). Primary tumors of 143 consecutive patients with NSCLC resected completely and without overt distant metastases (pT1-4, pN0-2, M0, R0) were examined for MMP-9 expression using immunohistochemistry with a polyclonal, affinity-purified rabbit antibody that recognizes both latent and active MMP-9. Immunohistochemical staining of tumor cells was evaluated in comparison to normal bronchiolar epithelium that served as an internal positive control. MMP-9 expression was categorized into negative, 5% and <95% tumor cells stained; and homogeneous, >or=95% tumor cells stained at least as intensively as bronchiolar epithelium. The median follow-up period was 72 months (range = 12-144 months). Homogeneous expression of MMP-9 was observed in 26 of 143 patients (18.2%) and did not correlate with clinicopathological parameters. Relapse defined as diagnosis of distant metastasis or local recurrence was observed in 78 of the 130 (60%) patients eligible for clinical follow up analysis. Relapse led to cancer-related death in all of the 78 patients within the observation period. Kaplan-Meier analysis showed a significant association between homogeneous MMP-9 expression and shortened cancer-related survival (log-rank p = 0.016). Multivariate regression analysis including pT-status, pN-status, tumor histology and tumor grading showed an independence of this prognostic impact of homogeneous MMP-9 expression (p = 0.045). Thus, immunohistochemical evaluation of MMP-9 expression may provide a basis for the preselection of patients to be included in trials investigating specific protease inhibitor therapy after surgical resection of NSCLC.