Human cholangiocarcinomas express somatostatin receptors and respond to somatostatin with growth inhibition

Cholangiocarcinoma, a malignancy of biliary epithelia, is usually fatal because of absence of tests for early detection and lack of effective therapy. Somatostatin (SS) receptors are expressed in several malignancies and in rodent biliary epithelia. We tested the hypothesis that SS receptors are present in cholangiocarcinomas. We examined tissue from seven surgically resected human cholangiocarcinomas and a human bile duct cancer cell line for the messenger RNA for one subtype of SS receptors (SSTR2) and studied binding and growth-active properties of SS and its analogues. SSTR2 messenger RNA was expressed in all seven human cholangiocarcinoma specimens. Experiments with the human cholangiocarcinoma cell line showed specific, saturable binding of an SS analogue (MK-678) with high affinity for SSTR2 on cholangiocarcinoma membranes; inhibition in vitro of tumor cell proliferation by SS-14 and its analogue, octreotide; and inhibition in vivo of tumor growth in athymic mice implanted with human cholangiocarcinoma cells and treated with lanreotide, another SS analogue. Experiments to elucidate a possible mechanism of growth inhibition by SS showed it was not through changes in cellular cyclic adenosine monophosphate or calcium levels. Using gamma camera imaging with an ¹¹¹In-SS analogue, we localized a histologically proven cholangiocarcinoma in a patient. These results suggest that SS analogues may be useful for diagnostic localization and treatment of biliary tract malignancies.     

Aminopeptidase N is involved in cell motility and angiogenesis: its clinical significance in human colon cancer

BACKGROUND & AIMS: The molecular basis of cell motility is highly complex and is controlled by a number of molecular systems, whereas angiogenesis is an important biological component of tumor progression. The aims of this study were to investigate the possible involvement of proteins at the cell surface in controlling cell motility and angiogenesis, and to identify the cell surface molecules involved in gastrointestinal tumors. METHODS: We addressed these issues using functional monoclonal antibodies, which inhibit cell motility, endothelial cell migration, and tube formation. Furthermore, we investigated the relationship between this antigen and colon cancer, and showed the prognostic significance in human colon cancer. RESULTS: We established a murine monoclonal antibody MH8-11, which inhibits cell motility and in vitro angiogenesis. This epitope was a 165-kilodalton protein, and the sequencing analysis revealed that it was almost identical to aminopeptidase N (APN)/cluster of differentiation (CD) 13. APN/CD13 expression was associated with tumor status (P = 0.025). The disease-free and overall survival rate for patients with positive APN/CD13 expression tumors was significantly lower than that for patients with negative APN/CD13 expression tumors (P = 0.014, 0.033, respectively). Among 47 node-positive patients, the survival rate of patients with negative APN/CD13 expression was better than that of those with positive APN/CD13 expression. CONCLUSIONS: Our data suggest that APN/CD13 is involved in cell motility and angiogenesis, and APN/CD13 expression may be a useful indicator of a poor prognosis for node-positive patients with colon cancer.     

Bone morphogenetic protein 4 expressed in esophagitis induces a columnar phenotype in esophageal squamous cells

BACKGROUND & AIMS: Barrett's esophagus (BE) is a metaplastic condition in which normal squamous esophageal epithelium is replaced by columnar epithelium. It is proposed that one of the possible mechanisms is dedifferentiation of squamous epithelium into columnar epithelium. The pathophysiology through which this metaplasia occurs is unknown. A recent study by serial analysis of gene expression showed that bone morphogenetic protein 4 (BMP-4) is uniquely expressed in BE. In this study, the role of the BMP pathway in the metaplastic transformation of normal squamous cells into columnar cells was examined. METHODS: Tissues from patients with esophagitis and BE and in an esophagitis-BE rat model were examined for the activation of the BMP pathway. Short-term cultures of primary normal squamous esophageal cells were treated with BMP-4, and cell biological changes were examined by Western blot analysis, immunohistochemistry, and microarrays. RESULTS: In both human and rat tissues, the BMP pathway proved to be activated in esophagitis and BE. Upon incubation of squamous cell cultures with BMP-4, the cytokeratin expression pattern showed a shift that was consistent with columnar epithelium. Involvement of the BMP pathway was suggested by up-regulation of Phosphorylated-Smad 1/5/8 (P-Smad 1/5/8) that was effectively blocked by Noggin, a BMP antagonist. Comparison of the gene expression profiles of squamous cells, BMP-4-treated squamous cells, and BE cells showed a significant shift in the profile of the BMP-4-treated squamous cells toward that of the cultured BE cells. CONCLUSIONS: These results suggest that the BMP pathway could play a role in the transformation of normal esophageal squamous cells into columnar cells.     

畸胎瘤细胞源性生长因子和血管内皮生长因子在食管鳞癌中的表达及临床意义

目的 探讨食管鳞癌(ESCC)中畸胎瘤细胞源性生长因子(PCDGF)、血管内皮生长因子(VEGF)的表达与肿瘤临床病理参数之间的关系,明确PCDGF和VEGF在血管生成中的作用.方法 以免疫组化方法检测郑州大学第一附属医院2005年7月至2006年5 月收治的50例食管鳞癌患者手术切除标本PCDGF与VEGF的表达,并以CD105抗体标记肿瘤组织血管内皮细胞,计算肿瘤间质微血管密度(MVD).结果 食管鳞癌中PCDGF、VEGF的表达较正常食管上皮明显增加(P＜0.01);PCDGF和VEGF与肿瘤的浸润深度、TNM分期和淋巴结转移呈正相关(P均＜0.05);PCDGF、VEGF的表达与MVD值呈显著正相关(P＜0.01);PCDGF的表达与VEGF的表达呈正相关(P＜0.05).结论 PCDGF标记癌组织的敏感性较高,有望成为一种新的食管鳞癌肿瘤标志物.食管鳞癌中PCDGF、VEGF的表达与血管生成关系密切,可能通过促进肿瘤新生血管生成参与肿瘤的生长、浸润和转移.     

Correlation of matrix metalloproteinase suppressor genes RECK, VEGF, and CD105 with angiogenesis and biological behavior in esophageal squamous cell carcinoma

AIM： To explore the expression of reversion inducing cysteine-rich protein with Kazal motifs （RECK）, vascular endothelial growth factor （VEGF） and endoglin （CD105） protein and its correlation with occurrence, development, invasion and metastasis in esophageal squamous cell carcinoma （ESCC）. METHODS： Streptavidin-peroxidase （SP） immunohistochemistry was used to detect expression of RECK and VEGF in 62 cases of ESCC, 31 cases of adjacent atypical hyperplastic epithelium and 62 cases of normal esophageal epithelium. CD105 Mb was used to assess microvessel density （MVD）. RESULTS： The expression of RECK was closely correlated with histological grade, infiltrative depth and lymphatic metastasis in ESCC （P 〈 0.05）. The expression of RECK decreased during cancer development： normal esophageal epithelium （85.5%, 53/62）, adjacent atypical hyperplastic epithelium （71.0%, 22/31）, and carcinoma （59.7%, 37/62）. There was a significant difference among the groups （P 〈 0.05）. The expression of VEGF protein was closely correlated with infiltrative depth and lymphatic metastasis in ESCC （P 〈 0.05）. The expression of VEGF protein increased during cancer development： normal esophageal epithelium （29.0%, 18/62）, adjacent atypical hyperplastic epithelium （54.8%, 17/31）, and carcinoma （67.7%, 42/62）. There was a significant difference among the groups （P 〈 0.05）. MVDCD105 increased in accordance with histological grade, butthere was no significant difference （grade Ⅰ, 36.92 ± 10.85; grade Ⅱ, 37.65 ± 9.50; and grade Ⅲ, 38.06 ± 12.19）. The MVDCD105 was closely correlated with infiltration and lymphatic metastasis in ESCC （P 〈 0.05）. The expression of RECK was inversely correlated with the expression of VEGF and CD105.CONCLUSION： RECK, VEGF and CD105 play important roles in the infiltration, metastasis and carcinogenesis in esophageal carcinoma. Angiogenesis in ESCC may be promoted by over-expression of CD105.     

食管鳞状细胞癌中VEGF与TIMP-2、MVD、树突状细胞的关系及对浸润转移的影响

目的探讨与VEGF相关的TIMP-2表达、血管形成、肿瘤免疫异常对食管鳞癌浸润转移的影响.方法运用原位杂和免疫组织化学方法检测46例食管鳞癌组织中VEGF蛋白和mRNA的表达、TIMP-2的表达、CD34标记的微血管密度、S-100标记的肿瘤浸润性树突状细胞的密度.结果食管鳞癌组织中VEGF蛋白阳性表达率为58.7%(27/46),其阳性表达与浸润深度、转移状态有关;TIMP-2表达与分化、浸润深度有关,VEGF与TIMP-2之间没有相关关系;VEGF表达阳性组MVD(50.71±12.16/单位面积)显著高于阴性组(31.74±17.93/单位面积);食管鳞癌中S-100 树突状细胞平均密度与分化程度、浸润深度的有关,VEGF表达与树突状细胞密度之间存在显著的负相关性(r=-0.462).结论VEGF蛋白表达具有促进食管鳞癌浸润转移的作用,免疫组化方法较原位杂交更适合临床作为判断食管鳞癌浸润转移潜能的手段;TIMP-2可抑制食管鳞癌的浸润,其表达与VEGF表达之间无相关关系;MVD值可作为判断食管鳞癌浸润、转移及预后的参考指标;VEGF影响鳞癌组织中树突状细胞的密度,进而可能影响宿主的抗肿瘤免疫能力和肿瘤的浸润转移过程.     

癌胚抗原相关细胞黏附分子1和血管内皮生长因子在HBV相关性肝癌血管生成中的作用

目的 研究癌胚抗原相关细胞黏附分子1 (CEACAMl)和血管内皮生长因子(VEGF)在HBV相关性肝癌组织中的表达与HBV相关性肝癌血管生成的关系.方法 采用免疫组织化学SP法检测81例HBV相关性肝癌组织中CEACAM1、VEGF的表达,光镜下计数CD105标记的微血管密度(MVD).结果 81例HBV相关性肝癌组...     

食管鳞癌中微小核糖核酸-21的作用及对靶蛋白的影响

目的 探讨微小RNA(miRNA)-21对食管鳞癌细胞Eca109和哈萨克族食管癌的促增殖作用及对程序性细胞死亡基因4(PDCD4)表达的影响。方法将食管鳞癌细胞系Eca109分为miRNA-21 Mimics组（转染序列5′-UCAACAUCAGUCUGAUAAGCUA-3′）、miRNA-21抑制剂组（转染序列5′-UAGCUUAUCAGACUGAUGUUGA-3′）、阴性对照组（转染随机序列）和正常对照组（不予转染等任何处理）。细胞按4.5×105个/孔接种于6孔板,基于RNA干扰(RNAi)技术、使用脂质体2000试剂进行细胞转染。采用细胞计数法检测转染后各组细胞的增殖情况。使用实时荧光定量聚合酶链反应(qRT-PCR)检测各组细胞的miRNA-21转录水平。采用Western印迹技术检测转染后各组PDCD4蛋白表达情况。检测18对哈萨克族食管癌及其对应的癌旁正常食管组织中miRNA-21转录和PDCD4蛋白表达情况。结果 转染48 h后,与正常对照组比较,miRNA-21Mimics组Eca109细胞数增加36％(P=0.002),miRNA-21抑制剂组细胞数减少28％(P=0.002),阴性对照组细胞数变化不明显(P=0.515)。转染48 h后,miRNA-21抑制剂组、miRNA-21 Mimics 组、阴性对照组、正常对照组miRNA-21的相对表达量分别为0.37±0.10、9.17±1.08、0.74±0.23和1.04±0.34,PDCD4蛋白相对表达量分别为1.47±0.11、0.61±0.09、0.89±0.12、0.79±0.02。与正常对照组比较,miRNA-21抑制剂组miRNA-21相对表达量下降(P=0.031)、PDCD4蛋白相对表达量上调（P=0.001）,miRNA-21 Mimics组miRNA-21相对表达量上升(P=0.001)、PDCD4相对表达量下调(P=0.030),阴性对照组则差异无统计学意义（P值分别=0.272和0.541）。18对哈萨克族食管癌组织中16对的miRNA-21相对表达量(0.11±0.09)高于其对应的癌旁正常食管组织(0.03±0.03,P=0.001),PDCD4蛋白相对表达量（0.92±0.39）低于其对应的癌旁正常食管组织（1.57±0.80,P=0.004）,且癌组织中miRNA-21相对表达水平越高,PDCD4蛋白相对表达水平越低（r=-0.538,P=0.046）。结论miRNA-21可能通过抑制PDCD4蛋白表达而促进食管鳞癌细胞Eca109增殖,并且参与哈萨克族食管癌的发生。     

The role of matrix metalloproteinase-7 in redefining the gastric microenvironment in response to Helicobacter pylori

BACKGROUND & AIMS: Interactions between epithelial and stromal cells are important determinants of mucosal organization, but the signaling mechanisms are understood incompletely. Matrix metalloproteinase (MMP)-7 is produced uniquely in epithelia, may act on growth factors and matrix proteins, and in the stomach is increased with Helicobacter pylori infection. We have studied the role of MMP-7 in signaling between epithelial cells and a key stromal cell type, the myofibroblast. METHODS: Immunohistochemistry and Western blotting were applied to gastric corpus biopsy specimens; primary cultures of human gastric glands and myofibroblasts were used to study the role of MMP-7 in regulating proliferation and migration of the latter, and MMP-7 substrates were identified by proteomic methods. RESULTS: Increased abundance of the myofibroblast marker alpha-smooth muscle actin was identified in H. pylori-positive biopsy specimens. Media from H pylori-infected gastric epithelial cultures stimulated proliferation and migration of primary human gastric myofibroblasts and antisense oligonucleotide treatment indicated a role for MMP-7. Proteomic methods identified insulin-like growth factor binding protein (IGFBP)-5 as a substrate for MMP-7 in medium from gastric myofibroblasts. Knockdown of IGFBP-5 by small interfering RNA or immunoneutralization of IGF-II, abolished myofibroblast responses to MMP-7. Proliferation of gastric epithelial cells also was stimulated by MMP-7-treated myofibroblasts via IGF-II. CONCLUSIONS: MMP-7 acts as an epithelial-derived signal increasing the bioavailability of IGF-II released from myofibroblasts. Because IGF-II acts on both stromal and epithelial cells, the findings suggest that increased MMP-7 expression contributes to redefining the niche occupied by dividing cells and leading to hyperproliferation in H pylori infection.     

Expression and association of CD44v6 with prognosis in T2-3N0M0 esophageal squamous cell carcinoma

Aim

To investigate the expression of CD44v6 in stage T2-3N0M0 esophageal squamous cell carcinoma (ESCC) and its prognostic significance.

Methods

The expression of CD44v6 in a series of 227 ESCC specimens was evaluated by immunohistochemistry (IHC). A reproducible semiquantitative method which took both staining percentage and intensity into account was applied for IHC scoring, and receiver operating characteristic (ROC) curve analysis was utilized to select the cut-off score for high or low IHC reactivity. Then, the correlations of CD44v6 expression with clinicopathological features of patients and its prognostic relevance were determined.

Results

In the present study, the proportion of low CD44v6 expression was found significantly lower in Grade 3 of ESCC, than that of Grade 1 and Grade 2 of ESCC. There are no significant correlations between CD44v6 expression and other clinicopathological parameters including gender, age, tumor size, tumor location, depth of invasion and pathological stage. The Kaplan-Meier survival curves showed that up-regulated expression of CD44v6 indicated a poorer post-operative survival for ESCC patients of stage T2-3N0M0 (P=0.009), especially for those with T2 lesions (P=0.044) or with stage IIB diseases (P=0.005). Multivariate analysis also confirmed that CD44v6 expression [relative risk, 1.639; 95% confidence interval (CI): 1.142-2.354, P=0.007] and depth of invasion (relative risk, 1.487; 95% CI: 1.063-2.080, P=0.020) were independent prognostic factors.

Conclusions

Elevated CD44v6 expression may be an adverse prognostic indicator for patients with stage T2-3N0M0 ESCC, especially for those with T2 lesions or stage IIB diseases.     

Congestive heart failure induces endothelial cell apoptosis: protective role of carvedilol

OBJECTIVES

The purposes of this study were to determine whether the serum of patients with congestive heart failure (CHF) can induce apoptosis of endothelial cells and to elucidate the underlying mechanisms. Moreover, the effect of the beta-blocker carvedilol was investigated.

BACKGROUND

Congestive heart failure is associated with impaired endothelial function in the peripheral systemic vasculature and with systemic release of inflammatory cytokines. Pro-inflammatory cytokines have been shown to induce endothelial cell apoptosis in vitro. Therefore, we hypothesized that CHF is associated with enhanced apoptosis of endothelial cells.

METHODS

Human umbilical vein endothelial cells were exposed to the serum of patients with CHF (n = 15) or healthy volunteers (n = 11), and apoptosis was determined by fluorescence staining of the nuclei and demonstration of deoxyribonucleic acid laddering. Moreover, apoptotic membrane particles were detected in plasma samples of patients with CHF.

RESULTS

The serum of patients with CHF revealed a significantly enhanced pro-apoptotic activity as compared with age- and gender-matched healthy volunteers (p < 0.001). Furthermore, patients with CHF revealed significantly elevated plasma concentrations of apoptotic membrane particles. Apoptosis of endothelial cells correlated with elevated tumor necrosis factor-alpha (TNF-alpha) (r = 0.585, P = 0.002) and soluble TNF receptor serum levels (r = 0.517, P = 0.007). Carvedilol completely suppressed the increase in apoptosis induced by the serum of patients with CHF. Moreover, carvedilol dose-dependently inhibited TNF-alpha–induced apoptosis. The antiapoptotic activity of carvedilol was mediated by reduced activation of the caspase cascade through inhibition of mitochondrial cytochrome c release. The suppression of apoptosis by carvedilol was due to its antioxidative rather than beta-blocking effects, as the analogue BM91.0228, which has no beta-blocking activity, exerted similar effects.

CONCLUSIONS

These findings indicate that endothelial cell apoptosis may play a role in the pathophysiology of heart failure. Inhibition of endothelial cell apoptosis by carvedilol may contribute to the beneficial effects of carvedilol in patients with heart failure.     

Characterization of the IGF axis in a rat liver-derived epithelial cell line

We have used the techniques of chemical cross-linking, Western blotting and immunoprecipitation-ligand blotting to demonstrate that insulin-like growth factor binding protein-2 (IGFBP-2) is associated with plasma membranes of an epithelial cell line derived from rat liver as well as being secreted into the medium by these cells. We demonstrate that these cells secrete IGF-I, but not IGF-II into serum free medium. Evidence from signalling, cell proliferation and cross-linking experiments indicate that these cells also express cell surface IGF-I receptors. Dose-response experiments indicate an enhanced biological activity of the IGF-I analogue des (1-3) IGF-I compared to wild-type IGF-I in both acute signalling experiments and longer-term (24 h) mitogenic assays. As this IGF-I analogue has lower affinity for IGFBPs, we believe that in this cell culture system, activity of IGF-I may be attenuated in the long and short term by the accumulation of IGFBP-2 in conditioned medium and by the presence of IGFBP-2 associated with the cell membrane and/or ECM.     

Uridine triphosphate (UTP) induces profibrotic responses in cardiac fibroblasts by activation of P2Y2 receptors

Cardiac fibroblasts (CFs) play a key role in response to injury and remodeling of the heart. Nucleotide (P2) receptors regulate the heart but limited information is available regarding such receptors in CFs. We thus sought to determine if extracellular nucleotides regulate fibrotic responses (e.g., proliferation, migration and expression of profibrotic markers) of CFs in primary culture. UTP increased rat CF migration 3-fold (p < 0.001), proliferation by 30% (p < 0.05) and mRNA expression of profibrotic markers: alpha smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), transforming growth factor beta, soluble ST2, interleukin-6 and monocyte chemoattractant protein-1 (MCP-1) by 3.0-, 15-, 2.0-, 7.6-, 11-, and 6.1-fold, respectively (p < 0.05). PAI-1 protein expression induced by UTP was dependent on protein kinase C (PKC) and extracellular signal-regulated kinase (ERK), based on blockade by the PKC inhibitor Ro-31-8220 and the ERK inhibitor U0126, respectively. The rank order for enhanced expression of PAI-1 and α-SMA by nucleotides (UTPγS> > UDPβS> > ATPγS), the expression of P2Y2 receptors as the most abundantly expressed P2Y receptor in rat CFs and a blunted response to UTP in P2Y2^-/- mice all implicate P2Y2 as the predominant P2Y receptor that mediates nucleotide-promoted profibrotic responses. Additional results indicate that P2Y2 receptor-promoted profibrotic responses in CFs are transient, perhaps as a consequence of receptor desensitization. We conclude that P2Y2 receptor activation is profibrotic in CFs; thus inhibition of P2Y2 receptors may provide a novel means to diminish fibrotic remodeling and turnover of extracellular matrix in the heart.