KAI1基因转染对乏氧培养下胰腺癌MiaPaCa-2细胞增殖、迁移及VEGF表达的影响

目的 观察转染KAI1基因的人胰腺癌MiaPaCa-2细胞在乏氧条件下培养后细胞增殖、迁移、侵袭能力的变化,探讨其可能机制.方法 应用KAl1基因过表达质粒转染乏氧条件培养后的人胰腺癌MiaPaCa-2细胞,采用蛋白质印迹法检测转染细胞KAI1、VEGF-C、VEGF-A蛋白的表达,四甲基偶氮唑蓝（MTT）法检测转染细胞的增殖,细胞划痕及Transwell小室实验观察转染细胞的迁移及侵袭能力,酶联免疫吸附测定法检测培养上清液中人VEGF-C、VEGF-A含量.结果 转染KAI1基因后的MiaPaCa-2-K细胞的KAI1蛋白表达量较未转染细胞显著增加[（0.549 ±0.021）比0].乏氧条件培养后转染细胞的增殖无明显变化,但它的迁移距离明显缩短,穿膜细胞数显著减少[（14.0±5.8）比（43.0±14.4）个,P＜0.05];细胞的VEGF-C表达显著降低[（0.218±0.043）比（0.745±0.069）.P＜0.05],但VEGF-A表达变化不显著;细胞培养上清液中VEGF-C含量显著减少[（1236±247）比（2045±221） pg/ml,P＜0.01].结论 转染KAl1基因的MiaPaCa-2细胞在乏氧条件下培养后的细胞迁移、侵袭能力减弱,其机制可能是通过下调VEGF-C的表达来抑制胰腺癌淋巴转移的.     

星形细胞肿瘤组织中VEGF—A、VEGF—C的表达变化及其与肿瘤血管生成的关系

目的观察血管内皮生长因子（VEGF）．A和VEGF—C在星形细胞肿瘤组织中的表达变化,分析两指标间及与肿瘤血管生成的关系。方法留取93例星形细胞肿瘤患者肿瘤组织及9例接受高血压脑出血开颅手术患者的正常脑组织,用免疫组织化学（sP）方法检测VEGF-A、VEGF-C蛋白表达和微血管密度（MVD）,RT-PCR法检测VEGF．A、VEGF．CmRNA表达,对两VEGF间及与肿瘤WHO分级、MVD的关系进行统计学分析。结果星形细胞肿瘤组织中VEGF．A、VEGF．C蛋白阳性表达率及MVD均显著高于正常脑组织,且均随肿瘤恶性程度增高而增强（P均〈0．叭）;星形细胞肿瘤组织中VEGF—A、VEGF-C蛋白表达及两者与MVD均呈显著相关（P均〈0．01）。星形细胞肿瘤组织中VEGF—A和VEGF．CmRNA表达均显著高于正常脑组织,且均随肿瘤恶性程度增高而增强（P均〈0．05）;肿瘤组织中VEGF．A和VEGF．CmRNA表达呈显著相关（P均〈0．05）。结论VEGF—A、VEGF—C在星形细胞肿瘤组织中的表达上调并与肿瘤恶性程度有关,两者间及与肿瘤血管生成均关系密切。     

表没食子儿茶素没食子酸酯对常氧及低氧状态下胰腺癌细胞的作用

目的:探讨表没食子儿茶素没食子酸酯(EGCG)对在常氧及低氧诱导下人胰腺癌细胞株BxPC-3的增殖、凋亡的影响及其相关基因的表达。方法:采用氯化钴(CoCl2)建立低氧模型,在常氧及低氧状态下,研究不同浓度EGCG对人胰腺癌细胞株BxPC-3的作用。采用四唑氮蓝(MTT)还原法检测细胞活力;流式细胞仪检测细胞凋亡;逆转录聚合酶链反应(RT-PCR)检测细胞基质金属蛋白酶2(MMP-2)和血管内皮生长因子A(VEGF-A)mRNA水平的表达变化;蛋白质印迹法(Western-blot)检测MMP-2和VEGF-A蛋白水平的表达变化。结果:低浓度的EGCG短时间内对BxPC-3细胞的生长无显著抑制作用,但随着作用时间的延长和剂量的增加,EGCG显示了细胞生长抑制作用;流式细胞仪结果显示EGCG可诱导胰腺癌细胞凋亡,低氧状态下其抑制作用低于常氧状态。EGCG可显著抑制MMP-2和VEGF-A蛋白的表达,下调MMP-2和VEGF-A mRNA的表达。结论:EGCG无论在常氧或低氧环境下均可以显著抑制胰腺癌BxPC-3细胞的生长,促进其凋亡,低氧状态下其抑制作用低于常氧状态下,提示在临床上胰腺癌化学治疗效果差可能与其肿瘤内低氧状态有关。其可能相关机制与下调VEGF、MMP-2表达水平有关。     

Vascular endothelial growth factor 165b expression in stromal cells and colorectal cancer

AIM:To characterize the implications of vascular endothelial growth factor(VEGF)-A in stromal cells and colorectal cancer and the expression of VEGF-A splice variants.METHODS:VEGF-A expression in tumor and stromal cells from 165 consecutive patients with colorectal cancer was examined by immunohistochemistry.The association between VEGF-A expression status and clinicopathological factors was investigated.Twenty freshfrozen samples were obtained for laser capture microdissection to analyze the splice variant...     

The importance of angiogenesis markers in the outcome of patients with diffuse large B cell lymphoma: a retrospective study of 97 patients

Purpose The role of angiogenesis has been extensively evaluated in solid tumors and more recently in hematologic malignancies. Several surrogate markers of angiogenesis including tumor VEGF, VEGF receptors, and microvessel density have correlated with outcome in some lymphoma studies. This is a single institution retrospective study evaluating the role of angiogenesis markers in the clinical outcome of patients with diffuse large B cell lymphoma (DLBCL). Patients and methods A total of 97 patients with DLBCL diagnosed and managed at Indiana University between 1993 and 2001 were included. Archived tumor samples were stained for VEGF-A, VEGF-C, VEGF-R1, and CD31 and graded as negative or positive (1+, 2+, 3+). The relationship between the expression of these markers and the international prognostic variables as well as the progression free survival (PFS) and the overall survival (OS) was evaluated. Results VEGF-A, VEGF-C, VEGF-R1 were expressed in 77, 98, and 18% of tumors, respectively. VEGF-A negative patients had an improved OS compared to VEGF-A (1+) (P = 0.0502). VEGF-C correlated with both LDH (r = 0.28, P = 0.0502) and IPI score (r = 0.25, P = 0.013). VEGF-R1 negative patients had a superior survival compared to those with VEGF-R1 (2+) (P = 0.0154). Conclusions The presence of tumor associated angiogenesis may alter the outcome of patients with DLBCL and could be a prognostic factor. Further clinical studies are needed to correlate the degree of angiogenesis with response to anti-angiogenesis agents. Supported in part by funds from Indiana University Cancer Center, Indianapolis, IN, USA.     

VEGF-D expression correlates with colorectal cancer aggressiveness and is downregulated by cetuximab

AIM： To gain mechanistic insights into the role played by epidermal growth factor receptor （EGFR） in the regulation of vascular endothelial growth factors （VEGFs） in colorectal cancer （CRC）. METHODS： The impact of high-level expression of the growth factor receptors EGFR and VEGF receptor （VEGFR）3 and the VEGFR3 ligands VEGF-C and VEGF-D on disease progression and prognosis in human CRC was investigated in 108 patients using immu- nohistochemistry. Furthermore, the expression of the lymphangiogenic factors in response to the modulation of EGFR signalling by the EGFR-targeted monoclonal antibody cetuximab was investigated at the mRNA and protein level in human SW480 and SW620 CRC cell lines and a mouse xenograft model. RESULTS： Human CRC specimens and cell lines displayed EGFR, VEGF-C and VEGF-D expression with varying intensities. VEGF-C expression was associated with histological grade. Strong expression of VEGF-D was significantly associated with lymph node metas- tases and linked to a trend for decreased survival in lymph node-positive patients. EGFR blockade with cetuximab resulted in a significant decrease of VEGF-D expression in vitro and in vivo. CONCLUSION： In conclusion, the expression of VEGF-D in colorectal tumours is significantly associated with lymphatic involvement in CRC patients and such expression might be blocked effectively by cetuximab.     

Altered expression and localization of the tight junction protein ZO-1 in primary and metastatic pancreatic cancer

INTRODUCTION: ZO-1 is a tight junction membrane protein that plays a critical role in cell-cell interaction, proliferation, and differ entiation. AIM: To localize and evaluate the expression of ZO-1 in the normal human pancreas, in pancreatic ductal adenocarcinoma (PDAC), and in chronic pancreatitis (CP). METHODOLOGY AND RESULTS: Northern and Western blot analysis revealed ZO-1 expression in all six tested pancreatic cancer cell lines. Expression of ZO-1 mRNA was increased sixfold in PDAC samples in comparison with normal samples (p = 0.04). Confocal microscopy revealed the presence of ZO-1 in the apical and apicolateral areas of ductular cells in the normal pancreas. Similarly, in CP, ZO-1 was localized at apical and apicolateral areas of small proliferating ductular cells and large metaplastic ducts. In PDAC, however, ZO-1 expression was observed irrespective of whether the cancer cells formed duct-like structures or exhibited a diffuse infiltrating pattern. Metastatic pancreatic cancer cells within lymph nodes displayed variable staining patterns, ranging from apical and apicolateral to a diffuse membranous staining. CONCLUSION: These observations suggest that ZO-1 is overexpressed in PDAC and raise the possibility that this overexpression may confer a metastatic advantage to pancreatic cancer cells.     

Accelerated lymphangiogenesis in malignant lymphoma: possible role of VEGF-A and VEGF-C

There is little information regarding the lymphangiogenesis of malignant lymphoma. In this study, we evaluated the lymphangiogenesis and angiogenesis in 44 lymph nodes of 39 malignant lymphomas and five non-reactive normal lymph nodes, based on the lymphatic vessel density (LVD) and microvessel density (MVD) calculated by the computer-assisted assessment of vessel density. The LVD of malignant lymphomas was significantly higher than that of non-reactive normal lymph nodes, irrespective of subtypes (P = 0.00077). On the contrary, there was no difference in MVD between malignant lymphomas and non-reactive normal lymph nodes, except for diffuse large B cell lymphomas, which had a significantly low value of MVD, in comparison with non-reactive normal lymph nodes (P = 0.009). We further examined the expression of vascular endothelial growth factor (VEGF)-C and VEGF-A, which function on lymphangiogenesis in lymph node samples. VEGF-C was expressed in 36 of 39 malignant lymphomas. All 39 of the malignant lymphoma samples expressed VEGF-A. Furthermore, the level of LVD and VEGF-A or VEGF-C was positively correlated. These findings suggest that lymphangiogenesis is actively developed in lymph nodes of malignant lymphomas and it may be induced by both VEGF-A and VEGF-C secreted from lymphoma cells.     

Measurement of circulating levels of VEGF-A, -C, and -D and their receptors, VEGFR-1 and -2 in gastric adenocarcinoma

AIM： TO analyze the serum levels and prognostic significance of vascular endothelial growth factor （VEGF） -A, -C, and -D, and their receptors, VEGFR-1 and -2 in gastric adenocarcinomas. METHODS： The serum levels of VEGF family members were measured in 76 control subjects and 76 patients with gastric adenocarcinoma using an enzyme-linked immunosorbent assay （ELISA）. These measurements were correlated with clinco-pathological features and survival rates. RESULTS： The serum levels of VEGF-A and its receptor, VEGFR-1, were significantly higher in patients with gastric cancer than in healthy donors （t = 2.3, P = 0.02 and t = 4.2, P 〈 0.0001, respectively）. In contrast, the serum levels of VEGF-D were significantly higher in control subjects than in patients （t = 2.9, P = 0.004）. There was no significant difference in serum levels of VEGF-C and VEGFR-2 between patients and controls. VEGF-C was associated with advanced tumor stage and presence of metastasis. VEGFR-1 was associated with metastasis, advanced overall stage,tumor differentiation and survival. VEGFR-2 levels were associated with poor tumor differentiation. There was no significant prognostic value for any of the VEGF family members or their receptors except for VEGFR-1 where high levels were associated with a poor overall survival. CONCLUSION： Serum VEGF levels vary significantly in the same cohort of patients with variable clinicopathological features and prognostic values. The simultaneous measurement of VEGF receptors levels in sera may overcome the limitations of a single biomarker assay.     

Metastasis-associated protein 1 induces VEGF-C and facilitates lymphangiogenesis in colorectal cancer

AIM:To study the correlation between high metastasisassociated protein 1(MTA1)expression and lymphangiogenesis in colorectal cancer(CRC)and its role in production of vascular endothelial growth factor-C(VEGF-C). METHODS:Impact of high MTA1 and VEGF-C expression levels on disease progression and lymphovasculardensity(LVD,D2-40-immunolabeled)in 81 cases of human CRC was evaluated by immunohistochemistry. VEGF-C mRNA and protein expressions in human LoVo and HCT116 cell lines were detected by real-time polymer...     

胰岛素经VEGF-A/VEGFR2途径促进恒河猴视网膜血管内皮细胞的血管新生

用人胰岛素处理恒河猴视网膜血管内皮细胞系RF/6A,测定其增殖、迁移、管腔形成情况和血管内皮生长因子A(VEGF-A)及其受体(VEGFR)的表达与磷酸化.与空白对照组相比,胰岛素促进RF/6A细胞增殖、迁移和管腔形成(均P＜0.01)、促进VEGF-A mRNA的表达和蛋白的活性(均P＜0.05);促进VEGFR2 mRNA的表达和蛋白的磷酸化(均P＜0.01),而对VEGFR1 mRNA的表达影响无统计学意义(P＞0.05)     

Study on relationship between angiogenesis and micrometastases of peripheral blood in breast cancer

Purpose  To investigate the relationship between microvessel density, expression of vascular endothelial growth factor A (VEGF-A) and micrometastases in peripheral blood of patients with breast cancer. Method  Microvessel density (MVD) and expression of VEGF-A were detected by immunohistochemistry S-P. Nested RT-PCR was introduced to detect the expression of hMAM mRNA in peripheral blood of all cases. Result  Average MVD was 28.95 ± 6.95 microvessels/100× and positive rate of VEGF-A was 64.0% (32/50) in 50 cases with breast cancer. MVD count and expression of VEGF-A were related to tumor size, metastasis of axillary lymph nodes and clinical stages (P < 0.05), independent of age and histological classification (P > 0.05). The positive rate of hMAM mRNA in peripheral blood was 34.0% (17/50), which correlated with lymphatic metastasis and clinical stages (P < 0.05), independent of pathological category, menopause and hormone receptor (P > 0.05). MVD count and positive rate of VEGF-A in breast cancer with positive expression of hMAM mRNA was obviously higher than those without hMAM mRNA expression (χ ² = 5.766, P = 0.032; t = 5.37, P = 0.002). Conclusions  MVD count and positive expression of VEGF-A closely correlated to hMAM mRNA released from tumor cells in the circulation. hMAM mRNA is expected to become a valuable marker for further study on micrometastases of breast cancer.     

Suppression of heat shock protein 27 induces long-term dormancy in human breast cancer

The mechanisms underlying tumor dormancy have been elusive and not well characterized. We recently published an experimental model for the study of human tumor dormancy and the role of angiogenesis, and reported that the angiogenic switch was preceded by a local increase in VEGF-A and basic fibroblast growth factor. In this breast cancer xenograft model (MDA-MB-436 cells), analysis of differentially expressed genes revealed that heat shock protein 27 (HSP27) was significantly up-regulated in angiogenic cells compared with nonangiogenic cells. The effect of HSP27 down-regulation was further evaluated in cell lines, mouse models, and clinical datasets of human patients with breast cancer and melanoma. Stable down-regulation of HSP27 in angiogenic tumor cells was followed by long-term tumor dormancy in vivo. Strikingly, only 4 of 30 HSP27 knockdown xenograft tumors initiated rapid growth after day 70, in correlation with a regain of HSP27 protein expression. Significantly, no tumors escaped from dormancy without HSP27 expression. Down-regulation of HSP27 was associated with reduced endothelial cell proliferation and decreased secretion of VEGF-A, VEGF-C, and basic fibroblast growth factor. Conversely, overexpression of HSP27 in nonangiogenic cells resulted in expansive tumor growth in vivo. By clinical validation, strong HSP27 protein expression was associated with markers of aggressive tumors and decreased survival in patients with breast cancer and melanoma. An HSP27-associated gene expression signature was related to molecular subgroups and survival in breast cancer. Our findings suggest a role for HSP27 in the balance between tumor dormancy and tumor progression, mediated by tumor-vascular interactions. Targeting HSP27 might offer a useful strategy in cancer treatment.     

OPN和VEGF-C在胃癌中的表达及其临床意义

目的:评价骨桥蛋白(osteopontin,OPN)和血管内皮生长因子C(vascular endothelial growth factor C,VEGF-C)在胃癌中表达及与临床病理参数之间相关性,进一步探讨二者的共表达在胃癌淋巴结转移中的机制.方法:收集复旦大学附属中山医院手术切除胃癌组织标本及患者相关临床资料,对纳入研究的93例胃癌原发灶标本采用免疫组织化学染色法(EnVision二步法),检测OPN与VEGF-C蛋白的表达.结果:93例胃癌组织中OPN与VEGF-C的阳性表达率分别为64.5%(60/93)和69.9%(65/93),在非肿瘤性胃黏膜中均未见其阳性表达;OPN和VEGF-C的表达与胃癌浆膜侵犯、TNM分期以及淋巴结转移呈明显相关(P<0.05).蛋白之间的相关性分析显示,OPN与VEGF-C之间存在正相关(r=0.493,P<0.01).结论:联合检测OPN与VEGF-C有助于阐述胃癌发生发展、浸润转移的机制,OPN可能通过上调VEGF-C的表达促进胃癌的淋巴结转移.     

Clinicopathological and prognostic significance of galectin-1 and vascular endothelial growth factor expression in gastric cancer

AIM: To evaluate the expression of galectin-1 and vascular endothelial growth factor (VEGF) in gastric cancer and investigate their relationships with clinicopathologic factors and prognostic significance. METHODS: Galectin-1 and VEGF were immunohistochemically investigated in tumor samples obtained from 214 gastric cancer patients with all tumor stages. Immunohistochemical analyses for galectin-1 and VEGF expression were performed on formalin-fixed, paraffin-embedded sections of surgical specimens. The relationship between the expression and staining intensity of galectin-1 and VEGF, clinicopathologic variables, and patient survival were analyzed. All patients underwent follow-up until cancer-related death or more than five years after tumor resection. P values < 0.05 were considered statistically significant.RESULTS: Immunohistochemical staining demonstrated that 138 of 214 gastric cancer samples (64.5%) were positive for galectin-1, and 116 out of 214 gastric cancer samples (54.2%) were positive for VEGF. There was a significant association between galectin-1 and VEGF expression; VEGF was detected in 60.1% of galectin-1-positive samples and 43.4% of galectin-1-negative samples (P < 0.05). Galectin-1 expression was associated with tumor size, tumor location, stage, lymph node metastases, and VEGF expression (all P < 0.05). VEGF expression was related to tumor size, stage, and lymph node metastases (all P < 0.05). The 5-year survival rate was 56.6% for galectin-1-positive patients and 69.2% for galectin-1-negative patients, and the prognosis for galectin-1-positive patients was significantly poorer compared with galectin-1-negative patients (χ 2 = 13.880, P = 0.000). The 5-year survival rates for VEGF-positive and VEGF-negative patients were 53.4% and 70.5%, respectively (χ2 = 4.619, P = 0.032). The overall survival rate of patients with both galectin-1 and VEGF overexpression in gastric cancer tissue samples was significantly poorer than other groups (both P < 0.05).CONCLUSION: Galectin-1 expr     

Expression of vascular endothelial growth factor-C and angiogenesis in esophageal squamous cell carcinoma

AIM: To investigate the expression of vascular endothelial growth factor-c (VEGF-C) mRNA and microvessel density (MVD) in human esophageal squamous cell carcinoma (ESCC) and its relationship with clinical significance. METHODS: Specimens obtained from 43 patients undergoing surgical resection for ESCC were used in this study. The expression of VEGF-C mRNA was examined by in situ hybridization. Tumor MVD was determined immunohistochemically with anti-CD31 antibody and estimated by image analysis. Ten sections of adjacent normal mucosa were also examined. RESULTS: VEGF-C mRNA expression was detected in cytoplasm of carcinoma cells. Of the 43 ESCC patients studied, 18 cases (41.9%) were positive for VEGF-C mRNA. No VEGF-C mRNA expression was observed in normal esophageal mucosa. VEGF-C mRNA expression correlated significantly with lymph node metastasis, TNM stage and depth of invasion (P < 0.05). Furthermore, histological grade (differentiation) tended to correlate with VEGF-C mRNA expression, but was not statistically significant (P > 0.05). In tumor lesions, the MVD was significantly greater than that in normal esophageal mucosa. MVD correlated significantly with lymph node metastasis, TNM stage and depth of invasion (P < 0.05), but not with histological grade (differentiation) (P > 0.05). Lesions with VEGF-C mRNA expression had a significantly higher MVD than that of those without VEGF-C mRNA expression (P < 0.05). CONCLUSION: VEGF-C plays a role in lymphatic metastasis via lymphangiogenesis and angiogenesis in ESCC. VEGF-C is one of the important predictors of the biological behavior in ESCC.     

染色体缺失磷酸酶及张力蛋白同源物对胰腺癌细胞系细胞周期和增殖能力的影响

目的探讨10号染色体缺失的磷酸酶及张力蛋白同源物(PTEN)对胰腺癌细胞质(ASPC-1)血管内皮生长因子(VEGF)蛋白表达、细胞周期和增殖的影响.方法将质粒pEAK8-PTEN和pEAK8分别转染指数生长期的胰腺癌细胞株(ASPC-1),挑选阳性细胞克隆,扩增培养,用RT-PCR、Western印迹、流式细胞术、生长速率等方法分别检测PTEN对ASPC-1细胞VEGF蛋白、细胞周期及增殖能力的影响.结果ASPC-1细胞转染后,PTEN mRNA表达量是转染前的3倍;ASPC-1、ASPC-1-pEAK8、ASPC-1-pEAK8-PTEN细胞PTEN蛋白表达量分别为13.2、12.6和21.6,VEGF蛋白表达量分别为17.2、16.5和13.1,转染后较转染前降低.细胞周期显示,ASPC-1-pEAK8-PTEN细胞较ASPC-1、ASPC-1-pEAK8细胞G2/M、S期细胞增多,Gl期细胞减少,并出现少量凋亡细胞(P＜0.01).ASPC-1-pEAK8-PTEN细胞生长曲线平缓,生长速度较另两种细胞明显降低.结论 PTEN可使ASPC-1细胞VEGF蛋白表达下降,细胞阻滞在G2/M期,抑制肿瘤细胞增殖.