Inhibition of Cyclooxygenase‐2 Suppresses Lymph Node Metastasis via VEGF‐C

Most experimental work addressing cyclooxygenase‐2 (COX‐2) inhibitor has focused on suppressing hematogenic spread. Little is known about the mechanism by which this inhibitor can also block lymphatic metastasis. Here, the effects of COX‐2 inhibitor on vascular endothelial growth factor‐C (VEGF‐C) expression, lymphangiogenesis and lymph node metastasis were investigated. Utilizing the highly metastatic human lung adenocarcinoma cell line Anip973 and its parental line AGZY83‐a, which has a low metastatic capacity, we found elevated VEGF‐C and COX‐2 immunoreactivity in Anip973 cells compared with AGZY83‐a cells. Celecoxib down‐regulated expression of VEGF‐C mRNA and protein in Anip973 cells while PGE₂ up‐regulated expression of VEGF‐C mRNA and protein in AGZY83‐a cells in a concentration‐dependent manner. The expression of COX‐2 and VEGF‐C was significantly increased in xenografted Anip973 tumors compared with AGZY83‐a tumors. The Anip973 tumors showed more lymphatic vessels and lymph node metastasis than the AGZY83‐a tumors. In vivo, celecoxib decreased VEGF‐C expression in Anip973 tumor‐treated mice to a similar level to that in the AGZY83‐a tumor‐treated mice. Consistent with this decrease in VEGF‐C expression, the density of lymphatic vessels and lymph node metastasis in Anip973 tumor‐treated mice were suppressed to approximately that found in the AGZY83‐a tumor‐treated ones. Taken together, our results suggest that the differential expression of COX‐2 and VEGF‐C might help explain the different metastasis phenotype of lung adenocarcinoma cancer, and that COX‐2 inhibitor mediates VEGF‐C to block lymphangiogenesis and lymph node metastasis. Thus, COX‐2 may be a potential therapeutic target for blocking lymph node metastasis in lung adenocarcinoma. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

Expression of VEGF‐C and VEGF‐D as Significant Markers for Assessment of Lymphangiogenesis and Lymph Node Metastasis in Non‐Small Cell Lung Cancer

Vascular endothelial growth factor (VEGF)‐C and VEGF‐D induce lymphangiogenesis through activation of VEGF receptor 3 (VEGFR‐3) and have been implicated in tumor spread to the lymphatic system. Lymph node dissemination critically determines clinical outcome and therapeutic options of patients with non‐small cell lung cancer (NSCLC). However, the relationship of VEGF‐C, VEGF‐D, and lymph node metastasis in cancers, including NSCLC, is still controversial. To evaluate the relationship between lymphangiogenesis and lymph node metastasis, the expression of VEGF‐C and VEGF‐D in NSCLC tumors were detected by immunohistochemistry and quantitative real‐time polymerase chain reaction (QRT‐PCR). QRT‐PCR revealed that in marginal region VEGF‐C and VEGF‐D mRNA was significantly higher than in tumor center, and VEGF‐D mRNA was also higher than that in peritumoral lung tissue. Immunohistochemically, we observed the same heterogeneous expression of VEGF‐C and VEGF‐D proteins. The group with high expression of VEGF‐C and VEGF‐D in marginal region had a higher incidence of lymph node metastasis compared with the group with low expression. Furthermore, the group with high expression of VEGF‐D in marginal region had a higher incidence of lymphatic invasion. The group with high peritumoral lymphatic vessel density (LVD) had higher expression of VEGF‐C and VEGF‐D mRNA compared with the group with low peritumoral LVD. Our studies suggested that the expression of VEGF‐C and VEGF‐D at invasive edge was significantly associated with lymph node metastasis or lymphatic invasion in patients with NSCLC and may be involved in regulation of lymphangiogenesis and lymph node metastasis in NSCLC. Anat Rec, 2010. © 2010 Wiley‐Liss, Inc.     

Lentivirus‐Mediated Small Interfering RNA Targeting VEGF‐C Inhibited Tumor Lymphangiogenesis and Growth in Breast Carcinoma

Lymph node metastasis is a major prognostic factor for patients with breast cancer. The activation of vascular endothelial growth factor (VEGF)‐C plays a key role in lymph node metastasis through promoting lymphangiogenesis. Thus, we attempted to elucidate whether small interfering RNAs (siRNA) targeting VEGF‐C could suppress lymphangiogenesis and lymph node metastasis in vivo. A lentivirus‐based VEGF‐C siRNA vector was infected into breast cancer cells and a xenograft model. The expression of VEGF‐C mRNA and protein were quantified by quantitative real‐time polymerase chain reaction (QRT‐PCR), immunohistochemistry, and western blot analysis. The effect of VEGF‐C siRNA on breast cancer cells was investigated by an invasion assay. Lymphangiogenesis was analyzed with anti‐LYVE‐1 and anti‐D2‐40 by immunohistochemical analysis. Lentivirus‐mediated VEGF‐C siRNA stably reduced VEGF‐C mRNA and protein expression. VEGF‐C siRNA inhibited the invasive ability of breast cancer cells in vitro. Five weeks after intratumoral injection, the tumor volume was significantly smaller in the VEGF‐C siRNA group than in the control scramble siRNA group in the MDA‐MB‐231 cell xenograft model. The numbers of LYVE‐1 and D2‐40 positive vessels per microscopic field were significantly decreased in the VEGF‐C siRNA group, which indicates that VEGF‐C siRNA inhibited lymphangiogenesis. Moreover, lymph node metastasis was significantly suppressed by VEGF‐C siRNA in vivo. In conclusion, these results indicate that lentivirus‐mediated VEGF‐C siRNA offers a new approach for therapeutic intervention to prevent tumor growth and lymphatic metastasis of breast cancer. Anat Rec, 292:633–639, 2009. © 2009 Wiley‐Liss, Inc.     

Smad4 Inhibits VEGF‐A and VEGF‐C Expressions via Enhancing Smad3 Phosphorylation in Colon Cancer

Smad4 is a critical factor in the TGF‐β pathway and is involved in tumor progression and metastasis, but the role of Smad4 in colon cancer cells is unclear. The aim of this study is to explore the effect and the underlying mechanism of Smad4 on the growth, migration and apoptosis of colon cancer cells as well as vascular endothelial growth factor (VEGF)‐A and VEGF‐C secreted by these cells. In this study, we showed that Smad4, VEGF‐A, and VEGF‐C are independent prognostic factors of colon cancer, and Smad4 expression was negatively correlated with VEGF‐A and ‐C in samples. We found that Smad4 mRNA and protein levels in colon cancer cells, particularly in HCT‐116 cells, were significantly lower than those in the human intestinal epithelial cell line (HIEC). Smad4 overexpression promoted tumor cell apoptosis, inhibited VEGF‐A and ‐C expression in vitro and in vivo, but had no effect on cell proliferation and migration. Tail vein injection of the virus inhibited xenograft growth in nude mice. Importantly, we also demonstrated that Smad4 could increase the phosphorylation level of Smad3, but not Smad2, which may be one of the mechanisms underlying these effects of Smad4 in colon cancer. Therefore, Smad4 may be a new target for the treatment of colon cancer. Anat Rec, 300:1560–1569, 2017. © 2017 Wiley Periodicals, Inc.     

Integrin α4 Induces Lymphangiogenesis and Metastasis via Upregulation of VEGF‐C in Human Colon Cancer

Vascular endothelial growth factor‐C (VEGF‐C) is a key regulator in lymphangiogenesis, and is overexpressed in various malignancies. Integrin α4β1, a new member of the VEGF‐C/VEGF receptor pathway, was found to be overexpressed in melanoma tumors. However, little is known regarding the potential role of integrin α4β1 in lymphangiogenesis and other solid tumors. The aim of this study was to investigate the expression patterns of integrin α4 and VEGF‐C in relation to lymphangiogenesis and clinicopathological parameters in human colon cancer. The expression of integrin α4, VEGF‐C, and VEGFR‐3 was assessed in 71 human colon cancer tissues and 30 paracancerous normal tissues by immunohistochemical staining. Lymphatic microvessel density (LMVD) was measured after D2‐40‐labeling, and the correlations among different factors were statistically analyzed. The expression of integrin α4, VEGF‐C, VEGFR‐3, and LMVD was higher in colon cancer tissues compared with the normal paracancerous colon tissues. There was a positive correlation between the expression of integrin α4 and VEGF‐C. Integrin α4 and VEGF‐C were significantly associated with the clinicopathological parameters (LMVD, Duke's stage, and lymph node metastasis). Kaplan–Meier analyses indicated that patients with high integrin α4 or VEGF‐C expression had significantly shorter overall survival and tumor‐free survival time. Multivariate analyses suggested that integrin α4 and VEGF‐C may serve as independent prognostic factors for human colon cancer. Both integrin α4 and VEGF‐C are involved in lymphangiogenesis and lymphatic metastasis. Our results demonstrated that integrin α4 is a novel prognostic indicator for human colon cancer. Anat Rec, 299:741–747, 2016. © 2016 Wiley Periodicals, Inc.     

VEGF‐C,VEGF‐D and VEGFR‐3 expression in peripheral neuroblastic tumours

Ramani P, Nash R, Radevsky L, Patel A, Luckett M & Rogers C
(2012) Histopathology
VEGF‐C, VEGF‐D and VEGFR‐3 expression in peripheral neuroblastic tumours Aims: More than 50% of neuroblastomas (NBs) present with haematogenous and/or lymphatic metastasis; however, little is known about the clinicopathological significance in NBs of the key lymphangiogenesis growth factors vascular endothelial growth factor (VEGF)‐C and VEGF‐D and the receptor VEGFR‐3. Methods and results: Ninety‐three NBs and nine ganglioneuromas (GNs) were immunostained for VEGF‐C, VEGF‐D and VEGFR‐3. VEGF‐C and VEGF‐D were present in 76% and 82% of the NBs, respectively. There was no significant difference in VEGF‐C expression between NBs and GNs. VEGF‐D expression was significantly higher in NBs compared with GNs and in MYCN‐amplified NBs. VEGFR‐3 tumoral cell expression (VEGFR‐3c), present in 48% of the NBs, was significantly higher in NBs from children ≥18 months at presentation and those belonging to a high‐risk group. VEGFR‐3 lymphovascular density was increased significantly in NBs compared with GNs and in NBs associated with adverse clinicopathological and biological factors. Lymphovascular invasion, assessed in VEGFR‐3‐stained vessels, was present in ～50% of NBs. Cox regression analyses demonstrated that VEGFR‐3c expression was associated with a significantly shorter event‐free survival and that its effect was independent of the important pathological variable, mitosis–karyorrhexis index. Conclusions: VEGF‐D and VEGFR‐3 up‐regulation support tumour progression in NB and VEGFR‐3c may provide a useful prognostic marker in NBs.     

Prognostic value of COX‐2 immunohistochemical expression evaluated by quantitative image analysis in colorectal cancer

Epidemiological studies have shown that the inducible form of cyclooxygenase (COX‐2) may be involved in colorectal carcinogenesis, but it is controversial whether its expression is a prognostic factor for colorectal cancer. The aim of the study was to examine the expression of COX‐2 in colorectal cancer and investigate its prognostic relevance. Tissue sections of primary tumors from 132 patients undergoing curative resection for colorectal cancer were immunohistochemically examined for COX‐2 expression. The levels of intensity and extent of COX‐2 staining were quantified by use of a computerized image analysis system and correlated with various clinicopathological characteristics and survival. COX‐2 immunoreactivity was observed in the cytoplasm of tumour epithelial cells of all colorectal cancer tissues examined. No significant correlation was found between levels of intensity and extent of COX‐2 staining and various clinicopathological characteristics, including age, gender, tumor location, tumor size, tumor grade, depth of invasion, lymph node status and TNM stage. There was an inverse correlation between intensity and extent of COX‐2 staining scores (Spearman's rho=?0.414; p<0.001). To analyze the prognostic value of intensity and extent of COX‐2 staining, the patients were divided into four groups with respect to quartiles (≤25; >25 to ≤50; >50 to ≤75; and >75). No significant disease‐specific survival difference among the quartiles was found based on analysis of intensity (p=0.689) and extent (p=0.975) of COX‐2 staining. These results suggest that the expression of COX‐2 protein has no significant impact on the outcome of patients with colorectal cancer.     

Triggering of DC migration by dengue virus stimulation of COX‐2‐dependent signaling cascades in vitro highlights the significance of these cascades beyond inflammation

A term “bone‐breaking fever” is used in Chinese medicine to describe the symptoms of patients infected with dengue virus (DV). We examined the significance of the COX‐prostaglandin pathway in human DC infected by DV. We show that DV infection induced the expression of COX‐2 and the production of prostaglandin E₂ (PGE₂) in DC, and stimulated the DNA binding of NF‐κB and the kinase activity of both IκBα kinase (IKK) α and β. DV infection also activated MAPK and AP‐1 signaling. Both IκBα kinase‐NF‐κB and MAPK‐AP‐1 were upstream of COX‐2 activation. Our investigation into the significance of COX‐2‐PGE₂ pathway also revealed that DV infection enhances DC migration by inducing CC chemokine receptor 7 (CCR7) expression, and that blocking COX‐2 or MAPK activity suppresses DV‐induced DC migration. Our data also suggest that PGE₂ can induce CCR7 expression on DC and that antagonists of the PGE₂ receptors EP2 and EP4 suppress DV‐induced DC migration. We further show that the increased CCR7 expression was observed in both DV‐infected and bystander DC, suggesting the presence of secondary effects in inducing CCR7 expression. Collectively, this study reveals not only the pathways involved in COX‐2 synthesis in DV‐infected DC but also the autocrine action of PGE₂ on the migration of DV‐infected DC.     

Improved Regeneration of Autologous Transplanted Lymph Node Fragments by VEGF‐C Treatment

Secondary lymphedema is a common complication after removal of lymph nodes in combination with radiation therapy in the treatment of breast cancer, cervical cancer, and melanomas. Only symptomatic therapies are available at the moment, and lymphedema is for most patients a lifelong condition involving psychological and physical disabilities. Animal models exist to study the pathophysiology of lymphedema but not to study surgical treatments. The aim of this study was to show that regeneration of autologous transplanted lymph node fragments is possible in rats that were irradiated previously locally in the groin and to examine the effects of vascular endothelial growth factor (VEGF)‐C injections on the rate of regeneration of transplanted lymph nodes. In all of the animals, inguinal and popliteal lymph nodes and adjacent lymphatic vessels were unilaterally removed and the inguinal region irradiated by a single dose of 15 Gy. Afterward, lymph node fragments were transplanted subcutaneously in the irradiated region. Half of the animals were treated by local VEGF‐C injections after transplantation. Four weeks after transplantation, drainage of the leg was tested by injection of blue dye, and the transplanted fragments were removed and examined immunohistologically. We could show that regeneration of autologous transplanted lymph node fragments is possible in areas treated with radiotherapy in the rat. We also documented that transplants can achieve a connection to the lymphatic collectors of the leg. The results suggest that the outcome of regeneration can be improved by injection of VEGF‐C in the transplantation area. Thus, lymph node fragment regeneration may be relevant for lymphedema prevention and therapy. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

VEGF‐D promotes pulmonary oedema in hyperoxic acute lung injury

Leakage of fluid from blood vessels, leading to oedema, is a key feature of many diseases including hyperoxic acute lung injury (HALI), which can occur when patients are ventilated with high concentrations of oxygen (hyperoxia). The molecular mechanisms driving vascular leak and oedema in HALI are poorly understood. VEGF‐D is a protein that promotes blood vessel leak and oedema when overexpressed in tissues, but the role of endogenous VEGF‐D in pathological oedema was unknown. To address these issues, we exposed Vegfd‐deficient mice to hyperoxia. The resulting pulmonary oedema in Vegfd‐deficient mice was substantially reduced compared to wild‐type, as was the protein content of bronchoalveolar lavage fluid, consistent with reduced vascular leak. Vegf‐d and its receptor Vegfr‐3 were more highly expressed in lungs of hyperoxic, versus normoxic, wild‐type mice, indicating that components of the Vegf‐d signalling pathway are up‐regulated in hyperoxia. Importantly, VEGF‐D and its receptors were co‐localized on blood vessels in clinical samples of human lungs exposed to hyperoxia; hence, VEGF‐D may act directly on blood vessels to promote fluid leak. Our studies show that Vegf‐d promotes oedema in response to hyperoxia in mice and support the hypothesis that VEGF‐D signalling promotes vascular leak in human HALI. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

细胞色素P450 2C19基因多态性与肺癌遗传易感性

目的 通过对肺癌组及对照组细胞色素P450 2C19(cytochrome P450 2C19,CYP2C19)等位基因进行基因分型,探讨CYP2C19基因中G681A和G636A多态性的分布与肺癌易感性的关系.方法 提取293例肺癌患者和300例健康对照组患者的外周血基因组DNA,用实时荧光PCR方法对CYP2C19进行基因分型.对肺癌组和健康对照组基因分布进行比较.结果 肺癌组CYP2C19* 2/*3基因型显著高于正常对照组,差异有统计学意义(18.77％ vs 5.67％;P=0.000),对照组CYP2C19* 1/*2基因型显著高于肺癌组,差异有统计学意义(30.67％ vs 13.99％;P=0.000).两组病例CYP2C19代谢型分布比较结果,肺癌组慢代谢型频率超过对照组一倍以上(33.45％ vs 15.33％).肺癌与各相关指标的多因素Logistic 相关分析结果显示,CYP2C19慢代谢型可能是肺癌发生的独立危险因素(P=0.000,OR:2.755,95％ CI:1.748 ～4.343).结论 CYP2C19慢代谢型可能与肺癌的发生有关.     

CYP2C19基因多态性与肺鳞癌发病的相关性

目的 探讨CYP2C19基因多态性与肺鳞癌发病的相关性.方法 选取2015年6月至2015年12月军事医学科学院附属医院收治的肺癌患者.入院后完善病例资料,检测CYP2C19基因表型,单因素及多因素分析CYP2C19基因与肺鳞癌发病的相关性,分析其是否为肺鳞癌发病的独立危险因素.结果 本研究共入组200例肺癌患者,其中肺鳞癌59例.肺鳞癌中,快代谢型患者显著多于其它类型的肺癌患者(66.10% vs.46.10%).多因素分析结果表明快代谢型患者与慢代谢型患者相比,其发生肺鳞癌的风险增大约5.9倍(OR=5.987,95%CI:1.118~3.272).结论 CYP2C19快代谢型改变是肺鳞癌发病的独立危险因素.     

Caveolin‐1 is an Important Factor for the Metastasis and Proliferation of Human Small Cell Lung Cancer NCI‐H446 Cell

Caveolin‐1 (CAV‐1) has been reported to play an important role in the development of a variety of human cancers. CAV‐1 expression is revealed to be reduced or absent in the malignant tumor cells of small cell lung cancers (SCLC). This study was performed to investigate the influences of the stable expression of CAV‐1 on the metastasis and proliferation of SCLC in vitro. The wild‐type CAV‐1 gene was successfully transfected into the NCI‐H446 cells and was stably expressed in the NCI‐H446 cells. The effects of CAV‐1 on the morphology, proliferation, and metastasis potential for NCI‐H446 cell were evaluated by crystal violet staining, MTT analysis, transwell assay, and scratch wound assay, respectively. Western blot and gelatin zymography were used to examine the expression changes of the metastasis‐related MMP‐3 and E‐cadherin. Stable expression of CVA‐1 was observed in the H446‐CAV‐1 cells, which enlarged the cell shape with filopodia. The proliferation of H446‐CAV‐1 was inhibited, while its migration and invasion abilities were promoted in vitro. The re‐expression of CAV‐1 reduced the expression of E‐cadherin, while it increased the protein expression and enzyme activity of MMP‐3. Taken together, the cellular proliferation of the NCI‐H446 could be inhibited by the re‐expression of CAV‐1. CAV‐1 might increase the cell metastasis potential through the interaction with E‐cadherin and MMP‐3 genes. These in vitro findings confirm the involvement of CAV‐1 in the proliferation and metastasis of SCLC. Anat Rec, 2009. © 2009 Wiley‐Liss, Inc.     

肺癌间质血管与肺癌的转移

我们对32例肺鳞癌及癌周正常肺组织的免疫组化研究表明:肺癌间质血管内皮细胞Ⅷ因子表达明显增强,主要组织相容抗原(HLA-DR)几乎不表达,Ⅳ型胶原在基底膜上的表达与鳞癌的分化程度密切相关,分化越低,其间质血管基底膜越不完整,这些形态和功能的改变都有利于肺癌细胞的浸润生长和转移。但从核仁形成区的研究才明:无论肺癌的分化程度如何,其间质血管内皮细胞核内均为一个Ag-NORs颗粒。推测间质血管内皮细胞的生物学行为并没有改变。     

盆腔淋巴结转移与宫颈癌临床病理特征的关系研究

目的 研究宫颈癌临床病理特征与盆腔淋巴结转移的关系.方法 回顾性分析从2013年5月至2015年5月于菏泽市立医院手术治疗且病例资料完整的宫颈癌患者100例,分析其肿瘤的期别、组织学类型、生长类型和盆腔淋巴结转移情况.结果 100例患者中有盆腔淋巴结转移者18例,转移率为18％.18例中有绝经期8例,占8/18 (44.4％),非绝经期12例,占10/18(55.6％),数据差异有统计学意义;高危HPV阳性18例,占100％,阴性0例,数据差异有统计学意义;Ⅰ b11例,占11/18(61.1％),Ⅱa有7例,占7/18(38.1％),差异有统计学意义(P＜0.05);肿瘤≤4cm有14例,占14/18(77.8％),肿瘤＞ 4cm有4例,占4/18(22.2％),差异有统计学意义.(P＜0.05);接受化疗的患者有5例,占5/18(27.8％),未接受化疗的有13例,占13/18(72.2％),差异有统计学意义(P＜0.05).而盆腔淋巴结转移与年龄、病理类型、组织学分级差异无统计学意义(P＞0.05).结论 盆腔淋巴结转移和宫颈癌与高危HPV、临床分期、术前化疗、肿瘤大小有关;且与年龄、病理类型、组织学分级无关.盆腔淋巴结状态结合临床病理因素在临床上个体化治疗具有意义.