Adherence of ovarian cancer cells induces pleural mesothelial cell (PMC) permeability

Malignant pleural effusion (MPE) carries a grave prognosis with median survival after diagnosis being 5 months.The major causes of MPE are lung, breast, ovary,and gastric cancer. It is still unclear how cancer cells penetrate the pleural mesothelial monolayer and reach the pleural space. In this study we examined the effect of ovarian epithelial cancer cells on a confluent pleural mesothelial cell (PMC) monolayer. We demonstrate that ovarian cancer cells adhere to the mesothelial monolayer in a time-dependent manner and induce PMC barrier dysfunction as evidenced by a drop in electrical resistance on electrical cell substrate impedance-sensing system (ECIS) and increased protein permeability. Barrier dysfunction is attenuated by addition of vascular endothelial growth factor (VEGF) antibody. Significant release of VEOF was noted when ovarian cancer cells were cocultured with PMC. Electron microscopy demonstrated gap formation in PMC monolayer only at the site of cancer cell attachment with surrounding areas remaining confluent.     

Autocrine IL-6-induced Stat3 activation contributes to the pathogenesis of lung adenocarcinoma and malignant pleural effusion

Malignant pleural effusion (MPE) is a poor prognostic sign for patients with non-small-cell lung cancer (NSCLC). The generation of MPE is largely regulated by vascular endothelial growth factor (VEGF), and upregulation of VEGF by Stat3 has been observed in several types of tumor cells. In this study, we demonstrate constitutively activated Stat3 in several human lung cancer cell lines and in tumor cells infiltrated in the pleurae of patients with adenocarcinoma cell lung cancer (ADCLC) and MPE. The observations suggest that activated Stat3 plays a role in the pathogenesis of ADCLC. In PC14PE6/AS2 cells, a Stat3-positive human ADCLC cell line, autocrine IL-6 activated Stat3 via JAKs, not via Src kinase. PC14PE6/AS2 cells express higher VEGF mRNA and protein than do Stat3-negative PC14PE6/AS2/dnStat3 cells. In an animal model, PC14P6/AS2/dnStat3 cells produced no MPE and less lung metastasis than did PC14P6/AS2 cells. PC14PE6/AS2 cells also expressed higher VEGF protein, microvessel density, and vascular permeability in tumors than did PC14P6/AS2/dnStat3 cells. Therefore, we hypothesize that autocrine IL-6 activation of Stat3 in ADCLC may be involved in the formation of malignant pleural effusion by upregulating VEGF. Higher levels of IL-6 and VEGF were also found in the pleural fluids of patients with ADCLC than in patients with congestive heart failure. The autocrine IL-6/Stat3/VEGF signaling pathway may also be activated in patients with ADCLC and MPE. These findings provide novel targets for the management of MPE.     

Tumor necrosis factor-alpha promotes malignant pleural effusion

Tumor necrosis factor (TNF)-alpha is present in the microenvironment of human tumors, including malignant pleural effusion (MPE). Although the cytokine is produced in the pleural cavity by both tumor and host cells, its effects on MPE formation are unknown. In these studies, we sought to determine the role of TNF-alpha in the pathogenesis of MPE and to assess the therapeutic effects of its neutralization in a preclinical model. For this, MPEs were generated in immunocompetent mice using intrapleural injection of mouse lung adenocarcinoma cells. The roles of tumor- and host-derived TNF-alpha were assessed using combined experimentation with TNF-alpha gene-deficient mice and in vivo TNF-alpha neutralization. To expand the scope of preclinical data, TNF-alpha and vascular endothelial growth factor (VEGF) expression were determined in human cancer cell lines and human MPE. In the MPE model, TNF-alpha of host and tumor origin was present. TNF-alpha neutralization significantly limited tumor dissemination, effusion formation, vascular hyperpermeability, TNF-alpha and VEGF expression, and angiogenesis, thereby improving survival. In contrast, these variables were not different between TNF-alpha gene-sufficient and TNF-alpha gene-deficient mice. In mouse cancer cells, TNF-alpha functioned via nuclear factor-kappaB- and neutral sphingomyelinase-dependent pathways to induce TNF-alpha and VEGF, respectively. These results were recapitulated in human cancer cells, and a correlation was detected between TNF-alpha and VEGF content of human MPE. We conclude that tumor-derived TNF-alpha is important in the development of MPE in mice, and provide preclinical evidence supporting the efficacy of TNF-alpha blockade against malignant pleural disease.     

Prognostic Values of VEGF and Endostatin with Malignant Pleural Effusions in Patients with Lung Cancer

Aims: Angiogenesis is important in malignant pleural effusion (MPE) formation and it is regulated by anumber of pro- and anti-angiogenic cytokines. The purpose of this study was to evaluate the prognostic valueof angiogenic factor vascular endothelial growth factor (VEGF) and angiogenesis inhibitor endostatin in lungcancer patients with MPE, and investigate the relationship between these two kinds of agent. Methods: Usingenzyme-linked immunoadsorbent assay, the concentrations of VEGF and endostatin were measured in pleuraleffusions (PE) and serum from a total of 70 lung cancer patients with MPE and 20 patients with tuberculosis.Results: Compared to patients with tuberculosis, the levels of VEGF and endostatin in both PE and serum weresignificantly higher in patients with lung cancer. There were statistically significant correlations between VEGFlevels in PE and serum (r=0.696, p<0.001), endostatin levels in PE and serum (r=0.310, p=0.022), and VEGF andendostatin levels in PE (r=0.287, p=0.019). Cox multivariate analysis revealed that elevated pleural VEGF andendostatin levels and serum endostatin level were independent predictors of shorter overall survival. Conclusion:Both pro- and anti-angiogenic factors are likely contributors to PE formation. Our results suggest that the levelsof VEGF and endostatin in PE, together with endostatin in serum, may be potential prognostic parameters forlung cancer patients with MPE.     

抗血管生成治疗在恶性胸腔积液中的应用进展CSCD

恶性胸腔积液(malignant pleural effusions,MPE)是晚期肿瘤的常见并发症,肺癌和恶性胸膜间皮瘤(malignant pleural mesothelioma,MPM)是MPE最常见的病因。MPE的治疗原则是在针对病因的全身治疗的基础上对胸腔进行局部治疗。血管内皮生长因子(vascular endothelial growth factor,VEGF)在MPE形成中的多个环节起着关键作用。贝伐珠单抗能抑制VEGF的活性,减少MPE的形成,改善患者预后。本综述系统回顾了贝伐珠单抗及其他抗血管生成药物在非小细胞肺癌(non-small cell lung cancer,NSCLC)和MPM相关MPE中的研究进展,阐述了不同抗血管生成药物对MPE的临床疗效和安全性。     

The Role of Vascular Endothelial Growth Factor in the Pathogenesis, Diagnosis and Treatment of Malignant Pleural Effusion

Malignant pleural effusions (MPEs) are a significant source of cancer-related morbidity. Over 150,000 patients in the United States suffer from breathlessness and diminished quality of life due to MPE each year. Current management strategies are of mostly palliative value and focus on symptom control; they do not address the pathobiology of the effusion, nor do they improve survival. Further elucidation of the pathophysiological mechanisms, coupled with the development of novel treatments such as intrapleural chemotherapeutics targeting this process, has the potential to greatly improve the efficacy of our current management options. Vascular endothelial growth factor-A (VEGF-A) has been implicated as a critical cytokine in the formation of malignant pleural effusions. Elevated levels of VEGF produced by tumor cells, mesothelial cells, and infiltrating immune cells result in increased vascular permeability, cancer cell transmigration, and angiogenesis. Therefore antiangiogenic therapies such as Bevacizumab, a monoclonal antibody targeting VEGF-A, may have a potential role in the management of malignant pleural effusions. Herein we review the pathogenesis and potential treatment strategies of malignant pleural effusions, with a focus on angiogenesis and antiangiogenic therapeutics.     

Apolipoprotein E expression promotes lung adenocarcinoma proliferation and migration and as a potential survival marker in lung cancer

Many human lung cancer cell lines express apolipoprotein E (ApoE), especially cells derived from malignant pleural effusions (MPE) in patients with lung adenocarcinoma. This study aimed to investigate the influence of ApoE expression on lung cancer. In lung cancer tissues, ApoE expression was more frequently found in malignant pleural effusions (MPE)-associated lung adenocarcinoma than in lung adenocarcinoma or squamous cell carcinoma without MPE (P < 0.05), indicating that ApoE is associated with the pathogenesis of MPE in patients with lung adenocarcinoma. Next, we examined the roles of ApoE in an MPE-derived lung adenocarcinoma cell line that endogenously over-expresses ApoE, PC14PE6/AS2 (AS2). In that experiment we inhibited ApoE expression by transfection of a plasmid carrying ApoE siRNAs into AS2 cells to generate AS-S2 and AS-S3 cells. Compared to vector-control cells and parental AS2 cells, AS2-S2 and AS2-S3 cells grew slower (P < 0.05), were more sensitive to cisplatin, and had significantly impaired cellular migration (P < 0.05). Furthermore, over-expression of ApoE was independently associated with poor survival in lung adenocarcinoma patients who had MPE at the time of diagnosis (P < 0.001). Conclusively, ApoE over-expression promotes cancer proliferation and migration and contributes to an aggressive clinical course in patients with lung adenocarcinoma and MPE.     

Interleukin 22-producing CD4(+) T cells in malignant pleural effusion

Th22 cells have been reported to be involved in human cancers. However, differentiation and immune regulation of Th22 cells in malignant pleural effusion (MPE) remain unknown. We noted that Th22 cell numbers were increased in MPE, and that IL-22 substantially promoted the proliferation and migratory activity of A549 cells. Moreover, IL-22 could strongly facilitate intercellular adhesion of A549 cells to pleural mesothelial cell monolayers. Our data revealed that the increase in Th22 cells in MPE was due to pleural cytokines and chemokines, and that Th22 exerted an important immune regulation on cancer cells in human pleural malignant environment.     

Regulation of tumor angiogenesis by integrin-linked kinase (ILK)

We show that integrin-linked kinase (ILK) stimulates the expression of VEGF by stimulating HIF-1alpha protein expression in a PKB/Akt- and mTOR/FRAP-dependent manner. In human prostate cancer cells, knockdown of ILK expression with siRNA, or inhibition of ILK activity, results in significant inhibition of HIF-1alpha and VEGF expression. In endothelial cells, VEGF stimulates ILK activity, and inhibition of ILK expression or activity results in the inhibition of VEGF-mediated endothelial cell migration, capillary formation in vitro, and angiogenesis in vivo. Inhibition of ILK activity also inhibits prostate tumor angiogenesis and suppresses tumor growth. These data demonstrate an important and essential role of ILK in two key aspects of tumor angiogenesis: VEGF expression by tumor cells and VEGF-stimulated blood vessel formation.     

Deguelin—An inhibitor to tumor lymphangiogenesis and lymphatic metastasis by downregulation of vascular endothelial cell growth factor‐D in lung tumor model

Deguelin, a rotenoid of the flavonoid family, has been reported to possess antiproliferative and anticarcinogenic activities in several cell lines and tumor models. However, it is still unclear whether deguelin effectively inhibits tumor‐associated lymphangiogenesis and lymphatic metastasis. Since tumor production of vascular endothelial cell growth factor (VEGF)‐D was associated with tumor lymphangiogenesis and lymphatic metastasis, we established the mouse lymphatic metastasis model by transfecting high expression VEGF‐D into LL/2 Lewis lung cells (VEGF‐D‐LL/2) and explored the effects of deguelin on lymphatic metastasis in the immunocompetent C57BL/6 mice. Our results indicated that deguelin inhibited proliferation, migration of VEGF‐D‐LL/2 cells via downregulating AKT and mitogen‐activated protein kinase pathway and interfered tube formation of lymphatic vascular endothelial cells on matrigel at nanomolar concentrations. Deguelin significantly downregulated the expression of VEGF‐D both at mRNA and protein levels in VEGF‐D‐LL/2 cells in a dose‐dependent manner. In the in vivo study, intraperitoneal administration of deguelin (4 mg/kg) remarkably inhibited the tumor‐associated lymphangiogenesis and lymphatic metastasis. The rates of lymph node and lung metastasis in deguelin‐treated mice were 0 and 16.7% compared with 58.3 and 83.3% in control group mice, respectively. Deguelin also resulted in a remarkable delay of tumor growth and prolongation of life span. Immunohistochemical staining with antibodies against VEGF‐D, LYVE‐1 and VEGFR‐3 revealed fewer positive vessel‐like structures in deguelin‐treated mice compared with control group mice. Taken together, we demonstrate for the first time that deguelin suppresses tumor‐associated lymphangiogenesis and lymphatic metastasis by downregulation of VEGF‐D both in vitro and in vivo.     

Abrogation of the interaction between osteopontin and alphavbeta3 integrin reduces tumor growth of human lung cancer cells in mice

Osteopontin (OPN) is a multifunctional cytokine involved in cell signaling by interacting with alphavbeta3 integrins. Recent clinical studies have indicated that OPN expression is associated with tumor progression and poor prognosis among patients with lung cancer. However, the biological role of OPN in human lung cancer has not yet been well-defined. The purpose of this study is to investigate and provide evidence for the causal role of OPN regarding tumor growth and angiogenesis in human lung cancer. In this study, we developed a stable OPN transfectant from human lung cancer cell line SBC-3 which does not express the intrinsic OPN mRNA. To reveal the in vivo effect of OPN on tumor growth of human lung cancer, we subcutaneously injected OPN-overexpressing SBC-3 cells (SBC-3/OPN) and control cells (SBC-3/NEO) into the nude mice. Transfection with the OPN gene significantly increased in vivo tumor growth and neovascularization of SBC-3 cells in mice. These in vivo effects of OPN were markedly suppressed with administration of anti-alphavbeta3 integrin monoclonal antibody or anti-angiogenic agent, TNP-470. Furthermore, recombinant OPN protein enhanced human umbilical vein endothelial cell (HUVEC) proliferation in vitro, and this enhancement was significantly inhibited with the addition of anti-alphavbeta3 integrin antibody. Taken together, these results suggest that OPN plays a crucial role for tumor growth and angiogenesis of human lung cancer cells in vivo by interacting with alphavbeta3 integrin. Targeting the interaction between OPN and alphavbeta3 integrin could be effective for future development of anti-angiogenic therapeutic agents for patients with lung cancer.     

胸腔热灌注化疗治疗肺癌胸腔积液的临床观察

目的 观察胸腔热灌注化疗对比胸腔置管化疗治疗肺癌所致恶性胸腔积液的临床疗效及不良反应.方法 纳入103例肺癌胸腔积液患者,胸腔热灌注化疗组65例,胸腔置管化疗组38例.观察胸腔积液控制率、胸腔积液进展时间、胸腔积液中血管内皮生长因子(VEGF)浓度与疗效的关系以及不良反应发生情况.结果 热灌注化疗组和置管化疗组的胸腔积液控制率分别为81.5％和52.6％,差异有统计学意义(x2=9.834,P=0.002).两组患者的中位胸腔积液进展时间分别为3.10个月和2.15个月,差异有统计学意义(x2=10.512,P=0.001).胸腔积液中VEGF低浓度患者接受胸腔热灌注化疗和腔内置管化疗后的中位胸腔积液进展时间分别为3.34个月和2.20个月,差异有统计学意义(x2=9.409,P=0.002),但VEGF高浓度亚组,两种治疗方法的中位胸腔积液进展时间分别为2.85个月和2.10个月,差异无统计学意义(x2=2.429,P=0.119).两组患者的不良反应主要为消化道不良反应、乏力及血液毒性,热灌注化疗组患者乏力较置管化疗组常见(67.7％∶13.2％;x2 =28.595,P＜0.001).结论 胸腔热灌注化疗治疗肺癌所致恶性胸腔积液较常规腔内置管化疗提高了胸腔积液控制率,且可延长胸腔积液进展时间,不良反应易于耐受,胸腔积液进展时间延长在胸腔积液VEGF低浓度亚组中尤为明显,VEGF可作为胸腔热灌注化疗的疗效预测因素.     

康莱特联合奥沙利铂胸腔灌注治疗恶性胸腔积液及对RCAS1、VEGF表 达的影响

目的:探讨康莱特联合奥沙利铂胸腔灌注治疗恶性胸腔积液的临床价值以及灌注治疗对恶性胸腔积液中SiSo细胞上的受体结合肿瘤抗原(RCAS1)、血管内皮生长因子(VEGF)表达的影响。方法:选取110例非小细胞肺癌并胸腔积液患者,分为康莱特组35例,奥沙利铂组35例及联合用药组40例,分别收集胸腔注药前及每次注药48h后的胸水标本,采用酶联免疫吸附测定（ELISA）方法检测胸水标本中的RCAS1、VEGF水平。结果:三组疗效比较,联合组优于康莱特组（P<0.01）。生活质量改善方面,联合组优于康莱特组与奥沙利铂组（P<0.05）。三组治疗后的毒副反应比较,康莱特组低于联合组及奥沙利铂组（P<0.05）。三组治疗前、第1周期治疗后、第2周期治疗后胸水RCAS1及VEGF表达水平均有逐渐下降趋势（P<0.05）,联合用药组较其它两组下降更明显（P<0.001）。结论:康莱特联合奥沙利铂胸腔灌注对控制恶性胸腔积液具有一定的临床价值,检测胸水RCAS1、VEGF表达水平可评估康莱特联合奥沙利铂灌注治疗效果。     

RNA干扰MDM2下调人乳腺癌细胞中VEGF的合成并抑制肿瘤血管的生成

目的 探讨小干扰RNA（small interfering RNA, siRNA）抑制人乳腺癌细胞中鼠双微粒体2 （murine double minute 2, MDM2）的表达对癌细胞中血管内皮生长因子（vascular endothelial growth factor, VEGF）合成以及裸鼠移植瘤组织内血管生成的影响。方法 根据MDM2已知的cDNA序列设计并转录合成特异性siRNA,转染高表达MDM2的人乳腺癌MDA-MB-468细胞,低氧培养后应用蛋白质印迹法和ELISA法检测肿瘤细胞及其上清液中VEGF的水平。构建裸鼠乳腺癌移植瘤模型,观察MDM2沉默后肿瘤生长情况,蛋白质印迹法、免疫组织化学及ELISA方法检测荷瘤组织和裸鼠血清标本中的VEGF含量,并以CD34标记血管内皮细胞计数微血管密度（microvessel density, MVD）。结果 siRNA抑制MDM2表达后,MDA-MB-468细胞分泌的VEGF蛋白显著减少（P=0.006）。裸鼠移植瘤模型显示,封闭MDM2表达使荷瘤组织生长变慢（P=0.008）,且荷瘤小鼠血清VEGF水平明显减低（P=0.008）,荷瘤组织内MVD也明显降低（P=0.003）。结论 MDM2 siRNA能有效减低乳腺癌细胞中VEGF的合成,并抑制裸鼠移植瘤组织内新生血管的生成,为MDM2/VEGF途径抗肿瘤血管生成作用的研究及靶向药物开发提供了新的思路。     

BPTF promotes tumor growth and predicts poor prognosis in lung adenocarcinomas

BPTF, a subunit of NURF, is well known to be involved in the development of eukaryotic cell, but little is known about its roles in cancers, especially in non-small-cell lung cancer (NSCLC). Here we showed that BPTF was specifically overexpressed in NSCLC cell lines and lung adenocarcinoma tissues. Knockdown of BPTF by siRNA significantly inhibited cell proliferation, induced cell apoptosis and arrested cell cycle progress from G1 to S phase. We also found that BPTF knockdown downregulated the expression of the phosphorylated Erk1/2, PI3K and Akt proteins and induced the cleavage of caspase-8, caspase-7 and PARP proteins, thereby inhibiting the MAPK and PI3K/AKT signaling and activating apoptotic pathway. BPTF knockdown by siRNA also upregulated the cell cycle inhibitors such as p21 and p18 but inhibited the expression of cyclin D, phospho-Rb and phospho-cdc2 in lung cancer cells. Moreover, BPTF knockdown by its specific shRNA inhibited lung cancer growth in vivo in the xenografts of A549 cells accompanied by the suppression of VEGF, p-Erk and p-Akt expression. Immunohistochemical assay for tumor tissue microarrays of lung tumor tissues showed that BPTF overexpression predicted a poor prognosis in the patients with lung adenocarcinomas. Therefore, our data indicate that BPTF plays an essential role in cell growth and survival by targeting multiply signaling pathways in human lung cancers.     

Treatment for malignant pleural effusion of human lung adenocarcinoma by inhibition of vascular endothelial growth factor receptor tyrosine kinase phosphorylation.

Malignant pleural effusion (PE) is associated with advanced human lung cancer. We found recently, using a nude mouse model, that vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is responsible for PE induced by non-small cell human lung carcinoma cells. The purpose of this study was to determine the therapeutic potential of a VEGF/VPF receptor tyrosine kinase phosphorylation inhibitor, PTK 787, against PE formed by human lung adenocarcinoma (PC14PE6) cells. PTK 787 did not affect the in vitro proliferation of PC14PE6 cells, whereas it specifically inhibited proliferation of human dermal microvascular endothelial cells stimulated by VEGF/VPF. A specific platelet-derived growth factor receptor tyrosine kinase inhibitor, CGP57148 (used as a control because PTK 787 also inhibits platelet-derived growth factor receptor tyrosine kinases), had no effect on proliferation of PC14PE6 or human dermal microvascular endothelial cells. i.v. injection of PC14PE6 cells into nude mice produced lung lesions and a large volume of PE containing a high level of VEGF/VPF. Oral treatment with CGP57148 had no effect on PE or lung metastasis. In contrast, oral treatment with PTK 787 significantly reduced the formation of PE but not the number of lung lesions. Furthermore, treatment with PTK 787 significantly suppressed vascular hyperpermeability of PE-bearing mice but did not affect the VEGF/VPF level in PE or expression of VEGF/VPF protein and mRNA in the lung tumors of PC14PE6 cells in vivo. These findings indicate that PTK 787 reduced PE formation mainly by inhibiting vascular permeability, suggesting that this VEGF/VPF receptor tyrosine kinase inhibitor could be useful for the control of malignant PE.     

The expression of vascular endothelial growth factor C and its receptors in non-small cell lung cancer.

Expression of vascular endothelial growth factor (VEGF)-C and that of its receptors were assessed in non-small cell lung cancer. Immunohistochemistry revealed positive VEGF-C expression in 38.7% (24/62) of the patients studied. A significant positive correlation was found between VEGF-C in cancer cells and VEGF receptor-3 (VEGFR-3) in vascular endothelial cells, but not between VEGF-C in cancer cells and VEGFR-2 in endothelial cells. In this cohort of lung cancer patients, VEGF-C expression was significantly associated with lymph node metastasis, lymphatic vessel invasion, and worse outcomes after the operation. Although the independent prognostic impact of VEGF-C and VEGFR-3 was not clear, VEGFR-2 expression in endothelial cells retained the independency as the prognostic indicator. In light of these findings, we conclude that VEGF-C plays an important role in lymphatic invasion/metastasis and tumour progression in non-small cell lung cancer.     

Heregulin selectively upregulates vascular endothelial growth factor secretion in cancer cells and stimulates angiogenesis

The interaction between the erbB tyrosine kinase receptors and their ligands plays an important role in tumor growth via the regulation of autocrine and paracrine loops. We report the effect of heregulin beta1, the ligand for erbB-3 and erbB-4 receptors, on the regulation of vascular endothelial growth factor (VEGF) expression, using a panel of breast and lung cancer cell lines with constitutive erbB-2 overexpression or engineered to stably overexpress the erbB-2 receptor. We demonstrate that heregulin beta1 induces VEGF secretion in most cancer cell lines, while no significant effect was observed in normal human mammary and bronchial primary cells. Overexpression of erbB-2 receptor results in induction of the basal level of VEGF and exposure to heregulin further enhances VEGF secretion. This is associated with increased VEGF mRNA expression. In contrast, VEGF induction is significantly decreased in a T47D cell line where erbB-2 is functionally inactivated. Conditioned media from heregulin-treated cancer cells, but not from normal cells, stimulates endothelial cell proliferation; this paracrine stimulation is inhibited by co-exposure to a specific VEGF neutralizing antibody. Furthermore, heregulin-mediated angiogenesis is observed in the in vivo CAM assay. This study reports the first evidence of VEGF regulation by heregulin in cancer cells. Oncogene (2000) 19, 3460 - 3469