凝血酶对A549细胞增殖和顺铂凋亡效应影响的实验研究

目的:研究凝血酶在体外对非小细胞肺癌细胞(A549)增殖、化疗药物顺铂疗效的影响及其机理.方法:非小细胞肺癌细胞株A549与不同浓度的凝血酶(0.05-3U/ml)共培养,按不同分组加入顺铂(30mg/ml)和水蛭素(0.05-10U/ml).在不同的时间,采用MTT比色法检测细胞生长活性,细胞增殖程度;采用流式细胞术检测凝血酶对顺铂杀伤肿瘤细胞作用的影响.结果:凝血酶在一定浓度范围内(0.05-1.5 U /ml)可促进A549增殖,72 h后增殖效应明显;在增殖浓度范围内可抑制顺铂对肿瘤细胞A549的凋亡作用.结论: 凝血酶在一定浓度范围内诱导A549细胞增殖,并减弱化疗药物顺铂对该细胞的杀伤作用.     

肺癌细胞自分泌VEGF对mCRPs的调控及其机制

目的：研究人非小细胞肺癌A549细胞自分泌VEGF对膜结合补体调节蛋白(membranebounal complement regulatory proteins, mCRPs)的调控及其机制。方法: RTPCR法检测CD46、CD55、CD59、VEGF及其受体(KDR和FLT1)和 IL8及其受体(CXCR1 CXCR2) mRNA在人非小细胞肺癌A549细胞的表达。MTT法检测抗VEGF抗体和抗IL8抗体对A549细胞增殖的影响,流式细胞术检测抗VEGF抗体和抗IL8抗体对A549细胞mCRPs表达水平的影响,Western blotting检测抗VEGF抗体对转录因子KLF2和磷酸化NFκB p65蛋白表达的影响。结果：A549细胞表达膜结合型CD46、CD55和CD59 mRNA,亦表达VEGF及其受体（KDR和FLT1）和 IL8及其受体（CXCR1和CXCR2） mRNA。抗VEGF抗体明显抑制A549细胞的增殖（P<0.05）。终质量浓度0.1 μg/ml抗VEGF抗体封闭72 h,CD55和CD59 mRNA表达下降,膜结合的CD55和CD59蛋白分子表达降低(均P<0.05);胞质和核KLF2蛋白相对定量值分别从063和0.88下降至0.42和0.66,胞质和胞核磷酸化NFκB p65蛋白相对定量值分别从0.44和0.28下降至0.37和019。结论：A549细胞可能通过自分泌VEGF增加NFκB p65和KLF2转录因子水平,从而上调CD55和CD59的表达。     

VEGF促进肺癌细胞侵袭性的自分泌机制初探

目的 通过研究VEGF、MMP-9之间的关系,初步探讨VEGF促进肺癌细胞侵袭性的自分泌机制,以期为肿瘤的抗VEGF治疗提供一定的理论依据.方法 在体外用条件培养基培养人肺腺癌细胞A549,经细胞免疫化学染色、侵袭实验方法来测定VEGF对肺腺癌细胞A549的MMP-9表达及侵袭性的影响.结果 (1)各实验组的A549细胞在不同浓度的rh VEGF165和不同时间的作用下,细胞内的MMP-9的表达有差异;(2)A549细胞侵袭数在和照组间差异有显著性(P＜0.001).结论 (1)VEGF能上调A549细胞内的MMP-9表达,且促进作用具有时间-剂量依赖性;(2)VEGF能促进A549细胞的侵袭性;(3)肺癌细胞中存在有自分泌机制,VEGF可通过自分泌机制上调肺癌细胞的MMP-9表达,进而促进肿瘤的浸润转移.     

水蛭素对肝细胞癌HepG2细胞抑制作用机制探讨

目的 研究水蛭素对肝细胞癌（hepatocellular carcinoma,HCC,简称肝癌）HepG2细胞抑制作用及其抗肝癌的分子机制。方法 将含不同浓度（1 U/mL、2 U/mL、4 U/mL和8 U/mL）水蛭素的培养液作用于肝癌HepG2细胞,采用MTT法检测水蛭素对肝癌HepG2细胞增殖的影响,流式细胞仪检测水蛭素对肝癌HepG2细胞凋亡的影响,Transwell法检测水蛭素对肝癌HepG2细胞迁移、侵袭的影响,荧光定量PCR检测血管内皮生长因子（vascular endothelial growth factor,VEGF）基因的mRNA表达水平,Western blot检测VEGF基因的蛋白表达水平。结果 与空白对照组比较,肝癌HepG2细胞增殖抑制率随水蛭素浓度（1 U/mL、2　U/mL、4 U/mL和8 U/mL）增加及作用时间（24　h、48　h、72　h）延长而增加,呈剂量-时间依赖效应（P<0.05）。不同浓度（2 U/mL、4 U/mL和8 U/mL）水蛭素作用48 h后,肝癌HepG2细胞凋亡率分别为（28.37±1.16）%、（40.27±0.97）%、（76.17±1.5）%,细胞侵袭个数分别为（204±9）个、（163±6）个、（94±4）个,细胞迁移个数分别为（86±5）个、（54±7）个、（20±5）个,细胞凋亡率、侵袭及迁移个数随水蛭素浓度增加而增加,呈浓度依赖性（P<0.05）。VEGF mRNA、蛋白的表达量随水蛭素浓度增加而明显下调（P<0.05）。结论 水蛭素能抑制肝癌HepG2细胞增殖、凋亡、迁移及侵袭,其机制可能是通过下调VEGF表达。     

Avastin阻断VEGF促横纹肌肉瘤细胞株RH4增殖的作用

背景与目的：血管内皮生长因子（vascular endothelial growth factor,VEGF）及其通路的研究是目前研究的热点之一,但其在横纹肌肉瘤中的研究鲜有报道．其作用机制尚不完全清楚。本研究通过检测横纹肌肉瘤细胞株RH4中VEGF及其受体的表达情况,以了解VEGF在横纹肌肉瘤中的作用及机制,并研究Avastin阻断VEGF促细胞增殖的作用。方法：使用RT-PCR方法检测RH4细胞株中VEGF、VEGFR1、2、3mRNA的表达：使用ELISA方法检测培养液上清中VEGF的分泌水平;使用Western blot方法检测VEGFR1蛋白的表达水平;使用直接计数的方法检测VEGF、Avastin对RH4细胞增殖的影响。结果：RT-PCR和ELISA结果显示在RH4细胞中存在VEGF的表达和分泌;在RH4细胞中仅有VEGFR1表达,VEGFR2和VEGFR3均为阴性：Western blot的结果进一步证实了VEGFR1在RH4中的表达。VEGF可直接促进RH4细胞的增殖,在0～100ng／ml的区间其促进增殖的作用随着浓度的增加而增加。10～40μg／ml Avastin可以阻断VEGF的促增殖作用。结论：在横纹肌肉瘤细胞RH4中VEGF可以通过自分泌的方式作用于自身,并通过VEGFR1促进肿瘤细胞的增殖,Avastin可以阻断VEGF的促增殖作用。     

2-甲基萘[2,3-b]呋喃-4,9-酮(FNQ3)对肺腺癌细胞株(A549)细胞Fas受体表达和细胞凋亡的影响

目的探讨2-甲基萘[2,3-b]呋喃-4,9-酮(FNQ3)对A549细胞表面Fas受体表达和细胞凋亡的调节效应。方法给A549细胞投与FNQ3,从药物的浓度依赖性及投药的时间依赖性两方面来观察药物对A549细胞表面Fas受体表达及诱导凋亡的作用。用半定量RT-PCR法检测FasmRNA,用流式细胞仪检测A549细胞表面的Fas受体表达和细胞凋亡。结果A549细胞表面Fas受体表达和A549细胞FasmRNA及细胞凋亡敏感度都随着药物浓度和投药时间的增加而增加。在研究药物浓度依赖性时,FNQ3浓度为2.5μg/ml时FasmRNA产量相对较高,为75000分子/μl;A549细胞表面Fas受体表达相对较多;细胞凋亡率达到37.26%(P<0.01)。在研究药物时间依赖性时,FNQ3(0.5μg/ml)投药时间为7天时,FasmRNA产量相对较高,为150000分子/μl;A549细胞表面Fas受体表达相对较多;细胞凋亡率30.45%(P<0.02)。结论FNQ3作为1种抗肿瘤药物能够诱导A549肿瘤细胞表面Fas受体表达,并上调由Fas受体介导的细胞凋亡。     

NSCLC细胞株A549和巨噬细胞株U937共培养对IL-8促表达和分泌的作用

目的:观察肿瘤相关巨噬细胞(tumor-associated macrophages,TAMs)U937和非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞株A549在体外不同的共培养条件下,对趋化因子白细胞介素-8(interleukin-8,IL-8)促表达和分泌的作用。方法:原位杂交法分别测定浓度为1×105mL-1的A549细胞和0、1×105、2×105、4×105和8×105mL-1的U937细胞共培养24h后,A549细胞中IL-8 mRNA的阳性表达。ELISA法分别测定浓度均为1×105mL-1的A549细胞和U937细胞共培养0、4、8、16、24和48h后,共培养物上清液中IL-8的含量。结果:随着U937细胞浓度逐渐增高,癌细胞A549中IL-8 mRNA表达的阳性系数均值也相应地增高,最高为3.88±0.35,明显高于U937细胞浓度为0×105mL-1共培养组的阳性系数均值0.25±0.46。共培养物上清液中IL-8的含量随共培养时间的延长而增加,在48h达到最高为5422.75±4442.55,明显高于共培养0h段的含量13.97±16.63和对照组各时段的含量,0h为5.06±4.62,48h为967.15±475.62,IL-8在后两者中也有一定的自分泌。结论:TAMs细胞U937和NSCLC细胞株A549共培养能够促进癌细胞IL-8 mRNA的阳性表达和共培养物上清液中IL-8分泌量的增加,并可能与两者相互作用的浓度和时间有关。     

瘦素对人肺腺癌A549细胞增殖及p-ERK1/2、VEGF表达的影响

 目的观察瘦素对人肺腺癌 A549 细胞增殖及p-ERK1/2、VEGF 表达的影响。初步探讨瘦素在促进肿瘤血管生成中的机制。方法 对人肺腺癌 A549 细胞采用细胞培养,MTT 法检测不同浓度瘦素对人肺腺癌A549细胞增殖影响,免疫组织化学法检测VEGF表达、Western blot 方法检测瘦素对p-ERK1/2、VEGF表达的影响。结果 在一定范围内,瘦素呈时间、剂量依赖性促进A549细胞的生长(P<0.05);与阴性对照组相比,不同浓度瘦素作用48h 后p-ERK1/2、VEGF 蛋白表达明显增加并具有剂量依赖性(P<0.05);p-ERK1/2和VEGF 之间呈正相关性(r=0.694,P<0.05)。结论 在体外瘦素对人肺腺癌A549 细胞有明显增殖作用,通过促进p ERK1/2和VEGF的表达,瘦素在促进肺癌血管的生长机制中可能具有一定作用。     

肝癌细胞中血管内皮生长因子的自分泌及其意义

目的 研究人肝癌细胞中血管内皮生长因子(VEGF)的自分泌及其意义。方法：反转录PCR(RTPCR)检测体外培养的人肝细胞肝癌细胞系SMMC7721、HHCC和HepG2中VEGF受体的表达。采用人工合成的反义寡核苷酸(ODN)抑制肝癌细胞VEGF的表达后，分别检测肝癌细胞增殖活性和超微结构的变化，流式细胞仪分析肝癌细胞周期以及VEGF’受体蛋白表达的变化。结果：VEGF受体1(Flt-1)在SMMC7721细胞中有表达，而HHCC和HepG2细胞则表达VEGF受体2(KDR)。反义ODN对HHCC细胞的增殖有抑制作用，且与ODN的作用浓度呈线性关系，浓度为2．5μmol／L、5 μmol／L和10μmol/L时细胞增殖抑制率分别为26％、41％和50％。反义ODN对SMMC7721细胞则无此作用，而正义ODN对上述2种细胞均无作用。反义ODN作用后，HHCC细胞出现S期比率下降，并且在细胞周期曲线中出现凋亡前期峰，而SMMC7721细胞无上述变化。反义0DN能降低HHCC细胞膜表面KDR的表达(阳性率分别为：反义组19.2％，正义组29.8％，对照组31.2％)，对于SMMC7721细胞Flt-1的膜表面表达则无明显影响。结论：人肝癌细胞中存在VEGF的自分泌途径，其机制可能为通过诱导肝癌细胞膜表面KDR的表达及其信号转导作用，从而调节肝癌细胞的分裂增殖。     

透明质酸寡糖调节A549／DDP多药耐药作用的研究

目的：通过研究透明质酸寡糖对人肺腺癌细胞系A549／DDP的P糖蛋白和多药耐药相关蛋白（MRP）、肺耐药蛋白（LRP）表达的影响,探讨透明质酸在引起肿瘤细胞多药耐药中的作用。方法：将CD44单抗或透明质酸寡糖与A549／DDP细胞共培养48小时,放免法检测培养基中细胞所分泌透明质酸的含量,流式细胞仪检测经上述处理的A549／DDP表面MDR1、MRP、LRP的表达率,MTT法检测在不同浓度顺铂作用下,各组细胞的存活率。结果：经透明质酸寡糖处理后A549／DDP,分泌透明质酸较未处理组明显减少（P〈0．05）;且细胞表面与多药耐药相关的MDR1、MRP、LRP的表达率均降低（P〈0．05）。另外,处理后的细胞,在不同浓度顺铂作用时,细胞凋亡率明显增加。结论：体外条件下,透明质酸寡糖能逆转A549／DDP的耐     

The role of thrombin in the neo-vascularization of malignant gliomas: an intrinsic modulator for the up-regulation of vascular endothelial growth factor

Thrombin is a key enzyme in the blood coagulation system where it converts fibrinogen to fibrin. It participates in a variety of biological processes such as the induction of mitogenesis and of morphological changes, the production of cytokines and growth factors, and apoptosis. To clarify the role of thrombin in the proliferation of human malignant gliomas, we investigated its effect on the expression of vascular endothelial growth factor (VEGF) in vitro and determined its intrinsic expression in human glioma tissues. In 3 human glioma cell lines tested, U-87 MG, U-251 MG, and U-105 MG, thrombin induced the VEGF mRNA expression and protein in a dose- and time-dependent manner. The thrombin receptor expression was detectable by RT-PCR and immunoblot. The secretion of VEGF protein in glioma cells was stimulated by the thrombin receptor agonist peptide and the induction of VEGF was significantly blocked by the thrombin inhibitor hirudin, indicating that the up-regulation of VEGF was mediated by the thrombin/thrombin receptor pathway. Immunoblot analysis demonstrated that prothrombin, the precursor of thrombin, was distributed in all 10 glioma tissues examined. In situ hybridization and immunohistochemical analysis revealed the co-localization of prothrombin mRNA-positive and GFAP-positive cells in the glioma tissues. Although various factors may be involved in the up-regulation of VEGF, our results suggest that human gliomas per se express prothrombin, and that thrombin, converted from prothrombin in glioma tissues, substantially stimulates angiogenesis in an autocrine fashion.     

Growth-regulated oncogene is pivotal in thrombin-induced angiogenesis

The mechanism of thrombin-induced angiogenesis is poorly understood. Using a gene chip array to investigate the pro-malignant phenotype of thrombin-stimulated cells, we observed that thrombin markedly up-regulates growth-regulated oncogene-alpha (GRO-alpha) in several tumor cell lines as well as endothelial cells by mRNA and protein analysis. Thrombin enhanced the secretion of GRO-alpha from tumor cells 25- to 64-fold. GRO-alpha is a CXC chemokine with tumor-associated angiogenic as well as oncogenic activation following ligation of its CXCR2 receptor. GRO-alpha enhanced angiogenesis in the chick chorioallantoic membrane assay 2.2-fold, providing direct evidence for GRO-alpha as an angiogenic growth factor. Anti-GRO-alpha antibody completely inhibited the 2.7-fold thrombin-induced up-regulation of angiogenesis, as well as the 1.5-fold thrombin-induced up-regulation of both endothelial cell cord formation in Matrigel and growth in vitro. Thrombin as well as its PAR-1 receptor activation peptide [thrombin receptor activation peptide (TRAP)] as well as GRO-alpha all markedly increased vascular regulatory proteins and growth factors: matrix metalloproteinase (MMP)-1, MMP-2, vascular endothelial growth factor (VEGF), angiopoietin-2 (Ang-2), CD31, and receptors KDR and CXCR2 in human umbilical vein endothelial cells. All of the thrombin/TRAP gene up-regulations were completely inhibited by anti-GRO-alpha antibody and unaffected by irrelevant antibody. Similar inhibition of gene up-regulation as well as thrombin-induced chemotaxis was noted with small interfering RNA (shRNA) GRO-alpha KD 4T1 breast tumor and B16F10 melanoma cells. In vivo tumor growth studies in wild-type mice with shRNA GRO-alpha KD cells revealed 2- to 4-fold impaired tumor growth, metastasis, and angiogenesis, which was not affected by endogenous thrombin. Thus, thrombin-induced angiogenesis requires the up-regulation of GRO-alpha. Thrombin up-regulation of GRO-alpha in tumor cells as well as endothelial cells contributes to tumor angiogenesis.     

Vascular endothelial groth factor induces tissue factor and matrix metalloproteinase production in endothelial cells: Conversion of prothrombin to thrombin results in progelatininase a activation and cell proliferation

Production of vascular endothelial growth factor (VEGF) by cancer cells at invasive and metastatic sites is an important aspect of tumor angiogenesis. Although known primarily as a mitogen and a vascular permeability factor (VPF) for endothelial cells, VEGF/VPF has been proposed to induce the expression of procoagulant factors in endothelial cells. In this study, we have explored the ramifications of VEGF induction of tissue factor (TF) in human umbilical vein endothelial cells (HUVECs) and subsequent activation of progelatinase A. Within 3 hr of incubation with VEGF/VPF, endothelial cells accelerate TF generation as measured using chromogenic substrate assays for coagulation factors Xa and thrombin. Incubation of VEGF/VPF-pre-treated cells with prothrombin and factors X, Va, and VIIa at 37°C and subsequent generation of thrombin resulted in activation of secreted endothelial progelatinase A as demonstrated by gelatin zymography. Anti-thrombin III or antibodies to TF inhibited thrombin generation and progelatinase A activation. VEGF/VPF also directly increased HUVEC secretion of interstitial collagenase, tissue inhibitor of metalloproteinases (TIMP-1) and, to a lesser extent, gelatinase A. The effect of thrombin on endothelial proliferation in serum-free media was examined. Thrombin was a growth factor for HUVECs at a lower dose than that required for progelatinase A activation. Whereas TIMP-2 abrogated thrombin-induced progelatinase A activation, it had no significant effect on thrombin-induced endothelial cell growth. We propose that an early step in tumor angiogenesis involves VEGF-induced thrombin generation and increased MMP production with subsequent activation of endothelial progelatinase A and degradation of the underlying basement membrane. Int. J. Cancer 75:780–786, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Expression of protease-activated receptor 1 in oral squamous cell carcinoma.

Protease-activated receptor 1 (PAR-1) is a G-coupled membrane protein. In this study, we analyzed the expression of PAR-1 in oral squamous cell carcinomas (SCCs). PAR-1 was expressed in oral SCCs, but the level of PAR-1 protein was lower in non-metastatic cells than in metastatic cells. Thrombin stimulated the growth of metastatic cells, and both thrombin and thrombin receptor activation peptide (TRP) enhanced the adhesion of these cells to fibronectin, but had no effect on non-metastatic cells. Thrombin and TRP also induced matrix metalloproteinase (MMP)-2 and MMP-9 activities in metastatic cells. These results suggest that PAR-1 may contribute to the growth and invasive potential of oral SCC.     

Paracrine interactions of vascular endothelial growth factor and platelet-derived growth factor in endothelial and lung cancer cells

While the effects of single growth factors on endothelial cells (ECs) have been extensively studied, the importance of induction of growth factors such as PDGF-BB (platelet derived growth factor) in ECs and its impact on tumor cell functions are only partly understood. Human umbilical vein endothelial cells (HUVECs) were cultured under serum-free conditions and stimulated by 20 ng/ml VEGF (vascular endothelial growth factor) or 20 ng/ml bFGF (basic fibroblastic growth factor). As determined by real-time PCR, both VEGF and bFGF induced a significant (up to 4-fold) increase in PDGF-B RNA expression which was time- and dose-dependent (p<0.05). Similarly, conditioned medium (CM) from lung cancer cells (A549) which is known to contain multiple growth factors including VEGF and bFGF also induced PDGF-B RNA expression. Using ELISA assays, VEGF and bFGF significantly increased PDGF-BB protein secretion in HUVECs (p<0.01). By addition of BIBF 1000, a novel inhibitor of the VEGF and bFGF receptor kinases, the effect of VEGF on PDGF-B RNA induction was significantly antagonized (p<0.01). Furthermore, we studied the biological significance of EC-derived PDGF-BB on lung cancer cells. Interestingly, HUVEC-derived CM significantly stimulated migration of A549 cells (p<0.001) with a trend to further increased migration with the use of VEGF-stimulated (PDGF-BB rich) CM (p=0.2). Collectively, endothelial and lung cancer cells seem to interact via various paracrine pathways, e.g. by the reciprocal induction of VEGF and PDGF-BB. Thus, targeting key molecules would result in expression alterations of multiple factors and alter the biological functions of both stromal and tumor cells.     

Prognostic role of protease-activated receptors 1 and 4 in resected stage IB non-small-cell lung cancer

BACKGROUND: Protease-activated receptor (PAR)-1 and PAR-4 are involved in extracellular matrix invasion and angiogenesis. PATIENTS AND METHODS: A series of 60 resected stage IB non-small-cell lung cancers (NSCLCs), including 30 adenocarcinomas (ADCs) and 30 squamous cell carcinomas (SCCs), were processed by immunohistochemistry with antibodies to PAR-1, PAR-4, vascular endothelial growth factor (VEGF), and CD34. RESULTS: Protease-activated receptor-1 was expressed in 37 cases (62%) and PAR-4 in 39 (65%). Adenocarcinomas were significantly more positive than SCC for PAR-1 (17 vs. 8 cases) and PAR-4 (10 vs. 5 cases). Vascular endothelial growth factor was expressed in 42 cases (70%): 22 ADC and 20 SCC. A significant correlation emerged between PAR-1 and/or PAR-4 expression and VEGF but not with microvessel density. Median follow-up was 38 months; actuarial 5-year survival was 43%. At univariate analysis, 3-year survival was shorter in patients expressing PAR-4 versus negative cases (29% vs. 60%; P = 0.002). In 46 patients expressing PAR-1 and/or PAR-4, 3-year survival was 30% versus 68% in 14 patients with no PAR expression (P = 0.002). A trend toward shorter 3-year survival was seen in PAR-1-positive versus PAR-1-negative cases (34% vs. 46%; P = 0.06). Multivariate analysis identified expression of PAR-1 and/or PAR-4 as an independent prognostic factor for reduced survival in resected stage IB NSCLC. CONCLUSION: Expression of PAR-1 and PAR-4 in early-stage NSCLC could be included in a molecular algorithm for the selection of patients eligible for adjuvant studies.     

Molecular polarity in endothelial cells and tumor-induced angiogenesis

Endothelial cells expose receptors for vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) at the abluminal, basal surface that work as basic regulators of tumor-induced angiogenesis. Their specific localization makes them susceptible to the activity of tumor-released stimulatory factors, like VEGF/VPF, which induce proliferation of the endothelial cell toward the extracellular matrix. At the same time, VEGF/VPF stimulates endothelial cells to expose tissue factor (TF), the high-affinity transmembrane receptor and cofactor for cellular initiation of the plasma coagulation protease cascades through the extrinsic pathway, so generating thrombin. Thrombin exerts a number of activities: it forms an extracellular fibrin barrier from the VEGF/VPF-dependent fibrinogen extravasation; it activates progelatinase-A (pro-MMP-2), which destroys the basal membrane, allowing proliferation of endothelial cells (ECs) in the novel tumoral fibrin matrix; finally, it induces EC proliferation, potentiating the VEGF effect. Another important factor exposed at the abluminal endothelial cell surface is membrane type 1 matrix metalloproteinase (MT1-MMP), a membrane-bound metalloproteinase, which also activates progelatinase-A, allowing an alternative pathway to that of thrombin to destroy the basal membrane. In addition, we will see that MT1-MMP is also engaged in a direct, cell-associated fibrinolytic activity, essential for tubulogenesis of the novel outsprouting capillary.     

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