Cannabinoids inhibit the vascular endothelial growth factor pathway in gliomas

Cannabinoids inhibit tumor angiogenesis in mice, but the mechanism of their antiangiogenic action is still unknown. Because the vascular endothelial growth factor (VEGF) pathway plays a critical role in tumor angiogenesis, here we studied whether cannabinoids affect it. As a first approach, cDNA array analysis showed that cannabinoid administration to mice bearing s.c. gliomas lowered the expression of various VEGF pathway-related genes. The use of other methods (ELISA, Western blotting, and confocal microscopy) provided additional evidence that cannabinoids depressed the VEGF pathway by decreasing the production of VEGF and the activation of VEGF receptor (VEGFR)-2, the most prominent VEGF receptor, in cultured glioma cells and in mouse gliomas. Cannabinoid-induced inhibition of VEGF production and VEGFR-2 activation was abrogated both in vitro and in vivo by pharmacological blockade of ceramide biosynthesis. These changes in the VEGF pathway were paralleled by changes in tumor size. Moreover, intratumoral administration of the cannabinoid Delta9-tetrahydrocannabinol to two patients with glioblastoma multiforme (grade IV astrocytoma) decreased VEGF levels and VEGFR-2 activation in the tumors. Because blockade of the VEGF pathway constitutes one of the most promising antitumoral approaches currently available, the present findings provide a novel pharmacological target for cannabinoid-based therapies.     

Bevacizumab for glioblastoma refractory to vascular endothelial growth factor receptor inhibitors

Glioblastoma (GBM) is a highly vascular tumor dependent on angiogenesis through the vascular endothelial growth factor (VEGF) signaling cascade. Inhibition of VEGF signaling is an important therapeutic strategy. We report our experience with bevacizumab (BEV), a VEGF targeting antibody, following failure of a VEGF receptor targeting tyrosine kinase inhibitor (TKI). We retrospectively identified patients treated on clinical trials with VEGFR–TKIs for recurrent GBM followed by BEV at next recurrence. Survival was estimated by the Kaplan–Meier method. Fourteen patients were identified (six women; median age 57). All received VEGFR–TKIs (sunitinib 11, cediranib 2, sorafenib 1) then BEV at next recurrence. There were no radiographic responses to VEGFR–TKIs; best response was stable disease in 50% (7/14). Patients received BEV alone (21%, 3/14) or in combination with chemotherapy (79%, 11/14). On BEV, 29% (4/14) had a partial response, and 36% (5/14) stabilized. Of evaluable patients, 42% (5/12) had neurological improvement and 56% (5/9) reduced corticosteroid requirement. Median survival on BEV was 7.8 months (95% CI 4.0–15.8), median progression-free survival (PFS) was 4.0 months (95% CI 1.6–10.5), and the 6-month PFS rate was 29% (95% CI 9–52). Our radiographic and survival outcomes with BEV following progression after VEGFR–TKIs are similar to data from studies of BEV as initial salvage therapy, although our sample size was small. Prior exposure to VEGFR–TKIs may not preclude response to BEV, but sensitivity to BEV may be lower following more robust VEGFR inhibition.     

Green tea catechins inhibit vascular endothelial growth factor receptor phosphorylation.

Vascular endothelial growth factor (VEGF) receptors (VEGFR) play a major role in tumor angiogenesis and, thus, represent attractive targets for the development of novel anticancer therapeutics. In this work, we report that green tea catechins are novel inhibitors of VEGFR-2 activity. Physiological concentrations (0.01-1 microM) of epigallocatechin-3 gallate, catechin-3 gallate, and, to a lesser extent, epicatechin-3 gallate induce a rapid and potent inhibition of VEGF-dependent tyrosine phosphorylation of VEGFR-2. The inhibition of VEGFR-2 by epigallocatechin-3 gallate was similar to that induced by Semaxanib (SU5416), a specific VEGFR-2 inhibitor. The inhibition of VEGFR-2 activity by the catechins displayed positive correlation with the suppression of in vitro angiogenesis. These observations suggest that the anticancer properties of green tea extracts may be related to their inhibition of VEGF-dependent angiogenesis.     

Use of soluble recombinant decoy receptor vascular endothelial growth factor trap (VEGF Trap) to inhibit vascular endothelial growth factor activity

Primary tumors and metastases require blood vessel formation to support their continued growth and eventual metastasis. They use existing vasculature during initial growth but eventually must orchestrate the development and maintenance of new vessels--a process termed angiogenesis--to grow beyond a small size and spread. Angiogenesis is regulated by a number of soluble factors, the relative proportions of which can exacerbate or inhibit the process. Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis, produced by the majority of human solid tumors. Inhibitors of VEGF might have an impact on the growth and metastasis of these cancers. The relevance of this strategy to the treatment of colorectal cancer was first successfully demonstrated in human clinical trials using a monoclonal antibody against VEGF. A potent antiangiogenic soluble recombinant decoy, VEGF Trap is a protein constructed from VEGF receptor-binding domains linked to an immunoglobulin G(1) constant region. It possesses an affinity for VEGF that is significantly higher than that of the monoclonal antibody. VEGF Trap has demonstrated marked efficacy in halting angiogenesis and shrinking tumors in preclinical animal models and is currently being studied in phase I clinical trials in humans with advanced solid malignancies.     

CXCL12 and vascular endothelial growth factor synergistically induce neoangiogenesis in human ovarian cancers

Ovarian carcinomas have a poor prognosis, often associated with multifocal i.p. dissemination accompanied by intense neovascularization. To examine tumor angiogenesis in the tumor microenvironment, we studied malignant ascites and tumors of patients with untreated ovarian carcinoma. We observed that malignant ascites fluid induced potent in vivo neovascularization in Matrigel assay. We detected a sizable amount of vascular endothelial cell growth factor (VEGF) in malignant ascites. However, pathologic concentration of VEGF is insufficient to induce in vivo angiogenesis. We show that ovarian tumors strongly express CXC chemokine stromal-derived factor (SDF-1/CXCL12). High concentration of CXCL12, but not the pathologic concentration of CXCL12 induces in vivo angiogenesis. Strikingly, pathologic concentrations of VEGF and CXCL12 efficiently and synergistically induce in vivo angiogenesis. Migration, expansion, and survival of vascular endothelial cells (VEC) form the essential functional network of angiogenesis. We further provide a mechanistic basis for explaining the interaction between CXCL12 and VEGF. We show that VEGF up-regulates the receptor for CXCL12, CXCR4 expression on VECs, and synergizes CXCL12-mediated VEC migration. CXCL12 synergizes VEGF-mediated VEC expansion and synergistically protects VECs from sera starvation-induced apoptosis with VEGF. Finally, we show that hypoxia synchronously induces tumor CXCL12 and VEGF production. Therefore, hypoxia triggered tumor CXCL12 and VEGF form a synergistic angiogenic axis in vivo. Hypoxia-induced signals would be the important factor for initiating and maintaining an active synergistic angiogeneic pathway mediated by CXCL12 and VEGF. Thus, interrupting this synergistic axis, rather than VEGF alone, will be a novel efficient antiangiogenesis strategy to treat cancer.     

Shikonin and its derivatives inhibit the epidermal growth factor receptor signaling and synergistically kill glioblastoma cells in combination with erlotinib

Overexpression and mutation of the epidermal growth factor receptor (EGFR) gene play a causal role in tumorigenesis and resistance to treatment of glioblastoma (GBM). EGFR inhibitors such as erlotinib are currently used for the treatment of GBM; however, their efficacy has been limited due to drug resistance. New treatment strategies are therefore urgently needed. Shikonin, a natural naphthoquinone, induces both apoptosis and necroptosis in human glioma cells, but the effectiveness of erlotinib‐shikonin combination treatment as well as the underlying molecular mechanisms is unknown yet. In this study, we investigated erlotinib in combination with shikonin and 14 shikonin derivatives in parental U87MG and transfected U87MG.ΔEGFR GBM cells. Most of the shikonin derivatives revealed strong cytotoxicity. Shikonin together with five other derivatives, namely deoxyshikonin, isobutyrylshikonin, acetylshikonin, β,β‐dimethylacrylshikonin and acetylalkannin showed synergistic cytotoxicity toward U87MG.ΔEGFR in combination with erlotinib. Moreover, the combined cytotoxic effect of shikonin and erlotinib was further confirmed with another three EGFR‐expressing cell lines, BS153, A431 and DK‐MG. Shikonin not only dose‐dependently inhibited EGFR phosphorylation and decreased phosphorylation of EGFR downstream molecules, including AKT, P44/42MAPK and PLCγ1, but also together with erlotinib synergistically inhibited ΔEGFR phosphorylation in U87MG.ΔEGFR cells as determined by Loewe additivity and Bliss independence drug interaction models. These results suggest that the combination of erlotinib with shikonin or its derivatives might be a potential strategy to overcome drug resistance to erlotinib.     

PTEN restoration and PIK3CB knockdown synergistically suppress glioblastoma growth in vitro and in xenografts

Glioblastoma is the most frequent and malignant glioma in adults. To develop an effective gene therapy strategy for glioblastoma, we investigated the anti-proliferative effects of phosphatase and tensin homolog (PTEN) restoration and siRNAs specifically targeting PIK3CB and PIK3CA on PTEN-deficient glioblastoma cells in vitro and in subcutaneous xenografts. Restoration of PTEN or knockdown of PIK3CB, but not PIK3CA, in glioblastoma cells markedly down-regulates the phosphorylation level of AKT, inhibits cell proliferation and colony formation, arrests the cell cycle at the G0/G1 stage, and promotes caspase-dependent apoptosis. Combined treatment with PTEN restoration and PIK3CB knockdown shows strong synergy. PTEN restoration or PIK3CB knockdown is also able to efficiently inhibit the growth of human U251 glioblastoma xenografts in nude mice, while tumor growth is entirely suppressed by a combination of the two treatments. In addition, we found that the mRNA levels of inhibitors of apoptosis proteins (IAPs) are reduced in U251 cells by PTEN restoration, suggesting that combined antitumor effects may also be partly attributed to the inhibition of the IAP pathway by PTEN restoration. Collectively, our results demonstrate that PI3 K isoforms play specific roles in tumorigenesis, and that combined treatment of PTEN restoration and PIK3CB siRNA is a promising gene therapy strategy for PTEN-deficient gliomas.     

血管内皮生长因子及其受体与骨肉瘤关系的研究进展

血管内皮生长因子（ＶＥＧＦ）是一种序列高度保守、高度特异性的促血管内皮细胞生长因子,广泛分布于人和动物体内的大脑、肾脏、肝脏、脾脏、胰腺和骨骼等组织中,对内皮细胞具有强烈的促有丝分裂作用,刺激血管内皮细胞增殖和血管通透性增加,促进新生血管形成。ＶＥＧＦ通过与血管内皮细胞表面受体（ＶＥＧＦＲ）特异性结合发挥生物学效应。抑制ＶＥＧＦ及ＶＥＧＦＲ的活性可以减缓或阻滞骨肉瘤侵袭和转移。研究表明,ＶＥＧＦ及ＶＥＧＦＲ对肿瘤血管及淋巴管的生成及肿瘤侵袭和转移起重要作用。本文对ＶＥＧＦ及ＶＥＧＦＲ与骨肉瘤血管与淋巴管生成及其侵袭与转移的关系作一综述。     

Histone deacetylase inhibitors such as sodium butyrate and trichostatin A inhibit vascular endothelial growth factor (VEGF) secretion from human glioblastoma cells

We investigated the effects of histone deacetylase (HDAC) inhibitors such as sodium butyrate (SB) and trichostatin A (TSA) on the expression of vascular endothelial growth factor (VEGF) by human glioblastoma T98G, U251MG, and U87MG cells. The glioblastoma cells secreted three VEGF isoforms, VEGF (189), (165), and (121), although the expression levels of VEGF differed between the cell types. Treatment with either 5mM SB or 100ng/ml TSA reduced VEGF secretion in conditioned media and reduced VEGF mRNA expression. We also studied the expression of VEGF-B,-C, and-D mRNA in human glioblastoma cells and their modulation by HDAC inhibitors. The PCR products of VEGF-B (357 bp), VEGF-C (501 bp), and VEGF-D (484 bp) were amplified in all glioblastoma cells examined. Treatment with SB reduced the expression of VEGF-D mRNA in U251MG cells and the expression of VEGF-B mRNA in U87MG cells. TSA treatment reduced the expression of VEGF-D in U251MG cells. These results suggest that HDAC inhibitors reduce VEGF secretion and modulate the expression of the other VEGF family members, and therefore may inhibit angiogenesis in glioblastoma tissues.     

Urokinase receptor and vascular endothelial growth factor are synergistically associated with the liver metastasis of colorectal cancer.

Considering recent findings that the urokinase plasminogen activation (PA) system is involved in invasion and vascular endothelial growth factor (VEGF) is involved in angiogenesis of colorectal cancer, we evaluated these factors in the liver metastasis of primary colorectal cancer. Cancer tissues from 71 colorectal cancer patients were assayed quantitatively for antigen levels of urokinase type plasminogen activator (uPA), uPA receptor (uPAR), and plasminogen activator inhibitor-1 and -2 (PAI-1, PAI-2), and were also assayed immunohistochemically for expression of VEGF protein. Among the PA system factors, both the levels of uPAR and PAI-1 were significantly higher in larger tumors than in smaller ones, and were also significantly higher in tumors that invaded subserosa, serosa or adjacent organs than in mucosal, submucosal tumors or in tumors that invaded the muscle layer. The uPAR levels were significantly higher in tumors with liver metastasis than in those without. VEGF overexpression was significantly more frequent in tumors with lymph node involvement or liver metastasis than in those without. Among the PA system factors, the uPAR levels were significantly higher in tumors with VEGF overexpression and a multivariate analysis revealed that high uPA level and VEGF overexpression were independent risk factors for liver metastasis. The combination of high uPAR level and overexpression of VEGF was associated with the worst prognosis in patients with colorectal cancer. These results suggest that uPAR and VEGF might contribute synergistically to the liver metastasis of colorectal cancer.     

Inhibitors of the vascular endothelial growth factor receptor

The inhibitors of VEGF-mediated signaling continue to wind their way through extensive preclinical and clinical development paths. Whereas the first phase III trial did not meet its endpoints, one hopes that the others will. As we learn more about the VEGF pathways in the laboratory and the clinic, we can interpret with greater certainty what role these drugs or their successors will have in the treatment of human cancers.     

Pancreatic carcinoma cells express neuropilins and vascular endothelial growth factor, but not vascular endothelial growth factor receptors

BACKGROUND: Neuropilins (NRPs) are characterized as coreceptors of vascular endothelial growth factor (VEGF). In the current study, the authors assessed the expression of NRPs, VEGF, and vascular endothelial growth factor receptors (VEGFRs), as well as VEGF-induced cell proliferation, in pancreatic carcinoma cell lines and tissue specimens. METHODS: Human pancreatic carcinoma cell lines (Panc-1 and MIA PaCa-2), normal human pancreatic ductal epithelial cells (HPDE), and human umbilical vein endothelial cells (HUVECs) were cultured. Human pancreatic adenocarcinoma tissue specimens were also studied. Expression levels of NRPs, VEGFRs, and VEGF were determined by real-time polymerase chain reaction analysis and immunostaining. Cell proliferation was examined using a [3H]thymidine incorporation assay. RESULTS: Both NRP-1 and NRP-2 were expressed in Panc-1 cells, HPDE cells, and HUVECs but were expressed minimally in MIA PaCa-2 cells. Panc-1 expressed 30 times more NRP-1 mRNA than NRP-2 mRNA. NRP-1 levels in Panc-1 cells were 5.3 times higher than in HPDE cells but were similar to NRP-1 levels in HUVECs. NRP-2 levels in Panc-1 cells were similar to NRP-2 levels in HPDE cells but lower than NRP-2 levels in HUVECs. Expression of all three VEGFRs was observed only in HUVECs. However, VEGF mRNA was detected in all cell types except for HUVECs. NRP-1 immunoreactivity levels were much higher than NRP-2 immunoreactivity levels in Panc-1 and human pancreatic adenocarcinoma tissue specimens, whereas VEGFRs were not detected in either of these two settings. In response to VEGF165, [3H]thymidine incorporation in Panc-1 cells increased significantly (by 61%; P < 0.01). A monoclonal antibody against human NRP-1 significantly blocked VEGF-induced cell proliferation in Panc-1 cells. CONCLUSIONS: The pancreatic carcinoma cell line Panc-1 and adenocarcinoma tissue specimens expressed high levels of NRP-1 and VEGF, but not VEGFRs, and exogenous VEGF significantly increased NRP-1-mediated, but not VEGFR-mediated, Panc-1 cell proliferation. These data suggested that NRP-1 may be involved in the pathogenesis of pancreatic carcinoma.     

Inhibition of glioblastoma angiogenesis and invasion by combined treatments directed against vascular endothelial growth factor receptor-2, epidermal growth factor receptor, and vascular endothelial-cadherin.

PURPOSE: Inhibition of angiogenesis can influence tumor cell invasion and metastasis. We previously showed that blockade of vascular endothelial growth factor receptor-2 (VEGFR-2) with the monoclonal antibody DC101 inhibited intracerebral glioblastoma growth but caused increased tumor cell invasion along the preexistent vasculature. In the present study, we attempted to inhibit glioma cell invasion using a monoclonal antibody against the epidermal growth factor receptor (EGFR), which in the context of human glioblastomas, has been implicated in tumor cell invasion. In addition, we analyzed whether blockade of vascular endothelial (VE)-cadherin as a different antiangiogenic target could also inhibit glioblastoma angiogenesis and growth. EXPERIMENTAL DESIGNS: Nude mice who received intracerebral glioblastoma xenografts were treated using monoclonal antibodies against VEGFR-2 (DC101), EGFR (C225), and VE-cadherin (E4G10) either alone or in different combinations. RESULTS: Increased tumor cell invasion provoked by DC101 monotherapy was inhibited by 50% to 66% by combined treatment with C225 and DC101. C225 inhibited glioblastoma cell migration in vitro, but had no effect on the volume of the main tumor mass or on tumor cell proliferation or apoptosis in vivo, either alone or in combination with DC101. The anti-VE-cadherin monoclonal antibody E4G10 was a weaker inhibitor of tumor angiogenesis and growth than DC101, and also caused a weaker increase in tumor cell invasion. CONCLUSIONS: Inhibition of angiogenesis achieved by blocking either VEGFR-2 or VE-cadherin can cause increased glioma cell invasion in an orthotopic model. Increased tumor cell invasion induced by potent inhibition of angiogenesis with DC101 could be inhibited by simultaneous blockade of EGFR.     

Antitumor treatment efficacy by targeting epidermal growth factor receptor and vascular endothelial growth factor receptor-2 in an orthotopic human glioblastoma model

Using an orthotopic intracerebral model from our established HM55-BGIV-101 tumor line, we investigated the antitumor effect on the angiogenesis and growth of human glioblastoma after treatment with monoclonal antibody DC101 against the vascular endothelial growth factor receptor-2 and monoclonal antibody C225 against the epidermal growth factor receptor. Nude mice bearing intracerebral glioblastoma xenografts were treated intraperitoneally with DC101 and C225 either alone or in combination. Histopathological analysis of solid tumor volume, satellite tumor number, microvessel density, tumor cell proliferation, and apoptosis was performed. In the DC101-treated group, solid tumor volume and microvessel density were reduced by 59.7 and 64%, respectively; tumor cell proliferative activity was reduced by 53.2% and the apoptotic index (AI) was increased by 66.7%; satellite tumor number was enhanced by 84.4%. C225 alone reduced satellite tumor number by 43.3%, but had no effect on solid tumor volume, microvessel density, tumor cell proliferation, and apoptosis. C225 combined with DC101 not only reduced solid tumor volume, microvessel density, tumor cell proliferative activity, and increased AI, but also reduced satellite tumor number. Inhibition of angiogenesis achieved by DC101 can cause increased tumor cell invasiveness. In our studies this increased tumor cell invasiveness was inhibited simultaneously by C225, which provides a theoretical basis for treatment of glioblastoma by the method of combining drugs with different pharmacological activity.     

Porphyrin analogues as novel antagonists of fibroblast growth factor and vascular endothelial growth factor receptor binding that inhibit endothelial cell proliferation, tumor progression, and metastasis

Fibroblast growth factors (FGFs) and vascular endothelial growth factor (VEGF) play a pivotal role in the multistep pathway of tumor progression, metastasis, and angiogenesis. We have identified a porphyrin analogue, 5,10,15,20-tetrakis(methyl-4-pyridyl)-21H,23H-porphine-tetra -p-tosylate salt (TMPP), as a potent inhibitor of FGF2 and VEGF receptor binding and activation. TMPP demonstrated potent inhibition of binding of soluble FGF receptor 1 (FGFR1) to FGF2 immobilized on heparin at submicromolar concentrations. TMPP inhibits binding of radiolabeled FGF2 to FGFR in a cell-free system as well as to cells genetically engineered to express FGFR1. Furthermore, TMPP also inhibits the binding of VEGF to its tyrosine kinase receptor in a dose-dependent manner. In an in vitro angiogenic assay measuring the extent of endothelial cell growth, tube formation, and sprouting, TMPP dramatically reduced the extent of the FGF2-induced endothelial cell outgrowth and differentiation. In a Lewis lung carcinoma model, mice receiving TMPP showed a marked inhibition of both primary tumor progression and lung metastases development, with nearly total inhibition of the metastatic phenotype upon alternate daily injections of TMPP at 25 microg/g of body mass. Finally, novel meso-pyridylium-substituted, nonsymmetric porphyrins, as well as a novel corrole-based derivative, with >50-fold increase in activity in vitro, had a significantly improved efficacy in blocking tumor progression and metastasis in vivo.     

血管内皮细胞生长因子与穿膜蛋白在骨肉瘤中的表达及相关性研究

目的：研究血管内皮细胞生长因子(vascularendothelialgrowthfactor,VEGF)与穿膜蛋白(delta like 1 homolog,DLK1)在骨肉瘤中的表达,并探讨二者的表达与骨肉瘤分级、转移和预后的关系及其二者的相关性,为骨肉瘤侵袭和转移调控机制的研究打基础。方法应用微波链霉菌抗生物素蛋白－过氧化物酶连结( streptavidin perosidase,SP )免疫组化法检测42例骨肉瘤患者(实验组)组织中的VEGF和DLK1的表达水平,同时设30例良性的骨软骨瘤作为阴性对照组。结合骨肉瘤的生物学行为及临床随访资料对 VEGF 和DLK1的表达与骨肉瘤分级、转移和预后的相关性及骨肉瘤细胞中 VEGF 和 DLK1表达的相关性进行分析。结果骨肉瘤中VEGF和DLK1的阳性表达率分别是76.2%和66.7%,骨软骨瘤中VEGF和DLK1的阳性表达率都为0,且骨肉瘤中VEGF和DLK1的阳性表达率显著高于对照组( P＜0.05)。VEGF在不同病理分级(高、中、低分化)及Enneking分期( I～II A期、II B～III期)骨肉瘤组织中的阳性表达不同,差异有统计学意义( P＜0.05),且随着病理分级和Enneking外科分期的升高,VEGF的阳性表达率也呈增强趋势;DLK1在不同病理分级的骨肉瘤组织中的阳性表达不同,差异有统计学意义( P＜0.05),且随着病理分级的升高,DLK1阳性表达强度也随之增强。VEGF与DLK1有相关性,二者呈正相关( r＝0.8957,P＜0.05)。VEGF、DLK1的表达与骨肉瘤转移均呈显著正相关性( r＝0.6782、r＝0.7643,P＜0.05)。VEGF、DLK1的表达与骨肉瘤预后呈显著正相关性(r＝0.8462、r＝0.8758,P＜0.05)。结论 VEGF与DLK1在骨肉瘤细胞中均高度表达;VEGF和DLK1表达阳性者骨肉瘤患者的肿瘤恶性程度高、转移率高、预后差;VEGF和DLK1表达阳性是骨肉瘤的分级、转移和预后不良的重要生物学指标,二者在骨肉瘤的发生和转移上可能具有相互协同作用。     

青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子及其受体表达的影响

 目的　研究青蒿琥酯对急性单核细胞白血病SHI-1细胞株血管内皮生长因子（VEGF）及其受体（VEGFR）的影响。方法　酶联免疫吸附法检测非细胞毒性浓度（5、10、20 ng／ml）青蒿琥酯作用SHI-1细胞后培养上清液VEGF浓度,流式细胞术检测有或无青蒿琥酯作用时,SHI-1细胞表面VEGFR-1及VEGFR-2阳性表达率。结果　培养24、48 h后,无青蒿琥酯作用的 SHI-1细胞培养上清液VEGF质量浓度分别为（980.3±2.2）、（982.4±2.3）pg／ml,VEGFR-1表达率分别为（5.40±3.11）%和（4.45±2.85）%,VEGFR-2表达率分别为（13.90±2.26）%和（13.95±1.96）%。 5、10 、20 ng／ml青蒿琥酯作用24 h后,SHI-1细胞培养上清液VEGF质量浓度分别为（234.6±1.8）、（114.9±1.6）、（108.8±1.5）pg／ml,作用48 h后分别为（62.3±1.7）、（60.9±1.6）、（32.7±1.7）pg／ml,与培养相同时间无青蒿琥酯组相比,VEGF浓度明显下降（均P＜0.05）,且相同浓度青蒿琥酯作用24 h与48 h间差异亦有统计学意义（均P＜0.05）。5、10 、20 ng／ml青蒿琥酯作用24 h,VEGFR-1阳性率分别为（4.30±2.21）%、（4.20±1.37）%和（3.90±1.86）%,作用48 h后分别为（3.80±2.87）%、（3.60±1.73）%和（3.00±1.82）%,相同作用时间不同浓度青蒿琥酯组间及相同浓度作用不同时间组间VEGFR-1阳性率差异均无统计学意义（均P＞0.05）;作用24 h后,SHI-1细胞VEGFR-2阳性率分别为（4.40±1.15）%、（3.10±0.68）%和（1.10±0.72）%,作用48 h后分别为（3.00±1.68）%、（2.20±0.93）%和（0.60±0.92）%,3个不同浓度青蒿琥酯作用相同时间后VEGFR-2表达率降低（均P＜0.05）,相同浓度作用24与48 h间差异均无统计学意义（均P＞0.05）。结论　SHI-1细胞株高分泌VEGF,青蒿琥酯可下调VEGF分泌及VEGFR-2的表达,而对VEGFR-1表达的调节作用不显著。     

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.