Increased endothelial uptake of paclitaxel as a potential mechanism for its antiangiogenic effects: potentiation by Cox-2 inhibition

Paclitaxel has antiangiogenic properties, but the mechanisms for the enhanced sensitivity of endothelial cells (ECs) to this drug are not established. The aims of our study were to compare the distribution of paclitaxel into ECs with other cell types, to assess the effects of low doses of paclitaxel on Cox-2 expression and to determine the combined effects of paclitaxel and Cox-2 inhibitors on angiogenesis in vitro and in patients with cancer. Upon exposure to low (5 nM) concentrations of [3H]-paclitaxel, uptake of radioactivity was more than 5 times higher in ECs than other cell types. Exposing human umbilical vein ECs to low nanomolar (1-50 nM) concentrations of paclitaxel enhanced Cox-2 expression more than 2-fold, as measured by ELISA. Combined treatment with paclitaxel and the Cox-2 inhibitor NS-398 resulted in increased antiendothelial effects as compared to each agent alone. To assess the biologic effects of the combined treatment in vivo, 4 cancer patients were treated with a prolonged intravenous infusion of paclitaxel (10 mg/m2/day) and the Cox-2 inhibitor celecoxib (400 mg p.o. BID), and plasma angiogenic activity and drug levels were measured. The treatment was well tolerated, providing steady-state concentrations of paclitaxel in plasma near 10 nM and potent plasma antiendothelial effects were observed. These findings suggest that antiangiogenic effects of paclitaxel may be due its preferential accumulation in ECs. Low dose paclitaxel in combination with a Cox-2 inhibitor is an attractive antiangiogenic and antitumor strategy that deserves further evaluation in clinical trials.     

Combined histone deacetylase and cyclooxygenase inhibition achieves enhanced antiangiogenic effects in lung cancer cells

Prostaglandin E2 (PGE2) is an important pro‐angiogenic and pro‐proliferative cytokine and the key enzymes modulating its levels, cyclooxygenase (COX)‐2 and 15‐hydroxyprostaglandin dehydrogenase (15‐PGDH) play important opposing roles in carcinogenesis. Previously we found loss of 15‐PGDH expression in lung cancer and its reactivation leads to strong in vivo tumor‐suppressive effect via an antiangiogenic mechanism. Here, we find that HDAC inhibitors (HDACI), such as trichostatin A (TSA) and vorinostat could reactivate 15‐PGDH expression but overall induce PGE2 generation and this is the result of concomitant induction of COX‐1 and ‐2 leading to functional promotion of endothelial cell proliferation and capillary formation. Direct TSA treatment inhibits endothelial cell proliferation and capillary formation in our study in line with prior reports as HDACIs have been shown to directly inhibit angiogenesis. The elevation of PGE2 levels induced by HDACI is potently neutralized by indomethacin (INN) or Celecoxib co‐treatment and accordingly, angiogenesis is more effectively inhibited when using conditioned medium of co‐treatment than either alone confirming that this effect is mediated via the PGE2 axis. Accordingly, blockage of EP2/4 receptors mitigates the stimulation of angiogenesis by excessive PGE2 generation mediated by TSA. In this study, we identify a potentially adverse effect of HDACIs through induction of both 15‐PGDH and COX‐2 leading to elevated PGE2 levels and thereby stimulation of angiogenesis. Co‐treatment of TSA and INN shows more potent anti‐angiogenic effects by inducing 15‐PGDH and inhibiting COX‐2. Overall, our results suggest that combined HDACI and COX inhibition should be explored clinically to achieve more meaningful benefits from HDACI therapy in lung cancer. © 2011 Wiley Periodicals, Inc.     

Selective inhibition of cyclooxygenase (COX)-2 inhibits endothelial cell proliferation by induction of cell cycle arrest

High-level expression of cyclooxygenase (COX)-2 is reported in 80-90% of colorectal adenocarcinomas. Selective inhibition of COX-2 was shown to reduce colorectal tumorigenesis in different models of carcinogenesis and to prevent metastasis in xenograft tumor models, as well as to suppress in vitro induced angiogenesis. Recently, COX-2 was reported to be expressed not only in malignant epithelial cells, but also in the neovasculature that feeds the tumor in a variety of solid human cancers. Thus, one of the possible mechanisms by which selective COX-2 inhibitor reduces tumor growth and metastasis is through inhibition of tumor angiogenesis. Although a report suggested a possible role of endothelial COX-1 in the process of angiogenesis, in a recent study, the selective inhibition of COX-2 was shown to strongly inhibit angiogenesis by inducing endothelial cell (EC) apoptosis. In the present study, using human umbilical vein endothelial cells (HUVECs) as a model of angiogenesis, we investigated the potential antiangiogenic effect of the selective COX-2 inhibitor and its mechanism of action, and clearly demonstrated that selective inhibition of COX-2 caused a dose-dependent decrease in the proliferative activity of ECs, as well as an inhibition of capillary-like tube formation. The inhibitory effect on EC proliferation was dependent on the cell cycle arrest to the G1 phase and not on cell apoptosis.     

非小细胞肺癌环氧合酶-2表达与血管生成之间的关系

背景与目的:我们以前的研究发现,环氧合酶-2(COX-2)在非小细胞肺癌(NSCLC)上调表达是患者独立预后的不良因素。COX-2参加结肠癌等实体瘤的血管生成,是否参与NSCLC血管生成目前尚有争议。本研究的目的是探讨COX-2表达与血管生成和血管内皮细胞生长因子(VEGF)-A表达之间的关系。方法:用脂质体将表达COX-2反义核酸的质粒和空质粒分别稳定转染H1299细胞(H1299-AS和H1299-P),采用RT-PCT和W esternB lot法检测细胞株COX-2和VEGF-A的水平。应用免疫组织化学染色法检测60例NSCLC肿瘤组织COX-2、VEGF-A和CD31表达情况,用χ2检验分析这些参数间相互关系。结果:H1299-AS细胞株COX-2 mRNA和蛋白水平较H1299-P细胞明显下降;VEGF-A蛋白表达水平亦同步下降。肺腺癌COX-2表达率为77%,显著高于肺鳞癌的37%(P<0.05)。NSCLC中COX-2和VEGF-A表达与肿瘤微血管密度明显相关(r分别为0.55和0.42,P<0.05)。COX-2表达阴性患者肿瘤微血管密度为45.7±15.6根/200×视野,显著高于不表达患者的37.4±9.7根/200×视野(P<0.05)。NSCLC组织中COX-2表达水平和微血管密度与患者术后生存期明显相关(P<0.05)。结论:NSCLC存在COX-2和VEGF-A共表达,COX-2可能通过诱导VEGF-A表达来发挥促血管生成样作用。     

骨髓来源内皮前体细胞促进肿瘤新生血管生成

Objective:Neovascularization of tumor is a complex process.In this study,we aimed to reveal whether the bone marrow-originated endothelial progenitor calls (EPCs) contributed to neovasculature in tumor and the angiogenesis-associated factors,VEGF and B-FGF,enhanced this process.Methods:We had established a mouse model,which were deprived of bone marrow by radiation and transplanted with bone marrow of syngenetic GFP (Green Fluorescence Protein)-transgened mice,then implanted Lewis calls.Immunohistochemical and immunoflourensenca proved the EPCs location in tumors by indentifying colocalization of GFP expression in cells staining with endothelial progenitor cell markers,CD 133,ICAM-1,CD31.The growth statue and MVD of tumor was observed after injection of VEGF or B-FGF.ICAM-1 and VE-cadherin in tumor were detected by Western blot.Results:By immunohistochemical and immunoflourensence,we proved part of bone marrow precursors located in area of tumor angiogenesis and VEGF or B-FGF increased the MVD of tumor.In Western blot,it was found and VEGF or B-FGF up-regulate the expression of ICAM-1,VE-Cadherin.Conclusion:Bone marrow-derived endothelial progenitor cell seem to be recruited in neovasculature induced by tumor.VEGF and B-FGF are key regulators of this process.     

环氧合酶-2对胃癌血管内皮生长因子及血管形成的影响

 目的　探讨环氧合酶 2 (COX 2 )对胃癌血管内皮生长因子 (VEGF)的表达及血管生成的影响。方法　应用免疫组织化学技术检测胃癌组织中COX 2 ,VEGF表达和微血管密度 (MVD)。结果　COX 2在62 .2 %胃癌组织中表达增高 ,COX 2表达与VEGF表达显著相关 (γS=0 .5 85 ,P <0 .0 1 ) ,且COX 2和VEGF均阳性的胃癌组织MVD(64.0± 2 5 .4)亦明显高于两者均阴性者 (3 0 .7± 1 1 .5 ) (P <0 .0 1 )。结论　胃癌组织中存在COX 2的高表达 ,COX 2通过增加VEGF表达而促进肿瘤血管形成     

Cyclooxygenase-2 expression is critical for chronic UV-induced murine skin carcinogenesis

While it has been established that both the constitutive and inducible forms of cyclooxygenase (COX-1 and COX-2, respectively) play important roles in chemical initiation-promotion protocols with phorbol ester tumor promoters, the contribution of these two enzymes to ultraviolet (UV) light-induced skin tumors has not been fully assessed. To better understand the contribution of COX-1 and COX-2 to UV carcinogenesis, we transferred the null allele for each isoform onto the SKH-1 hairless strain of mouse. Due to low viability on this background with complete knockout of COX-2, heterozygous mice were used in UV carcinogenesis experiments. While the lack of one allele of COX-1 had no effect on tumor outcome, the lack of one allele of COX-2 resulted in a 50-65% reduction in tumor multiplicity and a marked decrease in tumor size. Additionally, transgenic SKH-1 mice that overexpress COX-2 under the control of a keratin 14 promoter developed 70% more tumors than wild-type SKH-1 mice. The lack of one allele of either COX-1 or COX-2 reduced prostaglandin (PG) E2 levels in response to a single UV treatment. The proliferative response to UV was significantly reduced in COX-2, but not COX-1, heterozygous mice. UV-induced apoptosis, however, was greater in COX-2 heterozygous mice. Collectively, these results clearly establish the requirement for COX-2 in the development of skin tumors.     

JTE-522, a selective COX-2 inhibitor, interferes with the growth of lung metastases from colorectal cancer in rats due to inhibition of neovascularization: a vascular cast model study

The lung is a frequent site of metastasis from colorectal cancer, but angiogenesis of lung metastases has not been clarified. Some COX-2 inhibitors prevent tumor growth, although the inhibitory mechanism at the metastatic site is obscure. We investigated the microvascular structure of small lung metastases and the effect of JTE-522, a selective COX-2 inhibitor, on the angiogenesis of pulmonary metastases from colorectal cancer in rats. The tail veins of 25 male F344/DuCrj rats, aged 5 weeks, were injected with a tumor suspension containing 5 x 10(6) RCN-9, a rat colon cancer cell line. Three weeks later, pulmonary vascular resin corrosion casts were taken and the vascularity of metastases was studied using stereo and scanning electron microscopes. We investigated the effect of 0, 10 and 30 mg/kg/day of JTE-522 on the angiogenesis of pulmonary metastases in 3 groups of 5 male rats out of 25. JTE-522 reduced the diameter of tumor vessels as well as the number and size of metastatic tumors. The diameter of tumor vessels and the size of lung metastases significantly and positively correlated with neovascularization in the control group, but not in the JTE-522-treated groups. JTE-522 also affected type of vasculature of metastases, which differed depending on their size. JTE-522 interfered with the growth of hematogenous metastatic tumors by disrupting neovascularization. However, JTE-522 may have some important mechanisms other than inhibition of neovascularization. JTE-522 may be one of the therapeutic agents for the treatment of hematogenous metastasis of colorectal cancer.     

环氧合酶-2与肿瘤的放射治疗

环氧合酶-2(COX-2)在多种恶性肿瘤组织中呈高表达,其表达与肿瘤放疗密切相关,通过放射线对COX-2的诱导表达,可以降低肿瘤对放疗的敏感性,预后较差.临床上可根据检测COX-2蛋白的表达水平来评估肿瘤患者对放疗是否抵抗,以预测患者的放疗效应,并据此制定个体化治疗方案.     

SiRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth

In the past few years, several laboratories have developed antiangiogenic molecules that starve tumors by targeting their vasculature and we have shown that, when produced in tumors, the antiangiogenic molecule thrombospondin-1 (TSP1) reduces the vascularization and delays tumor onset. Yet over time, tumor cells producing active TSP1 do eventually form exponentially growing tumors. These tumors are composed of cells secreting unusually high amounts of the angiogenic stimulator vascular endothelial growth factor (VEGF) that are sufficient to overcome the inhibitory TSP1. Here, we use short double-stranded RNA (siRNA) to trigger RNA interference and thereby impair the synthesis of VEGF and ask if this inability to produce VEGF prevents the development of TSP1 resistance. Systemic in vivo administration of crude anti-VEGF siRNA reduced the growth of unaltered fibrosarcoma tumor cells, and when the anti-VEGF siRNA was expressed from tumor cells themselves, such inhibition was synergistic with the inhibitory effects derived from TSP1 secretion by the tumor cells. Anti-VEGF siRNA delayed the emergence of TSP1-resistant tumors and strikingly reduced their subsequent growth rate.     

Dual targeting of vascular endothelial growth factor and bone morphogenetic protein‐9/10 impairs tumor growth through inhibition of angiogenesis

Clinical development of anti‐angiogenic agents has been a major landmark in cancer therapy for several types of cancers. Signals mediated by both vascular endothelial growth factor (VEGF) and bone morphogenetic protein (BMP)‐9 and 10 have been implicated in tumor angiogenesis. However, previous studies have shown that targeting the individual signals was not sufficiently effective in retarding tumor growth in certain preclinical and clinical conditions. In the present study, we developed a novel decoy chimeric receptor that traps both VEGF and BMP‐9/10. Single targeting of either VEGF or BMP‐9/10 signals significantly reduced the formation of tumor vessels in a mouse xenograft model of human pancreatic cancer; however, it did not show significant therapeutic effects on tumor growth. In contrast, dual targeting of the angiogenic signals resulted in more significant inhibition of tumor angiogenesis, leading to delay of tumor growth. Our findings suggest that simultaneous blockade of VEGF and BMP‐9/10 signals is a promising therapeutic strategy for the cancers that are resistant to anti‐VEGF and BMP‐9/10 therapies.     

Total cyclooxygenase-2 mRNA levels correlate with vascular endothelial growth factor mRNA levels, tumor angiogenesis and prognosis in non-small cell lung cancer patients

Interaction between cancer cells and adjacent stromal cells is important to promote tumor development. Our aim was to study total COX-2 mRNA expression in both cancer cells and surrounding stromal cells and its association with angiogenic factor VEGF mRNA expression, tumor angiogenesis and prognosis in patients with NSCLC. COX-2 mRNA expression in both cancer cells and stromal tissue was analyzed using real-time quantitative (RTQ) RT-PCR in 60 NSCLC surgical specimens. Immunohistochemistry (IHC) was used to localize COX-2 protein in tumor specimens. Correlations between tumoral total COX-2 mRNA expression and VEGF mRNA expression (measured by RTQ RT-PCR), intratumoral microvessel counts (evaluated by IHC), other clinicopathologic variables, survival and relapse were tested. COX-2 protein expression was found in cancer as well as the surrounding stromal cells (including infiltrating inflammatory cells and endothelial cells of tumor-associated microvessels). VEGF protein expression was mainly located in cancer cells. There was a significant association between high tumoral total COX-2 mRNA expression and high VEGF mRNA expression (p = 0.01) or high intratumoral MVC (p < 0.001) but not other clinicopathologic variables, including tumor status and lymph node metastasis. Patients with higher tumoral total COX-2 mRNA expression had a statistically shorter survival time (median 15.0 +/- 2.61 months) and relapse time (median 5.0 +/- 1.37 months) than those with lower tumoral total COX-2 mRNA expression (median 40.0 +/- 3.12 and 34.0 +/- 3.11 months; p < 0.0001 and p < 0.0001, respectively, log-rank test). A significant difference in survival and relapse time was also seen between patients with high and low tumoral VEGF mRNA expression and between those with high and low intratumoral MVC (p = 0.0046 and p = 0.0038, respectively). After stratification by disease stage or histologic subtype, the prognostic significance of high total COX-2 mRNA expression was still apparent in both stage I and stage II-IV and in both squamous cell carcinoma and adenocarcinoma (p < or = 0.01 for all). Multivariate analysis using the Cox regression model with backward elimination showed that tumoral total COX-2 mRNA expression and lymph node status were the 2 most important independent prognostic predictors for survival and disease relapse. We report that total COX-2 mRNA expression in cancer cells and surrounding stromal cells correlates strongly and positively with VEGF mRNA expression, intratumoral MVC and adverse prognosis in NSCLC patients. This implies that COX-2 expression in both cancer cells and stromal cells within the tumor microenvironment may play an important role in upregulating the expression of the angiogenic factor VEGF and tumor angiogenesis in NSCLC and explains, in part, the adverse prognostic effect of COX-2 overexpression in patients with NSCLC.     

Inhibition of human vascular endothelial cells proliferation by terbinafine

We have demonstrated previously that terbinafine (TB), an oral antifungal agent used in the treatment of superficial mycosis, suppresses proliferation of various cultured human cancer cells in vitro and in vivo by inhibiting DNA synthesis and activating apoptosis. In our study, we further demonstrated that TB at a range of concentrations (0-120 microM) dose-dependently decreased cell number in cultured human umbilical vascular endothelial cells (HUVEC). Terbinafine was not cytotoxic at a concentration of 120 microM, indicating that it may have an inhibitory effect on the cell proliferation in HUVEC. The TB-induced inhibition of cell growth rate is reversible. [(3)H]thymidine incorporation revealed that TB reduced the [(3)H]thymidine incorporation into HUVEC during the S-phase of the cell-cycle. Western blot analysis demonstrated that the protein levels of cyclin A, but not cyclins B, D1, D3, E, CDK2 and CDK4, decreased after TB treatment. The TB-induced cell-cycle arrest in HUVEC occurred when the cyclin-dependent kinase 2 (CDK2) activity was inhibited just as the protein level of p21 was increased and cyclin A was decreased. Pretreatment of HUVEC with a p21 specific antisense oligonucleotide reversed the TB-induced inhibition of [(3)H]thymidine incorporation. Taken together, these results suggest an involvement of the p21-associated signaling pathway in the TB-induced antiproliferation in HUVEC. Capillary-like tube formation and chick embryo chorioallantoic membrane (CAM) assays further demonstrated the anti-angiogenic effect of TB. These findings demonstrate for the first time that TB can inhibit the angiogenesis.     

IGF binding protein-6 expression in vascular endothelial cells is induced by hypoxia and plays a negative role in tumor angiogenesis

Hypoxia stimulates tumor angiogenesis by inducing the expression of angiogenic molecules. The negative regulators of this process, however, are not well understood. Here, we report that hypoxia induced the expression of insulin-like growth factor binding protein-6 (IGFBP-6), a tumor repressor, in human and rodent vascular endothelial cells (VECs) via a hypoxia-inducible factor (HIF)-mediated mechanism. Addition of human IGFBP-6 to cultured human VECs inhibited angiogenesis in vitro. An IGFBP-6 mutant with at least 10,000-fold lower binding affinity for IGFs was an equally potent inhibitor of angiogenesis, suggesting that this action of IGFBP-6 is IGF-independent. The functional relationship between IGFBP-6 and vascular endothelial growth factor (VEGF), a major hypoxia-inducible angiogenic molecule, was examined. While VEGF alone increased angiogenesis in vitro, co-incubation with IGFBP-6 abolished VEGF-stimulated angiogenesis. The in vivo role of IGFBP-6 in angiogenesis was tested in flk1:GFP zebrafish embryos, which exhibit green fluorescence protein in developing vascular endothelium, permitting visualization of developing blood vessels. Injection of human IGFBP-6 mRNA reduced the number of embryonic inter-segmental blood vessels by ～40%. This anti-angiogenic activity is conserved in zebrafish because expression of zebrafish IGFBP-6b had similar effects. To determine the anti-angiogenic effect of IGFBP-6 in a tumor model, human Rh30 rhabdomyosarcoma cells stably transfected with IGFBP-6 were inoculated into athymic BALB/c nude mice. Vessel density was 52% lower in IGFBP-6-transfected xenografts than in vector control xenografts. These results suggest that the expression of IGFBP-6 in VECs is up-regulated by hypoxia and IGFBP-6 inhibits angiogenesis in vitro and in vivo.     

Tumor skewing of CD34+ progenitor cell differentiation into endothelial cells

Tumor production of granulocyte-macrophage colony-stimulating factor (GM-CSF) results in the mobilization of CD34(+) progenitor cells into the peripheral blood and tumor tissue. Using the Lewis lung carcinoma (LLC) model, in vitro studies showed that LLC cells could chemoattract CD34(+) cells predominantly through tumor production of VEGF. Addition of LLC-conditioned medium to CD34(+) cells that were cultured under conditions that support myeloid lineage cells skewed the differentiation of these precursor cells toward endothelial cells expressing CD31 and CD144. This differentiation of CD34(+) cells toward endothelial cells was attributed predominantly to angiopoietin-1 in the tumor-conditioned medium. The CD34(+) cells expressed the angiopoietin receptor Tie-2 and their differentiation into endothelial cells was blocked with neutralizing angiopoietin-1 antibodies. In vivo studies showed that infusion of lacZ(+) CD34(+) cells from the bone marrow of transgenic mice into wild-type mice bearing LLC tumors resulted in the accumulation of lacZ(+) cells within the tumor mass, particularly at the tumor's periphery. That these infused CD34(+) progenitor cells could develop into endothelial cells of the tumor vasculature was supported by their acquisition of the endothelial cell markers CD31 or CD144 within the tumor tissue. These studies demonstrate the capacity of tumor to attract CD34(+) cells to the tumor site and to direct the differentiation of these CD34(+) cells into endothelial cells that can become a component of the tumor vasculature.