黏附分子αvβ3和αvβ5介导肿瘤细胞与肿瘤内皮细胞的黏附

目的 体外分析黏附分子αvβ3和αvβ5及其配体Del-1、L1在肿瘤细胞-内皮细胞黏附中的作用.方法 应用逆转录聚合酶链反应(RT-PCR)和流式细胞分析比较正常肝窦内皮细胞(LSEC)和肝癌的血管内皮细胞T3A上细胞间黏附分子1(ICAM-1)、αvβ3和αvβ5的表达以及缺氧对其表达的调控.分别用RT-PCR和Western blot方法分析6种肿瘤细胞Del-1和L1的表达及缺氧对其表达的调控.使用连续光谱荧光测定仪定量肿瘤细胞在LSEC和T3A上的黏附,并分析抗不同黏附分子的抗体和siRNA对肿瘤细胞一内皮细胞黏附的阻断作用.结果 T3A细胞αvβ3和αvβ5的基础表达高于LSEC,而ICAM-1的基础表达低于LSEC.缺氧上调两种内皮细胞αvβ3和αvβ5的表达,而ICAM-1表达仅在LSEC中升高.在不同肿瘤细胞中,Del-1和L1的基础表达存在明显差异,并且在缺氧条件下受到明显不同的调节.Del-1和L1高表达的肿瘤细胞在T3A细胞的黏附明显高于LSEC,并且在缺氧条件下黏附明显增加,这种黏附可以被抗αvβ3、αvβ5的抗体或沉默β3和β5的小干扰RNA(siRNA)阻断.结论 细胞黏附分子αvβ3、αvβ5及其配体在肿瘤细胞-肿瘤内皮细胞的黏附过程中起重要介导作用.     

Tumor cell-tumor endothelial cell adhesion mediated by alphavbeta3 and alphavbeta5 molecules

OBJECTIVE: To investigate the role of adhesion molecules alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 in the tumor-endothelial cell adhesion in vitro. METHODS: The expression of alphavbeta3, alphavbeta5 and ICAM-1 in liver sinusoidal endothelial cells (LSEC) and liver cancer endothelial cells (T3A) cultured under normoxia or hypoxia were analyzed by RT-PCR and fluorescent activated cell sorter (FACS). The expression of Del-1 and L1 in six tumor cell lines under normoxia or hypoxia were analyzed by RT-PCR and Western blot, respectively. The adhesion of dye-labeled tumor cells and endothelial LSEC and T3A cells was measured by a fluorescence plate reader after their culture. RESULTS: The expression of alphavbeta3 and alphavbeta5 were higher in T3A cells than that in LSEC cells, and were upregulated under hypoxia, while the expression of ICAM-1 was lower in T3A cells than that in LSEC cells, and was upregulated under hypoxia only in LSEC. The expression of Del-1 and L1 molecules were obviously different in various tumor cell lines and were differentially regulated under hypoxia. The adhesion of tumor cells with Del-1 or L1 expression was higher in T3A cells than that in LSEC cells, and was significantly increased under hypoxia condition. Furthermore, the adhesion of tumor cells to T3A could be inhibited by antibodies against alphavbeta3 and alphavbeta5, or SiRNAs for beta3 and beta5. CONCLUSION: alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 may play an important role in tumor cell migration.     

Inhibition of angiogenesis and tumor growth by SCH221153, a dual alpha(v)beta3 and alpha(v)beta5 integrin receptor antagonist

New blood vessel formation is essential for tumor growth and metastatic spread. Integrins alpha(v)beta3 and alpha(v)beta5 are arginine-glycine-aspartic acid-dependent adhesion receptors that play a critical role in angiogenesis. Hence, selective dual alpha(v)beta3 and alpha(v)beta5 antagonists may represent a novel class of angiogenesis and tumor-growth inhibitors. Here, an arginine-glycine-aspartic acid-based peptidomimetic library was screened to identify alpha(v)beta3 antagonists. Selected compounds were then modified to generate potent and selective dual inhibitors of alpha(v)beta3 and alpha(v)beta5 receptors. One of these compounds, SCH 221153, inhibited the binding of echistatin to alpha(v)beta3 (IC50 = 3.2 nM) and alpha(v)beta5 (IC50 = 1.7 nM) with similar potency. Its IC50 values for related alpha(IIb)beta3 and alpha5beta1 receptors were 1294 nM and 421 nM, respectively, indicating that SCH 221153 is highly selective for alpha(v)beta3 and alpha(v)beta5 receptors. In cell-based assays, SCH 221153 inhibited the binding of echistatin to alpha(v)beta3- and alpha(v)beta5-expressing 293 cells and blocked the adhesion of endothelial cells to immobilized vitronectin and fibroblast growth factor 2 (FGF2). SCH 221153, but not the inactive analogue SCH 216687, was effective in inhibiting FGF2 and vascular endothelial growth factor-induced endothelial cell proliferation in vitro with an IC50 equal to 3-10 microM. Angiogenesis induced by FGF2 in the chick chorioallantoic membrane assay was also inhibited by SCH 221153. Finally, SCH 221153 exerted a significant inhibition on tumor growth induced by intradermal or s.c. injection of human melanoma LOX cells in severe combined immunodeficient mice.     

Integrins alpha(v)beta3 and alpha(v)beta5 are expressed by endothelium of high-risk neuroblastoma and their inhibition is associated with increased endogenous ceramide

Inhibition of the RGD-binding integrins, alpha(v)beta3 and alpha(v)beta5, prevents endothelial cell anchorage and induces endothelial apoptosis, which results in disruption of tumor angiogenesis and inhibition of tumor growth in animal models. In this study, we demonstrate by immunohistochemical analysis that integrin alpha(v)beta3 was expressed by 61% (mean) of microvessels in high-risk neuroblastomas (stage IV and MYCN-amplified stage III; n = 28) but only by 18% (mean) of microvessels in low-risk tumors (stages I and II and non-MYCN-amplified stage III; n = 12). Integrin alpha(v)beta5 was found on 60% (mean) of microvessels in 21 Stage IV tumors. These data suggest that neuroblastomas may be targeted for antiangiogenic treatment directed against endothelial integrins alpha(v)beta3 and alpha(v)beta5. In cell culture, inhibition of integrin-dependent endothelial cell anchorage to vitronectin by RGDfV, an RGD function-blocking cyclic peptide, induced apoptosis in bovine brain endothelial cells compared with the control peptide, RADfV (37.5% versus 8.7%, respectively), as detected by chromatin condensation and nuclear fragmentation. Treatment with RGDfV but not with RADfV, which prevented attachment of endothelial cells to vitronectin or fibronectin, was associated with up to a 50% increase in endogenous ceramide, a lipid second messenger that can mediate cell death. Furthermore, exogenous C2-ceramide was cytotoxic to bovine brain endothelial cells and induced activation of C-jun N-terminal kinase (JNK), a MAP kinase that can be activated in stress-induced apoptosis pathways. This suggests that ceramide may function in detachment-induced endothelial cell apoptosis, originating from inhibition of vitronectin binding to integrins such as alpha(v)beta3 and alpha(v)beta5. This is the first report to demonstrate expression of integrins alpha(v)beta3 and alpha(v)beta5 by microvascular endothelium of a childhood tumor and association of their expression with neuroblastoma aggressiveness. Furthermore, our data provide the first suggestion that inhibition of endothelial cell anchorage, resulting from specific blockade of RGD-binding integrins, increases endogenous ceramide, which may contribute to endothelial cell death.     

Integrin alpha v beta 3 antagonist Cilengitide enhances efficacy of radiotherapy in endothelial cell and non-small-cell lung cancer models

PURPOSE: Integrins alpha v beta 3 and alpha v beta 5 are important in tumor growth and angiogenesis and have been recently explored as targets for cancer therapy. Radiotherapy also inhibits tumor growth and affects vasculature. We explored the combination of integrin antagonist Cilengitide (EMD 121974) and ionizing radiation. METHODS AND MATERIALS: Levels of alpha v beta 3 were determined for human umbilical vein endothelial cells (HUVEC), as well as H157 and H460 human non-small-cell lung cancer cells, using FACS analysis and immunofluorescence imaging. Clonogenic assays, Western immunoblots probed for cleaved caspase 3, and Annexin-V probing were used to evaluate cell survival and apoptosis. A cell detachment assay and matrigel assay were used to further examine the effects of treatment. RESULTS: Human umbilical vein endothelial cells had the highest alpha v beta 3 level, followed by H157, and H460. Interestingly, we found that 5 Gy irradiation induced expression of alpha v beta 3 in all cell lines. Clonogenic assays showed a radiosensitizing effect with Cilengitide, and calculation of the dose enhancement ratio showed that the effect was highest in HUVECs (1.38), followed by H157 (1.19), and H460 (1.10), corresponding to the levels of target expression. There was an increase in apoptotic cells after combination treatment with Cilengitide and radiation, and there was an increase in detached cells after treatment with Cilengitide. Additionally, there was decreased endothelial tubule formation after combination treatment. CONCLUSIONS: We conclude that radiation induces expression of alpha v beta 3 integrin in endothelial and non-small-cell lung cancer models, and that integrin antagonist Cilengitide is a radiosensitizer in proportion to the levels of target integrin expression.     

Overexpression of platelet-type 12-lipoxygenase promotes tumor cell survival by enhancing alpha(v)beta(3) and alpha(v)beta(5) integrin expression

Arachidonic acid metabolism leads to the generation of biologically active metabolites that regulate cell growth and proliferation, as well as survival and apoptosis. We have demonstrated previously that platelet-type 12-lipoxygenase (LOX) regulates the growth and survival of a number of cancer cells. In this study, we show that overexpression of platelet-type 12-LOX in prostate cancer PC3 cells or epithelial cancer A431 cells significantly extended their survival and delayed apoptosis when cultured under serum-free conditions. These effects were shown to be a result of enhanced surface integrin expression, resulting in a more spread morphology of the cells in culture. PC3 cells transfected with 12-LOX displayed increased alpha(v)beta(3) and alpha(v)beta(5) integrin expression, whereas other integrins were unaltered. Transfected A431 cells did not express alpha(v)beta(3); however, alpha(v)beta(5) integrin expression was increased. Treatment of both transfected cell lines with monoclonal antibody to alpha(v)beta(5) (and in the case of PC3 cells, anti-alpha(v)beta(3)) resulted in significant apoptosis. In addition, treatment with 100 nM 12(S)-hydroxy-eicosatetraenoic acid, the end product of platelet-type 12-LOX, but not other hydroxy-eicosatetraenoic acids, enhanced the survival of wild-type PC3 and A431 cells and resulted in increased expression of alpha(v)beta(5). Furthermore, Baicalein or N-benzyl-N-hydroxy-5-phenylpentamide, specific 12-LOX inhibitors, significantly decreased alpha(v)beta(5)-mediated adhesion and survival in 12-LOX-overexpressing cells. The results show that 12-LOX regulates cell survival and apoptosis by affecting the expression and localization of the vitronectin receptors, alpha(v)beta(3) and alpha(v)beta(5), in two cancer cell lines.     

Prostatic carcinoma cell migration via alpha(v)beta3 integrin is modulated by a focal adhesion kinase pathway

The highly invasive human prostate cancer PC3 cell line was found to express the alpha(v)beta3 integrin; in contrast, the noninvasive LNCaP prostate cancer cell line did not express alpha(v)beta3. PC3 cells adhered to and migrated on vitronectin (VN), an alpha(v)beta3 ligand expressed in mature bone where prostate cancer cells preferentially metastasize. In contrast, LNCaP cells did not adhere to or migrate on VN. Analysis of primary human prostate cancer cells isolated from 16 surgical specimens, showed that these cells expressed alpha(v)beta3, whereas normal prostate epithelial cells did not. In addition, only primary prostate cancer cells adhered to and migrated on VN. The role of alpha(v)beta3 in mediating prostate epithelial cell migration was confirmed using LNCaP cell transfectants expressing beta3 (beta3-LNCaP). Exogenous expression of alpha(v)beta3 induced LNCaP cells to adhere to and migrate on VN. In response to alpha(v)beta3 engagement, increased tyrosine phosphorylation of focal adhesion kinase (FAK), a signaling molecule activated by integrins and able to modulate cell migration, was detected. Transfection of FAK-related nonkinase, known to compete with FAK for its correct localization and phosphorylation, caused inhibition of beta3-LNCaP cell migration, specifically on VN. These data indicate that de novo expression of alpha(v)beta3 integrin in prostate cancer cells generates a migratory phenotype that is modulated by a FAK signaling pathway. This study points to alpha(v)beta3 as potential target in prostate cancer cell invasion and metastasis.     

Fever-range hyperthermia stimulates alpha4beta7 integrin-dependent lymphocyte-endothelial adhesion.

Migration of blood-borne lymphocytes into lymphoid tissues is initiated by the L-selectin and alpha4beta7 integrin adhesion molecules. Previous studies have shown that L-selectin adhesion is dynamically regulated by febrile temperatures. It is now reported that fever-range hyperthermia also acts directly on lymphocytes to enhance selected adhesive functions of alpha4beta7 integrin. Fever-range hyperthermia treatment in vitro (40 degrees C, 12 h) of murine TK1 lymphoma cells and human peripheral blood lymphocytes (PBL) stimulates alpha4beta7 integrin-dependent adhesion to high endothelial venules (HEV) in Peyer's patch and mesenteric lymph node frozen sections. TK1 cells are alpha4beta7hi L-selectin(lo), allowing for the analysis of alpha4beta7 integrin without contributions from L-selectin. Adhesion was further shown to involve alpha4beta7 integrin and its endothelial counter-receptor, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) using function-blocking antibodies (i.e. DATK32, HP2/1, MECA-367). Fever-range hyperthermia also promotes alpha4beta7 integrin-mediated aggregation of TK1 cells. In sharp contrast, hyperthermia fails to increase alpha4beta7 integrin adhesion to fibronectin by TK1 cells. Expression of the alpha4beta7 heterodimer on TK1 cells or human PBL is not altered by hyperthermia, suggesting that hyperthermia stimulates adhesion by enhancing alpha4beta7 integrin avidity rather than its cell surface density. These results provide a mechanism whereby febrile temperatures during infection or clinical hyperthermia potentially amplify the immune response by stimulating L-selectin and alpha4beta7 integrin-dependent homing of immune effector cells to lymphoid tissues.     

Fever-range hyperthermia stimulates alpha4beta7 integrin-dependent lymphocyte-endothelial adhesion

Migration of blood-borne lymphocytes into lymphoid tissues is initiated by the Lselectin and alpha4beta7 integrin adhesion molecules. Previous studies have shown that L-selectin adhesion is dynamically regulated by febrile temperatures. It is now reported that fever-range hyperthermia also acts directly on lymphocytes to enhance selected adhesive functions of alpha4beta7 integrin. Fever-range hyperthermia treatment in vitro (40^oC, 12h) of murine TK1 lymphoma cells and human peripheral blood lymphocytes (PBL) stimulates alpha4beta7 integrin-dependent adhesion to high endothelial venules (HEV) in Peyer's patch and mesenteric lymph node frozen sections. TK1 cells are alpha4beta7^hi L-selectin^lo, allowing for the analysis of alpha4beta7 integrin without contributions from L-selectin. Adhesion was further shown to involve alpha4beta7 integrin and its endothelial counter-receptor, mucosal addressin cell adhesion molecule-1 (MAdCAM-1) using function-blocking antibodies (i.e. DATK32, HP2/1, MECA-367). Fever-range hyperthermia also promotes alpha4beta7 integrin-mediated aggregation of TK1 cells. In sharp contrast, hyperthermia fails to increase alpha4beta7 integrin adhesion to fibronectin by TK1 cells. Expression of the alpha4beta7 heterodimer on TK1 cells or human PBL is not altered by hyperthermia, suggesting that hyperthermia stimulates adhesion by enhancing alpha4beta7 integrin avidity rather than its cell surface density. These results provide a mechanism whereby febrile temperatures during infection or clinical hyperthermia potentially amplify the immune response by stimulating L-selectin and alpha4beta7 integrin-dependent homing of immune effector cells to lymphoid tissues.     

人肝癌微血管内皮细胞黏附分子表达特点及其对外周血单个核细胞黏附和跨内皮迁移的影响

目的:研究人肝癌微血管内皮细胞(human liver cancer microvascular endothelial cell,HLCMVEC)上黏附分子表达的特点及其对免疫细胞黏附和迁移的影响.方法:分离培养HLCMVEC,以人肝窦内皮细胞系(liver sinusoid endothelial cell, LSEC)作为正常对照.通过细胞ELISA方法比较多种黏附分子在两种内皮细胞上的表达.外周血单个核细胞(peripheral blood mononuclear cell,PBMC)用荧光染料BCECF标记,将其与HLCMVEC或LSEC共培养,随后采用倒置荧光显微镜和连续光谱荧光仪检测PBMC在两种内皮细胞上的黏附和跨内皮迁移能力;此外,共培养前分别预加各种黏附分子的功能抗体,然后分析各种黏附分子在PBMC与肿瘤微血管内皮细胞黏附和迁移中的作用.结果:HLCMVEC表达CD31、CD34和细胞间黏附分子-3(intercellular adhesion molecule-3,ICAM-3)的水平低于LSEC(P＜0.05),表达ICAM-1和血管细胞黏附分子-1(vascular cell adhesion molecule-l,VCAM-1)的水平明显低于正常LSEC(P＜0.01),但表达整合素αvβ3和αvp5明显高于LSEC(P＜0.01).PBMC在HLCMVEC上的黏附和趋化剂诱导下的跨内皮迁移均显著低于LSEC[黏附:(205.5±46.0) vs (330.5±48.4)个,迁移:(49.0±10.6) vs(110.0±19.2)个,均P＜0.01];该黏附和迁移可被ICAM-1、ICAM-3、VCAM-1抗体明显阻断(P＜0.01),抗CD31抗体对黏附阻断不明显但能阻断跨内皮迁移(P＜0.05).结论:HLCMVEC特有的黏附分子表达特点抑制了PBMC的黏附和跨内皮迁移.     

A chimeric cell adhesion molecule mediates homing of lymphocytes to vascularized tumors.

To facilitate tumor colonization by adoptively transferred cells of the immune system, we created a chimeric cell adhesion molecule that mediates tumor-specific homing by binding to the integrin alpha(v)beta3 on angiogenic endothelial cells. A high-affinity cell adhesion molecule for integrin alpha(v)beta3 was generated by fusing the disintegrin kistrin to the transmembrane adhesion molecule CD31/PECAM-1. This chimeric cell adhesion molecule, termed KISS31, mediates adhesion of lymphoid cells to soluble recombinant integrin alpha(v)beta3 and to endotheliomal monolayers in vitro. KISS31-expressing lymphoid cells accumulate in angiogenic tumors in two in vivo models, in B16/129 melanoma xenografts on the chick chorioallantois and in s.c. growing Lewis lung carcinoma in mice. Our data indicate that expression of KISS31 on lymphoid cells confers tumor-specific homing. This is, to our knowledge, the first example of an experimental mechanism that targets living cells to tumors by redirecting their homing pattern.     

The role of alpha(v)beta(3) in prostate cancer progression

Integrin alpha(v)beta(3) is involved in varied cell biological activities, including angiogenesis, cell adhesion, and migration on several extracellular matrix components. Although alpha(v)beta(3) is not typically expressed in epithelial cells, it is expressed in macrophages, activated leukocytes, cytokine-stimulated endothelial cells, osteoclasts, and certain invasive tumors. Interestingly, the adhesion and migration of breast cancer cells on bone matrix are mediated, in part, by alpha(v)beta(3). Similar to breast cancer cells, prostate cancer cells preferentially metastasize to the bone. The biological events that mediate this metastatic pattern of prostate cancer are not well defined. This review discusses the role alpha(v)beta(3) plays in prostate cancer progression, with specific emphasis on bone metastasis and on alpha(v)beta(3) signaling in prostate cancer cells. The data suggest that alpha(v)beta(3), in part, facilitates prostate cancer metastasis to bone by mediating prostate cancer cell adhesion to and migration on osteopontin and vitronectin, which are common proteins in the bone microenvironment. These biological events require the activation of focal adhesion kinase and the subsequent activation of PI-3 kinase/Akt signaling pathway.     

Inhibition of alpha(v)beta3 integrin survival signaling enhances antiangiogenic and antitumor effects of radiotherapy.

The involvement of alpha(v)beta3 and alpha(v)beta5 integrins in angiogenesis and the use of integrin antagonists as effective antiangiogenic agents are documented. Radiotherapy is an important therapy option for cancer. It has been shown that ionizing radiation exerts primarily antiangiogenic effects in tumors but has also proangiogenic effects as the reaction of the tumor to protect its own vasculature from radiation damage. Here, we show that combined treatment with S247, an Arg-Gly-Glu peptidomimetic antagonist of alpha(v)beta3 integrin, and external beam radiotherapy are beneficial in local tumor therapy. We found that radiation up-regulates alpha(v)beta3 expression in endothelial cells and consecutively phosphorylates Akt, which may provide a tumor escape mechanism from radiation injury mediated by integrin survival signaling. In the presence of S247, the radiation-induced Akt phosphorylation is strongly inhibited. Our studies on endothelial cell proliferation, migration, tube formation, apoptosis, and clonogenic survival show that the radiosensitivity of endothelial cells is enhanced by the concurrent administration of the integrin antagonist. The in vitro data are successfully translated into human glioma (U87), epidermoid (A431), and prostate cancer (PC3) xenograft models growing s.c. on BALB/c-nu/nu mice. In vivo, the combination of S247 treatment and fractionated radiotherapy (5 x 2.5 Gy) leads to enhanced antiangiogenic and antitumor effects compared with either monotherapies. These results underline the importance of alpha(v)beta3 integrin when tumors protect their microvasculature from radiation-induced damage. The data also indicate that the combination of integrin antagonists and radiotherapy represents a rational approach in local cancer therapy.     

The small alpha5beta1 integrin antagonist, SJ749, reduces proliferation and clonogenicity of human astrocytoma cells

The potential role of alpha5beta1 integrins in cancer has recently attracted much interest. However, few alpha5beta1-selective antagonists have been developed compared with other integrins. The most specific nonpeptidic alpha5beta1 antagonist described thus far, SJ749, inhibits angiogenesis by affecting adhesion and migration of endothelial cells. We investigated the effects of SJ749 in two human astrocytoma cell lines, A172 and U87, which express different levels of alpha5beta1. SJ749 dose-dependently inhibited adhesion of both cell types on fibronectin. Application of SJ749 to spread cells led to formation of nonadherent spheroids for A172 cells but had no effect on U87 cell morphology. SJ749 also reduced proliferation of A172 cells due to a long lasting G0-G1 arrest, whereas U87 cells were only slightly affected. However, under nonadherent culture conditions (soft agar), SJ749 significantly reduced the number of colonies formed only by U87 cells. As U87 cells express more alpha5beta1 than A172 cells, we specifically examined the effect of SJ749 on A172 cells overexpressing alpha5. Treatment of alpha5-A172 cells with SJ749 decreased colony formation similarly to that observed in U87 cells. Therefore, in nonadherent conditions, the effect of SJ749 on tumor cell growth characteristics depends on the level of alpha5beta1 expression. Our study highlights the importance of alpha5beta1 as an anticancer target and shows for the first time that a small nonpeptidic alpha5beta1-specific antagonist affects proliferation of tumor cells.     

Engaged urokinase receptors enhance tumor breast cell migration and invasion by upregulating alpha(v)beta5 vitronectin receptor cell surface expression

We have previously shown that urokinase receptor physically and functionally interacts with alpha(v)beta5 vitronectin receptor, leading to tumor breast cell migration and invasion. Here, the link between these 2 receptors was further investigated by analyzing the expression levels of urokinase receptor and alpha(v)beta5 integrin in 35 human breast carcinomas and 5 benign breast lesions. The occurrence of a positive correlation between urokinase receptor and alpha(v)beta5 protein levels in benign and malignant tumor specimens prompted us to investigate whether engaged urokinase receptors might modulate alpha(v)beta5 expression. Here, we report the receptor-dependent ability of catalytically inactive urokinase to upregulate the alpha(v) and beta5 chains in MDA-MB-231 and MCF-7 breast carcinoma cell lines in a time- and concentration-dependent manner. This effect is dependent on protein kinase C activity and requires new protein synthesis. Accordingly, the availability of assembled alpha(v)beta5 receptors on the cell surface increases upon urokinase treatment, as shown by immunoprecipitation and immunocytochemical analyses. Exposure to urokinase leads to enhanced tumor cell migration and invasion, which is prevented by the "phosphorylation-like" urokinase receptor antagonist His-uPA(138E/303E), the DNA-binding drug mithramycin, the protein kinase C inhibitor calphostin C and anti-alpha(v)beta5 antibodies. Finally, urokinase enables benign breast MCF-10A cells to cross Matrigel in a alpha(v)beta5- and urokinase receptor-dependent manner, indicating that urokinase controls a regulatory circuitry crucial to breast tumor progression.     

Identification of CD47/integrin-associated protein and alpha(v)beta3 as two receptors for the alpha3(IV) chain of type IV collagen on tumor cells.

Previous studies from our laboratories demonstrated that a peptide from the noncollagenous domain of the alpha3 chain of basement membrane collagen (COL IV), comprising residues 185-203, inhibits polymorphonuclear leukocyte activation and melanoma cell proliferation independently of its ability to promote cell adhesion; these properties require the presence of the triplet -SNS- at residues 189-191 (J. C. Monboisse et al., J. Biol. Chem., 269: 25475-25482, 1994; J. Han et al., J. Biol. Chem., 272: 20395-20401, 1997). More recently, we demonstrated that native COL IV and -SNS-containing synthetic peptides (10 microg/ml) added to culture medium inhibit the proliferation of not only melanoma cells but also breast, pancreas, and stomach tumor cells up to 82% and prostate tumor cells by 15%. This inhibition was shown to be dependent on a COL IV- or peptide-induced increase in intracellular cAMP (T. A. Shahan et al., Connect. Tissue Res., 40: 221-232, 1999). Attempts to identify the putative receptor(s) on tumor cells led to the isolation of five proteins (Mr 33,000, 52,000, 72,000, 95,000, and 250,000) from melanoma and prostate cells by affinity purification with the alpha3(IV)179-208 peptide. The Mr 52,000, 95,000, and 250,000 proteins were shown to be CD47/integrin-associated protein(IAP), the integrin beta3 subunit, and the alpha(v)beta3 integrin complex, respectively. The Mr 33,000 and 72,000 proteins have not yet been identified. To confirm the specificity of ligand binding to the receptors, cell membranes from either melanoma or prostate tumor cells were pretreated with the unlabeled ligand alpha3(IV)187-191 (-YYSNS-); alternatively, the peptide was pretreated with a peptide-reactive monoclonal antibody (A5D7) before receptor isolation. These treatments inhibited the purification of CD47/IAP, the integrin beta3 subunit, and the alpha(v)beta3 integrin complex from tumor cells. Furthermore, cells treated with CD47/IAP- or the alpha(v)beta3 integrin-reactive antibodies prevented the alpha3(IV)185-203 peptide from inhibiting cell proliferation and the subsequent rise in intracellular cAMP. Pretreating cells with the alpha3(IV)187-191 (-YYSNS-) peptide also inhibited their adhesion to the alpha3(IV)185-203 peptide substrate, whereas the inactive alpha1(IV)185-203 peptide, from the same region of the alpha1 chain as the alpha3(IV)185-203 peptide, had no effect. Incubation of cells with either CD47/IAP and/or alpha(v)beta3 integrin-reactive antibodies inhibited their adhesion to the alpha3(IV)185-203 peptide, whereas antibodies to the beta1 and beta2 integrin subunits were without effect. These data suggest that ALC-COL IV, through its alpha3(IV) chain, inhibits tumor cell proliferation using the receptors CD47/IAP and the alpha(v)beta3 integrin.     

alpha(v) integrins regulate cell proliferation through integrin-linked kinase (ILK) in ovarian cancer cells

Integrins regulate both adhesion and signaling processes involved in proliferation and survival. alpha(v)beta(3) and alpha(v)beta(5) integrins have been shown to mediate cell adhesion and migration. Here we used human ovarian cancer cell lines (IGROV1, SKOV-3) that express alpha(v)beta(3) and alpha(v)beta(5) to study their role in cell proliferation and the signaling pathways involved. We found that alpha(v) integrins regulate cell proliferation through activation of integrin-linked kinase (ILK). An anti-alpha(v)-blocking antibody specifically inhibits the growth of IGROV1 and SKOV-3. The inhibition of cell proliferation involves alpha(v)beta(3) in IGROV1 cells, and both alpha(v)beta(3) and alpha(v)beta(5) in SKOV-3 cells. The reduced growth rate induced by alpha(v) integrin blockade is linked in both cell lines to G1/S cell cycle arrest. alpha(v) integrin blockade by neutralizing antibody as well as cyclic-RGD peptide caused an inhibition of ILK activity and phosphorylation of PKB/Akt on serine-473 but not on threonine-308, and was accompanied by an increase in p27(Kip1) expression. Overexpression of wild-type ILK rescued the phosphorylation of PKB/Akt on serine-473 in cells treated with anti-alpha(v) antibody. Inhibition of ILK by a pharmacological inhibitor results in inhibition of cell proliferation, PKB/Akt phosphorylation and increase of p27(Kip1). These results demonstrate that alpha(v) integrins regulate ovarian cancer cell proliferation through ILK.     

The pattern of metastasis of human melanoma to the central nervous system is not influenced by integrin alpha(v)beta(3) expression

We investigated the effect of integrin alpha(v)beta(3) expression on the metastatic pattern of human melanoma cells in the central nervous system (CNS). For this purpose, we developed a hematogenous CNS melanoma metastasis model in nude mice using a modified internal carotid artery infusion technique. This protocol revealed 2 different patterns of CNS metastasis. The integrin alpha(v)beta(3)-expressing melanoma lines Mel57 and Zkr nearly exclusively produced metastases in the brain parenchyma, whereas cells of the BLM and MV3 lines, devoid of integrin alpha(v)beta(3) expression, preferentially metastasized to dura mater and leptomeninges. Treatment with hyaluronidase to obtain single BLM cell suspensions did not influence the metastatic pattern, indicating that this was not simply the result of entrapment of tumor cell aggregates in large-sized leptomeningeal vessels. The role of integrin alpha(v)beta(3) expression in the process of metastasis was tested by transfection of BLM, but did not lead to an altered pattern of metastasis. We did observe, however, slower growth of the transfected tumors, although the in vitro growth rate was unaltered, indicating a reduction in tumorigenicity. We conclude from our findings that CNS metastasis of melanoma cells in the mouse xenograft model occurs in at least 2 different but very reproducible patterns. Although it is predicted that adhesion of tumor cells to endothelial cells plays a role in this phenomenon, tumor cell integrin alpha(v)beta(3) expression per se does not explain the difference in metastatic behavior in the CNS. We assume that other, as yet unknown factors, must be involved.     

Dissecting the role of matrix metalloproteinases (MMP) and integrin alpha(v)beta3 in angiogenesis in vitro: absence of hemopexin C domain bioactivity, but membrane-Type 1-MMP and alpha(v)beta3 are critical

Matrix metalloproteinase (MMP)-2 and its hemopexin C domain autolytic fragment (also called PEX) have been proposed to be crucial for angiogenesis. Here, we have investigated the dependency of in vitro angiogenesis on MMP-mediated extracellular proteolysis and integrin alpha(v)beta3-mediated cell adhesion in a three-dimensional collagen I model. The hydroxamate-based synthetic inhibitors BB94, CT1399, and CT1847 inhibited endothelial cell invasion, as did neutralizing anti-membrane-type 1-MMP (MT1-MMP) antibodies and tissue inhibitor of MMP (TIMP)-2 and TIMP-3 but not TIMP-1. This confirmed the pivotal importance of MT1-MMP over other MMPs in this model. Invasion was also inhibited by a nonpeptidic antagonist of integrin alpha(v)beta3, EMD 361276. Although PEX strongly inhibited pro-MMP-2 activation, when contaminating lipopolysaccharide was neutralized, PEX neither affected angiogenesis nor bound integrin alpha(v)beta(3). Moreover, no specific binding of pro-MMP-2 to integrin alpha(v)beta3 was found, whereas only one out of four independently prepared enzymatically active MMP-2 preparations could bind integrin alpha(v)beta3 , and this in a PEX-independent manner. Likewise, integrin alpha(v)beta3 -expressing cells did not bind MMP-2-coated surfaces. Hence, these findings show that endothelial cell invasion of collagen I gels is MT1-MMP and alpha(v)beta3 - dependent but MMP-2 independent and does not support a role for PEX in alpha(v)beta3 integrin binding or in modulating angiogenesis in this system.     

Selectins induced by interleukin-1beta on the human liver endothelial cells act as ligands for sialyl Lewis X-expressing human colon cancer cell metastasis

We have previously reported that colon cancer cells metastasized to the liver expressed an increased amount of sialyl Lewis X (SLeX) antigen compared to their corresponding primary lesions. It is now well known that SLeX antigen and sialyl Lewis A (SLeA) antigen are ligands for the selectins expressed on the endothelial cells. Therefore, it is assumed that SLeX-rich colon cancer cells could be easily adhered to the endothelial cells that express selectins. In this report we have tried to induce selectin expression on the human liver sinusoidal endothelial cells and have examined the adhesion of SLeX-high or -low expressing colon cancer cells to the interleukin-1beta (IL-1beta)-treated liver specimens using Stamper-Woodruff assay. These human colon cancer cells are termed KM12HX or KM12LX cells, respectively. A significantly increased number of KM12HX cells adhered to the IL-1beta-treated liver specimens compared to KM12LX cells. The adhesion of KM12HX cells was inhibited by the pretreatment of tumor cells with anti-SLeX antibody or by the pretreatment of liver specimens with anti-selectin antibodies. Selectin expression on the liver sinusoidal endothelial cells and endothelial cells of blood vessels after IL-1beta treatment was confirmed by immunohistochemically using anti-selectin monoclonal antibodies (MAbs). These findings strongly suggest that SLeX-expressing cancer cells could adhere to the sinusoidal endothelial cells via an SLeX-selectin interaction system and this could be a first step for colon cancer cells that metastasize to the liver. The mechanism by which these selectins can be induced in vivo is the next problem to be considered.