Studies on the expression of alpha(v)beta3 integrin receptors in non-malignant and malignant human cervical tumor tissues

Integrins are group of cell surface receptors, which mediate adhesion between cell-cell and cell-extracellular matrix proteins of the basement membrane. Integrins play an important role in cellular growth, development, morphology, signalling and also in tumor development. Among the integrin group of cell surface receptors one of the most important member is alpha(v)beta3 integrin receptor. Evidences say that the expression of this integrin receptor is regulated during tumor development. Large numbers of studies have been done to establish the role of alpha(v)beta3 integrin receptor in human melanoma systems. Expression of this receptor in metastatic but not benign melanomas suggests a role for this integrin in the regulation of tumor proliferation. Alpha(v)beta3 integrin receptor also plays a significant role in tumor angiogenesis, apoptosis and signal transduction process. Recent studies show that collagenase MMP-2 binds directly to integrin alpha(v)beta3 on the surface of invasive tumor cells and facilitates tumor cell invasion. In this present communication we studied the expression of alpha(v)beta3 integrin receptor in malignant and non-malignant cervical tumor tissues. Because receptor ligand interaction is a cell surface phenomenon the membrane fraction of tumor tissues was separated and the expression of alpha(v)beta3 integrin receptor was assayed by ELISA and immunoprecipitation from membrane extracted protein fraction. Comparative ELISA and immunoprecipitation clearly demonstrates much higher expression of alpha(v)beta3 vitronectin integrin receptor in the membrane fraction of malignant human cervical tumor tissues than nonmalignant tissue membrane fractions.     

重组人内皮抑素30肽的抗肿瘤作用研究

目的：探讨重组人内皮抑素30肽对肝癌细胞的生物活性及其可能的作用途径。方法：建立HepG2肝癌细胞的单层细胞和多细胞球模型,采用MTT方法和流式细胞技术检测重组人内皮抑素30肽对单层细胞和多细胞球的生长抑制率和细胞调亡率;采用免疫组化和图像分析技术了解重组人内皮抑素30肽与肝癌细胞表面整合素结合的情况。结果：MTT实验表明,在二维细胞中,重组人内皮抑素30肽对HepG-2肝癌细胞表现出明显的抑制作用。在单层细胞和多细胞球中,重组人内皮抑素30肽均可导致细胞凋亡。免疫组化结果显示,无论在单层细胞还是多细胞球中,使用重组人内皮抑素30肽组HepG-2肝癌细胞表面的αtβ1整合素和αvβ3整合素的表达均比对照组明显降低（P〈0．05）。结论：重组人内皮抑素30肽对HepG2肝癌细胞具有直接的杀伤作用。重组人内皮抑素30肽可与HepG-2肝癌细胞表面的αtβ1整合素和αvβ3整合素结合发挥其抗肿瘤作用。     

Bimolecular interaction of insulin-like growth factor (IGF) binding protein-2 with alphavbeta3 negatively modulates IGF-I-mediated migration and tumor growth

Both the integrin and insulin-like growth factor binding protein (IGFBP) families independently play important roles in modulating tumor cell growth and progression. We present evidence for a specific cell surface localization and a bimolecular interaction between the alpha v beta 3 integrin and IGFBP-2. The interaction, which could be specifically perturbed using vitronectin and alpha v beta 3 blocking antibodies, was shown to modulate IGF-mediated cellular migration responses. Moreover, this interaction was observed in vivo and correlated with reduced tumor size of the human breast cancer cells, MCF-7 beta 3, which overexpressed the alpha v beta 3 integrin. Collectively, these results indicate that alpha v beta 3 and IGFBP-2 act cooperatively in a negative regulatory manner to reduce tumor growth and the migratory potential of breast cancer cells.     

The alpha 3 beta 1 integrin is associated with mammary carcinoma cell metastasis, invasion, and gelatinase B (MMP-9) activity

The alpha 3 beta 1 integrin is elevated in several types of metastatic tumor and has been associated with increased migration and invasion. Our analysis of a series of mammary carcinomas of different histotypes and their corresponding metastases demonstrated significantly increased expression of alpha 3 beta 1 in the tumor metastases. We therefore studied alpha 3 beta 1 expression of several human breast carcinoma cell lines and its association with the invasive phenotype. The MDA-MB-231 cell line expressed high levels of the beta1, alpha 2, alpha 3, alpha 5, and alpha 6 integrin subunits along with moderate levels of the alpha v beta 3 integrin. This line was highly migratory and the most invasive using a chemo-invasion assay. In contrast, the other lines tested, MDA-MB-145, MCF-7, and SK-BR-3, showed lower migratory and invasive activity and reduced alpha 3 integrin subunit expression. Metalloproteases capable of degrading collagen IV are necessary for the invasive process. RT-PCR showed that MDA-MB-231 cells expressed MMP-9, but not MMP-2, gelatinase/collagenase IV. Gelatin zymography demonstrated that invading MDA-MB-231 cells released high levels of MMP-9 gelatinase activity. A direct role for this gelatinase in MDA-MB-231 cell invasion was confirmed by inhibition of invasion using the metalloprotease inhibitor Batimastat. Treatment of MDA-MB-231 cells with a function-blocking anti-alpha 3 antibody strongly inhibited migration and invasion. This correlated with a marked reduction in MMP-9 activity produced by MDA-MB-231 cells, suggesting a role for alpha 3 beta 1 ligand binding in cell signaling and regulation of extracellular matrix degradation.     

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.     

A novel disintegrin salmosin inhibits tumor angiogenesis.

Salmosin is a snake venom-derived novel disintegrin that antagonizes platelet aggregation. In this study, we investigated its functional specificity in tumor angiogenesis. Salmosin significantly inhibited bovine capillary endothelial cell proliferation induced by basic fibroblast growth factor but had no effect on normal growth of the cell. The basic fibroblast growth factor-induced in vivo angiogenesis in the chorioallantoic membrane was disrupted by salmosin treatment without affecting normal embryonic angiogenesis. Adhesion of the bovine capillary endothelial cells to vitronectin was also inhibited by the binding of salmosin to the alpha(v)beta3 integrin. Both the metastatic-tumor growth and the solid-tumor growth that developed in mice were effectively suppressed by salmosin treatment. Several lines of experimental evidence strongly suggest that the tumor-specific antiangiogenic activity of salmosin disrupts tumor growth by blocking the alpha(v)beta3 integrin that is expressed on the vascular endothelial cell surface.     

Thiolutin, an inhibitor of HUVEC adhesion to vitronectin, reduces paxillin in HUVECs and suppresses tumor cell-induced angiogenesis.

Recent studies have shown that integrin alpha v beta 3, a receptor for vitronectin, plays an important role in tumor-induced angiogenesis and tumor growth and that antagonists of alpha v beta 3 inhibit angiogenic processes including endothelial cell adhesion and migration. On the other hand, most inhibitors of integrin alpha v beta 3 are peptide antagonists that include the Arg-Gly-Asp (RGD) motif. We therefore reasoned that non-peptide inhibitors of endothelial cell adhesion to vitronectin might be useful for inhibition of tumor angiogenesis in vivo. We screened for low-molecular-weight natural products able to inhibit adhesion of human umbilical vein endothelial cells (HUVECs) to vitronectin, and pyrrothine group compounds including aureothricin, thioaurin and thiolutin were isolated from microbial culture broths. Of these compounds, thiolutin inhibited adhesion of HUVECs to vitronectin the most effectively (IC(50), 0.83 microM). In vivo experiments showed that thiolutin significantly suppressed angiogenesis induced by tumor cells (S-180), a pathological form of neovascularization, in a mouse dorsal air sac assay system. To explore the mechanism of inhibition of HUVEC adhesion to vitronectin by thiolutin, we examined the effect of this agent on intracellular cell adhesion signaling. We found that the amount of paxillin in HUVECs was significantly reduced by thiolutin treatment, while those of other focal adhesion proteins including vinculin and focal adhesion kinase (FAK) were not. Metabolic labeling experiments showed that thiolutin enhanced degradation of paxillin in HUVECs. Protease inhibitors (MG115 and E64-D) decreased the rate of degradation of the paxillin induced by thiolutin and partially restored thiolutin-induced inhibition of HUVEC adhesion to vitronectin. Based on these findings, we concluded that thiolutin, an inhibitor of HUVEC adhesion to vitronectin, reduces the paxillin level in HUVECs and suppresses tumor cell-induced angiogenesis in vivo.     

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.     

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.     

Integrin alpha(v)beta3 promotes M21 melanoma growth in human skin by regulating tumor cell survival.

Growth and dissemination of malignant melanoma has a profound impact on our population, and little is known concerning the mechanisms controlling this disease in humans. Evidence is provided that integrin alpha(v)beta3 plays a critical role in M21 melanoma tumor survival within human skin by a mechanism independent of its known role in angiogenesis. Antagonists of alpha(v)beta3 blocked melanoma growth by inducing tumor apoptosis. Moreover, M21 melanoma cell interactions with denatured collagen, a known ligand for alpha(v)beta3, caused a 5-fold increase in the relative Bcl-2:Bax ratio, an event thought to promote cell survival. Importantly, denatured collagen colocalized with alpha(v)beta3-expressing melanoma cells in human tumor biopsies, suggesting that alpha(v)beta3 interaction with denatured collagen may play a critical role in melanoma tumor survival in vivo.     

Expression and function of CYR61, an angiogenic factor, in breast cancer cell lines and tumor biopsies

We have previously shown that expression of heregulin (HRG) is closely correlated with breast cancer progression. We have subsequently isolated Cyr61, a ligand for the alpha(v)beta3 integrin that is differentially expressed in HRG-positive cells, and have shown that it is expressed in all of the invasive and metastatic breast cancer cell lines tested. Preliminary evaluation of Cyr61 expression in breast tumor biopsies revealed expression of Cyr61 in about 30% of invasive breast carcinomas. Significantly, we demonstrated that Cyr61 is a downstream effector of HRG action, because a Cyr61-neutralizing antibody abolished the ability of HRG-expressing cells to migrate in vitro. Furthermore, we have shown that HRG-expressing cells denote higher levels of alpha(v)beta3 expression, and we have established that Cyr61 action is mediated, at least in part, through its receptor alpha(v)beta3, because a functional blocking antibody of the alpha(v)beta3 blocked the Matrigel outgrowth of HRG-expressing cells. These results strongly suggest that Cyr61 is necessary for HRG-mediated chemomigration and that Cyr61 plays a functional role in breast cancer progression, possibly through its interactions with the alpha(v)beta3 receptor.     

Expression of alpha(v)beta6 integrin in oral leukoplakia

The distribution of alpha(v)beta6 integrin was examined in oral leukoplakia, lichen planus and squamous cell carcinomas using immunohistochemistry. Controls included oral mucosal wounds, chronically inflamed and normal oral mucosa. Integrins beta1, beta3, beta4, beta5, fibronectin and tenascin were also studied. The integrin alpha(v)beta6 was highly expressed throughout the whole lesion of 90% of the squamous cell carcinomas but was not present in any of the normal specimens. alpha(v)beta6 integrin was also expressed in 41% of the leukoplakia specimens, and 85% of the lichen planus samples, but in none of the tissues with inflammatory hyperplasia or chronic inflammation. The expression of beta1 integrins was localized in the basal layer, and that of the beta4 at the cell surface facing the basement membrane of all specimens. The integrins beta3 and beta5 were absent from all normal and leukoplakia specimens. Fibronectin and tenascin were present in the connective tissue underneath the epithelium of all the sections, and their expression was similar in both alpha(v)beta6-positive and alpha(v)beta6-negative tissues. A group of 28 leukoplakia patients were followed 1-4 years after first diagnosis. In this group, initially alpha(v)beta6 integrin-positive leukoplakia specimens had high tendency for disease progression while alpha(v)beta6-negative specimens did not progress. These results suggest that the expression of alpha(v)beta6 integrin could be associated in the malignant transformation of oral leukoplakias.     

Coordinated expression of integrin subunits,matrix metalloproteinases (MMP), angiogenic genes and Ets transcription factors in advanced-stage ovarian carcinoma: a possible activation pathway?

The events that mediate tumor progression in ovarian carcinoma are poorly understood to date. This review summarizes our results studying metastasis-associated molecules in advanced-stage ovarian carcinomas, details the co-expression of mRNA of these genes, and discusses their prognostic role. Fifty-five primary and metastatic FIGO stage III-IV ovarian carcinomas were analyzed for the expression of alpha v and beta1 integrin subunits, the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP, the MMP inhibitor TIMP-2, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin-8 (IL-8), PEA3 and Ets-1 using mRNA in situ hybridization. Tumor and adjacent stromal cell expression was scored. The association between integrin subunit expression and the expression of MMP, TIMP-2, angiogenic genes, PEA3 and Ets-1 was statistically analyzed. Alpha v integrin subunit mRNA expression in carcinoma cells showed significant association with that of MMP-2 and IL-8 in this cellular compartment, while the presence of beta1 integrin subunit mRNA showed similar association with that of PEA3, Ets-1, IL-8, bFGF and MMP-2. Expression of beta1 integrin subunit mRNA in stromal cells was associated with that of TIMP-2 and Ets-1 in this compartment. In addition, significant intercellular associations were found between alpha v integrin subunit mRNA expression in carcinoma cells and stromal cell expression of Ets-1, as well as between stromal cell expression of alpha v integrin subunit and labeling for IL-8 in carcinoma cells. The presence of beta1 integrin subunit mRNA in carcinoma cells showed a significant association with that of Ets-1, IL-8 and bFGF in stromal cells, while the presence of beta1 integrin subunit mRNA in stromal cells was associated with tumor PEA3 mRNA expression. To the best of our knowledge, this is the first evidence for coordinated autocrine and paracrine expression of members of these four families of metastasis-associated genes in human cancer. The results of this analysis support experimental data regarding cross-talk between carcinoma cells and peritumoral fibroblasts. They also suggest the existence of a putative activation sequence of metastatic genes, involving the beta1 (and possibly alpha v) integrin subunits, IL-8, PEA3, Ets-1 and MMP in ovarian carcinoma.     

The role of adhesion molecules, alpha v beta 3, alpha v beta 5 and their ligands in the tumor cell and endothelial cell adhesion.

Tumor metastasis is a complex process involving the interaction between tumor cells and endothelial cells in which some adhesion molecules play an important role. It was our aim to investigate the role of the adhesion molecules, alpha v beta 3 and alpha v beta 5 and their ligands, developmental endothelial locus-1 (Del-1) and L1, in tumor cell adhesion to endothelial cells in vitro. In this study, the expression and regulation of alpha v beta 3, alpha v beta 5 and intercellular adhesion molecule -1 on liver sinusoidal endothelial cells and liver cancer endothelial cells (T3A) were analyzed by real-time PCR and fluorescent-activated cell sorter. The expression and regulation of the integrin ligands, Del-1 and L1, in six tumor cell lines were analyzed by real-time PCR and western blot. We found the expressions of alpha v beta 3 and alpha v beta 5 were higher on T3A than that on liver sinusoidal endothelial cells, whereas expression of intercellular adhesion molecule-1 was lower on T3A than that on liver sinusoidal endothelial cells. After 24 h hypoxia, the expressions of alpha v beta 3 and alpha v beta 5 were upregulated on T3A and liver sinusoidal endothelial cells; the expression of intercellular adhesion molecule-1 was increased on liver sinusoidal endothelial cells, but remained unchanged on T3A. Del-1 and L1 expression levels were obviously diverse in various tumor cell lines and differentially modulated after 12 h hypoxia. The adhesion of tumor cells with Del-1 and L1 expression was higher in T3A than that in liver sinusoidal endothelial cells, and was significantly increased under hypoxic conditions. Interestingly, the tumor cell adherence could be inhibited by antibodies against alpha v beta 5 and alpha v beta 5, but not by an antibody against intercellular adhesion molecule-1. The adhesion of tumor cells without Del-1 and L1 expression was also higher on T3A than that on liver sinusoidal endothelial cells, but the adhesion could not be inhibited by antibodies against alpha v beta 5, alpha v beta 5 or intercellular adhesion molecule-1, suggesting that other receptors are involved. In conclusion, alpha v beta 5, alpha v beta 5 and their ligands Del-1 and L1 play an important role in the process of tumor cells moving from the original place.     

Fibronectin and fibronectin-receptors of the integrin type in normal colon mucosa, adenoma and carcinoma

Integrin alpha 3, alpha 4, alpha 5, and alpha v subunits heterodimerize with beta 1 and beta 3 chains to form functional fibronectin (FN) receptors. Two of them, alpha 4 beta 1 and alpha 5 beta 1, recognize FN as the only matrix molecule. Additional binding of laminin, collagen, and entactin is achieved by alpha 3 beta 1 while alpha v beta 1 and alpha v beta 3 also bind vitronectin. We investigated immunohistochemically the tissue distribution of FN and the expression of FN receptor alpha- and beta-chains in 20 normal colon tissues, 10 adenomas, 90 carcinomas, and 10 liver metastases derived thereof. In normal and adenomatous colon tissues FN was detected in association with reticular and fibrillar structures of the gut wall. The tumor stroma of carcinomas and liver metastases contained abnormally high amounts of FN which were detectable as chaotic deposits. Normal and adenomatous mucosa were alpha 3(+), alpha 4(-), alpha 5(-), alpha v(+/-), beta 1(+), beta 3(-) indicating that only promiscuous FN receptors were expressed. 21 of 90 carcinomas had a focal or complete loss of alpha 3 which was more often found in Dukes C/D tumors (p<0.005). The liver metastases more often had lower levels of alpha 3 expression compared to the primary tumor. The alpha v-expression was inconsistent and often weak. These losses of FN receptor constituents in carcinomas were not paralleled by any noticeable differences in stromal content or distribution pattern of FN, Enhanced expression/induction of FN receptors was found in only 2 of 90 carcinomas which focally neo-expressed the alpha 5-chain. This aberrant alpha 5 expression, however, was flow cytometrically found in 3/4 colon carcinoma cell lines which otherwise had receptor profiles corresponding to the in situ situation. FN-adherence of cell lines was variable. Anti-alpha 3 had no blocking effect on FN adherence of SW480, SW620, and COLO 205 but a minor effect on FN binding of HT-29. Anti-alpha 5 blocked FN adhesion whenever alpha 5 was expressed. Anti-av had no blocking effect while anti-beta 1 reduced FN adherence of all cell lines. The net biological effect(s) of aberrant expression of integrin type FN receptors in colon carcinomas is unpredictable as yet.     

Noninvasive imaging of alpha(v)beta3 integrin expression using 18F-labeled RGD-containing glycopeptide and positron emission tomography

The alpha(v)beta3 integrin is an important cell adhesion receptor involved in tumor-induced angiogenesis and tumor metastasis. Here we describe the 18F-labeling of the RGD-containing glycopeptide cyclo(-Arg-Gly-Asp-D-Phe-Lys(sugar amino acid)-) with 4-nitrophenyl 2-[18F]fluoropropionate and the evaluation of this compound in vitro and in tumor mouse models. Binding assays with isolated immobilized alpha(v)beta3, alpha(v)beta5, and alpha(IIb)beta3 as well as in vivo studies using alpha(v)beta3-positive and -negative murine and xenotransplanted human tumors demonstrated receptor-specific binding of the radiolabeled glycopeptide yielding high tumor:background ratios (e.g., 120 min postinjection: tumor:blood, 27.5; tumor:muscle, 10.2). First imaging results using a small animal positron emission tomograph suggest that this compound is suitable for noninvasive determination of the alpha(v)beta3 integrin status and therapy monitoring.     

Cell growth and matrix invasion of EBV-immortalized human B lymphocytes is regulated by expression of alpha(v) integrins

alpha(v) integrins have been shown to play an important role in epithelial-derived cell migration, cell growth and tumor invasion/metastasis, however their role on cells of hematopoietic origin is less clear. Epstein-Barr virus (EBV), a human herpesvirus associated with several lymphoproliferative disorders in man, induces expression of alpha(v) integrins on transformed B lymphocytes. In the studies reported here, we show that EBV infection increases alpha(v), beta3 and beta5 integrin subunit mRNAs as well as upregulates the expression of the alphavbeta3 integrin protein on human B cells. Among the nine different EBV proteins expressed in latently infected B cells (nuclear and plasma membrane-associated), only LMP1, LMP2A and EBNA2 were shown to selectively transactivate the alpha(v) integrin promoter. Treatment of EBV-transformed B cells with alpha(v) antisense oligonucleotides specifically reduced cell surface expression of alpha(v) integrins, inhibited cell growth in low serum, reduced cell invasion in matrigels and decreased expression of metalloprotease, MMP9. These studies indicate that alpha(v) integrins play a significant role in EBV-induced B-lymphocyte proliferation and invasion. Strategies to interfere with alphav integrin expression and/or function may therefore be of potential value in the treatment of EBV-associated lymphoproliferative disorders.     

Comparison of a monomeric and dimeric radiolabeled RGD-peptide for tumor targeting

The alpha v beta 3 integrin, a transmembrane heterodimeric protein expressed on sprouting endothelial cells, binds to the arginine-glycine-aspartic acid (RGD) amino acid sequence of extracellular matrix proteins such as vitronectin. Growing malignant tumors continuously require angiogenesis. As a result, alpha v beta 3 is preferentially expressed in growing tumors and is a potential target for radiolabeled RGD-peptides. In this study we compared the tumor targeting characteristics of a monomeric radiolabeled RGD-peptide with those of a dimeric analogue. Both peptides were radiolabeled with 99mTc via the hydrazinoni-cotinamid (= HYNIC) moiety to form 99mTc-HYNIC-c(RGDfK) and 99mTc-HYNIC-E-[c(RGDfK)]2. In vitro, the IC50 showed a 10-fold higher affinity of the dimer for the alpha v beta 3 integrin as compared to the monomer (0.1 vs. 1.0 nM). In athymic female BALB/c mice with subcutaneously growing OVCAR-3 ovarian carcinoma xenografts, tumor uptake peaked at 5.8 +/- 0.7% ID/g and 5.2 +/- 0.6% ID/g for the dimer and the monomer, respectively. At 1, 2, and 4 h postinjection (p.i.) uptake of the dimer in the tumor was significantly higher than that of the monomeric analogue. Tumor-to-blood ratios were highest at 24 h p.i. at a value of 63 for both compounds. At all timepoints kidney retention of the dimer was significantly higher as compared to kidney retention of the monomer. In conclusion, in this mouse model the dimeric RGD-peptide showed better retention in the tumor than the monomeric analogue, most likely due to the bivalent interaction with the target cell. Furthermore, kidney retention of the dimeric peptide was higher than that of the monomeric peptide.     

Neovascular targeting with cyclic RGD peptide (cRGDf-ACHA) to enhance delivery of radioimmunotherapy

Radioimmunotherapy (RIT) has been hampered by delivery of only a small fraction of the administered dose of radiolabeled MAb to tumor. A strategy for creating and controlling tumor vascular permeability would enable more effective RIT. The alpha v beta 3 integrin receptor is an appealing target for strategies designed to enhance permeability of tumor vessels because it is highly and preferentially expressed in most tumors. In human tumor mouse models, apoptosis of neovascular endothelial cells has been demonstrated after treatment with alpha v beta 3 antagonists. Since this apoptotic effect could transiently increase permeability of tumor blood vessels, radiolabeled antibodies (MAb) circulating during this period would have increased access to extravascular tumor. To determine if this hypothesis was correct, a pharmacokinetic study of an immunospecific MAb given after an alpha v beta 3 antagonist was performed in nude mice bearing human breast cancer xenografts. The alpha v beta 3 antagonist, cyclic RGD pentapeptide (c-RGDf-ACHA; cyclo arginine glycine aspartic acid D-phenylalanine -1 amino cyclohexane carboxylic acid), inhibits alpha v beta 3 binding to its vitronectin ligand at nanomolar levels. Cyclic RGD peptide (250 micrograms i.p.) given 1 hour before 111In-ChL6 MAb resulted in a 40-50% increase in tumor uptake (concentration), when compared to the control tumor uptake, of MAb 24 hours after administration. When cyclic RGD peptide was given as a continuous infusion (17.5 micrograms/hr) for 1 or 24 hours before 111In-ChL6, tumor uptake of 111In-ChL6 was increased less, and, these data were not statistically different from the control data. There were no differences for any of the groups in the groups in the concentrations of 111In-ChL6 in normal organs or blood when compared to the control group. The results suggest that cyclic RGD peptide provided a temporary, selective increase in tumor vascular permeability, that allowed a larger fraction of the 111In-ChL6 to accumulate in the tumor.     

Design, synthesis, and evaluation of radiolabeled integrin alpha v beta 3 receptor antagonists for tumor imaging and radiotherapy

The goal of this research is the development of tumor imaging and radiotherapeutic agents based on targeting of the integrin alpha(v)beta(3) (vitronectin receptor). Macrocyclic chelator DOTA has been conjugated to peptidomimetic vitronectin receptor antagonist SH066 to give TA138. TA138 and (89)Y-TA138 retain antagonist properties and high affinity for integrin alpha(v)beta(3) (IC(50) = 12 and 18 nM, respectively), and good selectivity versus integrin alpha(IIb)beta(3) (IC(50) > 10,000 nM). TA138 forms stable complexes with (111)In and (90)Y in > 95% RCP. (111)In-TA138 demonstrates high tumor uptake in the c-neu Oncomouse (Charles River Laboratories [Charles River, Canada]) mammary adenocarcinoma model (9.39% ID/g at 2 hours PI) and low background activity. Blood clearance is rapid and excretion is renal. Tumors are visible as early as 0.5 hours PI. Radiotherapy studies in the c-neu Oncomouse model demonstrated a slowing of tumor growth at a dose of 15 mCi/m(2), and a regression of tumors at a dose of 90 mCi/m(2).