Integrin alphaVbeta3 contains a cell surface receptor site for thyroid hormone that is linked to activation of mitogen-activated protein kinase and induction of angiogenesis.

Integrin alpha(V)beta(3) is a heterodimeric plasma membrane protein whose several extracellular matrix protein ligands contain an RGD recognition sequence. This study identifies integrin alpha(V)beta(3) as a cell surface receptor for thyroid hormone [L-T(4) (T(4))] and as the initiation site for T(4)-induced activation of intracellular signaling cascades. Integrin alpha(V)beta(3) dissociably binds radiolabeled T(4) with high affinity, and this binding is displaced by tetraiodothyroacetic acid, alpha(V)beta(3) antibodies, and an integrin RGD recognition site peptide. CV-1 cells lack nuclear thyroid hormone receptor, but express plasma membrane alpha(V)beta(3); treatment of these cells with physiological concentrations of T(4) activates the MAPK pathway, an effect inhibited by tetraiodothyroacetic acid, RGD peptide, and alpha(V)beta(3) antibodies. Inhibitors of T(4) binding to the integrin also block the MAPK-mediated proangiogenic action of T(4). T(4)-induced phosphorylation of MAPK is inhibited by small interfering RNA knockdown of alpha(V) and beta(3). These findings suggest that T(4) binds to alpha(V)beta(3) near the RGD recognition site and show that hormone-binding to alpha(V)beta(3) has physiological consequences.

Antiangiogenesis and anticancer efficacy of TA138, a novel alphavbeta3 antagonist.

BACKGROUND: Angiogenesis is a complex process involving endothelial cell migration, proliferation, invasion, and tube formation. Inhibition of these processes might have implications in various angiogenesis-mediated disorders. MATERIALS AND METHODS: The antiangiogenic efficacy of the novel alphavbeta3 antagonist TA138 was examined using in vivo and in vitro model systems. RESULTS: The in vitro studies demonstrated the ability of TA138 and RP747 (conjugated TA138) to inhibit endothelial cell migration toward vitronectin, with an IC50=0.04 and 0.045 microM, respectively. Furthermore, utilizing the chick chorioallantoic membrane models, TA138 inhibited basic fibroblast growth factor-induced neovascularization. CONCLUSION: TA138 might be a useful tool for the inhibition of angiogenesis associated with human tumor growth, or other pathological neovascularization processes. RP747 demonstrated antitumor efficacy in 1 spontaneous tumor model (c-neu oncomouse model, alphavbeta3 positive cells) and in 1 xenograft model (HCT116 human tumor colon carcinoma, alphavbeta3 negative cells) injected subcutaneously into nude mice.

Human alphavbeta3 integrin potency and specificity of TA138 and its DOTA conjugated form (89)Y-TA138.

The present study was undertaken to define the alphavbeta3-binding potency and specificity of TA138, a nonpeptide integrin antagonist, and its conjugated form, 89Y-TA138. Various integrin-specific binding and functional assays as well as cell-adhesion assays were used to determine the potency and integrin specificity for TA138 and 89Y-TA138. Both TA138 and 89Y-TA138 inhibited alphavbeta3-mediated [125I]echistatin binding to 293-beta3-transfected cells, with IC50 values of 0.046 and 0.059 microM, respectively, and IC50 values of 0.012 and 0.018 microM, respectively, in inhibiting an alphavbeta3 integrin-mediated 293-beta3-transfected cell adhesion to fibrinogen. TA138 inhibited human umbilical vein endothelial cell adhesion to fibrinogen, with an IC50 value of 0.052 +/- 0.006 microM. Both TA138 and 89Y-TA138 demonstrated a relatively high degree of specificity for human alphavbeta3 integrin as compared with other human integrins, including alphavbeta5, alphaIIbbeta3, and alpha5beta1 (IC50 > 10 microM). Both 89Y-TA138 and TA138 demonstrated comparable alphavbeta3 affinity and specificity as compared with other closely related human integrins such as alphavbeta5, alphaIIbbeta3, or alpha5beta1.

Overexpression of estrogen receptor-alpha in the endometrial carcinoma cell line Ishikawa: inhibition of growth and angiogenic factors.

BACKGROUND: A high level of estrogen receptor-alpha (ER-alpha) is believed to be favorable in the prognosis and treatment of endometrial, ovarian, and breast cancer. High levels of ER-alpha have been shown to inhibit the growth and invasive, metastatic potential of breast cancer cell lines. To bring about these inhibitory effects, ER-alpha probably acts through other cellular factors involved in the regulation of cell growth. OBJECTIVE: To investigate the role of high levels ER-alpha in growth inhibition of endometrial cancer cells. METHODS: A human ER-alpha cDNA was stably overexpressed in an endometrial cancer cell line, namely, Ishikawa. ER-alpha-overexpressing, parent, and control Ishikawa cells were grown in vitro and their growth rates were compared by cell count. ER-alpha-overexpressing and parent Ishikawa cells were also grown in vitro as tumors in a chicken chorioallantoic membrane (CAM) model, and tumor growth and angiogenesis was measured. Finally, levels of angiogenesis-modulating factors, nitric oxide synthase (NOS), and vascular endothelial growth factor (VEGF) were examined in relation to ER overexpression. RESULTS: The growth of Ishikawa cells was found inhibited in culture as well as in the CAM model. Angiogenesis of CAM tumors was also found inhibited in ER-overexpressing cells. Angiogenic factor VEGF was inhibited whereas the activity of NOS was found elevated following ER overexpression. CONCLUSION: Our work on the Ishikawa cell line indicates that high levels of ER-alpha in endometrial cancer may inhibit cancer growth by modulating angiogenic factors, thereby limiting the blood supply to the growing tumor. Our results support the earlier data from other groups that have shown a positive correlation between high ER content and better prognosis of endometrial cancers.

Anti-angiogenic mechanisms and efficacy of the low molecular weight heparin, tinzaparin: anti-cancer efficacy.

Inhibitors of angiogenesis are potential anti-cancer agents in that they deprive tumors of the blood necessary for growth and metastasis. The anti-angiogenic efficacy of tinzaparin, a known anticoagulant low molecular weight heparin (LMWH), was examined in vitro in endothelial cell tube formation assay and in vivo in the chick chorioallantoic membrane model. The observed anti-angiogenic effects of tinzaparin were shown to be dose-related and dependent on the relatively higher molecular weight tinzaparin fragments. These experiments demonstrated that tinzaparin is a potent inhibitor of angiogenesis (ED90-100 range, 0.05-0.1 mg) regardless of the angiogenic factor and suggest that its effect is mediated via cellular release of tissue factor pathway inhibitor (TFPI). This was evident by the reversal of either tinzaparin or r-TFPI anti-angiogenesis efficacy by a specific monoclonal TFPI antibody. The ED90-100 for the inhibition of angiogenesis for r-TFPI ranged from 0.01 to 0.03 mg in the chorioallantoic membrane model regardless of the proangiogenic factor. In addition, either tinzaparin or r-TFPI inhibited the growth of colon carcinoma tumors, human fibrosarcoma tumors, and human lung carcinoma in the chorioallantoic membrane tumor implant model. Thus, the LMWH tinzaparin, in addition to its anticoagulant effects, may offer important benefits in treatment of cancer and other disorders supported by pathologic angiogenesis.

Proangiogenic action of thyroid hormone is fibroblast growth factor-dependent and is initiated at the cell surface.

The effects of thyroid hormone analogues on modulation of angiogenesis have been studied in the chick chorioallantoic membrane model. Generation of new blood vessels from existing vessels was increased 3-fold by either l-thyroxine (T4; 10(-7) mol/L) or 3,5,3'-triiodo-l-thyronine (10(-9) mol/L). T4-agarose reproduced the effects of T4, and tetraiodothyroacetic acid (tetrac) inhibited the effects of both T4 and T4-agarose. Tetrac itself was inactive and is known to block actions of T4 on signal transduction that are initiated at the plasma membrane. T4 and basic fibroblast growth factor (FGF2) were comparably effective as inducers of angiogenesis. Low concentrations of FGF2 combined with submaximal concentrations of T4 produced an additive angiogenic response. Anti-FGF2 inhibited the angiogenic effect of T4. The proangiogenic effects of T4 and FGF2 were blocked by PD 98059, a mitogen-activated protein kinase (MAPK) pathway inhibitor. Endothelial cells (ECV304) treated with T4 or FGF2 for 15 minutes demonstrated activation of MAPK, an effect inhibited by PD 98059 and the protein kinase C inhibitor CGP41251. Reverse transcription-polymerase chain reaction of RNA extracted from endothelial cells treated with T4 revealed increased abundance of FGF2 transcript at 6 to 48 hours, and after 72 hours, the medium of treated cells showed increased FGF2 content, an effect inhibited by PD 98059. Thus, thyroid hormone is shown to be a proangiogenic factor. This action, initiated at the plasma membrane, is MAPK dependent and mediated by FGF2.