Near-infrared fluorescence optical imaging is a powerful technique for studying diseases at the molecular level in preclinical models. We recently reported that monomeric RGD peptide c(RGDyK) conjugated to the NIR fluorescent dye specifically targets integrin receptor both in cell culture and in living subjects. In this report, Cy5.5-conjugated mono-, di-, and tetrameric RGD peptides were evaluated in a subcutaneous U87MG glioblastoma xenograft model in order to investigate the effect of multimerization of RGD peptide on integrin avidity and tumor targeting efficacy. The binding affinities of Cy5.5-conjugated RGD monomer, dimer, and tetramer for α v β 3 integrin expressed on U87MG cell surface were determined to be 42.9 ± 1.2, 27.5 ± 1.2, and 12.1 ± 1.3 nmol/L, respectively. All three peptide-dye conjugates had integrin specific uptake both in vitro and in vivo. The subcutaneous U87MG tumor can be clearly visualized with each of these three fluorescent probes. Among them, tetramer displayed highest tumor uptake and tumor-tonormal tissue ratio from 0.5 to 4 h postinjection. Tumor-to-normal tissue ratio for Cy5.5-conjugated RGD monomer, dimer, and tetramer were found to be 3.18 ± 0.16, 2.98 ± 0.05, and 3.63 ± 0.09, respectively, at 4 h postinjection. These results suggest that Cy5.5-conjugated monomeric, dimeric, and tetrameric RGD peptides are all suitable for integrin expression imaging. The multmerization of RGD peptide results in moderate improvement of imaging characteristics of the tetramer, compared to that of the monomer and dimeric counterparts.

The stilbene resveratrol (RV) initiates p53-dependent apoptosis via plasma membrane integrin aVb3 in human cancer cells. A thyroid hormone (L-thyroxine, T 4 ) membrane receptor also exists on aVb3. Stilbene and T 4 signals are both transduced by extracellular-regulated kinases 1 and 2 (ERK1/2); however, T 4 promotes cell proliferation in cancer cells, whereas RV is proapoptotic. Thyroid hormone has been shown to interfere with RV-induced apoptosis. However, the mechanisms involved are not fully understood. In this study, we examined the mechanism whereby T 4 inhibits RV-induced apoptosis in glioma cells. RV activated conventional protein kinase C and ERK1/2 and caused nuclear localization of cyclooxygenase-2 (COX-2), consequent p53 phosphorylation and apoptosis. RV-induced ERK1/2 activation is involved in not only COX-2 expression but also nuclear COX-2 accumulation. NS-398, a COX-2 inhibitor, did not affect ERK1/2 activation, but reduced the nuclear abundance of COX-2 protein and the formation of complexes of nuclear COX-2 and activated ERK1/2 that are required for p53-dependent apoptosis in RVtreated cells. T 4 inhibited RV-induced nuclear COX-2 and cytosolic pro-apoptotic protein, BcLx-s, accumulation. Furthermore, T 4 inhibited RV-induced apoptosis by interfering with the interaction of nuclear COX-2 and ERK1/2. This effect of T 4 was prevented by tetraiodothyroacetic acid (tetrac), an inhibitor of the binding of thyroid hormone to its integrin receptor. Tetrac did not, in the absence of T 4 , affect induction of apoptosis by RV. Thus, the receptor sites on aVb3 for RV and thyroid hormone are discrete and activate ERK1/2-dependent downstream effects on apoptosis that are distinctive.

Abegrin TM (MEDI-522 or Vitaxin TM ), a humanized monoclonal antibody against human integrin A v B 3 , is in clinical trials for cancer therapy. In vivo imaging using Abegrin TM