MAB1976F Sigma-AldrichAnti-Integrin αVβ3 Antibody, clone LM609, FITC conjugated

We developed an in vitro contact through-feet blood brain barrier (BBB) model built using type IV collagen, rat astrocytes, and human umbilical vein endothelial cells (HUVECs) cocultured through Transwell porous polycarbonate membrane. The contact between astrocytes and HUVECs was demonstrated by electron microscopy: astrocytes endfeet pass through the 8.0 μm pores inducing HUVECs to assume a cerebral phenotype. Using this model we evaluated transmigration of melanoma cells from two different patients (M1 and M2) selected among seven melanoma primary cultures. M2 cells showed a statistically significant higher capability to pass across the in vitro BBB model, compared to M1. Expression of adhesion molecules was evaluated by flow cytometry: a statistically significant increased expression of MCAM, αvβ3, and CD49b was detected in M1. PCR array data showed that M2 had a higher expression of several matrix metalloproteinase proteins (MMPs) compared to M1. Specifically, data suggest that MMP2 and MMP9 could be directly involved in BBB permeability and that brain invasion by melanoma cells could be related to the overexpression of many MMPs. Future studies will be necessary to deepen the mechanisms of central nervous system invasion.

Targeted delivery of therapeutics to tumor neovasculature is potentially a powerful approach for selective cancer treatment. Integrins are heterodimeric transmembrane proteins involved in cell adhesion and cell signaling, and their expression is commonly upregulated in cancers and inflammatory diseases. The α(v)β(3) integrin is differentially upregulated on angiogenic endothelial cells as well as on many cancer cells. Here we demonstrate the differential targeting of cisplatin prodrug-encapsulated poly(d,l-lactic-co-glycolic acid)-block-polyethylene glycol (PLGA-PEG) nanoparticles (NPs) to the α(v)β(3) integrin on cancer cells using the cyclic pentapeptide c(RGDfK). Cisplatin is one of the most widely used anticancer drugs, and approaches that can improve its therapeutic index are of broad importance. The RGD-targeted Pt(IV)-encapsulated NPs displayed enhanced cytotoxicity as compared to cisplatin administered in its conventional dosage form in model prostate and breast cancer epithelial cells in vitro. Cytotoxicities were also elevated in comparison to those of previously reported systems, a small molecule Pt(IV)-RGD conjugate and a Pt(IV) nanoscale coordination polymer carrying RGD moieties. This result encouraged us also to evaluate the anticancer effect of the new construct in an animal model. The RGD-targeted PLGA-PEG NPs were more efficacious and better tolerated by comparison to cisplatin in an orthotopic human breast cancer xenograft model in vivo.

Cage-like protein nanoparticles are promising platforms for cell- and tissue-specific targeted delivery of imaging and therapeutic agents. Here, we have successfully modified the 12 nm small heat shock protein from Methanococcus jannaschii (MjHsp) to detect atherosclerotic plaque lesions in a mouse model system. As macrophages are centrally involved in the initiation and progression of atherosclerosis, targeted imaging of macrophages is valuable to assess the biologic status of the blood vessel wall. LyP-1, a nine residue peptide, has been shown to target tumor-associated macrophages. Thus, LyP-1 was genetically incorporated onto the exterior surface of MjHsp, while a fluorescent molecule (Cy5.5) was conjugated on the interior cavity. This bioengineered protein cage, LyP-Hsp, exhibited enhanced affinity to macrophage in vitro. Furthermore, in vivo injection of LyP-Hsp allowed visualization of macrophage-rich murine carotid lesions by in situ and ex vivo fluorescence imaging. These results demonstrate the potential of LyP-1-conjugated protein cages as nanoscale platforms for delivery of imaging agents for the diagnosis of atherosclerosis.

On the basis of the evidence that vacuolar H(+)-ATPase is implicated in the development of the metastatic phenotype, we have explored the possibility to target the enzyme function in an attempt to control the metastatic behavior of tumor cells. In this study, we used an indole derivative, NiK-12192 [4-(5,6-dichloro-1H-indol-2-yl)-3-ethoxy-N-(2,2,6,6-tetramethyl-piperidin-4-yl)-benzamide], recently identified as an effective inhibitor of vacuolar H(+)-ATPase, as a potential antimetastatic agent in the treatment of NSCLC H460 xenograft, which is able to induce lung metastases in mice. Oral administration of NiK-12192 caused a significant inhibition of formation of spontaneous metastases. In contrast, the drug exhibited a negligible effect on the development of artificial metastases (i.e., after i.v. injection of tumor cells), thus supporting that the drug affects the early events of the metastatic process (e.g., migration and invasion). Cellular effects are consistent with this interpretation. In conclusion, the available results show for the first time that a vacuolar H(+)-ATPase inhibitor is effective in modulation of the metastatic behavior of a lung carcinoma, supporting its potential therapeutic interest as a novel treatment approach.

Protein cages, including viral capsids, ferritins, and heat shock proteins (Hsps), can serve as nanocontainers for biomedical applications. They are genetically and chemically malleable platforms, with potential as therapeutic and imaging agent delivery systems. Here, both genetic and chemical strategies were used to impart cell-specific targeting to the Hsp cage from Methanococcus jannaschii. A tumor vasculature targeting peptide was incorporated onto the exterior surface of the Hsp cage. This protein cage bound to alpha(v)beta(3) integrin-expressing cells. Cellular tropism was also imparted by conjugating anti-CD4 antibodies to the exterior of Hsp cages. These Ab-Hsp cage conjugates specifically bound to CD4(+) cells. Protein cages have the potential to simultaneously incorporate multiple functionalities, including cell-specific targeting, imaging, and therapeutic agent delivery. We demonstrate the simultaneous incorporation of two functionalities, imaging and cell-specific targeting, onto the Hsp protein cage.

Detection of adenovirus DNA in human tonsillar T cells in the absence of active virus replication suggests that T cells may be a site of latency or of attenuated virus replication in persistently infected individuals. The lytic replication cycle of Ad5 in permissive epithelial cells (A549) was compared to the behavior of Ad5 in four human T-cell lines, Jurkat, HuT78, CEM, and KE37. All four T-cell lines expressed the integrin coreceptors for Ad2 and Ad5, but only Jurkat and HuT78 express detectable surface levels of the coxsackie adenovirus receptor (CAR). Jurkat and HuT78 cells supported full lytic replication of Ad5, albeit at a level approximately 10% of that of A549, while CAR-transduced CEM and KE37 cells (CEM-CARhi and KE37-CARhi, respectively) produced no detectable virus following infection. All four T-cell lines bind and internalize fluorescently labeled virus. In A549, Jurkat, and HuT78 cells, viral proteins were detected in 95% of cells. In contrast, only a small subpopulation of CEM-CARhi and KE37-CARhi cells contained detectable viral proteins. Interestingly, Jurkat and HuT78 cells synthesize four to six times more copies of viral DNA per cell than did A549 cells, indicating that these cells produce infectious virions with much lower efficiency than A549. Similarly, CEM-CARhi and KE37-CARhi cells, which produce no detectable infectious virus, synthesize three times more viral genomes per cell than A549. The observed blocks to adenovirus gene expression and replication in all four human T-cell lines may contribute to the maintenance of naturally occurring persistent adenovirus infections in human T cells.

Altered expression of alphav integrins plays a critical role in tumor growth, invasion, and metastasis. In this study, we show that normal human epithelial ovarian cell line, HOSE, and ovarian cancer cell lines, OVCA 429, OVCA 433, and OVHS-1, expressed alphav integrin and associated beta1, beta3, and beta5 subunits, but only ovarian cancer cell lines OVCA 429 and OVCA 433 expressed alphavbeta6 integrin. The expression of alphavbeta6 in OVCA 429 and OVCA 433 was far higher than alphavbeta3 and alphavbeta5 integrin and correlated with high p42/p44 mitogen activated protein kinase (MAPK) activity and high secretion of high molecular weight urokinase plasminogen activator (HMW-uPA), pro-metalloproteinase 2 and 9 (pro-MMP-9 and pro-MMP-2). In contrast to HOSE and OVHS 1, OVCA 433 and OVCA 429 exhibited approximately 2-fold more plasminogen-dependent [3H]-collagen type IV degradation. Plasminogen-dependent [3H]-collagen IV degradation was inhibited by inhibitor of uPA (amiloride) and MMP (phenanthroline) and by antibodies against uPA or MMP-9 or alphavbeta6 integrin, indicating the involvement of alphavbeta6 integrin, uPA and MMP-9 in the process. The alphavbeta6 correlated increase in HMW-uPA and pro-MMP secretion could be inhibited by tyrosine kinase inhibitor genistein or the MEK 1 inhibitor U0126, consistent with a role of active p42/44 MAPK in the elevation of uPA, MMP-9, and MMP-2 secretion. Under similar conditions, genistein and U0126 inhibited plasminogen-dependent [3H]-collagen type IV degradation. These data suggest that sustained elevation of p42/44 MAPK activity may be required for the co-expression of alphavbeta6 integrin, which in turn may regulate the malignant potential of ovarian cancer cells via proteolytic mechanisms.

Osteopontin is an adhesive glycoprotein implicated in numerous diseases associated with inflammation and remodeling. There are several structural domains in osteopontin that are of particular interest. The RGD motif is a cell attachment sequence shown to be critical for cell adhesion through alphav-containing integrins. In close proximity to the RGD domain is the thrombin cleavage site. Previous observations suggest that thrombin cleavage of osteopontin occurs in vivo and may be physiologically important. To study the functional significance of osteopontin cleavage by thrombin, we made glutathione S-transferase-osteopontin fusion proteins. These proteins contain either the N- or C-terminal domains expected to be formed following thrombin cleavage at the Arg169-Ser170 peptide bond. We compared these osteopontin fragments with native osteopontin in their ability to support adhesion of several different cell lines and identified the receptors mediating these interactions. Our data show that the N-terminal osteopontin fragment, which contains the RGD domain, supports adhesion of a melanoma cell line that is unable to bind native osteopontin. This suggests that osteopontin adhesive interactions may be regulated by thrombin cleavage. We also demonstrate that osteopontin contains a cryptic binding activity, which can be recognized by a novel osteopontin receptor. This receptor has been identified as the alpha9beta1 integrin.

Angiogenesis depends on the adhesive interactions of vascular cells. The adhesion receptor integrin alpha v beta 3 was identified as a marker of angiogenic vascular tissue. Integrin alpha v beta 3 was expressed on blood vessels in human wound granulation tissue but not in normal skin, and it showed a fourfold increase in expression during angiogenesis on the chick chorioallantoic membrane. In the latter assay, a monoclonal antibody to alpha v beta 3 blocked angiogenesis induced by basic fibroblast growth factor, tumor necrosis factor-alpha, and human melanoma fragments but had no effect on preexisting vessels. These findings suggest that alpha v beta 3 may be a useful therapeutic target for diseases characterized by neovascularization.