HDAC Inhibitors as Epigenetic Regulators

Representative samples (left), n=4 3-, n=4 3-all, n=5 3+ (right). bone metastases relative to primary tumors from your same patient (n=42). Mechanistic investigations revealed that TGF- signaling through SMAD2/SMAD3 was necessary for breast malignancy induction of 3 within the bone. Using a micelle-based nanoparticle therapy that recognizes integrin v3 (v3-MPs of ~12.5nm), we demonstrated specific localization to breast cancer bone metastases in mice. Using this system for targeted delivery of the chemotherapeutic docetaxel, we showed that bone tumor burden could be reduced significantly with less bone destruction and less hepatotoxicity compared to equimolar doses of free docetaxel. Furthermore, mice treated with v3-MP-docetaxel exhibited a significant decrease in bone-residing tumor cell proliferation compared to free docetaxel. Taken together, our results offer preclinical proof of concept for a method to enhance delivery of chemotherapeutics to breast cancer cells within the bone by exploiting their selective expression of integrin v3 at that metastatic site. and maintenance of growth characteristics. Murine malignancy models To establish orthotopic mammary excess fat pad (MFP) tumors, 0.1106 tumor cells in 50L PBS were injected into MFP tissue of 7-week-old female mice. To establish experimental secondary metastases, 0.05106 tumor cells in 50uL PBS were intracardiac injected into the left ventricular chamber of 6-week-old female, with one exception; in collaboration with Dr. Theresa Guise, human MDA-MB-231 tumor cells were intracardiac injected (0.1106 tumor cells in 100L PBS) into 4-week-old female athymic nude mice, as previously described (30). MX-69 Synthesis of v3-targeted micelle nanoparticles Phospholipid/polysorbate 80 micelle nanoparticles (MPs) were prepared as a microfluidized suspension of 20% (v/v), combining polysorbate tween 80 (Sigma Aldrich, Inc.) with a 2.0% (w/v) of a surfactant comixture, and 1.7% (w/v) glycerin in pH 6.5 carbonate buffer, as previously explained (29). Optionally, the surfactant comixture included 2.28mol% of docetaxel-prodrug (DTX-PD), and/or 0.15mol% of v3-targeted quinolone nonpeptide coupled to phosphatidylethanolamine-PEG2000, with the remaining mol% lecithin. Docetaxel was altered into an Sn2 lipase-labile phosphatidylcholine DTX-PD as previously explained (32). For fluorescent labeling, rhodamine-conjugated to phosphatidylethanolamine (0.1 mol%) was incorporated into the lipid surfactant. The surfactant components for each formulation were combined Nfia with the polysorbate, buffer, and glycerin mixtures and were homogenized at 20,000 psi for 4 moments at 4C with a microfluidics (M110s; Microfluidics, Inc). The nanoparticles were preserved under inert gas in sterile sealed vials until use. Dynamic light scattering showed a nominal particle size of 12.50.8 nm, with polydispersities 0.2900.03, and an average electrophoretic zeta potential of -3.821.23 mV. The nanoparticle-targeting quinolone nonpeptide specific for integrin v3 was originally developed by Bristol-Myers Squibb Medical Imaging (US patent 6,511,648 and related patents) and coupled to phosphatidylethanolamine-PEG2000, as previously explained (29). The quinolone nonpeptide was initially characterized as the 111In-DOTA conjugate RP478 and cyan 5.5 homologue TA145 (33). This v3-targeting ligand is usually selective for cells expressing v3 (IC50 = 12 nM), as compared to IC50>10 M for 5, 51, or II3 (34). Furthermore, the affinity of the 3-targeting ligand increases 15-fold for activated integrin v3 receptor in the presence of Mn2+ (33). specificity of this v3-targeting ligand has been further exhibited through competitive pretreatment with unlabeled v3-targeting ligand, which inhibited v3-targeted nanoparticle binding (35). Bioluminescence imaging (BLI) and BLI was performed on IVIS50 (PerkinElmer, Waltham, MA) as previously explained (20). Total photon flux (photons/sec) was measured from fixed regions of interest (ROIs) over entire mouse or manually around organs using Living Image 2.6, as indicated. Mice with outstanding chest BLI intensity indicative of a failed intracardiac injection or with ineffective D-luciferin administration were excluded from all analyses. Investigators were blinded to treatment groups during all BLI analyses. Rhodamine-labeled MP colocalization with breast cancer bone metastases Tumor-bearing mice day 8 post-intracardiac injected with PyMT-Bo1 cells, or age-matched tumor-free mice, were treated with a single dose of rhodamine-labeled MPs (either non-targeted or v3-targeted) at a nanoparticle dose of 2nmol/g mouse excess weight. Investigators were blinded to treatment groups during intravenous MP and v3-MP injections. MPs MX-69 were allowed to circulate for 3 hours comparisons of interest were between saline-treated mice as compared to DTX or v3-MP/DTX-PD, or between saline-treated mice compared to the nanoparticle control treatments. In collaboration MX-69 with Dr. Theresa Guise, histological bone sections were.