The press and PBS wash were combined and fluorescence was measured and reported as relative to vehicle. Scratch Wound Healing Assay PCa cells (LNCaP, C4-2B, ARCaP, PC3, VCaP MC-Val-Cit-PAB-vinblastine and DU145) were seeded into 24-well plates. and invasion of PCa cells. Additionally, DI7E6 decreased phosphorylation of FAK, Akt and ERK. These results indicate that inhibition of integrin alpha V with DI17E6 inhibits several pro-metastatic phenotypes of PCa cells and therefore provide a rationale for further evaluation of DI17E6 for diminishing PCa progression. Implications This work identifies that therapeutic targeting of integrins containing an alpha V integrin unit inhibits cancer progression and thus may be of clinical benefit. PCa models  leading to testing an integrin v inhibitor, abituzumab (DI17E6, EMD 525797), in clinical PCa . DI17E6 recognizes the extracellular domains of the integrin v-chain and inhibits ligand binding to all v heterodimers (v1, v3, v5, v6, v8) without cross-reacting with other members of the integrin family . Results from a Phase I trial in patients with progressive castration-resistant prostate cancer with bone metastases after chemotherapy showed DI17E6 to be well tolerated with potential antitumor activity . However, the mechanisms through which targeting v integrins could provide an antitumor effect in prostate cancer MC-Val-Cit-PAB-vinblastine have not been defined. To explore these mechanisms, we evaluated DI17E6 on PCa cell lines. Materials and Methods Cells and MC-Val-Cit-PAB-vinblastine cell culture Human PCa cell lines DU145, LNCaP and PC3 were obtained from the American Type Culture Collection (ATCC; Rockville, MD) and cultured in RPMI 1640 (Invitrogen Co., Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Life Technologies, Inc.). The C4-2B cell line, which is an LNCaP subline, and ARCaP (kindly provided by Dr. Leland Chung, Cedars Sinai, Hollywood, CA) were maintained in T medium [80% DMEM (Life Technologies, Inc.), 20% F12 (Invitrogen), 100 units/liter penicillin G, 100 mg/ml streptomycin, 5g/mL insulin, 13.6 pg/mL triiodothyronine, 5g/mL transferrin, 0.25g/mL biotin, and 25 g/mL adenine] supplemented with 10% FBS. VCaP cells (kindly provided by Dr. Kenneth Pienta, University of Michigan, Ann Arbor, MI) were maintained in DMEM (Life Technologies, Inc.) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Life Technologies, Inc.). The human fetal osteoblast line (hfOB1.19) was obtained from ATCC and was maintained in DMEM with F12 (DMEM+F12, Invitrogen) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Life Technologies, Inc.). HS-5 and Saos2 cells were obtained from the ATCC and were maintained in alpha-MEM (Gibco, NY) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Life Technologies, Inc.). Primary cultures of neonatal foreskin-derived human dermal microvascular endothelial cells (HDMEC) were obtained commercially (Lonza, Walkersville, MD, USA). For cell culture, endothelial cell basal medium (EBM; Cambrex, Wlakersville, MD, USA) was used and supplemented with 10% FBS (Life Technologies), epidermal growth factor (10 ng/ml; BD Biosciences, Bedford MA), hydrocortisone (HC, 1 em /em g/ml; Sigma-Aldrich, St. Louis, MO) and 1% penicillin-streptomycin (Life Rabbit Polyclonal to FPRL2 Technologies, Inc.). All cultures were maintained at 37C, 5% CO2, and 100% humidity. The identities of cell lines were confirmed using short tandem repeat fingerprinting. Antibodies DI1E76 (Merck KGaA, Darmstadt, Germany) is an anti-integrin v-chain antibody . Anti-Integrin alpha V antibody [“type”:”entrez-protein”,”attrs”:”text”:”EPR16800″,”term_id”:”523382855″,”term_text”:”EPR16800″EPR16800] (ab179475) (Abcam, Cambridge, MA) is a rabbit monoclonal antibody. Cell Proliferation LNCaP, C4-2B, ARCaP, VCaP, PC3 and DU145 cells were grown in 96-well plates. Cells were then treated with DI17E6 (0.1g/ml, 1g/ml, 10g/ml, 100 g/ml) or vehicle for 24hr, 48hr and 72hr. Cell proliferation reagent WST-1 was added and incubated at 37C and 5% CO2 for 4hr. Absorbance was then measured at 440 nm with a plate reader. Data are presented as meanSD from triplicate determinations. Caspase-3/7 Assay LNCaP, C4-2B, ARCaP, VCaP, PC3 and DU145 were cultured in white-walled 96-well plates at a concentration of 1104 per 50l per well. Cells were treated with 50l DI176E (0.1g/ml, 1g/ml, 10g/ml and 100g/ml final concentrations) or vehicle or etoposide (100nM final concentration) (Sigma) as a positive control and were allowed to grow for 24 hours at which point 100ul of Apo-ONE Caspase-3/7 Reagent (Promega, Madison, WI) was added to each well and the cells were incubated for extended periods ( 4 hours). Absorbance was determined at 499 MC-Val-Cit-PAB-vinblastine and 521 nm using a plate reader (Multi-Mode Microplate Reader, SpectraMax M5, Molecular Devices MDS Analytical Technologies). Cell Cycle Analysis LNCaP, C4-2B, ARCaP, VCaP, PC3 and DU145 were cultured in 100mm dishes at a concentration of 1105/dish in 10 ml. Cells were treated with DI176E (100g/ml) or vehicle for 72 hours. Cells were then collected and fixed with ice-cold 70% ethanol for overnight. After that, cell cycle analysis was performed using flow cytometry as previously described . Briefly cells were centrifuged and washed with DPBS twice for removing ethanol then 0.8 l of DNAse-free RNAse was added into each tube and incubated.