2.1. Reagents and supplies
An antibiotic mixture containing penicillin, streptomycin, and neomycin; α-minimum essential medium (α-MEM); and low-glucose Dulbecco’s modified Eagle’s medium (DMEM-LG) were obtained from Gibco (Grand Island, NY, USA). RPMI 1640 (without L-glutamine), Cellgro COMPLETE™ medium, were purchased from Mediatech, Inc (Herndon, VA, USA). Fetal bovine serum (FBS) was purchased from Hyclone (Logan, UT, USA). Recombinant human macrophage colony-stimulating factor (M-CSF) and RANKL were purchased from RDI (Flanders, NJ, USA). 4HPR was obtained from Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute. S1P was from Avanti Polar lipids, Inc (Alabaster, AL, USA).
2.2. Preparation of osteoclasts and osteoblasts
Cultures of multinucleated bone-resorbing osteoclasts were prepared as previously described [14
]. Signed Institutional Review Board–approved informed consent forms were kept on record. The cells were cultured at 2.5 × 106
/mL in α-MEM supplemented with 10% FBS, antibiotics, RANKL (50 ng/mL), and M-CSF (25 ng/mL) for 10 to 14 days, at which time they contained large numbers of multinucleatedosteoclasts with bone-resorbing activity.
Osteoblasts were prepared as previously described [15
]. Briefly, mesenchymal stem cells (MSCs) were cultured in DMEM-LG medium supplemented with 10% FBS, dexamethasone (100 nM), β-glycerophosphate (10 mM), and ascorbate (0.05 mM) (osteoblastic medium) for approximately 3 weeks.
2.3. MM cells lines and osteoclast co-cultures
Two stromal cell dependent cell lines (BN and JB)[16
] and two stromal independent MM cells lines (ARP-I, CAG) were maintained in our laboratory. MM cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS and antibiotics. Osteoclasts were washed three times with PBS to detach and remove non-adherent cells. MM cells were co-cultured with osteoclasts in RPMI 1640 medium with 10% FBS. At the end of each experiment, MM cells were collected and processed as previously reported [14
] for further assays. At the end of each experiment MM cell growth was determined by MTT assay [17
2.4. Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL)
MM cells were collected, cytospin slides prepared (40,000 cells/slide) and fixed in 10% phosphate-buffered formalin for 20 minutes in freshly prepared 4% paraformaldehyde (PFA). The TUNEL assay was performed using the Klenow FragEL™ DNA Fragmentation Detection Kit (Calbiochem, Darmstadt, Germany) according to the manufacturer’s instruction.
2.5. Annexin V/propidium iodide (PI) staining
MM cells treated for 24 hr with indicated concentrations of 4HPR were analyzed for apoptosis using Annexin V propidium iodide (PI) detection kit (Caltag Laboratories, Burlingame, CA, USA) by FacScan flow-cytometer (Becton Dickinson, San Jose, CA). Results were expressed as percent apoptotic annexin V+ cells. Adherent HUVEC cultured in 96-well plates were similarly stained for annexin V and total number of apoptotic annexin V+ cells counted using ZEISS AX10 Observer A1 microscope (Delta Optical Instruments, Thornwood, NY).
2.6. Western Blotting
Cytosolic and nuclear fractions were isolated with the use of the Nuclear/Cytosol Fractionation Kit (BioVision Research Products, Mountain View, CA). Equal amounts of lysate were separated by electrophoresis on 4% to 12% sodium dodecyl sulfate–polyacrylamide gels (Bio-Rad, Hercules, CA, USA), and Western blotting was carried out according to the Western Breeze chemiluminescent immunodetection protocol as described by the manufacturer (invitrogen; Carlsbad, California). Antibodies for caspase-3, 8, 9 (cell signaling technology; Danvers, MA, USA) were used.
2.7. Measurement of intracellular reactive oxygen species (ROS)
Production of ROS was detected using a 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) fluorescent probe obtained from invitrogen (Carlsbad, California). This compound is a cell-permeant indicator for ROS that is nonfluorescent until the acetate groups are removed by intracellular esterases and oxidation occurs within the cell. After treatment with 4HPR, H2DCFDA (5 μM) was added into the medium with MM cells in 24-well plate (0.2 × 106/well) 20 min before the end of treatment. Centrifuge cells and replace medium with 0.5 ml PBS, then analyze cells by flow cytometry (Becton Dickinson, San Jose, CA).
2.8. Matrigel Tube Formation Assay
BD Matrigel growth factor–reduced basement membrane matrix (Becton Dickinson, Franklin Lakes, NJ) was diluted on ice with DMEM medium (1:2 dilution factor), poured onto 96-well plates (100 μl/well), and incubated at 37°C for 30 minutes. Human umbilical vein endothelial cells (HUVEC; American Type Culture Collection, Manassas, VA) were cultured in Clonetics EBM-2 medium (Lonza Walkersville Inc., Walkersville, MD) supplemented with a cocktail of growth factors according the manufacturer’s instructions. For tube formation assay, HUVEC were trypsinized and seeded on Matrigel-containing chamber slides (15,000 cells/well) with indicated medium (100 μl/well) in the absence or the presence of 4HPR (1–10 μM) for 3 4 hours. Conditioned medium collected from a 48-hour culture of ARP-I or CAG MM cell lines (1.5 × 106 cells/mL) was used to test the effect of MM cells on tube formation. Tube-like structures per well were counted in triplicate with the use of a phase-contrast microscope. Images were acquired with a SPOT 2 digital camera and processed with Adobe Photoshop version 10.
2.9. Statistical analysis
Unless indicated otherwise, all values are expressed as mean±SEM. Student’s unpaired t-test was used to test the effect of 4HPR on cell growth, apoptosis, and numbers of tube-like shapes and osteoclasts.