Materials and Methods
Patients and cell cultures. MM patient samples were processed and used after achievement of written informed consent according to the declaration of Helsinki. The project outline was submitted and approved by the Ethic Committee of the Padova and Parma University Hospitals. Primary BM stromal cells (BMSCs) were obtained from the CD138-negativefraction of BM mononuclear cells. Cells were incubated in α-minimum essential medium (MEM) or Dulbecco's modified essential medium (DMEM) supplemented with 10 or 15% FBS and 2 mmol/L of glutamine until confluent for 2 weeks. Patient derived plasma cells were obtained as in (1).
The human bone marrow stromal cell line HS-5 was cultured as in (1). The interleukin-6 (IL-6)-dependent MM cell line INA-6 - cultured in RPMI 1640 supplemented with 10% fetal bovine serum and IL-6 5 ng/ml - was a gift of Dr. M. Gramatzki, University of Kiel, Germany. Co-cultures of HS-5/INA-6 or OCs/INA-6 were stabilized as previously described (1, 2) . Osteoblast cell lines HOB-01 and HOBIT were purchased from ATCC and cultured in DMEM supplemented with 10% FBS.
Osteoclastogenesis assay and Osteoclast/MM cells assay. CD14+cells isolated using anti-CD14 MACS (Miltenyi Biotec) from peripheral blood mononuclear cells (PBMCs) were cultured under osteoclastogenic conditions. CD14+cells were incubated with recombinant human monocyte-colony stimulating factor (rhM-CSF; 25 ng/mL) and recombinant human RANKL (rhRANKL; 60 ng/mL) (Peprotech, London, UK) in α-minimal essential medium (MEM) with 10% of FBS in the presence of CX-4945 (1, 2.5, 5 μM) or vehicle or 10 mM zoledronic acid (ZOL) in 96-well microtiter plates for 28 days, replacing media every 3–4 days. Osteoclasts (OCs) were identified and counted by light microscopy at the end of the culture period as multinucleated (>3) cells positive for tartrate resistant acid phosphatase (TRAP) assay (Sigma Aldrich, Milan, Italy). Co-cultures of INA-6 and osteoclasts were obtained by adding 5 x 105 INA-6 cells to establish mature OC-cultures. Fourty-eight hours later cells were harvested and scored for viability by Trypan blue exclusion microscopic assay.
Cytokines and chemicals. Interleukin-6 (IL-6) was purchased from Sigma-Aldrich, Italy. CX-4945 was from Activate-scientific, Germany. Zoledronic acid was from Novartis, Basel Switzerland.
mRNA silencing. RNA interference was performed using the lipofectamine reagent (Lifetechnologies, Italy), according to the manufacturer’s instruction, with siGLO Green scrambled siRNAs or the combination of siGLO Green and CK2a or CK2b targeting siRNAs (100 pmoles each) (Thermo Scientific). CK2a target sequences were: GCAUUUAGGUGGAGACUUC; GGAAGUGUGUCUUAGUUAC; GCUGGUCGCUUACAUCACU; AACAUUGUCUGUACAGGUU. CK2b target sequences were: CAACCAGAGUGACCUGAUU; GCAAGGAGACUUUGGUUAC; GCAAUGAAUUCUUCUGUGA; CCAAGUGCAUGGAUGUGUA;
Evaluation of apoptosis and flow cytometry. Apoptosis was assessed by Annexin V/Propidium Iodide (PI) staining according to the manufacturer’s instructions. In the experiments with the CK2 inhibitor, cells were labeled with FITC annexin , PI (Immunostep, Spain) and APC-conjugated anti-CD45 (Becton-Dickinson, Italy) or PECy5-conjugated anti-CD38 and PE-conjugated anti-CD138 antibodies. In siRNA experiments cells were labeled with PE annexin (Becton-Dickinson, Italy) and APC anti-CD45. Fluorescence Activated Cell Sorting (FACS) analysis was performed using a FACS-Calibur Cell Cytometer and the CellQuest® software (Becton-Dickinson, Italy). PE- conjugated anti-CXCR4 was from R&D Systems (USA).
Cytotoxicity assay. HOBIT and HOB-01 cell lines were incubated with different concentrations of CX-4945, ranging from 1 µM to 10 µM, or vehicle for 24-48 hours. The viability of HOBIT and HOB-01 cells was evaluated by means of a MTT (3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide)-based cytotoxicity assay in accordance with the manufacturer’s protocol (Cell Counting Kit-8; Alexis Biochemicals, Enzo Life Sciences, Plymouth Meeting, PA)
Western blot (WB) and ELISA. WB was performed according to standard protocols. Antibodies used were: CK2 (3); PARP, p-STAT3 Ser727, total STAT3, Bcl2, Mcl1, (Cell signaling Technology, MA, USA); total p65-NF-kB, (Abcam, UK); p-NF-kB p65 Ser529,
bactin (Santa-Cruz Biotechnology, CA, USA), GAPDH (Ambion, USA); Bak (Merck, MA, USA); Bax (Becton Dickinson, Italy); p-FAK Tyr397 and total FAK (Millipore, USA).
TNFa concentrations were determined in media from cell cultures through ELISA (e-bioscience, USA) according to the manufacturer’s instructions.
Real time quantitative polymerase chain reaction. Performed as described in (1). The primers used for IL-6 were Forward 5’-GGCACTGGCAGAAAACAACCTG-3’ and Reverse 5’-TCACCAGGCAAGTCTCCTCATTGAAT-3’, for TNFa Forward 5’-AGATCATCTTCTCGAACCCCG-3’ and Reverse 5’-GGGTTTGCTACAACATGGGCT-3’ and for b-actin: Forward 5’-CAGCTCACCATGGATGATG-3’ and Reverse 5’-ATGCCGGAGCCGTTGTC-3.
Migration assay
INA-6 cells were treated with CX-4945 5mM for 18h, washed in Hanks’ balanced salt solution (HBSS) and resuspended in RPMI 1640 with 0.1% bovine serum albumine (BSA). After 1.5h at 37°C and 5% CO2 cells were seeded (1.5 x105 in 100ml RPMI, 0.1% BSA , IL-6 2.5ng/ml) in the upper compartments of polycarbonate Transwell two-chamber migration plates (pore size 5mm), (Costar, Corning, USA). SDF1a 75ng/ml was added in a volume of 600 ml in the lower compartment. Migrated cells were counted after 5 h using FACSCanto Cytometer and the FACSDivat® software (Becton-Dickinson, Italy).
Statistical analysis. Data obtained were evaluated for their statistical significance with the two-tail paired Student’s t test . Values were considered statistically significant at p values below 0.05.
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