By western blotting analysis, we showed activation/cleavage of caspase-8, caspase-9 and caspase-3, as well as increased PARP cleavage, in U266/LR7 cells exposed to combination treatment (Fig

By western blotting analysis, we showed activation/cleavage of caspase-8, caspase-9 and caspase-3, as well as increased PARP cleavage, in U266/LR7 cells exposed to combination treatment (Fig. Finally, treatment of SCID/NOD mice bearing human melphalan-refractory MM xenografts with systemic LNA-i-miR-221 melphalan overcame drug-resistance, evidenced by growth inhibition with significant antitumor effects together with modulation of PUMA and IL-23A ABCC1 in tumors retrieved Revefenacin from treated mice. Conclusions Taken together, our findings provide the proof of concept that LNA-i-miR-221 can reverse melphalan-resistance in preclinical models of MM, providing the framework for clinical trials to overcome drug resistance and improve patient outcome in MM. and (34), and that naked LNA-inhibitors of miR-221 (LNA-i-miR-221) are suitable for systemic delivery in animals (35). Here we investigated the role of miR-221/222 in melphalan-refractory MM, and demonstrate restoration of melphalan-sensitivity in refractory cells after exposure of MM cells to a novel 13 mer LNA-i-miR-221. Our findings provide therefore the rationale for clinical trials investigating LNA-i-miR-221 melphalan in drug-refractory MM. Materials and Methods Cell cultures, reagents and drugs Multiple Myeloma cell lines NCI-H929 t(4;14), RPMI-8226 t(14;16) and U266 t(11;14) were purchased from DSMZ (Germany) which certified authentication performed by Short Tandem Repeats DNA typing. These cells were immediately frozen and used from the original stock within 6 months. Melphalan-resistant U266/LR7 t(11;14) cells were kindly provided by Dr. A. Pandiella (University of Salamanca, Spain). AMO1 t(12;14) and bortezomib-resistant AMO1 Abzb t(12;14) cells were kindly provided by Dr. C Driessen (University of Tubingen, Germany). U266/LR7, AMO1 and AMO1 Abzb were not further authenticated but confirmed for the described drug-resistant phenotype. All cells were cultured in RPMI-1640 (Gibco?, Life Technologies), as previously described (36, 37). Human stromal HS-5 cells were purchased from ATCC, which certify authentication by Short Tandem Repeats profiling. Also these cells were immediately frozen and Revefenacin used from the original stock within 6 months. HS-5 were cultured in Dulbeccos modified Eagles medium (Gibco?, Life Technologies) supplemented with 10% heath inactivated Fetal Bovine Serum (FBS) and 1% P/S (Penicillin/Streptomycin). Following informed consent and Istitutional Ethical Comeettee approval, peripheral blood mononuclear cells (PBMCs) and primary CD138+ MM cells from BM aspirates of 3 MM patients, were isolated as previously described (38). LNA-i-miR-221 was designed and synthesized as previously described (35). Melphalan and Bortezomib were purchased from Sigma Aldrich and Selleck Chemicals, respectively. transfection of MM cells Synthetic mirVana? miR-221 and miR-222 inhibitors or mimics were purchased (Life Technologies); mirVana? miRNA mimic and inhibitor Negative Control #1 (Life Technologies) were used as experimental negative controls (NC). A total of 1 1 x 106 MM cells were transfected at 100 nM miRNAs concentrations by the Neon? Transfection System (Life Technologies) (1050 v, 2 pulse, 30 a); transfection efficiency, evaluated by flow-cytometry analysis relative to a FAM dyeClabeled anti-miRCnegative control, reached 85% to 90%. Similar conditions were applied for transfection of MM cells with Silencer? Select siRNA for PUMA/BBC3 (siPUMA) or with Silencer? Select siRNA control (siCNT) (Life Technologies), which was used at final concentration of 50 nM even in co-transfection experiments with miRNAs inhibitors. Virus generation and infection of Human Stromal HS-5 cells HS-5 cells cells stably expressing green fluorescent protein transgene were obtained as previously described (39) (see Supplementary Methods for detailed information). Reverse transcription and quantitative real-time PCR Total RNA extraction from MM cells and quantitative real-time PCR were performed as previously described (see Supplementary Methods for detailed information). (38) Cell proliferation and survival assay Cell growth inhibition was evaluated by Cell Counting Kit-8 (CCK-8) colorimetric assay (Dojindo Molecular Technologies, Inc.), according to the manufacturers instructions. For melphalan dose-response experiments, MM cells were seeded in 24-well plates at a density of 2.5 x 105 cells per well in 1 ml of culture medium and incubated for 24 hours Revefenacin in the presence of different M melphalan concentrations; after incubation, MM cells were inoculated in 96-well plates for CCK-8 assay. Final optical density (O.D.) was measured at 450 nm using GloMax (Promega). Wells without cells (culture medium alone) were used as blank. For combination experiments with miRNAs, 1 x 106 electroporated cells with NC or miR-221/222 inhibitors were incubated for 24 hours in 6-well plates; after harvesting, cells were inoculated Revefenacin in 24-well plates at a density of 2.5 x 105 cells/ml and incubated in the presence or absence of different M melphalan concentrations. Twenty-four hours after beginning drug exposure, cells were seeded in 96-well plates for CCK-8 assay and O.D. measurement. For co-culture experiments, 2.5 x 105 U266/LR7 cells transfected with.