Briefly, a cohort of 24 mice for each study were subcutaneously inoculated with 5 x 106 U266/LR7 cells in 100 l of RPMI-1640 medium; treatments started when tumors became measurable, approximately 3 weeks after cells were injected
Briefly, a cohort of 24 mice for each study were subcutaneously inoculated with 5 x 106 U266/LR7 cells in 100 l of RPMI-1640 medium; treatments started when tumors became measurable, approximately 3 weeks after cells were injected. cells. Decreased MM cell growth induced by inhibition of miR-221/222 melphalan was associated with a marked upregulation of pro-apoptotic BBC3/PUMA protein, a miR-221/222 target, as well as with modulation of drug influx-efflux transporters SLC7A5/LAT1 and the ATP-binding cassette (ABC) transporter ABCC1/MRP1. 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 ABCC1 in tumors retrieved 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 Relebactam 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 Relebactam Repeats DNA typing. These cells were immediately freezing 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 or college of Salamanca, Spain). AMO1 t(12;14) and bortezomib-resistant AMO1 Abzb t(12;14) cells were kindly provided by Dr. C Driessen (University or college of Tubingen, Germany). U266/LR7, AMO1 and AMO1 Abzb were not further authenticated but confirmed for the explained drug-resistant phenotype. All cells were cultured in RPMI-1640 (Gibco?, Existence Technologies), mainly because previously explained (36, 37). Human being stromal HS-5 cells were purchased from ATCC, which certify authentication by Short Tandem Repeats profiling. Also these cells were immediately freezing and used from the original stock within 6 months. Mouse monoclonal to IgG2a Isotype Control.This can be used as a mouse IgG2a isotype control in flow cytometry and other applications HS-5 were cultured in Dulbeccos revised Eagles medium (Gibco?, Life Systems) supplemented with 10% heath inactivated Fetal Bovine Serum (FBS) and 1% P/S (Penicillin/Streptomycin). Following educated consent and Istitutional Ethical Comeettee authorization, peripheral blood mononuclear cells (PBMCs) and main CD138+ MM cells from BM aspirates of 3 MM individuals, were isolated as previously explained (38). LNA-i-miR-221 was designed and synthesized as previously explained (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 (Existence Systems); mirVana? miRNA mimic and inhibitor Bad Control #1 (Existence Technologies) were used as experimental bad controls (NC). A total of 1 1 x 106 MM cells were transfected at 100 nM miRNAs concentrations from the Neon? Transfection System (Life Systems) (1050 v, 2 pulse, 30 a); transfection effectiveness, evaluated by flow-cytometry analysis relative to a FAM dyeClabeled anti-miRCnegative control, reached 85% to 90%. Related conditions were applied for transfection of MM cells with Silencer? Select siRNA for PUMA/BBC3 (siPUMA) or with Silencer? Select siRNA control (siCNT) (Existence Technologies), which was used at final concentration of 50 nM actually in co-transfection experiments with miRNAs inhibitors. Virus generation and illness of Human being Stromal HS-5 cells HS-5 cells cells stably expressing green fluorescent protein transgene were acquired as previously explained (39) (observe 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 explained (observe Supplementary Relebactam Methods for detailed info). (38) Cell proliferation and survival assay Cell growth inhibition was evaluated by Cell Counting Kit-8 (CCK-8) colorimetric assay (Dojindo Molecular Systems, Inc.), according to the manufacturers instructions. For melphalan dose-response experiments, MM cells were seeded in 24-well plates at a denseness of 2.5 x 105 cells per well in 1 ml of culture medium and incubated for 24 hours in the presence of different M melphalan concentrations; after incubation, MM cells were inoculated in 96-well plates for CCK-8 assay. Final optical denseness (O.D.) was measured at 450 nm using GloMax (Promega). Wells without cells (tradition medium only) 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 in 24-well plates at a denseness 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.