Alkylating agents are widely used chemotherapeutics in the treatment of many cancers, including leukemia, lymphoma, multiple myeloma, sarcoma, lung, breast and ovarian cancer. to elucidate their potential to overcome melphalan resistance. Introduction Multiple myeloma (MM) is an incurable bone marrow disease and the second most common hematological cancer. The median age of onset is 65 years and progression often leads to severe complications including immunodeficiency, osteolytic bone disease and renal failure . Although current therapies may improve the patients survival, disease progression and acquired drug resistance remain unsolved issues. Since the 1960s, the alkylating drug melphalan (L-phenylalanine mustard) has been employed in combination with corticosteroids as first-line therapy for MM . Novel agents such as bortezomib and lenalidomide have recently been introduced, but melphalan remains the standard therapy for transplant-ineligible patients and is the basis for high-dose therapy associated with autologous stem cell transplant . Melphalans efficacy has been attributed to its ability to induce cytotoxic interstrand cross-links (ICLs) in DNA , but it may also induce other lesions in DNA , RNA, proteins and lipids . The mechanisms by which melphalan kills tumor cells thus remain elusive and identifying factors that attenuate melphalan sensitivity is crucial to improving therapeutic outcomes. Acquired melphalan resistance in MM has been associated with reduced drug uptake , increased drug detoxification [8,9], reduced ICL formation and enhanced DNA repair of ICL lesions [10C12], modulation of DNA base excision and strand break repair [13,14], adaptation to reactive oxygen species (ROS)  and decreased apoptosis ; however, there are no robust biomarkers that predict melphalan resistance. Here we have used transcriptomics and Puromycin 2HCl proteomics to investigate cellular changes associated with acquired melphalan resistance in Puromycin 2HCl the RPMI8226 multiple myeloma cell line. We observed a metabolic switch conforming to the Warburg effect in the melphalan-resistant cell line accompanied by an increased oxidative stress response and enhanced survival and proliferation signaling. The increased survival was partially mediated through VEGF- and IL8-induced PI3K/p38 signaling and upregulated expression of the AKR1C family of aldo-keto reductases. We demonstrate that targeting enzymes within the affected pathways by specific inhibitors can overcome acquired melphalan resistance. Materials and Methods Reagents and antibodies For Western analysis antibodies to AKR1C2 (H00001646-D01, Abnova), AKR1C3 (H00008644-B01, Abnova), AKR1C4 (H00001109-M01, Novus), AKT1 (#2967, Cell Signaling), Caspase3 (sc-7148, Santa Cruz), SLC16A3 (OAAB08662, Aviva Systems Biology) PARP-1 (sc-74470, Santa Cruz), STAT3 (sc-81385, Santa Cruz), pSTAT3 (S2690, Sigma) and -actin (ab8226, Abcam) primary antibodies and HRP-conjugated secondary antibodies (Dako) were used. Melphalan, ursodeoxyholate, indomethacin, flufenamic acid, dichloroacetic acid, 2-deoxy-D-glucose, sodium oxamate, metformin, oligomycin, antimycinA, FLLL31, wortmannin, rapamycin, methyl glyoxal, acetylsalicylic acid, ibuprofen, (Sigma Aldrich), tert-butyl peroxide (Fluka), LY294002, SB203580 and BIRB0796 (Cell Signaling) were used in viability assays. Cell lines and preparation of cell extracts MM cell lines RPMI8226 and RPMI8226-LR5 were kindly donated by Prof. William S. Dalton at the H. Lee Moffitt Cancer Center & Research Institute, Tampa, USA. Cells were maintained, treated with melphalan and cell extracts prepared as previously described . mRNA isolation and analysis mRNA was isolated from six batches each of control and melphalan-treated RPMI8226 and RPMI8226-LR5 cells using the mirVana mRNA isolation kit (Ambion). RNA concentration and quality were determined using NanoDrop ND-8000 (NanoDrop Technologies) and Agilent 2100 Bioanalyzer (Agilent). RIN values of RNA samples used for cRNA amplification ranged from 8.5 to 10 (mean = 9.49). The Illumina TotalPrep RNA amplification Kit (Ambion) was used to amplify mRNA for hybridization on Illumina BeadChips. First strand cDNA was synthesized from each mRNA sample. Following second strand synthesis and purification, cRNA was synthesized for 12 hours. Gene expression profiles were measured using Illumina HumanHT-12_V3 Expression BeadChip. Raw data were exported from Illumina GenomeStudio to R using the Bioconductor Mouse monoclonal to eNOS package `lumi`version 2.1.3. . Data were quantile normalized and log2 transformed. The groups were compared using a t-test with empirical Bayes correction from the Bioconductor package `Limma`. The fold change Puromycin 2HCl Puromycin 2HCl was used to demonstrate rate of changes in average gene expressions between studied groups. Statistical analyses were performed using the false discovery rate (FDR) with a significance threshold of 0.01. The transcriptomic data have been deposited to the GEO repository with the identifier “type”:”entrez-geo”,”attrs”:”text”:”GSE60970″,”term_id”:”60970″GSE60970 . Western analysis, viability assay and mROS analysis Western analysis and MTT assays were performed as described . mROS was analyzed using MitoSOX Red (Molecular Probes) according to the manufacturers protocol. Briefly, cells (0.5 106 cells/ml) were pretreated with inhibitors for 6 h, washed with.