Expression proteomics of acute promyelocytic leukaemia cells treated with methotrexate Nitin Kumar Agarwal a , Gerhard Anton Mueller a , Claudia Mueller b , Jan-Henrick Streich a , Abdul Rahman Asif c , Hassan Dihazi a, ⁎ a Department of Nephrology and Rheumatology, Center of Internal Medicine, Georg-August University Göttingen, Robert-Koch Strasse 40, D-37075, Göttingen, Germany b Section for Transplantation Immunology and Immunohematology, Eberhard-Karls University Tübingen, Waldhoernle Strasse 22, 72072, Tübingen, Germany c Department of Clinical Chemistry, Georg-August University Göttingen, Robert-Koch Strasse 40, D-37075, Göttingen, Germany abstract article info Article history: Received 8 September 2009 Received in revised form 7 December 2009 Accepted 4 January 2010 Available online 22 January 2010 Keywords: Methotrexate NF-κB Proteasome subunits Cysteine-labeled differential in-gel electrophoresis Acute promyelocytic leukaemia Methotrexate was first introduced as a cytotoxic agent that inhibits nucleotide biosynthesis in various cancer disorders; its molecular mechanism remains elusive. To understand the molecular mechanism by which methotrexate induces apoptosis, we analyzed the resulting intracellular protein changes in methotrexate- treated acute promyelocytic leukaemia (HL-60) cells by cysteine-labeled differential in-gel electrophoresis (CL-DIGE) combined with mass spectrometry. Initial CL-DIGE analysis revealed that 24 proteins were differentially expressed (p < 0.05) in the HL-60 cell proteome after treatment with 2.5 µM methotrexate for 72 h. We found that three structural α4, α5, α7 proteasome subunits, a non-catalytic β3 and two 26S regulatory proteasome subunits were down-regulated in methotrexate-treated HL-60 cells. Western blot analyses further showed that the inhibition of proteasome subunits is accompanied by suppression of NF-κB subunits and promotes the accumulation of ubiquitinated proteins. Furthermore, methotrexate activated unfolded protein response by inducing the expression of endoplasmic reticulum-resident proteins such as calreticulin, protein disulphide isomerase A3 and A4, and 78 kDa glucose regulated protein in a time- dependent manner. Altogether, our findings demonstrated that targeting NF-κB, structural and regulatory proteasome subunits with methotrexate may provide new insight into understanding methotrexate-induced apoptotic activities in HL-60 cells. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Methotrexate (MTX), a folate analogue, first synthesized by Sidney Farber in collaboration with Harriett Kilte and Lederle chemists. It was the first example of rational drug design and mainly developed for the treatment of acute lymphocytic leukaemia [1]. Moreover, it was reported to cure choriocarcinoma and trophoblastic cancer [2,3]. Beneficial effects were also observed in patients with the Burkitt's and other non-Hodgkin's lymphomas and carcinomas such as breast and head and neck [4]. Besides its anticancer applications, MTX is widely used in the treatment of rheumatoid arthritis [5] and others chronic inflammatory diseases [6]. Dihyrofolate reductase is the primary site of action for MTX. Inhibition of dihyrofolate reductase leads to toxic effects through partial depletion of tetrahydrofolate cofactors that are required for purine and thymidylate synthesis [6]. Accumulating evidences suggest that MTX inhibits the proliferation of malignant cells by inhibiting 5-aminoimidazole-4-carboxamide ribonucleotide transfor- mylase [7], isoprenylcysteine carboxyl methyltransferase [8] and NF- κB transcription factor [9]. NF-κB is a major transcription factor that regulates genes responsible for cell proliferation and cell survival [10]. It is mainly controlled by the inhibitory IκB proteins. On external stimulation, IκB is rapidly phosphorylated by IKK and degraded via the ubiquitin– proteasome pathway, permitting nuclear import of NF-κB [10]. There are sufficient evidences in the literature suggesting the ability of proteasome inhibitors in blocking NF-κB activation by preventing the degradation of IκB [8]. Majumdar and Aggarwal further showed that suppression of NF-κB subunits in MTX-treated Jurkat cells was associated with inhibition of IKBα phosphorylation and degradation [9]. These observations indicate that MTX could have additional molecular targets that are as yet unappreciated. To get insight into the complex molecular mechanisms of MTX- induced apoptosis in HL-60 acute promyelocytic leukaemia cells, we conducted an investigation incorporating cysteine-labeled differential in-gel electrophoresis (CL-DIGE) combined with mass spectrometry technology. Our results revealed that MTX potentially down-regulates structural and regulatory proteasome subunits in HL-60 cells. This down-regulation results in the accumulation of ubiquitinated proteins and promote the expression of endoplasmic reticulum (ER)-resident Biochimica et Biophysica Acta 1804 (2010) 918–928 ⁎ Corresponding author. Tel.: + 49 551 3991221; fax: + 49 551 3991039. E-mail address: dihazi@med.uni-goettingen.de (H. Dihazi). 1570-9639/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2010.01.002 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap