Proteome-wide Identification of New Molecular Targets Affected by Methotrexate in Acute Promyelocytic Leukaemia Cell Line

Proteome-wide Identification of New Molecular Targets Affected by Methotrexate in Acute Promyelocytic Leukaemia Cell Line

Nitin Kumar Agarwal

Doctoral thesis

Date of Examination: 2007-05-02

Date of issue: 2007-06-11

Advisor: Mueller, Gerhard Anton Prof. Dr.

Advisor: Dihazi, Hassan Dr.

Referee: Hardeland, Rüdiger Prof. Dr.

Referee: Doenecke, Detlef Prof. Dr.

Referee: Hirsch-Ernst, Karen Dr.

Persistent Address: http://hdl.handle.net/11858/00-1735-0000-000D-F227-3

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Abstract

English

Methotrexate (MTX) was first introduced as a cytotoxic agent that inhibits nucleotide biosynthesis in various cancer disorders. Accumulating evidences suggest that MTX inhibits the proliferation of malignant cells by inhibiting 5-aminoimidazole-4-carbox-amide ribonucleotide transformylase, isoprenylcysteine carboxyl methyltransferase and NF-kappaB transcription factor. These observations indicate that MTX could have additional molecular targets that are therefore unappreciated. To get insights into the complex molecular mechanisms of MTX induced apoptosis in acute promyelocytic leukemia cells (HL 60), we conducted an investigation incorporating cysteine labeled differential in gel electrophoresis combined with mass spectrometry. Initial experimental analysis revealed that 24 proteins were differentially expressed (p > 0.05) in HL 60 cell proteome after addition of 2.5 µM MTX for 72 hours. The majority of MTX induced proteins were ascribed to the endoplasmic reticulum (ER) chaperones, glycolytic enzymes and the mitochondrial transmembrane electron transport system (MTETS). In particular, we noted that three structural alpha4, alpha5, alpha7; a non-catalytically beta3 and two 26S regulatory proteasome subunits were significantly down regulated in MTX treated HL 60 cell. We further showed in HL 60 cells that MTX induces ER chaperones, suppresses the NF-kappaB subunit p65, disturbs the mitochondrial transmembrane potential (Äøm) and generates reactive oxygen species (ROS) in a time dependent manner. All together, our findings revealed that MTX alter the level of a variety of proteins involved in the NF-kappaB associated proteasome complex formation, ER stress, and the MTETS. Their identification as molecular targets of MTX may provide new impulse in the understanding of apoptotic activities in acute promyelocytic leukaemia cell line.

Keywords: Acute Promyelocytic Leukaemia cells; Methotrexate; Proteomics; Cysteine labelled differential in-gel electrophoresis; Proteasome subunits