TY - JOUR
T1 - Expression proteomics of acute promyelocytic leukaemia cells treated with methotrexate
AU - Agarwal, Nitin Kumar
AU - Mueller, Gerhard Anton
AU - Mueller, Claudia
AU - Streich, Jan Henrick
AU - Asif, Abdul Rahman
AU - Dihazi, Hassan
N1 - Funding Information:
This study was supported by Deutsche Forschungsgemeinschaft ( GRK 335 ). HL-60 cells were kindly donated by Prof. G. Wulf, Department of Hematology, University Klinikum, Goettingen. We are thankful to Dr. Ankur Goyal for her constructive criticism and comments. We furthermore acknowledge the technical assistance of Elke Brunst-Knoblich, Sophia Garten and S. Boguscha.
PY - 2010/4
Y1 - 2010/4
N2 - 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.
AB - 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.
KW - Acute promyelocytic leukaemia
KW - Cysteine-labeled differential in-gel electrophoresis
KW - Methotrexate
KW - NF-κB
KW - Proteasome subunits
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U2 - 10.1016/j.bbapap.2010.01.002
DO - 10.1016/j.bbapap.2010.01.002
M3 - Article
C2 - 20097313
AN - SCOPUS:76849100161
SN - 1570-9639
VL - 1804
SP - 918
EP - 928
JO - Biochimica et Biophysica Acta - Proteins and Proteomics
JF - Biochimica et Biophysica Acta - Proteins and Proteomics
IS - 4
ER -