Expression proteomics of acute promyelocytic leukaemia cells treated with methotrexate

Biochim Biophys Acta. 2010 Apr;1804(4):918-28. doi: 10.1016/j.bbapap.2010.01.002. Epub 2010 Jan 22.


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 2.5microM methotrexate for 72h. We found that three structural alpha4, alpha5, alpha7 proteasome subunits, a non-catalytic beta3 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-kappaB 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 78kDa glucose regulated protein in a time-dependent manner. Altogether, our findings demonstrated that targeting NF-kappaB, structural and regulatory proteasome subunits with methotrexate may provide new insight into understanding methotrexate-induced apoptotic activities in HL-60 cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antimetabolites, Antineoplastic / pharmacology*
  • Apoptosis / drug effects
  • Down-Regulation / drug effects
  • Endoplasmic Reticulum / drug effects
  • Endoplasmic Reticulum / metabolism
  • HL-60 Cells
  • Humans
  • Leukemia, Promyelocytic, Acute / drug therapy*
  • Leukemia, Promyelocytic, Acute / metabolism*
  • Leukemia, Promyelocytic, Acute / pathology
  • Methotrexate / pharmacology*
  • NF-kappa B / metabolism
  • Neoplasm Proteins / metabolism
  • Proteasome Endopeptidase Complex / chemistry
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Subunits
  • Proteomics
  • Ubiquitination / drug effects
  • Unfolded Protein Response / drug effects


  • Antimetabolites, Antineoplastic
  • NF-kappa B
  • Neoplasm Proteins
  • Protein Subunits
  • Proteasome Endopeptidase Complex
  • Methotrexate