Expression proteomics of acute promyelocytic leukaemia cells treated with methotrexate

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.