Analysis of changes in the proteome of HL-60 promyeloid leukemia cells induced by the proteasome inhibitor PSI

Abstract

Proteasome inhibitors display potent anti-neoplastic and anti-angiogenic properties both in vitro and in vivo. The mechanisms, however, by which proteasome inhibitors kill tumor cells are still fairly elusive as is the molecular basis of resistance to treatment. To address these questions, we employed a high-throughput Western blotting procedure to analyze changes in a subproteome of ~800 proteins in the promyelocytic leukemia cell line HL-60 upon treatment with the proteasome inhibitor PSI (ZIle-Glu(OtBu)-Ala-Leu-aldehyde) and correlated the changes of selected target proteins with the changes in two multidrug resistant HL-60 variants. In total, 105 proteins were upregulated more than 1.5-fold after PSI treatment, while 79 proteins were downregulated. Activation of caspases-3 and -8, modulation of members of the Bcl-2 family as well as stimulation of stress signaling pathways was prominent during HL-60 apoptosis. We also identified changes in the abundance of proteins previously not known to be affected by proteasome inhibitors. In contrast, two multidrug resistant HL-60 cell lines, overexpressing either MRP1 or P-glycoprotein were largely resistant to PSI-induced apoptosis and could not be resensitized by the pharmacological inhibitors of the drug efflux pumps MK571 or PSC833. Drug resistance was also independent from the upregulation of Bad. Overexpression of multidrug resistance proteins, P-glycoprotein and MRP-1 is thus not sufficient to explain resistance of HL-60 cells to treatment with proteasome inhibitor PSI, which remains more closely related to a low level of Bax expression and to the inability to activate JNK. Alternative routes to the acquisition of resistance to PSI have therefore to be considered. Classification(1) Antibiotics and Chemotherapeutics

Retaining cytotoxic activity of anthrapyridone CO1 against multidrug resistant cells is related to the ability to induce concomitantly apoptosis and lysosomal death of leukaemia HL60/VINC and HL60/DOX cells

“…These findings together with our previously results for anthraquinone antitumour agents (DOX, pirarubicin and benzoperimidine BP1) and data published by other groups (e.g. for curcumin, UVC irradiation and proteasome inhibitor Z‐Ile‐Glu(OtBu)‐Ala‐Leu‐aldehyde (PSI)) strongly suggest that even though the studied sensitive HL60 cells and MDR cells (HL60/VINC and HL60/DOX) undergo apoptosis in response to anthraquinone antitumour agents and other cellular stress stimuli, they are also able to activate non‐apoptotic cell death pathways in response to some anticancer compounds (e.g. CO1).…”

Differential impact of bortezomib on HL-60 and K562 cells

“…In agreement with previously published results (Choi et al 2008), we have also shown that the bortezomib induced proteolytic activation of procaspase 3 in HL-60 cells manifested by the increase of p17 fragment of active caspase 3. In K562 cells, the bortezomib-induced decrease of procaspase 3 was not associated with the production of p17 fragment of caspase 3.…”

“…Instead of this, we have shown that the bortezomib induced cleavage of HSP90β through the action of caspase 8. This is in agreement with previous studies that have documented activation of caspase 8 after the incubation of different cancer cells with proteasome inhibitors (Colado et al 2008;Laussmann et al 2011;Fiandalo et al 2013) including HL-60 cells (Choi et al 2008). Since it has been documented that HSP90β is a substrate of caspase 10 but not caspase 8 (Chen et al 2009), we assume that caspase 8 is the initiator of this process while caspase 10 is responsible for direct cleavage of HSP90β.…”

Determination of differentially regulated proteins upon proteasome inhibition in AML cell lines by the combination of large‐scale and targeted quantitative proteomics

“…So far, very few proteomic studies have attempted to investigate the global cellular response to proteasome inhibition . Immunoblotting techniques have been widely used to measure protein abundance upon proteasome inhibition, but this strategy is restricted to well‐known pathway due to the availability of specific antibodies . One of the first large‐scale mass spectrometry (MS)‐based proteomic studies to decipher mechanism of proteasome inhibitors was performed in our lab by Uttenweiller and colleagues, and reported the effect of bortezomib on both differentiated and undifferentiated Acute Promyelocytic Leukemia (APL) cells and revealed that the differential apoptotic effects of bortezomib on these particular cells are mainly due to distinct protein toxicity levels .…”