MDM2 inhibitors induce apoptosis by suppressing MDM2 and enhancing p53, Bax, Puma and Noxa expression levels in imatinib‑resistant chronic myeloid leukemia cells

Abstract

Activation of BCR::ABL1 tyrosine kinase is the main pathogenic mechanism underlying chronic myeloid leukemia (CML) in 90% of affected patients. The prognosis for individuals with CML who receive treatment with BCR::ABL1 tyrosine kinase inhibitors (TKIs) such as imatinib, is promising, with a 5-year survival rate of >90%. However, unfortunately, 20-30% of patients who are treated with imatinib may become resistant to the BCR::ABL1 TKIs. The objective of the present study was to determine whether inhibitors of E3 ubiquitin-protein ligase Mdm2 (MDM2), a regulator of p53 that promotes apoptosis and is highly expressed in CML, could induce cell death in imatinib-resistant CML cells. Apoptosis and cell viability were evaluated using Annexin-V-positive cell count and caspase-3 activity, as well as trypan blue dye exclusion assay. Expression levels of MDM2, p53, Bax, Puma, Noxa, p21, and cleaved caspase-3 were determined via western blotting. MDM2 levels in both the cytoplasm and nucleus were found to be ~3-fold higher in K562/IR cells compared with K562 cells, while the levels of p53 in both cell structures were markedly lower. In addition, an examination of a publicly accessible database revealed that the levels of MDM2 were evidently greater in patients who did not respond to imatinib compared with those who did respond to the drug. NSC-66811 and Nutlin-3, MDM2 inhibitors, increased the percentage of Annexin-positive cells in K562/IR cells by 43 and 62% at concentrations of 10 and 25 µM, respectively. Furthermore, the MDM2 inhibitors increased the levels of Bax, Puma, Noxa, and p21 by increasing the expression of p53 and decreasing the expression of MDM2 in K562/IR cells. Additionally, pifithrin-α, a p53 inhibitor, suppressed MDM2 inhibitor-induced cell death in K562/IR cells. Overall, the findings of the present study highlight the therapeutic potential of MDM2 inhibitors for imatinib-resistant CML.

Keywords: E3 ubiquitin-protein ligase Mdm2; chronic myeloid leukemia; imatinib resistance; p53.

Figure 1

Increased expression levels of MDM2 and decreased expression levels of p53 in K562/DR cells. (A) Following incubation for 2 days, cell lysates were assessed via immunoblotting with antibodies against MDM2, p53, β-actin, and lamin A/C. MDM2 and p53 levels were normalized to those of β-actin or lamin A/C. Results are representative of three independent experiments. ^*P<0.01 vs. K562 cells. (B) Comparison of MDM2 levels between imatinib responders and non-responders using the GSE33224 [Responders (N=12) and Non-responders (N=8)] and GSE14671 [Responders (N=24), and Non-responders (N=12)] datasets. (C) MDM2 expression in imatinib non-responders in GSE33224 and GSE14671 [Responders (N=36) and Non-responders (N=20)] datasets, with a mean value of 1 for imatinib responders. MDM2, E3 ubiquitin-protein ligase Mdm2.

Figure 2

NSC-66811 and Nutlin-3 induce the apoptosis of K562/IR cells. (A) Following treatment of K562/IR cells with NSC-66811 and Nutlin-3, cell viability was determined via trypan blue dye exclusion assay. The cells were treated with the indicated concentrations of NSC-66811 and Nutlin-3 for 3 days. Results are representative of five independent experiments. ^*P<0.01 vs. untreated cells (0.1% DMSO). (B) K562/IR cells were treated with the indicated concentrations of NSC-66811 and Nutlin-3 for 3 days. Caspase-3 activity was determined using the caspase-3/CPP32 fluorometric assay kit. Results are representative of four independent experiments. ^*P<0.01 vs. untreated cells (0.1% DMSO). (C) K562/IR cells were treated with the indicated concentrations of NSC-66811 and Nutlin-3 for 3 days and apoptosis was detected using the Annexin V-fluorescein isothiocyanate apoptosis detection kit. Results are representative of four independent experiments. ^*P<0.01 vs. untreated cells (0.1% DMSO).

Figure 3

NSC-66811 and Nutlin-3 increase the expression levels of p53 and decrease the expression levels of MDM2 in K562/IR cells. K562/IR cells were treated with the indicated concentrations of NSC-66811 and Nutlin-3 for 3 days. (A) Cell lysates were assessed via immunoblotting with antibodies against MDM2, p53, and β-actin. MDM2 and p53 expression levels were normalized to those of β-actin. Results are representative of three independent experiments. ^*P<0.01 vs. untreated cells (0.1% DMSO). (B) Cell lysates were assessed via immunoblotting with antibodies against Bax, Puma, Noxa, p21, cleaved caspase-3, and β-actin. The expression levels of Bax, Puma, Noxa, p21, and cleaved caspase-3 were normalized to those of β-actin. Results are representative of three independent experiments. ^*P<0.01 vs. untreated cells (0.1% DMSO). MDM2, E3 ubiquitin-protein ligase Mdm2.

Figure 4

Pifithrin-α attenuates MDM2 inhibitor-inducing cell death in K562/IR cells. K562/IR cells were treated with the indicated concentrations of NSC-66811, Nutlin-3, and pifithrin-α for 3 days. Each MDM2 inhibitor was added 2 h after the addition of pifithrin-α. (A) Cell lysates were assessed via immunoblotting with antibodies against MDM2, p53, and β-actin. The expression levels of MDM2 and p53 were normalized to those of β-actin. Results are representative of three independent experiments. ^*P<0.01 vs. untreated cells (0.2% DMSO). (B) K562/IR cells were treated with NSC-66811 or Nutlin-3 in combination with pifithrin-α, and cell viability was determined via trypan blue dye exclusion assay. Cells were treated with the indicated concentrations of NSC-66811, Nutlin-3, or pifithrin-α for 3 days. Results are representative of five independent experiments. ^*P<0.01 vs. untreated cells (0.2% DMSO). MDM2, E3 ubiquitin-protein ligase Mdm2.