Role and mechanism of decitabine combined with tyrosine kinase inhibitors in advanced chronic myeloid leukemia cells

Abstract

Patients with advanced chronic myeloid leukemia (CML) have a poor prognosis, with the use of tyrosine kinase inhibitors (TKIs) to treat CML demonstrating poor results. The results of the present study revealed that, following Cell Counting Kit-8 analysis, treatment of K562 cells with decitabine (DAC) combined with TKIs exhibits synergic effects. Co-immunoprecipitation indicated that tyrosine-protein phosphatase non-receptor type 6 (SHP-1) and BCR-ABL fusion protein (BCR-ABL) (p210) form a complex in the K562 cell line, and in the primary cells derived from patients with CML. These results suggested that SHP-1 serves a role in regulating the tyrosine kinase activity of BCR-ABL (p210). In addition, SHP-1 expression increased, while BCR-ABL expression decreased in the group treated with DAC and TKIs combined group compared with the TKI monotherapy group. Treatment with imatinib (IM) demonstrated no effect on SHP-1 methylation in the K562 cell line; however, the methylation of SHP-1 was not determined in the combined IM and DAC therapy group. Treatment with DAC demonstrated the ability to activate the expression of silenced SHP-1 through demethylation, thus decreasing BCR-ABL tyrosine kinase activity, resulting in an improved therapeutic effect on CML.

Keywords: chronic myeloid leukemia; demethylation; immunoprecipitation; tyrosine kinase inhibitors; tyrosine-protein phosphatase non-receptor type 6.

Figure 1.

Optical density values of K562 cells following 48 h drug-treatment with (A) DAC (EC₅₀, 6457 nM), (B) nilotinib (EC₅₀, 10.55 nM) and (C) IM (EC₅₀, 227.5 nM) were measured using a Cell Counting Kit-8. (D) K562 cells treated with 5 µM DAC combined with 0.1 µM IM or 5 nM nilotinib exhibited proliferation inhibition that was statistically significant compared with the IM or nilotinib monotherapy treatment groups. ***P<0.001. DAC, decitabine; IM, imatinib; EC₅₀, half maximal effective concentration.

Figure 2.

Western blotting analysis of SHP-1 expression. (A) Representative image and (B) quantitative analysis of SHP-1 expression in K562 cells, and CML-CP and CML-BP mononuclear cells through immunoprecipitation. (C) Representative image and (D) quantitative analysis of SHP-1 expression in K562 cells through immunoprecipitation following 48 h drug-treatment. *P<0.05, **P<0.01, ***P<0.001. DAC, decitabine; IM, imatinib; CML, chronic myeloid leukemia; CP, chronic phase; BP, blastic phase; SHP-1, tyrosine-protein phosphatase non-receptor type 6.

Figure 3.

RT-qPCR analysis of SHP-1 and BCR/ABL mRNA expression. (A) SHP-1 and BCR/ABL mRNA expression in K562 cells following 48 h drug-treated through RT-qPCR analysis. Expression of (B) SHP-1 and (C) BCR/ABL mRNA expression in chronic myeloid leukemia-chronic phase mononuclear cells following 48 h drug treatment through RT-qPCR analysis. *P<0.05, ***P<0.001. SHP-1, tyrosine-protein phosphatase non-receptor type 6; RT-qPCR, real-time quantitative polymerase chain reaction; mRNA, microRNA; DAC, decitabine; IM, imatinib; BCR/ABL, BCR-ABL fusion protein.

Figure 4.

Western blotting analysis of SHP-1 and BCR/ABL expression. (A) Representative image and (B) quantitative analysis of SHP-1 expression in CML-CP mononuclear cells following 48 h drug treatment through immunoprecipitation. (C) Representative image and (D) quantitative analysis of SHP-1 expression in CML-BP mononuclear cells following 48 h drug treatment through immunoprecipitation. SHP-1 protein expression increased in the combined groups compared with the monotherapy groups, while BCR-ABL expression decreased. CML, chronic myeloid leukemia; CP, chronic phase; BP, blastic phase; SHP-1, tyrosine-protein phosphatase non-receptor type 6; DAC, decitabine; IM, imatinib; BCR/ABL, BCR-ABL fusion protein.

Figure 5.

SHP-1 gene methylation levels of K562 cells were measured through methylation-specific PCR in the control, IM, DAC, and IM combined with DAC group. The results demonstrated the presence of SHP-1 gene methylation in the control and IM monotherapy group, while no methylation was demonstrated in the IM+DAC and DAC monotherapy group. DAC, decitabine; IM, imatinib; SHP-1, tyrosine-protein phosphatase non-receptor type 6.