AML sensitivity to YM155 is modulated through AKT and Mcl-1

Abstract

HL60 and U937 (acute myeloid leukemia (AML) cell lines) were assessed for sensitivity to YM155, and found to have distinct sensitive and resistant phenotypes, respectively. In HL60 cells, YM155 inhibition of growth proliferation was due to apoptosis which was measured by annexin V/PI staining. YM155 induced apoptosis through activation of intrinsic and extrinsic pathways that also culminated in caspase-3 activity and PARP cleavage. YM155 sensitivity was partially associated with this compound's ability to down-regulate survivin transcription since this was more pronounced in the HL60 cell line. However, marked differences were also observed in XIAP, Bcl-2, and Mcl-1L, and Mcl-1s. Furthermore, YM155 treatment completely inhibited production of total Akt protein in HL60, but not U937 cells. Importantly, Akt activity (pAkt-Ser473) levels were maintained in YM155 treated U937 cells which may help stabilize other anti-apoptotic proteins. Combination treatments with an Akt inhibitor, MK-2206, reduced levels of pAkt-Ser473 in U937 cells and synergistically sensitized them to YM155 cytotoxicity. Collectively our results indicate that Akt signaling may be an important factor mediating YM155 response in AML, and combinatorial therapies with Akt inhibitors could improve treatment efficacy in YM155-resistant cells.

Keywords: AML; Akt; Bcl-2; Mcl-1; XIAP; YM155.

Fig. 1

YM155 causes growth inhibition of HL60 and U937 cells in a dose- and time-dependent manner. AML cells were treated with YM155 for a) 24 hours, b) 48 hours, and 72 hours. Data represent mean ± SE from three independent experiments.

Fig. 2

HL60 cells are more susceptible to YM155 induced apoptosis than U937. Both intrinsic and extrinsic apoptoses were evaluated using caspase activity assays. (a) Annexin V/PI apoptotic assay of cells treated with YM155 for 24 hours; (b) activity of caspase 8, 9 and 3/7 in cells exposed to different doses of YM155 for 8 hours (RE = Relative Expression). Data represent the mean ± SD of three independent experiments (*p < .05, ** p < .01, *** p < .001). (c) Western blot analysis of PARP cleavage after 24 hour YM155 treatment.

Fig. 3

YM155 treatment up-regulates expression of the p21 cell cycle inhibitor and increases in the sub G0/G1 population in HL60 cells. (a) Cell cycle analysis of cells treated with YM155 for 24 hours. (b) Expression of p21 transcript in HL60 and U937 cells treated with YM155 for 8 hours. Data represent the mean ± SE from at least two independent experiments. (c) Western Blot analysis of p21 protein in cells treated with YM155 for 24 hours.

Fig. 4

YM155 differentially modulates pro-apoptotic Mcl-1_L and anti-apoptotic Mcl-1_S in HL60 and U937 cells. (a) Western Blot analysis of Mcl-1_L and Mcl-1_S in cells treated with YM155 for 24 hours. (b) Expression of Mcl-1_S and (c) Mcl-1_L transcripts in cells treated with YM155 for 8 hours. Data represent the mean ± SE from at least two independent experiments (*p < .05, n/s = not significant).

Fig. 5

YM155 causes a dose-dependent reduction of Bcl-2 and XIAP in HL60 cells. (a) Western Blot analysis of Bcl-2 protein in cells treated for 24 hours. (b) Expression of Bcl-2 transcripts in cells treated with YM155 for 8 hours. Data represent the mean ± SE from at least two independent experiments. (c) Western Blot analysis of XIAP protein in cells treated for 24 hours. (d) Expression of XIAP transcripts in cells treated with YM155 for 8 hours. Data represent the mean ± SE from at least two independent experiments.

Fig. 6

AKT expression and activity are highest in U937 cells and not inhibited by YM155 treatment. (a) Western Blot analysis of AKT activity (pAKT-Ser473) and total AKT protein in cells treated with YM155 for 24 hours. This representative Blot is selected from three independent experiments. (b) AKT transcript expression in HL60 and U937 cells treated with YM155 for 8 hours. Transcript levels are relative to those measured in HL60 control (untreated) and represent the mean ± SD of two independent experiments.

Fig. 7

Resistant U937 cells are sensitized to YM155 using an AKT inhibitor (MK-2206). (a) Synergistic growth inhibitory effects of the combination YM155+MK-2206 treatments for 72 hrs. (b) MTT assay of cell viability in cells exposed to YM155 (50 nM), MK-2206 (1000 nM) or both compounds for 72 hours. Data represent the mean ± SD of three independent experiments (*p < .05,*** p < .001). (c) Western Blot analysis of AKT activity (pAKT-Ser473) and total AKT protein in U937 cells treated for 72 hours (Y = 50 nM YM155; M = 1000 nM MK-2206). (d) Graphical representation of the ratio of pAKT-Ser473/AKT for each treatment condition.

Fig. 8

Schematic diagram of 4 major cellular pathways targeted by YM155 treatment in tumor cells [10]. Red molecules were shown to be differentially affected by YM155 exposure in this study, and represent the changes observed in YM155-sensitive HL60 cells (↑ = up-regulation; ↓ = down-regulation). Dashed lines represent a hypothetical mechanism by which constitutive AKT signaling in U937 cells may help protect specific YM155 targets [,,–42] resulting in a treatment resistant phenotype. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)