The apoptotic mechanism of action of the sphingosine kinase 1 selective inhibitor SKI-178 in human acute myeloid leukemia cell lines

Abstract

We previously developed SKI-178 (N'-[(1E)-1-(3,4-dimethoxyphenyl)ethylidene]-3-(4-methoxxyphenyl)-1H-pyrazole-5-carbohydrazide) as a novel sphingosine kinase-1 (SphK1) selective inhibitor and, herein, sought to determine the mechanism-of-action of SKI-178-induced cell death. Using human acute myeloid leukemia (AML) cell lines as a model, we present evidence that SKI-178 induces prolonged mitosis followed by apoptotic cell death through the intrinsic apoptotic cascade. Further examination of the mechanism of action of SKI-178 implicated c-Jun NH2-terminal kinase (JNK) and cyclin-dependent protein kinase 1 (CDK1) as critical factors required for SKI-178-induced apoptosis. In cell cycle synchronized human AML cell lines, we demonstrate that entry into mitosis is required for apoptotic induction by SKI-178 and that CDK1, not JNK, is required for SKI-178-induced apoptosis. We further demonstrate that the sustained activation of CDK1 during prolonged mitosis, mediated by SKI-178, leads to the simultaneous phosphorylation of the prosurvival Bcl-2 family members, Bcl-2 and Bcl-xl, as well as the phosphorylation and subsequent degradation of Mcl-1. Moreover, multidrug resistance mediated by multidrug-resistant protein1 and/or prosurvival Bcl-2 family member overexpression did not affect the sensitivity of AML cells to SKI-178. Taken together, these findings highlight the therapeutic potential of SKI-178 targeting SphK1 as a novel therapeutic agent for the treatment of AML, including multidrug-resistant/recurrent AML subtypes.

Fig. 1.

SKI-178 induces cell death in a range of AML cell lines, including multidrug-resistant HL-60/VCR. (A and C) Whole cell lysates from the indicated AML cell lines were subjected to Western blot analysis to assess SphK1 (A) and MDR-1 (C) expression. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as an equal loading control. (B) Whole cell lysates from the indicated AML cell lines were prepared in buffer selective for SphK1 activity, and SphK1 catalytic activity was subsequently determined by thin-layer chromatography. (D) HL-60 and HL-60/VCR cells were exposed to increasing concentrations of SKI-178 over a 48-hour time period and cytotoxicity was assessed by MTT assays. Error bars indicate S.D. of triplicate counts of 2500 cells.

Fig. 2.

SKI-178 induces apoptosis through the intrinsic apoptotic signaling pathway. (A) Cytometric analysis of Annexin V/7AAD-stained HL-60 and HL-60/VCR cells treated with SKI-178 (5 μM) for indicated time periods or vehicle (DMSO) control for 48 hours. Results shown are representative of three separate experiments. (B) Quantification of Annexin V–positive staining including both early (Q3) and late (Q2) stage apoptosis. Error bars indicate S.D. of triplicate counts of 5000 cells. Statistical significance was assessed by two-tailed paired Student’s t test. Asterisks indicate significance: *P ≤ 0.001; **P ≤ 0.0001. (C) HL-60 cells treated with SKI-178 (5 μM) for indicated time intervals. Western blot analysis was performed on whole cell lysates with proteins indicated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. PARP, poly(ADP-ribose) polymerase.

Fig. 3.

SKI-178–induced Bcl-2 phosphorylation and caspase-7 activation are blocked by JNK and CDK1 inhibitors. (A) HL-60 cells treated for 24 hours with various MAPK inhibitors or vehicle (DMSO) alone or in combination with SKI-178 (5 μM). Western blot analysis was performed on whole cell lysate using antibody for cleaved caspase-7. (B) HL-60 cells treated with SKI-178 (5 μM) for indicated time intervals. Western blot analysis was performed on whole cell lysates with pJNK (Thr183/Tyr185) antibody or for the proteins indicated. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C) HL-60 cells treated for 24 hours with vehicle (DMSO), SP600125, a JNK-specific inhibitor (AS601245), or a CDK1-specific inhibitor (RO3306) alone or in combination with SKI-178. Western blot analysis was performed on whole cell lysate using antibody for pBcl-2 (Ser70) and cleaved active caspase-7. GAPDH serves as a loading control. Results shown are representative of at least three independent experiments.

Fig. 4.

Prolonged mitosis precedes SKI-178–induced apoptotic cell death. (A) Flow cytometry analysis of cell cycle distribution in HL-60 cells treated with SKI-178 or vehicle (DMSO) control for indicated time intervals. Histograms are representative of three separate experiments. (B) Percentage of AML cell lines in G2/M after 16 hours of treatment with SKI-178 (5 μM) or vehicle (DMSO). Error bars indicate S.D. of triplicate counts of 25,000 cells. Statistical significance was assessed by two-tailed paired student’s t test. Asterisks indicate significance: *P ≤ 0.01.

Fig. 5.

SKI-178 induces sustained Bcl-2 phosphorylation during prolonged mitosis. (A and B) Bcl-2 phosphorylation dynamics during cell cycle progression of vehicle (DMSO) treated or SKI-178 (5 μM) treated cells. HL-60 cells were synchronized at G1/S phase transition using a double thymidine block. Cells were released from the block into media containing vehicle (DMSO) (A) or SKI-178 (5 μM) (B). Whole cell lysates were collected at indicated time points, and Western blot analysis was performed using antibodies for pBcl-2 (Ser70), pHistone H3 (Ser10), or cleaved active caspase-7. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as a loading control. Results shown are representative of at least three independent experiments.

Fig. 6.

Inhibition of CDK1 completely abrogates SKI-178–induced Bcl-2 phosphorylation and caspase-7 activation after mitotic arrest. HL-60 cells were synchronized at G1/S phase transition using a double thymidine block and released into either vehicle (A) or SKI-178 (5 μM) (B). Cells released into SKI-178 were subdivided into four additional treatments: (C) HL-60 cells released into SKI-178 and cotreated with AS601245 at the time of release; (D) HL-60 cells released into SKI-178 and cotreated with AS601245 14 hours after release; (E) HL-60 cells released into SKI-178 and cotreated with RO3306 14 hours after release. Whole cell lysates were collected at indicated time points and blot analysis was performed using antibodies for pBcl-2 (Ser70) or pHistone H3 (Ser10). (F) To directly compare pBcl-2 (Ser70), pHistone H3 (Ser10), and caspase cleavage between the various treatments, Western blot analysis was performed using indicated antibodies on the 26-hour time points from (A–E). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as a loading control. Results shown are representative of at least three independent experiments.

Fig. 7.

SKI-178 induces sustained CDK1 activation during mitosis. HL-60 cells were treated with either vehicle or SKI-178 (5 μM) for the indicated time points. Whole cell lysates were collected at indicated time points, and Western blot analysis was performed using antibodies for pCDK1 (Tyr15). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as a loading control. Results shown are representative of three independent experiments.

Fig. 8.

SKI-178–induced CDK1 activation results in MCL-1 degradation. (A) Whole cell lysates from the indicated AML cell lines were subjected to Western blot analysis to assess expression of various antiapoptotic family members (Bcl-2, Bcl-xl, and Mcl-1). (B) HL-60 and HL-60/VCR cells treated for 24 hours with SKI-178, RO3306, or a combination of SKI-178 and RO3306. Western blot analysis was performed on whole cell lysates using indicated antibodies. (C) HL-60/VCR cells were synchronized at the G1/S phase transition using a double thymidine block and released into either vehicle or SKI-178. Cells released into SKI-178 were either maintained in SKI-178 alone or cotreated with RO3306 14 hours after release. Whole cell lysates were collected at indicated time points, and Western blot analysis was performed using indicated antibodies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) serves as a loading control.