Dasatinib/VPA-induced apoptosis activates PARP and caspase-9, -3 and -7 in HL60 cells.

<p>Cells were collected and treated under the same conditions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098859#pone-0098859-g003" target="_blank">Figure 3</a>. The cells were intracellular stained with anti-human cleaved PARP (cPARP), anti-human cleaved caspase-3 (cCas-3) and anti-rabbit IgG-FITC, followed by flow cytometry analysis. (A) The expression of intracellular cPARP. (B) The expression of intracellular cCas-3. (C) The intracellular expression of cPARP and cCas-3 in the combination group was monitored by FlowSight analysis. (D) The expression of capsase-9, -3 and -7 and procapsase-9, -3 and -7 was then measured by Western blot analysis. The membrane was stripped and reprobed with anti-β-actin mAb to confirm equal loading. (E) Data show the band density of (D). Representative blots are shown from three independent experiments with almost identical results. These data represent the means ± SEM. Significantly different from control (*) or combination of VPA and dasatinib (#); #: <i>P</i><0.05; **, ##: <i>P</i><0.01; ***, ###: <i>P</i><0.001.</p

Dasatinib Accelerates Valproic Acid-Induced Acute Myeloid Leukemia Cell Death by Regulation of Differentiation Capacity

<div><p>Dasatinib is a compound developed for chronic myeloid leukemia as a multi-targeted kinase inhibitor against wild-type BCR-ABL and SRC family kinases. Valproic acid (VPA) is an anti-epileptic drug that also acts as a class I histone deacetylase inhibitor. The aim of this research was to determine the anti-leukemic effects of dasatinib and VPA in combination and to identify their mechanism of action in acute myeloid leukemia (AML) cells. Dasatinib was found to exert potent synergistic inhibitory effects on VPA-treated AML cells in association with G₁ phase cell cycle arrest and apoptosis induction involving the cleavage of poly (ADP-ribose) polymerase and caspase-3, -7 and -9. Dasatinib/VPA-induced cell death thus occurred via caspase-dependent apoptosis. Moreover, MEK/ERK and p38 MAPK inhibitors efficiently inhibited dasatinib/VPA-induced apoptosis. The combined effect of dasatinib and VPA on the differentiation capacity of AML cells was more powerful than the effect of each drug alone, being sufficiently strong to promote AML cell death through G₁ cell cycle arrest and caspase-dependent apoptosis. MEK/ERK and p38 MAPK were found to control dasatinib/VPA-induced apoptosis as upstream regulators, and co-treatment with dasatinib and VPA to contribute to AML cell death through the regulation of differentiation capacity. Taken together, these results indicate that combined dasatinib and VPA treatment has a potential role in anti-leukemic therapy.</p></div

Effects of VPA and dasatinib on the apoptotic cells.

<p>These data represent the means ± SEM. Significantly different from control (*) or combination of VPA and D (#); ***, ###: <i>P</i><0.001. **: <i>P</i><0.01. *, #: <i>P</i><0.05. VPA, Valproic acid; D, dasatinib.</p

Combination of dasatinib and VPA inhibits HL60 cell proliferation.

<p>Cells were stimulated with various concentrations of 0, 0.5, 1, 1.5 and 2 µM dasatinib for 72 hr. The cytotoxicity was then evaluated by an MTS assay. (A) Dose-dependent responses of VPA on cell viability. (B) Dose-dependent responses of dasatinib on cell viability. (C) Treatment of VPA and/or dasatinib at 72 hr. Representative data are shown for at least three independent experiments. These data represent the means ± SEM. Significantly different from the control (*) or combination of VPA and dasatinib (#); *: <i>P</i><0.05; ***, ###: <i>P</i><0.001.</p

Dasatinib/VPA-induced apoptosis is via a caspase-dependent pathway and depends on MEK/ERK and p38 MAPK.

<p>Cells were preincubated with caspase-3 inhibitor (10 µM Z-DEVD-FMK), caspase-9 inhibitor (10 µM LEHD-CHO), MEK/ERK inhibitor (5 µM U0126 and 10 µM PD98059), p38 MAPK inhibitor (10 µM SB203580) and JNK inhibitor (10 µM SP600125) for 1 hr prior to treatment with 0.5 mM of VPA and 5 µM of dasatinib for 72 hr. (A, D) Caspase-9 activity; (B, E) caspase-3 activity (C, F); apoptotic cells. These data represent the means ± SEM. Significantly different from the control (*) or combination of VPA and dasatinib (#); ***, ###: <i>P</i><0.001. Cas3i, caspase-3 inhibitor; cas9i, caspase-9 inhibitor; U, U0126; PD, PD98059; SB, SB203580; SP, SP600125.</p

Mechanism by which dasatinib potentiates VPA-treated AML cell death.

<p>The combination of dasatinib and VPA on AML cell differentiation capacity is more potent than that of each drug alone. The combination is enough to promote intensive AML cell death through G₁ cell cycle arrest and caspase-dependent apoptosis. In addition, MEK/ERK and p38 MAPK control dasatinib/VPA-evoked apoptosis as upstream regulators. Eventually, the regulation of cell differentiation capacity contributes to AML cell death.</p

Effects of VPA and dasatinib on the cell viability.

<p>These data represent the means ± SEM. Significantly different from control (*) or combination of VPA and D (#); ***, ###: <i>P</i><0.001. *: <i>P</i><0.05. VPA, Valproic acid; D, dasatinib.</p

Synergistic effects of dasatinib and VPA on G₁ phase cell cycle arrest.

<p>Cells were incubated with 0.5 µM of dasatinib for 72 hr. The cells were harvested at 24 hr (A), 48 hr (B) and 72 hr (C) and then stained with PI/RNase staining buffer and analyzed by flow cytometry. The expression of G₁ phase cell cycle regulatory proteins was then measured by Western blot analysis. The membrane was stripped and reprobed with anti-β-actin mAb to confirm equal loading. (D) The expression of p21^Cip1 and p27^Kip1. (E) The expression of CDK2, 4 and 6. (F) The expression of cyclin D1 and E. (G) The expression of p27^Kip1 on NB4, HepG2, and Hep3B. Representative blots are shown from three independent experiments with similar pattern results.</p

Effects of dasatinib and VPA on CD11b and CD14 expression in HL60 cells.

<p>Cells were incubated with 5 µM of dasatinib and 0.5 mM if VPA for 3 and 5 days. The cells were then harvested and immune stained with anti-human CD11b and CD14 mAb. The expression of CD11b and CD14 was then measured by flow cytometry. The filled histogram represents the isotype control, and the open histogram represents CD11b-positive cells treated with 5 µM if dasatinib alone at Day 3 (A) and Day 5 (B). The open histogram represents CD14-positive cells treated with 0.5 mM of VPA alone at Day 3 (C). These data represent the means ± SEM. Significantly different from the DMSO-treated control (*) or combination of VPA and dasatinib (#); ***, ###: <i>P</i><0.001. VPA, valproic acid; D, dasatinib.</p

Dasatinib induces apoptosis in VPA-treated AML cells.

<p>The cells were also collected and treated under the same conditions described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098859#pone-0098859-g003" target="_blank">Figure 3</a>. Cells were stained with annexin V-FITC and/or propidium iodide (PI) followed by flow cytometry analysis. (A) Annexin V/PI staining of HL60 cells. (B) Data show the percentage of annexin V-positive cells (apoptotic cells) on (A). (C) DRAQ5 nuclear staining following combination treatment in HL60 cells. Data show the percentage of apoptotic cells of PBMC (D) and BMC (E) in the AML patients. These data represent the means ± SEM. Significantly different from the control (*) or combination of VPA and dasatinib (#); ##: <i>P</i><0.01; ***, ###: <i>P</i><0.001.</p