Antileukemia activity of the combination of an anthracycline with a histone deacetylase inhibitor

Abstract

We studied the cellular and molecular effects of the combination of an anthracycline with 2 different histone deacetylase inhibitors (HDACIs): vorinostat (suberoylanilide hydroxamic acid) and valproic acid (VPA). The 10% inhibitory concentration (IC(10)) of idarubicin was 0.5 nM in MOLT4 and 1.5 nM in HL60 cells. Concentrations above 0.675 microM of vorinostat resulted in at least 80% loss of cell viability in both cell lines. Concentrations of 1.5 to 3 mM of VPA induced 50% to 60% loss in viability in HL60 and 80% in MOLT4 cells. The combination of idarubicin with vorinostat at 0.075 microM or VPA at 0.25 mM resulted in at least an additive loss of cell viability in both lines. Vorinostat (0.35 microM) and VPA (0.25 mM) in combination with idarubicin (0.5 nM) resulted in a significant increase in apoptotic cells in MOLT4 cells. The combination resulted in an increase in histone H3 and H4 acetylation at 24 hours, phosphorylated H2AX, as well as in the induction of p21(CIP1) mRNA. No effect on cell cycle transition was observed. Of importance, the cellular and molecular effects observed were independent of the sequence used. In summary, the combination of an anthracycline with an HDACI should have significant clinical activity in patients with leukemia.

Figures

Figure 1.

Effects of idarubicin, vorinostat, or VPA in HL60 and MOLT4 cell viability. Cell viability was studied using trypan blue assays. Numbers on the y-axis represent the total number of viable cells. (A-B) HL60 and MOLT4 cells were treated with increasing concentrations of idarubicin (ida; 0.5-20 nM) daily for 3 days. Subsequently, both cell lines were treated with increasing concentrations of (C-D) vorinostat (Vor; 0.3-3 μM) or (E-F) VPA (0.25-3 mM) both daily for 3 days. Bars indicate the standard deviation.

Figure 2.

Effects of vorinostat or VPA in combination with idarubicin on cell viability. Cell lines were treated with increasing doses of VPA (0.25-3 mM) or vorinostat (0.075-1 μM) concomitantly with idarubicin at 0.5 nM in MOLT4 or 1.5 nM in HL60 cells daily for 3 days. Numbers on the y-axis represent the percentage of viable cells. (A) MOLT4 cells. (B) HL60 cells. IDA indicates idarubicin; VPA, valproic acid; and Vor, vorinostat. Error bars indicate standard deviation.

Figure 3.

Induction of apoptosis. The percentage of apoptotic cells was determined using annexin V flow cytometry assays. Cell lines were treated with increasing doses of VPA (0.25-3 mM) or vorinostat (0.075-1 μM) in combination with idarubicin at 0.5 nM in MOLT4 or 1.5 nM in HL60 cells daily for 3 days. Numbers on the y-axis represent percentage of apoptotic cells. (A) MOLT4 cells. (B) HL60 cells. IDA indicates idarubicin; VPA, valproic acid; and Vor, vorinostat (suberoylanilide hydroxamic acid). Error bars indicate standard deviation.

Figure 4.

Effect of the sequence on cell viability and apoptosis. MOLT4 cells were treated with vorinostat at 0.3 μM and idarubicin at 0.5 nM concomitantly or IDA was added 4 hours before vorinostat (Vor) (IDA, A; vorinostat, B) or after vorinostat (IDA, B; vorinostat, A) daily for 3 days. (A) Numbers on the y-axis represent the number of viable cells. (B) Numbers on the y-axis represent the percentage of apoptotic cells. Error bars indicate standard deviation.

Figure 5.

Induction of histone H3 and H4 acetylation. HL60 cells were treated with (A) vorinostat (Vor) at increasing concentrations or (B) VPA. The effect on the sequence of IDA with vorinostat using concentrations of 1 μM of vorinostat and IDA at 1.5 nM administered concomitantly, or with IDA preceding vorinostat (IDA, A; vorinostat, A-B) or the reverse (IDA, B; vorinostat, A), were studied (C).

Figure 6.

H2AX phosphorylation. HL60 cells (A) and MOLT 4 cells (B) were treated for 8 hours with vorinostat (Vor) 2.5 μM or IDA 20 nM or the combination. H2AX phosphorylation was analyzed using immunohistochemistry in paraffin-embedded cell suspensions. Numbers at the top of each image represent the percentage of cells staining for γH2AX. Images were acquired using a Zeiss Axiovert S100 inverted microscope equipped with a 40×/0.65 objective lens (Zeiss, Thornwood, NY) and a Hamamatsu cooled CCD camera (Hamamatsu, Hamamatsu City, Japan) and were digitally stored with WebSlide Browser (Bacus Laboratory, Lombard, IL). Images were obtained using 100× magnification.

Figure 7.

DNA topoisomerase IIα gene expression and p21CIP1. The effect in HL60 cells of treatment for 3 days with vorinostat (Vor; 0.075-1 μM), VPA (0.25-3 mM), or IDA (1.5 nM) or the combination on p21CIP1 or DNA topoisomerase IIα mRNA expression was determined using real-time PCR assay. Numbers on the y-axis represent relative expression of delta CT. (A) p21CIP1. (B) DNA topoisomerase IIα. Error bars indicate standard deviation.