Combined treatment with low concentrations of decitabine and SAHA causes cell death in leukemic cell lines but not in normal peripheral blood lymphocytes

Abstract

Epigenetic therapy reverting aberrant acetylation or methylation offers the possibility to target preferentially tumor cells and to preserve normal cells. Combination epigenetic therapy may further improve the effect of individual drugs. We investigated combined action of demethylating agent decitabine and histone deacetylase inhibitor SAHA (Vorinostat) on different leukemic cell lines in comparison with peripheral blood lymphocytes. Large decrease of viability, as well as huge p21WAF1 induction, reactive oxygen species formation, and apoptotic features due to combined decitabine and SAHA action were detected in leukemic cell lines irrespective of their p53 status, while essentially no effect was observed in response to the combined drug action in normal peripheral blood lymphocytes of healthy donors. p53-dependent apoptotic pathway was demonstrated to participate in the wtp53 CML-T1 leukemic cell line response, while significant influence of reactive oxygen species on viability decrease has been detected in p53-null HL-60 cell line.

Figure 1

Effect of DAC and/or SAHA on cell viability during 48-hour treatment. Cells of leukemic cell lines CML-T1 (a, circles) and HL-60 (b, squares) or PBL (c, triangles) were treated by 1 μM DAC (full line), 1 μM SAHA (dash line), or 1 μM DAC + 1 μM SAHA (dotted line) in the 48 h time frame, and then their metabolic activity was measured by MTT test. Data are the average of at least five experiments and ±SEM are plotted as error bars.

Figure 2

Apoptotic features induced by DAC and/or SAHA treatment: appropriate fraction of control cells (empty) or cells treated for 48 h by 1 μM DAC (vertically shaded), 1 μM SAHA (horizontally shaded), or DAC + SAHA combination (filled) measured by flow cytometry. (a) Annexin V-FITC/PI staining of nonfixed cells: Annexin V+/PI− fraction; effect of 10 μM z-VAD-fmk (dotted) or 20 μM Q-VD-Oph (checked). (b) PI staining of ethanol-fixed cells: fraction of cells in subG1 phase. (c) MitoTracker Red fluorescence: fraction of cells having polarized mitochondrial membrane. Error bars represent ±SEM of at least 3 measurements. Statistical significance degree of difference between treated samples and the corresponding control: P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). (d) Immunoblots showing caspases-3 and -7 activation after 48 h DAC and/or SAHA treatment. (e) Fragmentation of PARP in CML-T1 and HL-60 cell lines or PBL treated by DAC and/or SAHA for 48 h. Images are representatives of at least three experiments.

Figure 3

Inhibition of executive caspases cleavage by caspase inhibitors z-VAD-fmk (20 μM) or Q-VD-OPh (10 μM) in CML-T1 cells treated by 1 μM DAC + 1 μM SAHA for 48 h.

Figure 4

(a) Distribution of cell cycle phases monitored by PI staining of ethanol-fixed cells: untreated (C) or treated by 1 μM DAC (D), 1 μM SAHA (S), or DAC + SAHA combination (DS). Data are averaged from at least three experiments. (b) Expression of cyclin-dependent kinase inhibitor p21WAF1 after 48 h of 1 μM DAC and/or 1 μM SAHA treatment of leukemic cell lines (CML-T1, HL-60) or PBL. Images are representatives of at least five experiments.

Figure 5

Production of reactive oxygen species. Control cells (empty) and cells treated for 48 h by 1 μM DAC (vertically shaded), 1 μM SAHA (horizontally shaded), or DAC + SAHA combination (filled) were stained by H₂DCFDA, and the mean value of flow-cytometer-detected fluorescence intensity was analyzed. 10 mM NAC was used for DAC + SAHA-induced ROS generation reduction (checked). Error bars represent ±SEM of four measurements. Statistical significance degree of difference between treated samples and the corresponding control: P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

Figure 6

Apoptotic effects of one shot versus sequential DAC treatment. (a) Control cells (C) were treated by 1 μM DAC for 48 h (D) or sequentially treated by two doses of 0.5 μM DAC (DD1) or 1 μM DAC (DD2) every 24 h in the presence or absence of 1 μM SAHA. Treated cells were fixed in EtOH and stained by PI and the fraction of cells in subG1 phase of cell cycle was analyzed. Error bars represent ±SEM of four measurements. Statistical significance degree of difference: P < 0.05 (*). (b) Immunoblots showing executive caspases activation in samples are described in (a).

Figure 7

Transcription levels of cyclin-dependent kinase inhibitor p21WAF1, tumor suppressor gene TP53, and several apoptosis-related genes in leukemic cell lines treated by 1 μM DAC and/or 1 μM SAHA monitored by qRT-PCR. Error bars represent ±SEM of three measurements. Statistical significance degree of difference between treated samples and the corresponding control: P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***).

Figure 8

Expression of tumor suppressor p53 and proteins related to apoptosis in control cells and after 48 h DAC, SAHA, or DAC + SAHA treatment of leukemic cell lines monitored by immunoblotting. Images are representatives of at least four experiments with similar results.

Figure 9

Intact cells CML-T1 and cells treated by DAC + SAHA combination for 48 h stained with anti-Bax (red, Alexa Fluor647) and mitochondrial anti-COX (green, Alexa Fluor488) antibodies. Nuclei are visualised by Hoechst 33342. Scale bar represents 10 μm.