Melatonin Can Modulate the Effect of Navitoclax (ABT-737) in HL-60 Cells

Abstract

Melatonin (N-acetyl-5-methoxytryptamine MEL) is an indolamine that has antioxidant, anti-inflammatory and anti-tumor properties. Moreover, MEL is capable of exhibiting both anti-apoptotic and pro-apoptotic effects. In the normal cells, MEL possesses antioxidant property and has an anti-apoptotic effect, while in the cancer cells it has pro-apoptotic action. We investigated the combined effect of MEL and navitoclax (ABT-737), which promotes cell death, on the activation of proliferation in acute promyelocytic leukemia on a cell model HL-60. The combined effect of these compounds leads to a reduction of the index of mitotic activity. The alterations in the level of anti- and pro-apoptotic proteins such as BclxL, Bclw, Mcl-1, and BAX, membrane potential, Ca²⁺ retention capacity, and ROS production under the combined action of MEL and ABT-737 were performed. We obtained that MEL in combination with ABT-737 decreased Ca²⁺ capacity, dropped membrane potential, increased ROS production, suppressed the expression of anti-apoptotic proteins such as BclxL, Bclw, and Mcl-1, and enhanced the expression of pro-apoptotic BAX. Since, MEL modulates autophagy and endoplasmic reticulum (ER) stress in cancer cells, the combined effect of MEL and ABT-737 on the expression of ER stress and autophagy markers was checked. The combined effect of MEL and ABT-737 (0.2 μM) increased the expression of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), leading to a decrease in the level of binding immunoglobulin protein (BIP) followed by an increase in the level of C/EBP homologous protein (CHOP). In this condition, the expression of ERO1 decreased, which could lead to a decrease in the level of protein disulfide isomerase (PDI). The obtained data suggested that melatonin has potential usefulness in the treatment of cancer, where it is able to modulate ER stress, autophagy and apoptosis.

Keywords: HL-60 cells; acute promyelocytic leukemia; apoptosis; autophagy; endoplasmic reticulum stress; melatonin; navitoclax (ABT-737); permeability transition pore.

Figure 1

Concentration dependence of the cytotoxic effects of melatonin (MEL) and ABT-737. Cells were seeded in a 96-well plate at a density of 5 × 10³ cells per well and treated with indicated concentrations of (a) MEL and (b) ABT-737 for 24 h. The data are presented as means ± S.D. of ten separate experiments.

Figure 2

The effect of MEL and ABT-737 on the viability and proliferation of HL-60 cells. Cells were treated with 0.7 µM ABT-737 (column 2), and 0.2 µM ABT-737 (columns 3) and 1 mM MEL (column 4), and MEL in combination with 0.2 µM ABT-737 (column 5); untreated cells (control, column 1). (a) Cell viability in % relative to the control. (b) Mitotic index was calculated in the presence of 0.7 µM ABT-737 (column 2), and 0.2 µM ABT-737 (columns 3) and 1 mM MEL (column 4), and MEL in combination with 0.2 µM ABT-737 (column 5); untreated cells (control, column 1). “+” means the presence of MEL. The data are presented as the means ± S.D. of six separate experiments. * p < 0.05 significant difference in values in comparison with the control, ^# p < 0.05 significant difference in values compared to the value obtained after the addition of ABT-737 alone (0.2 µM, column 3).

Figure 3

The effect of MEL and ABT-737 on the level of apoptosis-associated proteins—Bcl-xL (a) and Bcl-w (b), Mcl-1 (c), Bax (d) and Endoplasmic reticulum (ER) stress marker C/EBP homologous protein (CHOP) (e) in HL-60 cells. Cells were treated with 0.7 µM ABT-737 (column 2), and 0.2 µM ABT-737 (columns 3) and 1 mM MEL (column 4), and MEL in combination with 0.2 µM ABT-737 (column 5); untreated cells (control, column 1). The ration protein level to GAPDH was used as a loading control. “+” means the presence of MEL. The protein level in the cell lysate without any additives served as a control (100%). The data are presented as the means ± S.D. of three separate experiments. * p < 0.05 significant difference in the protein level compared with the corresponding control, ^# p < 0.05 significant difference in the protein level compared to ABT-737 alone (0.2 µM, column 3).

Figure 4

The effect of MEL and ABT-737 on the membrane potential (ΔΨ_m) (a), ROS production (b) and in the level of autophagy marker LC3A/B (c) in HL-60 cells. Cells were treated with 0.7 µM ABT-737 (column 2), and 0.2 µM ABT-737 (columns 3) and 1 mM MEL (column 4), and MEL in combination with 0.2 µM ABT-737 (column 5); untreated cells (control, column 1). (a) the alteration of ΔΨ_m in our experimental conditions. Saponin (0.5%) was used as a positive control; (b) the alteration of ROS production in our experimental conditions. H₂O₂ (1 mM) was used as a positive control; (c) western blot of autophagy marker LC3A/B. The ratio of protein levels to GAPDH was used as a loading control. The protein level in the cell lysate without any additives served as a control (100%). “+” means the presence of MEL, Saponin and H₂O₂, respectively. The data are presented as the means ± S.D. of five separate experiments. * p < 0.05 significant difference in values compared with the corresponding control (untreated cells), ^# p < 0.05 significant difference in values relative to ABT-737 alone (0.2 µM, column 3).

Figure 5

The effect of MEL and ABT-737 on the calcium retention capacity (CRC) in HL-60 cells. Cells were treated with 0.7 µM ABT-737 (column 2), and 0.2 µM ABT-737 (columns 3) and 1 mM MEL (column 4), and MEL in combination with 0.2 µM ABT-737 (column 5); untreated cells (control, column 1). (a) Ca²⁺ fluxes in HL-60 cells permeabilized with digitonin (0.007%). Arrows show the times at which CaCl₂ (50 nmol of Ca²⁺ per mg of protein) was added; (b) Quantitative analysis of Ca²⁺ retention capacity corresponding to the threshold concentration of Ca²⁺. “+” means the presence of MEL The data are presented as the means ± S.D. of five separate experiments. * p < 0.05 significant difference relative to the control (untreated cells), ^# p < 0.05 significant difference compared to MEL alone (column 4).

Figure 6

The combined effect of MEL and ABT-737 on the level of ER stress markers—Protein disulfide isomerase (PDI) (a), binding immunoglobulin protein (BIP) (b) ER oxidoreductin 1-Lα (ERO1-Lα) (c), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) (d) and the activating transcription factor ATF4 (e) in HL-60 cells. Cells were treated with 0.7 µM ABT-737 (column 2), and 0.2 µM ABT-737 (columns 3) and 1 mM MEL (column 4), and MEL in combination with 0.2 µM ABT-737 (column 5); untreated cells (control, column 1). The ratio of protein levels to GAPDH was used as a loading control. The protein level in the cell lysate without any additives served as a control (100%). “+” means the presence of MEL The data are presented as the means ± S.D. of three separate experiments. * p < 0.05 significant difference in the protein level relative to the control (untreated cells), ^# p < 0.05 significant difference in the protein level compared to ABT-737 alone (0.2 µM, column 3).