Oxidative stress-mediated intrinsic apoptosis in human promyelocytic leukemia HL-60 cells induced by organic arsenicals

Abstract

Arsenic trioxide has shown the excellent therapeutic efficiency for acute promyelocytic leukemia. Nowadays, more and more research focuses on the design of the arsenic drugs, especially organic arsenicals, and on the mechanism of the inducing cell death. Here we have synthesized some organic arsenicals with Schiff base structure, which showed a better antitumor activity for three different kinds of cancer cell lines, namely HL-60, SGC 7901 and MCF-7. Compound 2a (2-(((4-(oxoarsanyl)phenyl)imino)methyl)phenol) and 2b (2-methoxy-4-(((4-(oxoarsanyl)phenyl)imino)methyl)phenol) were chosen for further mechanism study due to their best inhibitory activities for HL-60 cells, of which the half inhibitory concentration (IC50) were 0.77 μM and 0.51 μM, respectively. It was illustrated that 2a or 2b primarily induced the elevation of reactive oxygen species, decrease of glutathione level, collapse of mitochondrial membrane potential, release of cytochrome c, activation of Caspase-3 and apoptosis, whereas all of the phenomena can be eliminated by the addition of antioxidants. Therefore, we concluded that compound 2a and 2b can induce the oxidative stress-mediated intrinsic apoptosis in HL-60 cells. Both the simplicity of structure with Schiff base group and the better anticancer efficiency demonstrate that organic arsenicals are worthy of further exploration as a class of potent antitumor drugs.

Figure 1. The synthesis of Schiff base derivatives.

(a) concentrated hydrochloric acid and methanol (v/v: 24 mL/30 mL), catalytic amount of KI, SO₂ (2 bubbles/s), RT, 30 min; (b) aqueous ammonia 10%, 0 °C, 15 min; (c) EtOH, 78–80 °C, 2–6 h, under N₂.

Figure 2. Induction of apoptosis in HL-60 cells in vitro.

(a) The results of apoptosis by Annexin V-FITC/PI double-staining assay. HL-60 cells were treated with compound 2a (1.5 μM and 3 μM) or 2b (1 μM and 2 μM) for 24 h, then stained by Annexin V-FITC and PI dye. There are four kinds of cell populations shown as follows: live cells population (lower left), early apoptotic cells (lower right), late apoptotic cells (upper right) and dead cells (upper left). (b) Analysis of morphology changes in HL-60 cells. HL-60 cells were treated with 2a (1 μM and 2 μM) or 2b (0.75 μM and 1.5 μM) for 24 h followed by Hoechst 33342 staining. Phase contrast (top) and fluorescence (bottom) images were acquired by fluorescence microscopy. The apoptotic cells are shown with white arrows. These experiment were performed more than three times.

Figure 3. Production and elimination of ROS in HL-60 cells measured by DCFH-DA.

(a) The levels of intracellular ROS in the presence of antioxidants and compound 2a or 2b. HL-60 cells were pre-incubated with 2 mM NAC or 1 mM DTT or 1 mM VC or 0.075 mM LA for 4 h followed by the addition of 2 μM 2a or 1.5 μM 2b for another 24 h and the fluorescence data were obtained by flow cytometry. (b) The corresponding quantification data are shown in the way of column figure and expressed as the mean ± SD of three independent samples. **P < 0.01 and ***P < 0.001 vs the control group. (c) The images about the accumulation and elimination of ROS in cells. HL-60 cells were treated with 0.075 mM LA in advance for 4 h before incubated with 2 μM 2a or 1.5 μM 2b for another 24 h. Then 1 mL FBS-free medium with 10 μM DCFH-DA was added into each sample for 30 min at 37 °C. Phase contrast (top) and fluorescence (bottom) images were acquired by fluorescence microscopy.

Figure 4. Assessment of total GSH in cells and the protective effect of GSH.

(a) The total GSH level in cells under the treatment of 0.075 mM LA and 2 μM 2a or 1.5 μM 2b. The data are expressed as the mean ± SD of three independent samples. (b) The change of GSH/GSSH. The ratio was calculated according to the reduced GSH and GSSG levels. Data are expressed as the mean ± SD of three experiments. (c) The elimination effect of GSH for ROS. HL-60 cells were pre-incubated with 2 mM GSH for 4 h followed by adding 2 μM 2a or 1.5 μM 2b for another 24 h and the fluorescence data were obtained by flow cytometry. (d) The quantification data of fluorescence intensity are shown in the way of bar figure. Data are expressed as the mean ± SD of three experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 vs the control group.

Figure 5. Assessment of mitochondrial membrane potential and release of cytochrome c.

(a) The collapse and recovery of mitochondrial membrane potential. HL-60 cells were incubated with 3 μM 2a or 2 μM 2b in the presence or absence of 2 mM NAC, 2 mM GSH or 1 mM DTT for 24 h, followed by JC-1 staining. Data are obtained by the use of flow cytometry. (b) The alteration of mitochondrial membrane potential in HL-60 cells with or without 0.075 mM LA measured by JC-1 dye. This assay was repeated more than three times. (c) The change of mitochondrial membrane potential in cells monitored by Rh123. The cells were treated with 3 μM 2a or 2 μM 2b in the presence or absence of 0.075 mM LA. Data are recorded by flow cytometry and shown in the way of bar figure. The quantification data are expressed as the mean ± SD of three independent samples. **P < 0.01 vs the control group. (d) The release of cytochrome c. The cells were treated with 3 μM 2a or 2 μM 2b in the presence or absence of 0.075 mM LA. Data are expressed as the mean ± SD of three independent samples. ***P < 0.001 vs the control group.

Figure 6. The effect of antioxidants on cell death.

(a) Assessment of cell viability with the addition of NAC, GSH or DTT. HL-60 cells were incubated with 2a or 2b in the presence or absence of 1 mM NAC, 1 mM GSH, 1 mM DTT for 24 h and data are expressed as the mean ± SD of three independent samples. (b) The protective effect for cell viability with the different concentrations of LA. HL-60 cells were incubated with 2a or 2b in the presence or absence of LA with different concentrations for 24 h and the data are expressed as the mean ± SD of three independent samples. (c) Assessing the proportion of apoptotic cells in the presence of NAC, GSH, DTT or VC. HL-60 cells were treated with 3 μM 2a or 2 μM 2b and antioxidants for 24 h, followed by Annexin V-FITC/PI double staining. (d) Determination of the apoptotic cells percentage in the presence of LA. HL-60 cells were treated with 3 μM 2a or 2 μM 2b and 0.075 mM LA for 24 h, followed by Annexin V-FITC/PI double staining. (e) The increase of Caspase-3 activity. HL-60 cells were treated with 3 μM 2a or 2 μM 2b and 0.075 mM LA for 24 h and Caspase-3 activity in the cell extracts was determined by a colorimetric assay. *P < 0.05, **P < 0.01 and ***P < 0.001 vs the control group.