Differential effect of ascorbic acid and n-acetyl-L-cysteine on arsenic trioxide-mediated oxidative stress in human leukemia (HL-60) cells

Abstract

Arsenic trioxide (ATO) has been recommended for the treatment of refractory cases of acute promyelocytic leukemia (APL). Recent studies in our laboratory indicated that oxidative stress plays a key role in ATO-induced cytotoxicity in human leukemia (HL-60) cells. In the present investigation, we performed the MTT assay and trypan blue exclusion test for cell viability. We also performed the thiobarbituric acid test to determine the levels of malondialdehyde (MDA) production in HL-60 cells coexposed to either ascorbic acid (AA) and ATO or to n-acetyl-L-cysteine (NAC) and ATO. The results of MTT assay indicated that AA exposure potentiates the cytotoxicity of ATO in HL-60 cells, as evidenced by a gradual increase in MDA levels with increasing doses of AA. In contrary, the addition of NAC to ATO-treated HL-60 cells resulted in a dose-dependent decrease of MDA production. From these results, we conclude that the addition of the AA to ATO-treated HL-60 cells enhances the formation of reactive oxygen species (ROS), whereas the addition of NAC under the same experimental condition significantly (p < .05) decreases the level of ROS formation. On the basis of these direct in vitro findings, our studies provide evidence that AA may extend the therapeutic spectrum of ATO. The coadministration of NAC with ATO shows a potential specificity for tumor cells, indicating that it may not enhance the clinical outcome associated with ATO monotherapy in vivo.

Figure 1

Effect of ascorbic acid (AA) to human leukemia (HL-60) cells. HL-60 cells were cultured with different doses of AA for 24 hr as indicated in the Materials and Methods. Cell viability was determined based on the MTT assay. Each point represents a mean value and standard deviation of 3 experiments with 6 replicates per dose.

Figure 2

Potential effect of co-administration of ascorbic acid (AA) and arsenic trioxide (ATO) to human leukemia (HL-60) cells. HL-60 cells were cultured in the absence or presence of AA and ATO or in combination of AA and ATO for 24 hr as indicated in the Materials and Methods. Cell viability was determined based on the MTT assay. Each point represents a mean value and standard deviation of 3 experiments with 6 replicates per dose. *Significantly different from the control by ANOVA Dunnett's test; p < 0.05. **Significantly different from ATO alone by ANOVA Dunnett's test; p < 0.05.

Figure 3

Effect of n-acetyl-l-cysteine (NAC) on human leukemia (HL-60) cells. HL-60 cells were cultured with different doses of NAC for 24 hr as indicated in the Materials and Methods. Cell viability was determined based on the MTT assay. Each point represents a mean value and standard deviation of 3 experiments with 6 replicates per dose. *Significantly different from the control by ANOVA Dunnett's test; p < 0.05.

Figure 4

Potential effect of co-administration of n-acetyl-l-cysteine (NAC) and arsenic trioxide (ATO) to human leukemia (HL-60) cells. HL-60 cells were cultured in the absence or presence of NAC and ATO or in combination of NAC and ATO for 24 hr as indicated in the Materials and Methods. Cell viability was determined based on the MTT assay. Each point represents a mean value and standard deviation of 3 experiments with 6 replicates per dose. *Significantly different from the control by ANOVA Dunnett's test; p < 0.05.

Figure 5

Potentiation effect of AA on ATO-induced oxidative stress in HL-60 cells. Cells were incubated for 24 hr with 6 μg/mL ATO and various concentrations of AA (25, 50, and 100 μM). Malondialdehyde formation was determined as described in Materials and Methods. *Significantly different from the control by ANOVA Dunnett's test; p < 0.05. **Significantly different from ATO alone by ANOVA Dunnett's test; p < 0.05. Data are representative of 3 independent experiments.

Figure 6

Protective effect of NAC on ATO-induced oxidative stress in HL-60 cells. Cells were incubated for 24 hr with 6 μg/mL ATO and various concentrations of NAC (25, 50, and 100 μM). Malondialdehyde formation was determined as described in Materials and Methods. *Significantly different from the control by ANOVA Dunnett's test; p < 0.05. **Significantly different from ATO alone by ANOVA Dunnett's test; p < 0.05. Data are representative of 3 independent experiments.