Basic mechanisms of arsenic trioxide (ATO)-induced apoptosis in human leukemia (HL-60) cells

Abstract

Background: Acute promyelocytic leukemia (APL) is a blood cancer that affects people of all ages and strikes about 1,500 patients in the United States each year. The standard treatment of APL has been based on the combined administration of all-trans retinoic acid and chemotherapy including anthracyclins and cytarabine. However, 10-20% of patients relapse, with their disease becoming resistant to conventional treatment. Recently the Food and Drug Administration has approved the use of arsenic trioxide (ATO) or Trisenox for the treatment of APL, based on clinical studies showing a complete remission, especially in relapsed patients. In a recently published study we demonstrated that ATO pharmacology as an anti-cancer drug is associated with its cytotoxic and genotoxic effects in human leukemia cells.

Methods: In the present study, we further investigated the apoptotic mechanisms of ATO toxicity using the HL-60 cell line as a test model. Apoptosis was measured by flow cytometry analysis of phosphatidylserine externalization (Annexin V assay) and caspase 3 activity, and by DNA laddering assay.

Results: Flow cytometry data showed a strong dose-response relationship between ATO exposure and Annexin-V positive HL-60 cells. Similarly, a statistically significant and dose-dependent increase (p <0.05) was recorded with regard to caspase 3 activity in HL60 cells undergoing late apoptosis. These results were confirmed by data of DNA laddering assay showing a clear evidence of nucleosomal DNA fragmentation in ATO-treated cells.

Conclusion: Taken together, our research demonstrated that ATO represents an apoptosis-inducing agent and its apoptotic mechanisms involve phosphatidylserine externalization, caspase 3 activation and nucleosomal DNA fragmentation.

Figure 1

Toxicity of arsenic trioxide to human leukemia (HL-60) cells. HL-60 cells were cultured with different doses of arsenic trioxide for 24 hr as indicated in the Materials and Methods. Cell viability was determined based on the MTT assay. Each point represents a mean ± SD of 3 experiments with 6 replicates per dose. *Significantly different (p <0.05) from the control, according to the Dunnett's test [22].

Figure 2

Representative flow cytometry analysis data from Annexin V-FITC/PI assay. The histograms show a comparison of the distribution of annexin V negative cells (M1) and annexin V positive cells (M2) after 24 h exposure to ATO. A-control; B-2 μg/mL; C-4 μg/Ml; D-6 μg/mL; E-8 μg/mL.

Figure 3

Representative flow cytometry analysis data from active caspase-3 assay. The histograms show the distribution of caspase-3 negative cells (M1) and caspase-3 positive cells (M2) after 24 h exposure to ATO. A-control; B-2 μg/mL; C-4 μg/Ml; D-6 μg/mL; E-8 μg/mL.

Figure 4

Arsenic trioxide (ATO)-induced DNA fragmentation in HL-60 promyelocytic leukemia cells. Lane 1: M-molecular weight marker; lane 2: control with no ATO treatment; lane 3: 2 μg/mL; lane 4: 4 μg/mL; lane 5: 6 μg/mL; and lane 6: 8 μg/mL ATO. Twelve (12) μL of each sample was electrophoresed on a 1.2% agarose. DNA was stained with ethidium bromide and then visualized under UV light.

Figure 5

Schematic representation of the apoptotic mechanisms of arsenic trioxide (ATO) as a therapeutic agent in the treatment of acute promyelocytic leukemia. ATO exerts a dual effect on HL-60 cells by inducing partial differentiation and apoptosis. As shown on Figure 5, the mechanisms by which ATO induces apoptosis is mediated through oxidative stress [13] that leads to DNA damage and cell death [44], up-regulation of p53 tumor suppressor protein and repression of the c-fos transcription factor [18], induction of phosphatidylserine externalization, caspase-3 activation, and nucleosomal DNA fragmentation.