Vitamin D3 potentiates the antitumorigenic effects of arsenic trioxide in human leukemia (HL-60) cells

Abstract

Background: Arsenic trioxide (ATO) is a novel form of therapy that has been found to aid acute promyelocytic leukemia (APL) patients. Our laboratory has demonstrated that ATO-induced cytotoxicity in human leukemia (HL-60) cells is mediated by oxidative stress. Pro-oxidants have been known to play a role in free radical-mediated oxidative stress. Vitamin D3, (Vit D3) an active metabolite of vitamin D has been reported to inhibit the growth of number neoplasms such as prostate, breast, colorectal, leukemia, and skin cancers. The goal of the present research was to use (HL-60) cells as an in vitro test model to evaluate whether low doses of Vit D3 potentiate the toxicity of ATO and whether this toxic action is mediated via apoptotic mechanisms.

Method: HL-60 cells were treated either with a pharmacologic dose of ATO alone and with several low doses of Vit D3. Cell survival was determined by MTT assay. Cell apoptosis was measured both by flow cytometry assessment, and DNA laddering assay.

Results: MTT assay indicated that Vit D3 co-treatment potentiates ATO toxicity in HL-60 cells in a dose dependent manner. A statistically significant and dose-dependent increase (p <0.05) was recorded in annexin V positive cells (apoptotic cells) with increasing doses of Vit D3 in ATO-treated cells. This finding was confirmed by the result of DNA laddering assay showing clear evidence of nucleosomal DNA fragmentation in vitamin and ATO co-treated cells.

Conclusion: The present study indicates that Vit D3 potentiates the antitumor effects of ATO. This potentiation is mediated at least in part, through induction of phosphatidylserine externalization and nucleosomal DNA fragmentation. These findings highlight the potential impact of Vit D3 in promoting the pharmacological effect of ATO, suggesting a possible future role of Vit D3/ATO combination therapy in patients with acute promyelocytic leukemia (APL).

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 [15].

Figure 2

Effect of vitamin D₃ (Vit D₃) on human leukemia (HL-60) cells. HL-60 cells were cultured with different doses of Vit D₃ 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 (p < 0.05) from the control, according to the Dunnett’s test.

Figure 3

Cytotoxic effect of VitD₃ and ATO combination treatment on human leukemia (HL-60) cells. HL-60 cells were cultured in the absence or presence of vitD3 and ATO or in combination of VitD₃ 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 4

Representative flow cytometry analysis data from Annexin V-FITC staining. The histogram shows a comparison of the distribution of negative annexin V cells (M1) and positive annexin V cells (M2) after 24 hr incubation in HL-60 cells. A = control; B =6 μg/mL ATO; C = 0.5 μM VitD₃ + 6 μg/mL ATO; D = 1.0 μM VitD₃ + 6 μg/mL ATO; E = 1.5 μM VitD₃ 6 μg/mL ATO.

Figure 5

Annexin V-FITC positive cells induced by either arsenic trioxide alone or vitamin D3 and arsenic trioxide combination in HL-60 cells. Each point represents the mean value and the standard deviation of three experiments, showing similar results. *Significantly different from control (0 μg/mL), p < 0.05.

Figure 6

Induction of DNA fragmentation in HL-60 cells. Lane 1: M-Molecular weight marker. Lane 2-Positive control marker/Camptothecin (5 μM). Lane 3- ATO alone (6 μg/mL). Lane 4- Control with no treatment. Lane 5- VitD₃ (0.5 μM) + ATO (6 μg/mL ATO); Lane 6- VitD₃ (1.0 μM) + ATO (6 μg/mL ATO); Lane 7- VitD₃ (1.5 μM) + ATO (6 μg/mL ATO). HL-60 celles were treated for 24 hr. The DNA gel was stained with SYBR Green 1 after electrophoresis on a 1% agarose gel and then analyzed on the Typhoon 9400 phospho imager.