Abstract
BackgroundArsenic 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.MethodHL-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.ResultsMTT 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.ConclusionThe 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).
Highlights
Arsenic trioxide (ATO) is a novel form of therapy that has been found to aid acute promyelocytic leukemia (APL) patients
Vitamin D3 potentiates the cytotoxicity of arsenic trioxide in HL-60 cells We have previously reported that physiologic doses of ATO increase cellular proliferation while pharmacologic doses of ATO were highly cytotoxic to HL-60 cells, showing a 24 hr LD50 of 6.4 ± 0.6 μg/mL [15]
We found that the viability of HL-60 cells declined from (62 ± 5)% in cells treated with ATO alone to (44 ± 3)% in cells co-treated with 1.5 μM Vit D3 and 6 μg/mL ATO with P < 0.05 (Figure 3)
Summary
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. Arsenic trioxide (ATO) is a new form of therapy that has been found to benefit APL patients. Both in vitro and in vivo studies have shown that ATO can induce clinical remission of de novo and relapsed APL patients [1,2]. Several studies have reported that ATO induces apoptosis in malignant cells including APL, non-Hodgkin’s lymphoma, multiple myeloma, and chronic lymphocytic leukemia cells [3,4,5]. ATO induced apoptosis is associated with the generation of reactive oxygen species that contribute significantly to cell killing [7,8,9] and inhibition of growth [10]
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