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. 2017;5(1):1101.
Epub 2017 Jan 4.

Therapeutic Potential of Arsenic Trioxide (ATO) in Treatment of Hepatocellular Carcinoma: Role of Oxidative Stress in ATO-Induced Apoptosis

Erika B Dugo  1 , Clement G Yedjou  1   2 , Jacqueline J Stevens  1   2 , Paul B Tchounwou  1
Affiliations

Therapeutic Potential of Arsenic Trioxide (ATO) in Treatment of Hepatocellular Carcinoma: Role of Oxidative Stress in ATO-Induced Apoptosis

Erika B Dugo et al. Ann Clin Pathol. 2017.

Abstract

Hepatocellular carcinoma (HCC), the dominant form of primary liver cancer, is the sixth most common cancer in the world with more than 700,000 people diagnosed annually. Arsenic trioxide (ATO) has been shown to be a potent anticancer agent in various carcinomas, proving particularly effective in the clinical treatment of relapsed and refractory acute promyelocytic leukemia. However, its bioactivity and molecular mechanisms against HCC has not been fully studied. Using human HCC (HepG2) cells as a test model, we studied the effects of ATO and examined the role of oxidative stress (OS) and apoptosis in cytotoxicity. OS biomarkers showed a significant increase (p< 0.05) of malondialdehyde concentrations, and a gradual decrease of antioxidant enzymes (GPx & CAT) activities with increasing ATO doses. Flow cytometry data showed a dose dependent increase in annex in V positive cells and caspase 3 activities. These results were confirmed by data of the DNA laddering assay showing a clear evidence of nucleosomal DNA fragmentation, as well as data from Western blotting showing a significant modulation of specific apoptotic related proteins, including the activation of p53 and p21 expression and the down-regulation of Bcl-2 expression in ATO-treated cells. Taken together, our research demonstrates that ATO has a potential therapeutic effect against HCC, and its cytotoxicity may be mediated via oxidative stress and activation of the mitochondrial or intrinsic pathway of apoptosis.

Keywords: ATO; Apoptosis; DNA fragmentation; HepG2 cells; Oxidative stress.

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Figures

Figure 1
Figure 1
MDA standard curve with the absorbance wavelength of 586 nm on the y-axis and the control and different concentrations of MDA expressed in micromolar (μM) on the x-axis. R2 = 0.998.
Figure 2
Figure 2
Effects of different concentrations of arsenic trioxide on MDA production in HepG2 cells treated for 24 hours. Data are representative of 3 independent experiments. * signifies data that are significantly different (p< 0.05) compared to the control by ANOVA.
Figure 3
Figure 3
Effect of various concentrations of arsenic trioxide on glutathione peroxidase activity in HepG2 cells treated for 24 hours. Concentrations found to be significantly different (p<0.05) compared to the control are denoted by (*) according to ANOVA Dunnett.
Figure 4
Figure 4
Formaldehyde standard curve showing absorbance values at 540 nm as a function of Formaldehyde concentration in μM.
Figure 5
Figure 5
Effect of various concentrations of arsenic trioxide on catalase activity in HepG2 cells treated for 24 hours. Concentrations found to be significantly different (p<0.05) compared to the control is denoted by (*) according to ANOVA Dunnett.
Figure 6
Figure 6
Representative flow cytometry analysis data from Annexin V/PI assay of HepG2 cells following 24h exposure. A, control; B, 0.5 μg/mL ATO; C, 1 μg/mL ATO; D, 2 μg/mL ATO; E, 4 μg/mL ATO; F, 8 μg/mL.
Figure 7
Figure 7
Induction of phosphatidylserine externalization by ATO in HepG2 cells. Percentage of Annexin V positive ATO-treated HepG2 cells undergoing early apoptosis following 24h exposure. * indicates significant difference (p<0.05) from the control, as well as, exposure time according to the ANOVA Dunnett’s test.
Figure 8
Figure 8
Representative flow cytometry analysis data from Caspase 3 assay in HepG2 cells following 24h exposure.A, control; B, 0.5 μg/mL ATO; C, 1 μg/mL ATO; D, 2 μg/mL ATO; E, 4 μg/mL ATO; F, 8 μg/mL.
Figure 9
Figure 9
Activation of Caspase-3 by ATO in HepG2 cells. Percentage of late apoptotic ATO-treated HepG2 cells following 24h exposure utilizing Caspase-3 analysis. * indicates significant difference (p<0.05) from the control according to the ANOVA Dunnett’s test.
Figure 10
Figure 10
DNA fragmentation by agarose gel electrophoresis. HepG2 cells were treated with various concentrations of ATO for 24h. Lane 1: DNA molecular weight marker (M); Lane 2: Positive control (C+) cells were induced for apoptosis by camptothecin; Lane 3: Control or untreated HepG2 cells; Lanes 4, 5, 6, 7, 8 and 9: HepG2 cells treated with 0.5, 1, 2, 4, 8, and 16 μg/mL of arsenic trioxide.
Figure 11
Figure 11
Western blot analysis of apoptotic protein expression (A–D) in 24h ATO-treated HepG2 cells. A – p53; B – p21; C – Bcl-2 and D – cytochrome c.
Figure 12
Figure 12
Densitometric analysis of p53 expression in 24h ATO-treated HepG2 cells.
Figure 13
Figure 13
Densitometric analysis of p21 expression in 24h ATO-treated HepG2 cells.
Figure 14
Figure 14
Densitometric analysis of Bcl-2 and cytochrome c expression in 24h ATO-treated HepG2 cells.
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