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 downregulation 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.

Dugo EB, Yedjou CG, Stevens JJ, Tchounwou PB (2017) Therapeutic Potential of Arsenic Trioxide (ATO) in Treatment of Hepatocellular Carcinoma: Role of Oxidative Stress in ATO-Induced Apoptosis. Ann Clin Pathol 5(1): 1101.

•    ATO
•    Oxidative stress
•    Apoptosis
•    DNA fragmentation
•    HepG2 cells

ANOVA: One Way Analysis of Variance; APL: Acute Promyelocytic Leukemia; APS: Ammonium Persulfate; ATCC: American Type Culture Collection; ATO: Arsenic Trioxide; ATRA: All Trans Retinoic Acid; BHT: Butylated Hydroxytoluene; CAT: Catalase; DMEM: Dulbecco’s Modified Eagle’s Medium; DMSO: Dimethylsulfoxide; DNA: Deoxyribonucleic Acid; DTT: Dithiothreitol; EDTA: Ethylene Diamine Tetraacetic Acid; FACS: Fluorescence Activated Cell Sorting System; FITC: Fluorescein Isothiocyanate; GST: Glutathione Transferase; Gpx: Glutathione Peroxidase; HCC: Hepatocellular Carcinoma; Hcl: Hydrochloric Acid; MDA: Malondialdehyde; MTT: 3-(4,5-Dimethyl-2-Thiazolyl)- 2,5-Diphenyl-2tetrazoliumbromide; PE: Phycoerythrin; PBS: Phosphate Buffer Saline; PI: Propidium Iodine; ROS: Reactive Oxygen Species; SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoris; SOD: Superoxide Dismutase; TBE: Tris-Borate EDTA; TEMED: Tetramethylethylenediamine; UV/Vis: Ultraviolet Visible Spectroscopy.

Hepatocellular carcinoma is the dominant form of primary liver cancer occurring in patients with chronic liver disease and cirrhosis. Compared to all cancer sites, liver cancer death rates and incidence rates have increased among both men and women. Primary liver cancer is the sixth most common cancer in the world and the second leading cause of cancer death in men [1,2]. Over 700,000 people are diagnosed with liver cancer annually. Men are twice as likely as women to be diagnosed with liver cancer and the risk tends to increase with age. Liver cancer incidence has more than tripled since 1980 with rates increasing by 2.7% annually from 2003-2012 [3]. According to 2012 U.S. statistics from the Center for Disease Control (CDC), Asian/Pacific Islander men had the highest liver cancer incidence rates followed by Hispanic and African American men [4].

Therapeutic treatment methods for hepatocellular carcinoma are determined by factors such as liver function, nodule quantity and size, age, presence of comorbidities and tumor extension. Presently, there are five treatment options for hepatocellular carcinoma but none have been shown to be a cure-all with regard to recurrence, patient survival and longevity [5]. Surgical treatments include resection or liver transplantation, but the best option between both depends on hepatic function and whether or not the patients have cirrhosis. Percutaneous ablation techniques include percutaneous ethanol injection and radiofrequency ablation which is most suitable for patients who have early stage hepatocellular carcinoma but are not candidates for resection or liver transplantation. Chemo embolization treatment is recommended for patients with intermediate stage, inoperable hepatocellular carcinoma whose liver function is preserved and has asymptomatic multinodular tumors that have not spread or invaded areas or vessels outside the liver. Radio embolization treatment is used in patients who are between intermediate and advanced stage hepatocellular carcinoma and are not suited for resection, liver transplantation or ablation [6,7]. Until 2007, there was no systemic treatment option for patients with advanced hepatocellular carcinoma. So far, sorafenib is the only systemic drug showing improved survival benefit in liver cancer patients [8-10]. However, better understanding of the mechanisms involved in hepatocellular carcinoma could lead to more novel therapeutic interventions. Our lab is focusing on the use of arsenic trioxide (ATO) as a possible therapeutic option for the treatment of hepatocellular carcinoma.

Recent reports by our laboratory show that oxidative stress and DNA damage play a key role in arsenic trioxide (ATO)-induced cytotoxicity in acute promyelocytic leukemia (HL-60) cells [11]. The premise for the present study is to further investigate the role of oxidative stress and apoptosis in ATO toxicity utilizing human hepatocellular carcinoma (HepG2 ) cells as a model. Lipid peroxidation occurs as cells are being stressed by reactive oxygen species leading to loss of membrane function and integrity [12], thereby causing oxidative stress damage. Oxidative stress mediates many nocent effects of metals. The overproduction of reactive oxygen species (ROS) results in a significant increase in intracellular ROS, which leads to cellular damage, including lipid peroxidation, oxidative DNA modifications, protein oxidation and enzyme inactivation [13,14]. The imbalance between free radical generation and antioxidant defense systems resulting from oxidative stress is usually maintained in mammalian cells by key enzymes such as, superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST), glutathione peroxidase (GPx) and HO-1 that regulate intracellular ROS levels [15-17].

The apoptotic effect of ATO has been directly ascribed to oxidative stress or reactive oxygen species formation and the regulation of the mitochondrial pathway of apoptosis [18- 20]. Initial stages of apoptosis are characterized by initiator caspase activation, alterations in the cellular redox potential, cell shrinkage, loss of membrane lipid asymmetry and chromatin condensation. Later stages associated with the execution phase of apoptosis are characterized by activation of execution caspases and endonucleases, apoptotic body formation and cell fragmentation [21,22]. Arsenic has been shown to increase the activities of caspases-3, -8 and -9, to suppress Bcl-2 expression, to increase p53 and p21 activity, to activate MAPKs (p-JNK and p38) and release cytochrome c, leading subsequently to apoptosis in several cell lines [23-28].

Few studies have reported that ATO may be a potential therapeutic agent for the treatment of liver cancer. Using MHCC97-H hepatocellular carcinoma cell line, Cui et al, [29], reported that ATO negatively affects HCC tumorigenesis through down regulation of B7-H4 protein expression and inhibition of JNK pathway. In a study with HepG2 cells, Jiang et al, [30], demonstrated the ATO induces oxidative stress and apoptosis. Using TUNNEL and DNA laddering assays Yu et al, [31], reported that ATO-induced apoptosis is dose-dependent and is associated with the degradation of PML protein in the nuclei of in HepG2 cells. Other studies have reported that ATO inhibits HCC cell migration and invasion through upregulation of mRNA-491 via a demethylation that blocks NFkB activation/signaling [32,33]. Meng et al, [34], also demonstrated that ATO alters miRNA expression profile in HepG2 cells, and miRNA-29a seems to play an important role in ATO therapy against liver cancer. ATO has also been found to induce inhibition of metastatic potential of mouse hepatoma H22 cells [35]. The current study was designed to further examine the molecular mechanisms of ATO toxicity and its potential application for treatment of liver cancer.

It has been pointed out that the therapeutic potential of ATO against HHC may be enhanced by pharmaceutical products such as metformin [36], shikonin [37], survavin T34A [38], oridonin [39], genistein [40], and icari in [41]. On the other hand, it has been reported that the anti-cancer effect of sorafenib which is used for the treatment of HHC, is potentiated by ATO through the inhibition of Akt activation [42].

Chemicals and culture media

Arsenic trioxide (ATO) with an active ingredient of 100% (w/v) arsenic in 10% nitric acid was purchased from Fisher Scientific in Houston, TX. Dulbecco’s Modified Eagle’s Medium (DMEM)-F12, G418, fetal bovine serum (FBS), and phosphate buffered saline (PBS) were purchased from American Type Culture Collection (ATCC) in Manassas, VA. Penicillin-Streptomycin was purchased from Gibco Invitrogen in Carlsbad, CA.

Cell culture and ATO treatment

The established human hepatocellular carcinoma cell line used in the study were HepG2 (ATCC CRL-11997) cells purchased from ATCC in Manassas, VA. The HepG2 cells were grown in 50 cm2 tissue culture flasks in Dulbeco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin and streptomycin and 0.4mg/mL G418 at 37°C in a 5% CO2 incubator until confluent. The cells were then trypsinized with 0.25% trypsin-0.03% EDTA, diluted, counted, and seeded at 5x106 cells for each concentration of ATO (0.5, 1, 2, 4, and 8 µg/ mL) for 24 hours at 37°C in a 5% CO2 incubator.

Lipid peroxidation assay

The concentration of MDA was measured using a lipid peroxidation assay kit from Oxford Biomedical Research in Oxford, MI. The HepG2 cells were treated with and without ATO according to the aforementioned concentrations for 24 hours. The cell suspensions were collected in 15 mL tubes, followed by low speed centrifugation. The cell pellets were resuspended in 0.5 mL Tris-HCl, pH 7.5 and lysed in the presence of 10µL Butylated hydroxytoluene (BHT) by the freeze-thaw method. The protein concentrations of the cell suspension were determined using the BioRad Dc Protein Assay kit from BioRad Hercules, CA [43]. A 200µL aliquot of the culture medium or 2mg of cell lysate protein were assayed for MDA utilizing the lipid peroxidation assay protocol as previously described [44,45]. The absorbance was measured at 586 nm and the concentration of MDA determined from the standard curve.

Glutathione peroxidase assay

Glutathione Peroxidase (GPx) activity was measured using a glutathione peroxidase assay kit from Calbiochem-EMD Biosciences in Gibbstown, NJ. The HepG2 cells were treated with and without ATO according to the aforementioned concentrations for 24 hours. The cells were then isolated by cell scraping, rinsed twice with PBS and digested in 1mL of ice cold 50mM Tris-HCl, 5mM EthylenediamineTetraacetate (EDTA) and 1mM DTT. Cellular debris was removed by centrifugation and lysate used to determine GPx activity by following the protocol previously described [46,47] with a few modifications. The absorbance was measured at 340 nm using the Lab systems Multiskan Ascent microplate reader. Rate of the reaction was determined by constructing a standard curve as a function of absorbance versus time, and the GPx activity was expressed as nmol/minute/mL.

Catalase assay

Catalase activity was measured using a catalase assay kit from Calbiochem-EMD Biosciences in Gibbstown, NJ. The HepG2 cells were treated with and without ATO according to the aforementioned concentrations for 24 hours. The cells were detached by cell scraping, rinsed twice with PBS and digested in 1mL of ice cold homogenization buffer (50mM potassium phosphate and 1mM EDTA). Cellular debris was removed by centrifugation and lysate used to determine catalase activity following the protocol described [48] with a few modifications. Measurement of catalase activity was based on the reaction of the enzyme with methanol in the presence of an optimal concentration of H2 O2 . The formaldehyde produced was measured spectrophotometrically at 540 nm using the Lab systems Multiskan Ascent microplate reader. One unit of catalase was defined as the formation of 1n mol of formaldehyde per minute per milliliter.

Annexin V/PI assay

HepG2 cells were treated with and without ATO according to the aforementioned concentrations for 24 hours. Following treatment, the cells were isolated by trypsinization and rinsed twice with ice-cold PBS. Detection of phosphatidylserine on the outer leaflet of apoptotic cells was performed using Annexin V-FITC Apoptosis Detection kit in combination with propidium iodide (PI) staining (BD Pharmigen, Indianapolis, IN) according to the manufacturer’s recommendations [49,50]. The samples were pelleted and resuspended in binding buffer at 1 X 106 cells/mL. Tubes were labeled for each sample with 5 µL of Annexin V FITC, 5 µL of dissolved PI and 100 µL of the cell suspension (100,000 cells) being added to each tube. Each sample was examined by flow cytometry using a FAC Sort (Becton Dickinson Co, San Diego, CA, USA). The percentages of apoptotic cells were determined by analysis of the dot plots using Cell Quest software.

Caspase-3 assay

HepG2 cells were treated with and without ATO according to the aforementioned concentrations for 24 hours. Following treatment, the cells were isolated by trypsinization and rinsed twice with PBS. Active form of caspase-3 was determined by flow cytometry as previously described [51,52]. Cells (5×105 ) were washed, then fixed and permeabilized using the Cytofix/CytopermTM kit (BD Pharmingen) for 20 min at room temperature. Cells were washed with Perm/WashTM buffer, then stained with phycoerythrin (PE)-conjugated anti-active caspase-3 monoclonal antibodies using 20µL/1×106 cells for 60 min at room temperature in the dark. Following incubation with the antibody, cells were washed in Perm/Wash TM buffer, resuspended in Perm/Wash TM buffer and analyzed using a FAC Sort (Becton Dickinson Co., San Diego, CA, USA) flow cytometer.

DNA laddering

HepG2 cells were treated with and without ATO according to the aforementioned concentrations for 24 hours. The apoptotic DNA ladder kit was purchased from Roche Diagnostics Corporation (Indianapolis, IN). 2 x 106 cells were washed in PBS. The cells were then re-suspended in 200µL PBS and lysed in 200µL binding buffer. Centrifugation was used to separate the DNA in the lysate (which binds to the glass fiber fleece) from unbound lysate components (which flow through the fleece into a collection tube) as previously described [53]. The bound DNA was washed twice and eluted from the filter tube by centrifugation into 1.5 mL microcentrifuge tubes. An aliquot of each sample was taken to measure the optimal density at wavelength 280nm on the 2800 UV/VIS Spectrophotometer to determine the amount of DNA lysate to be loaded on the agarose gel. Twenty µg of the eluted DNA samples, 15 µL DNA Molecular Weight Marker and 15 µL Camptothecin-treated positive control cells were mixed with loading buffer and applied to a 1% agarose gel. The gel was then electrophoresed in TBE (Tris-borate EDTA) buffer at 40 V. The gel was stained using ethidium bromide (Sigma) for 15 minutes and the DNA ladder pattern on the gel visualized on the Typhoon 9400 Scanner.

Western blot analysis of specific ellular proteins

HepG2 cells were treated with and without ATO according to the aforementioned concentrations for 24 hours. Protein lysate was prepared in lysis buffer and the protein concentration determined using the Bio Rad Dc Protein Assay kit (Hercules, CA). Western blot analysis was performed as previously described [54]. Samples with equal amount of protein was separated by SDS-PAGE with 4% stacking gel (2.4ml of distilled water, 1.8mL 4X upper gel buffer (1.5M Tris-Cl), 3.13mL of 30% acrylamide (BioRad), 75 μ l of 10% SDS, 37.5μ l of 10% (w/v) ammonium persulfate (APS) and 2.5μl of N,N,N′,N′- tetramethylethylenediamine) and 12% resolving gel (4.8ml of distilled water, 1.15ml of 30% acrylamide, 2ml of 4X lower gel buffer (0.5M Tris-Cl), 40μl of APS and 9.3μl of TEMED) running gel. The proteins were then transferred to a nitrocellulose membrane by semi-dry transfer system (BioRad). After blocking the non-specific space of the membrane with 10% non-fat milk in PBS-T (PBS with 0.5% Tween 20), the membranes were incubated with monoclonal antibodies (1:1000) against p53, p21, Bcl-2 and cytochrome c overnight at 4°C. The membranes were washed three times at 5-minute intervals with PBS-T, and incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000) for 1 hour at 4°C. The membranes were then washed five times at 10 minute intervals with PBS-T. Proteins were detected using enhanced chemiluminescence (ECL) detection reagents (Pierce Thermo Scientific). Relative band intensity was quantified using the Molecular Imager Gel Doc XR+ System and Image Lab Software Version 3.0 (Bio-Rad).

Statistical analysis

Statistical analysis was done using one way analysis of variance (ANOVA) for multiple samples and Student’s t-test for testing differences. The results were represented as means ± standard deviations. All p-values less than 0.05 were considered to be significant. Oxidative stress and apoptotic data were presented graphically in the form of histograms, using Microsoft Excel computer program to represent the dose-response relationship among the treatment groups.

Induction of lipid peroxidation in ATO-treated Hepg2 Cells

To elucidate the possible involvement of lipid peroxidation products in ATO induced toxicity, we performed the lipid peroxidation assay. The malondialdehyde (MDA) standard curve presented in Figure (1) shows a strong positive correlation with an r2 value of 0.9983, with data on the effect of ATO on MDA production in HepG2 cells presented in Figure (2). Upon 24 hours treatment with ATO, the MDA values were 1.55 ± 0.212, 4.75 ± 0.318, 7.25 ± 0.141, 9.15 ± 0.459, 10.6 ± 0.282, and 12.2 ± 0.106 mM in 0, 0.5, 1, 2, 4, and 8 µg/mL of ATO, respectively.

Induction of glutathione peroxidase in ATO-treated HepG2 Cells

In this assay, glutathione peroxidase activity was determined for each sample by taking the absorbance reading at one minute time intervals over five minutes. Figure (3) demonstrates the effect of ATO on glutathione peroxidase activity in HepG2 cells which revealed a dose dependent decrease in glutathione peroxidase activity with increasing ATO concentrations. There was a significant difference between the control and the treatment groups.

Induction of Catalase in ATO-treated HepG2 Cells

In the catalase assay, the standard curve data showing a strong positive correlation and r2 value of 0.9845 for formaldehyde concentration in Figure(4) was used to determine the catalase activity for HepG2 cells exposed to each ATO concentration. Figure (5) demonstrates the effect of ATO on catalase activity revealing a slight elevation in catalase activity at low level of ATO treatment. When cells were treated with ATO levels of 2 µg/mL and higher, catalase activity decreased in a concentration dependent manner with the highest concentration (8 µg/mL) being significantly different (p< 0.05) compared to the control.

Modulation of phosphatidylserine externalization by ATO in Hepg2 Cells

Flow cytometric analysis was performed to determine the effect of ATO on early apoptosis utilizing the Annexin V/ PI assay. Representative histograms are shown in Figure (6). Upon 24 hours of treatment, the results of the annexin V/PI staining showed that the percentages of early apoptotic cells (Annexin V positive cells) were 0.2 %, 1.6 %, 1.4 %, 21.4 %, 44.7 %, and 51.7 % in 0, 0.5, 1, 2, 4, and 8 µg/mL ATO in HepG2 cells, respectively. The percentages of viable cells were 99.4 %, 93.5 %, 84.9 %, 76.5 %, 52.7 % and 46.7 %, respectively. The lower left quadrant of the histogram represents the viable cells which are Annexin V negative and PI negative. The lower right quadrant of the histogram represents the early apoptotic cells which are Annexin V positive and PI negative. Figure (7) shows a significant (p< 0.05) gradual increase in Annexin V positive cells at doses between 1-8 µg/ml ATO in HepG2 cells compared to the control cells; indicative of a dose-dependent induction of apoptosis by ATO.

Activation of caspase-3 in ATO-treated Hepg2 Cells

Flow cytometry data from the PE Caspase 3 assay was used to determine whether ATO contributed to late apoptosis in HepG2 cells. Apoptosis is felt to be one of the key pathways of ATO mediated control of cancer cell growth in both solid tumors and leukemia. Loss of mitochondrial membrane potential, with subsequent increase in mitochondrial permeability, and activation of caspases by ATO contributes to the central trigger of the apoptotic processes [55-57]. Data in Figure (8) shows a dose dependent increase in Caspase 3 activity and ATO treatment. Figure (9) shows that the percentages of cells undergoing late apoptosis (Caspase 3 positive cells) were 4.5%, 9.8%, 14.5%, 16.6 %, 23.2%, and 31.1% in 0, 0.5, 1, 2, 4, and 8 µg/mL ATO in HepG2 cells following 24h exposure, respectively. Significant differences (p< 0.05) in the number of apoptotic (Caspase-3 positive) cells were observed between the ATO-treated HepG2 cells and the control cells.

Nucleosomal DNA fragmentation in ATO-treated HepG2 cells

One of the characteristics of apoptosis is nucleosomal DNA fragmentation. To further study the action mechanism of ATOinduced apoptosis, DNA fragmentation assay by agarose gel electrophoresis was performed. Our results showed a typical ladder pattern and induction of nucleosomal DNA fragmentation in HepG2 cells treated with ATO as presented in Figure (10).

Expression of Apoptotic-related genes in ATO-treated Hepg2 Cells

Western blot analysis was conducted to determine the expression of specific cellular proteins in hepatocellular carcinoma HepG2 cells treated with ATO. Data represented in Figure (11) shows the expression of apoptotic-related genes p53, p21, Bcl-2 and cytochrome c. In our study, densitometric analysis of p53 and p21 showed increased expression in ATO-treated HepG2 cells compared to control with p53 expression being significantly different in all ATO concentrations (Figure 12) and p21 expression being significantly different between 2-8 µg/ml ATO concentrations (Figure 13). Densitometric analysis of Bcl-2 showed that expression was reduced in ATO-treated HepG2 cells with significant (p< 0.05) down regulation at 0.5, 4 and 8 µg/ml ATO concentrations (Figure 14). Densitometric analysis showed an increase in cytochrome c expression in a dose dependent manner with ATO concentrations between 0.5-8 µg/ml being significantly (p< 0.05) different compared to the control (Figure 14).

Apoptosis or programmed cell death plays an important role in the physiological process and homeostasis of the cell [58].

Apoptosis is a highly organized process characterized by the progressive activation of precise pathways leading to specific biochemical and morphological alterations [59]. Recent studies in our laboratory indicate that ATO induces apoptosis in HL-60 and A549 cells [60,61]. The present study focuses on the role oxidative stress plays in arsenic-induced apoptosis in human hepatocellular carcinoma (HepG2 ) cells. Oxidative stress is an imbalance between the production and disposal of reactive oxygen which forms as a result of free radical formation capable of initiating lipid peroxidation and alterations in the antioxidant cellular defense system. The antioxidant response is one of the most efficient mechanisms of cell defense where superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST), glutathione peroxidase (GPx) and HO-1 are among the main enzymatic mechanisms involved in clearing and scavenging ROS to maintain low intracellular levels [62,63]. Data from our lipid peroxidation assay as seen in Figure (2) show that arsenic trioxide significantly increases (p< 0.05) malondialdehyde (MDA) levels in human hepatocellular carcinoma (HepG2 ) cells in a dosedependent manner. In addition when we looked at whether the detoxification systems and antioxidants were compromised, we found that ATO significantly decreases glutathione peroxidase activities with increasing ATO concentrations in HepG2 cells and results in a non-linear decrease of catalase activities with 8µg/ml ATO being the most significant (Figures 3 and 5). When comparing glutathione peroxidase and catalase activity in ATO-treated HepG2 cells, catalase activity was found to be higher, suggesting that it is a stronger scavenger for the decomposition of H2 O2 , one of the main ROS involved in arsenic-induced DNA damage [64,65]. Findings consistent with previous data suggesting that alteration of oxidative stress markers is due to overuse/failure of the antioxidant defense system secondary to reactive oxygen species production [66].

Mitochondria are an important organelle involved in arsenicinduced apoptosis [67,68]. There are two major pathways of apoptosis, the death receptor pathway (extrinsic) and the mitochondrial pathway (intrinsic) [69]. The mitochondrial pathway of apoptosis can be triggered by a variety of stimuli, including chemotherapeutic agents, UV radiation, and oxidative stress. One of the early events in apoptotic cell death is loss of asymmetry in the phospholipid bilayer of the plasma membrane with translocation of phosphatidylserine from the inner to the outer leaflet, which can be detected by increased binding of fluorescence tagged Annexin V to the external surface of the cells.

As seen in Figures (6) and (7), data from the Annexin V-PI staining showed an increase in the percentage of cells in the bottom right quadrants of the dot plot with increasing ATO concentrations, indicative of apoptosis. Similar results have been seen in studies of keratinocytes, human leukemia cells and peripheral blood mononuclear cells [70-72]. Activation of caspases appears to be directly responsible for many of the molecular and structural changes in apoptosis. Depending on the stimulus that initiates apoptosis, different caspase cascades are activated [73]. Caspase-3 plays an essential role as an executor in apoptosis [74,75]. Figures (8) and (9) show the activation of caspase-3 as assessed by flow cytometry to have a dose-dependent increase in the percentage of cells undergoing late apoptosis. Our results indicate ATO-induced hepatocyte death occurring in vitro to be apoptotic and caspase-mediated, which is a common and critical component of the apoptotic cell death pathway. In a similar study, Jiang et al [30] reported that ATO induced apoptosis in HepG2 cells is mediated via oxidative stress through the mitochondria pathway and activation of caspases.

One of the hallmarks of apoptosis is nucleosomal DNA fragmentation that occurs due to numerous events initiated by caspase 3 [76]. As seen in Figure (10), our results from the DNA fragmentation assay by agarose gel electrophoresis showed a typical ladder pattern in HepG2 cells treated with ATO. Earlier studies reported that arsenite concentrations above 0.25 mM induced DNA strand breakage in human leukemia cells and Chinese hamster ovarian cells [77]. Arsenite also induced deletion, aneuploidy, sister chromatid exchange and different chromosomal aberrations [78-80]. The DNA ladder pattern obtained from our results is highly specific for apoptotic mode of cell death, thereby confirming arsenic induced cellular apoptosis.

Arsenic has been found to induce gene expression in a number of apoptotic and stress response proteins and transcription factors [81-83]. p53 is a tumor suppressor gene that regulates the expression of genes involved in growth arrest, DNA damage and apoptosis [84,85]. In our study, we demonstrated an upregulation of p53 expression with respect to increasing ATO concentrations in HepG2 cells (Figures 11A and 12). Other studies also showed that p53 expression increased in response to ATO exposure in human leukemia cells, TM4 Sertoli cells and pancreatic cells [86-88]. Activation of p53 in our study may be a result of ROS generation, suggesting an important consequence of oxidant-induced activation of p53. Arsenic can induce, inhibit or have no influence on the p53 expression and its regulation of down-stream genes like p21 [89-91]. Arsenic has been reported to increase p21 expression in several cell lines revealing its role in the apoptotic signaling pathway [92-94]. The activation of p21 within a cell either helps to block cell cycle progression to allow time for DNA repair before replication or causes apoptosis via induction of the proapoptotic protein BAX and down regulation of the antiapoptotic protein Bcl-2 in heavily damaged cells [95,96]. Our results show a dose-dependent increase in p21 expression with respect to ATO exposure in HepG2 cells (Figures 11B and 13). Earlier studies have reported up regulation of p21 to be time and dose-dependent in human pancreatic cancer cells and A498 renal carcinoma cells [97, 98]. The up regulation of p21 in our study may play a role in the stress response to ATO and may serve as an executioner of apoptosis following caspase activation.

Bcl-2 is an anti-apoptotic protein that acts to stabilize the mitochondrial membrane, control the mitochondrial membrane permeability to prevent the release of cytochrome C, and suppresses the activation of the caspase-3 apoptotic cascade. P53 antagonizes the antiapoptotic activities of Bcl-2, Bcl-xl and Mcl-1 thus, allowing proapoptoticBax, Bak and Bid to trigger apoptosis [99,100]. Translocation of p53 to the mitochondria induces apoptosis via cytochrome c release and activation of the intrinsic pathway which in turn is also regulated [101,102]. In our study, we examined the effect of arsenic trioxide on Bcl-2 expression in HepG2 cells, and observed a biphasic response indicating an up-regulation at 1 µg/mL of ATO treatment followed by a significant dose-dependent down-regulation at higher doses of ATO exposure (Figures 11C and 14). The up regulation of Bcl-2 expression at lower level of exposure may be due to an arsenicinduced hormetic effect; a condition that has previously been characterized by a stimulation of cell proliferation or induction of mitogenic effect at low doses, followed by an inhibition of cell viability or induction of cytotoxicity at higher doses [60,103- 105]. This adaptive or protective response at low doses is characteristic of many oxidative stress causing compounds including chemotherapeutic agents [106]. Between 1 and 8 µg/ mL ATO, Bcl-2 expression was significantly down-regulated. Similar results have been found in gallbladder carcinoma cells and acute promyelocytic leukemia cells treated with ATO [107,108]. These results indicate that ATO exposure blocks Bcl2 from preventing apoptosis in HepG2 cells, thereby allowing cytochrome c to be released. Increased permeability of the mitochondrial membrane during apoptosis triggers the release of cytochrome c from mitochondria in the apoptotic process in response to proapoptotic stimuli. The release of cytochrome c into the cytoplasm activates caspases cascade [109-111]. Our results show an increase of cytochrome c expression in ATOtreated HepG2 cells compared to the control (Figures 11D and 14). Previous studies reported a release of cytochrome c into the cytosol of ATO-treated mouse embryonic fibroblasts [112]. The release of cytochrome c into the cytoplasm results in caspase 9 activation and subsequent activation of caspase 3, ultimately leading to apoptosis as evidenced by data presented in this paper.

Our study demonstrates that arsenic trioxide is highly toxic to human hepatocellular (HepG2 ) carcinoma cells resulting in oxidative stress and apoptosis. The generation of reactive oxygen species within the cell occurs as a result of oxidative stress. This statement is confirmed by the results from the lipid peroxidation assay showing an increase in MDA, as well as a decrease in the antioxidant enzyme activity of GPx and catalase in ATO-treated cells. In response to ROS, antioxidant defense enzymes are activated to metabolize these oxidants, however a higher level of ATO treatment, as indicated by our data, shows a loss of activation thus pointing to its inability to protect cells from oxidative stress. Flow cytometric analysis showed an increase of caspase 3 and annexin V activity with increasing ATO concentrations. Also, a characteristic DNA fragmentation was observed in the DNA laddering assay. Western blot analysis of ATO-treated HepG2 cells revealed a down regulation of Bcl-2 expression, and an upregulation of cytochrome c, p53 and p21 expression. Taken together, these findings provide further insights into the role oxidative stress plays in ATO-induced apoptosis; making its potential use for the treatment of hepatocellular carcinoma worth exploring further.

PBT, CGY and EBD conceived, and designed the study. EBD performed the experiments and drafted the manuscript. CGY supervised the experiments and assisted in performing data interpretation. JJS provided critical review and manuscript editing. PBT participated in the implementation of the study and reviewed the manuscript for submission. All authors read and approved the final draft of the manuscript. This research was supported by a grant from the National Institutes of Health / National Institute on Minority Health and Health Disparities (G12MD007581) through the RCMI Center for Environmental Health at Jackson State University.

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