Abstract
Background: Arsenic trioxide (As2O3), a major compound in traditional Chinese medicine, is known to be an effective anticancer agent in acute promyelocytic leukemia (APL). The effects of As2O3 on human hepatocellular carcinoma (HCC) SK-Hep-1 cells were studied employing proteomics-based methodologies. Materials and Methods: Using 1-dimensional electrophoresis (1DE) and liquid chromatography electrospray ionization quadruple time-of-flight analysis, the whole proteomes of the control and As2O3-treated cells were profiled. Results: In all, 207 and 62 proteins, which were specifically found in control and As2O3-treated cells, respectively, were classified with their biological processes by gene ontology (GO) annotation. The GO data indicated that 16 proteins were closely associated with apoptotic mechanisms. As2O3-induced DNA damage and oxidative stress that accompanied apoptosis in SK-Hep-1 cells were observed using comet assay and 5-and-6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate fluorescence microscopy, respectively. Conclusion: The anticancer activities of As2O3 may be mediated by DNA damage- and reactive oxygen species-induced apoptotic mechanisms which involve the proteins identified in this study.
Hepatocellular carcinoma (HCC) is a fatal disease with a 5-year survival rate of 5% in HCC patients with poor prognosis (1). HCC treatment involves the use of drugs such as doxorubicin and methotrexate. The side-effects of these drugs, however, are a cause of concern since they have been linked to hepatitis and heart damage in HCC patients. Therefore, the discovery of new effective drugs is essential for the treatment of HCC (2).
Recently, the search for new compounds for cancer therapy has been the focus of numerous studies, many of which have focused on compounds that have been widely used in traditional medicine (3). Arsenic trioxide (As2O3) is a major compound in traditional Chinese medicine used to cure hemorrhoids, acute ulcerative gingivitis, and asthma (2). Nevertheless, it has not been considered as a therapeutic agent because of its toxic side-effects when used long-term (4). However, arsenic compounds have been reevaluated for their anticancer properties (2, 5). It has been reported that As2O3 predominantly induced apoptosis in acute promyelocytic leukemia (APL) cells (6, 7), which was mediated by increased levels of intracellular reactive oxygen species (ROS), depolarization of mitochondrial membranes and DNA damage (6-8). The apoptotic effects of As2O3 in APL have led investigators to propose that As2O3 may also induce apoptotic mechanisms in other types of cancer cells (9-11). However, little is known about the molecular mechanisms of As2O3-induced apoptosis in these cancer cells.
Previous studies have shown that As2O3 significantly inhibited cell proliferation and induced apoptosis in HCC cell lines (12-14). Oketani et al. showed that As2O3 inhibited cell proliferation and induced apoptosis in Huh-7, HepG2 and SK-Hep-1 cells as demonstrated by the increased appearance of sub-G1 cells after treatment with As2O3 (13). The sensitivity of HCC cells to As2O3 was also shown to be inversely related to intracellular glutathione (GSH) concentrations and GSH synthesis levels (13).
In this present study, the molecular mechanisms of As2O3-induced apoptosis were investigated in SK-Hep-1 cells using proteomics-based methodologies.
Materials and Methods
Cell culture and treatment. The human HCC cell line SK-Hep-1 was purchased from the Korean Cell Line Bank. SK-Hep-1 cells were cultured in Dulbecco's modified Eagle's medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA) and penicillin (100 μg/ml) and kept in a humidified atmosphere at 37°C with 5% carbon dioxide. As2O3 was purchased from Sigma (Deisenhofen, Germany), prepared as a 10 mM stock solution dissolved in DMSO and stored at 4°C. The SK-Hep-1 cells were seeded in 100-mm culture dishes at a density of 5 mM 6 cells. As the cells reached 70% confluence, the medium was replaced by fresh medium containing As2O3 while the control cells were cultured in medium containing the same volume of DMSO without As2O3.
Cell proliferation assay. To assess the effect of As2O3 on cell viability, SK-Hep-1 cells (2×103) were seeded in a 48-well plate. After 24 h incubation, the cells were treated with different concentrations of As2O3 (0, 0.25, 0.75, 1, 2, 3, 5, 10 μM). After 24 h (or 48 h) of treatment, the medium was removed and each well was treated with 20 μl of 3-(4,5-dimethylthiazol-2-yl)-5-5-diphenyl-tetrazolium bromide (MTT; Sigma) solution at 37°C for 2 h, and solubilized using 100 μl of DMSO. The plate was then vortexed for 1 h and the level of formazan was measured by a spectrophotometer at 575 nm.
1-dimensional electrophoresis (1DE). Protein expression profiles of SK-Hep-1 cells before and after treatment with 2 μM As2O3 for 48 h were observed by 1-DE analysis. The cells were harvested and lysed in lysis buffer (10 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), 1 mM phenylmethylsulphonyl fluoride (PMSF), 1 μg/ml leupeptin, and 1 μg/ml aprotinin). The protein concentration of the lysates was determined by Bradford assay. For 1-DE, 50 μg of proteins were collected from the lysates of the control and As2O3-treated cells, and loaded into the 10% SDS polyacrylamide gels. The proteins were separated at 50 V for 30 min using a Mini-Protean II system (Bio-rad, Hercules, CA, USA). After electrophoresis, the polyacrylamide gel was stained using a Coomassie Brilliant Blue G-250 reagent (Bio-Rad). Each of the stained gels was sliced into 15 pieces and used for further mass spectrometry/mass spectrometry (MS/MS) analysis.
Liquid chromatography electrospray ionization quadruple time-of-flight (LC-ESI-Q-TOF). The LC MS/MS system comprised a Surveyor MS pump (Thermo Electron, San Jose, CA, USA), a Spark auto sampler (Spark, Emmen, the Netherlands), and a Finnigan LTQ linear ion trap MS (Thermo Electron) equipped with NSI sources. An aliquot of 10 μl of tryptic digested peptides was injected for the LC-MS/MS analysis. Digested samples were also injected directly into a peptide CapTrap cartridge for concentration and desalting and applied to a reversed phase (RP) column that was packed in-house with 5 μm, 300 Å pore size C18. The peptides were then separated on an RP column. The mobile phase A (H2O) and B (acetonitrile) solutions contained 0.1% v/v formic acid. The flow rate was maintained at 200 nl/min. The gradient was started at 5% B and a linear gradient up to 60% B was achieved in 49 min, then ramped to 80% B in 5 min and to 100% A in the next 15 min.
The MS was operated in a data-dependent mode (m/z 300-1800) in which each full MS scan was followed by five MS/MS scans where the five most abundant peptide molecular ions were dynamically selected from the prior scan for collision-induced dissociation (CID) using a normalized collision energy of 35%. The temperature of the heated capillary and electrospray voltage were 195°C and 1.9 kV, respectively.
Gene ontology (GO) annotation. The LC-ESI-Q-TOF data of total proteins have Uniprot accession numbers (Swiss-Prot numbers or Translated EMBL numbers). GO annotation was then assigned to the Uniprot accession numbers using the QuickGO web tool (http://www.ebi.ac.uk/ego/). Each Uniprot accession number was assigned to three categories: biological process, molecular function, and cellular component (15, 16). If a single protein was annotated by several processes, such as functions or components, a representative of such annotations was reflected in the data.
Comet assay. Comet assay (Trevigen, Gaithersburg, MD, USA) was performed to observe DNA damage of the As2O3-treated cells compared to that of the control. The cells treated with 2 μM As2O3 (or 1% DMSO) for 48 h were harvested using a scraper and washed once with ice-cold phosphate-buffered saline, then re-suspended to a density of 3×105 cells/ml. Aliquots (5 μl) of this suspension were added to 500 μl low melting agarose; subsequently, 75 μl of this were poured onto the provided microscope slides. The agarose solution was allowed to set at 4°C for 30 min and the slides were then immersed in lysis solution at 4°C for 1 h to dissolve cellular proteins and lipids. After placing in alkali solution for 1 h at room temperature in a dark environment, the slides were immersed in 1× Tris-borate-EDTA (TBE) buffer for 5 min twice and transferred to a horizontal electrophoresis apparatus containing 1× TBE buffer for 10 min at 25 V in the dark. The slides were gently washed twice in distilled water. After staining with Hoechst 33342 solution (Invitrogen), the slides were examined by fluorescence microscopy (Axiovert 200; Zeiss Co, Oberkochen, German) and analyzed using Comet Assay IV (Perceptive Instuments Ltd. Suffolk, UK). Three comet parameters were evaluated for each image: tail lenth (μm), tail intensity (DNA %), and tail moment.
ROS detection. To investigate the effect of As2O3 on intracellular ROS production in the SK-Hep-1 cells, fluorometric assay using 5-and-6-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) was used as a probe for ROS detection. SK-Hep-1 cells (1×106) were plated in 60-mm culture dishes, and treated with 0.1% DMSO or 2 μM As2O3 for 48 h. After treatment, the cells were rinsed with HBSS solution and incubated with a medium containing 20 μM DCF-DA for 15 min at 37°C in the dark. The DCF-DA loaded cells were rinsed with HBSS solution and observed by fluorescence microscopy. Fluorescence was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm, and fluorescent images were taken at 5-s intervals.
Results
Antiproliferative activity of As2O3. The MTT assay showed that As2O3 inhibited the growth of the SK-Hep-1 cells in a dose- and time-dependent manner (Figure 1). While a high dose of As2O3 (more than 2 μM) caused noticeable cytotoxicity within 1 day, a low dose of As2O3 (less than 1 μM) did not significantly inhibit the cell growth. The half maximal inhibitory concentration (IC50) value of As2O3 on SK-Hep-1 was about 4.5 μM for 24 h treatment and 2.3 μM for 48 h treatment.
Classification of identified proteins by GO analysis. Using LC-ESI-Q-TOF, in all, 733 and 558 proteins were identified from the control and As2O3-treated cells, respectively. Out of these, 471 proteins were found to be expressed in both groups, while 262 and 94 proteins were specifically expressed in the control or As2O3-treated cells, respectively. Based on GO annotation, 207 and 62 proteins were annotated and classified by biological processes, such as transport, translation, carbohydrate metabolic process, DNA replication, RNA splicing, transcription, cell proliferation, signal transduction (including Ras-related signal transduction), apoptosis and response to oxidative stress (Table I). The GO annotation data indicated that 94 (76 down-regulated proteins, 18 up-regulated proteins) of the total identified proteins were associated with the proliferation of cells, classified as translation, DNA replication, transcription, cell proliferation, apoptosis, Ras-related signal transduction, cell growth, oxidation reduction, response to oxidative stress, DNA repair and anti-apoptosis. Sixteen proteins were closely associated with DNA damage-induced apoptosis and regulation of intracellular ROS (Table II).
Effect of As2O3 on DNA damage and intracellular ROS level. Using the comet assay the As2O3-treated cells showed significant DNA strand breaks compared to the control (Figure 2, Table III). DCF-DA fluorescent microscopy showed that the production of intracellular ROS was significantly increased after treatment with As2O3, and indicated that the morphology of the cells was noticeably changed after the treatment with As2O3 (Figure 3).
Discussion
Many studies have indicated that the anticancer activities of As2O3 in HCC are significantly related to the apoptotic mechanisms, which are mediated by DNA damage (17, 18) and increased oxidative stress (19-21). In the present study, As2O3 inhibited cell proliferation in SK-Hep-1 (Figure 1). Among 269 differentially expressed proteins in the control and As2O3-treated cells, 94 were closely related to the anticancer activities of As2O3, including translation, DNA replication, transcription, cell proliferation, apoptosis, Ras-related signal transduction, cell growth, oxidation reduction, response to oxidative stress, DNA repair and anti-apoptosis. Interestingly, 16 proteins were closely related to the apoptotic mechanisms mediated by DNA damage and increased oxidative stress (Table II). The anticancer activity of As2O3 was associated with increased DNA damage (Figure 2) and ROS production (Figure 3) as observed by comet assay and ROS detection, respectively.
DNA damage-induced apoptosis is considered as an important effect of anticancer activity of As2O3 (17, 18). In this study, ten of the total identified proteins were directly associated with the induction of apoptosis: mitochondrial 28S ribosomal protein S29 (MRPS29), mitochondrial 28S ribosomal protein S30 (BM-047), mitochondrial apoptosis-inducing factor 1 (AIFM1), mitochondrial NADH dehydrogenase (ubiquinone) iron-sulfur protein 3 (NDUFS3), programmed cell death protein 5 (PDCD5), serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform (PPP2CA), eukaryotic translation elongation factor 1 epsilon-1 (MCA3), lactoyl glutathione lyase (LGUL), 26S protease regulatory subunit 8 (PSMC5) and cell division control protein 2 homolog (CDC2). MRPS29, BM-047, AIFM1, NDUFS3, PDCD5, PPP2CA, MCA3 and LGUL are regarded as apoptosis-inducing proteins, while PSMC5 and CDC2 are responsible for the inhibition of apoptotic mechanisms. Among the apoptosis-inducing proteins, the expression of MCA3 and LGUL was up-regulated after treatment with As2O3, while that of the other proteins was down-regulated. Moreover, the expression of PSMC5 and CDC2 was down-regulated after the treatment. This expression change suggested that the apoptotic mechanisms of As2O3 seem to be more directly related to the expression of these four proteins (MCA3, LGUL, PSMC5, and CDC2) than the other proteins. In addition, many studies have shown that the apoptotic mechanisms of As2O3 are related to the change in CDC2 expression (22-26). Cai et al. showed that As2O3-induced apoptosis and mitotic arrest in NB4 cells, an acute promyelocytic leukemia cell line, accompanied the inhibition of CDC2 expression (26).
Several studies have suggested that the change in intracellular ROS level is related to the As2O3-induced apoptosis in HCC (19, 20). In this proteomics-based study, six proteins, glutathione synthetase (GSS), cDNA FLJ46672 fis clone TRACH3009008 (highly similar to thioredoxin reductase [Q6ZR44]), serine/threonine-protein kinase OSR1(OXSR1), sepiapterin reductase (SPR), ubiquinol-cytochrome c reductase complex core protein 1 (UQCRC1) and cytochrome b-c1 complex subunit Rieske (UQCRFS1), which are considered to be responsible for the control of the intracellular ROS level, were found. Interestingly, the molecular function of these six proteins is categorized as “oxidoreductase activity”, which might be an important mechanism for the As2O3-induced oxidative stress. However, it appeared that only the up-regulated proteins (OXSR1, UQCRFS1) showed this oxidoreductase activity in the As2O3-treated cells. Down-regulation of the other proteins (GSS, Q6ZR44, SPR, and UQCRC1) might be related to the conditions for the production of ROS, which resulted in further apoptotic mechanisms. Moreover, several studies have shown that the depletion of GSS resulted in the decreased production of glutathione (GSH) (27, 28), which is one of the major antioxidant proteins that functions in the lungs against oxidative stress. A previous study (13) also showed that the sensitivity of SK-Hep-1 cells to As2O3 was increased after a decrease in the intracellular level of GSH. Thus, the decreased expression of GSS might be an important event in As2O3-induced apoptosis.
Overall, this proteomics-based study provides a better understanding of the As2O3-induced apoptotic mechanism, and suggests that As2O3 may have significant therapeutic value for the treatment of human solid tumors. Further studies need to be carried out focusing on the specific function of the identified proteins in As2O3-treated SK-Hep-1 cells.
In conclusion, many proteins identified in As2O3-treated SK-Hep-1 cells are associated with the anticancer activities of As2O3 and some are involved in apoptotic mechanisms. The effect of As2O3 is mediated by DNA damage and increased intracellular ROS level.
Acknowledgments
This study was supported by partial funding from Han Cell INC. (2007-1-1)
- Received July 7, 2009.
- Revision received September 4, 2009.
- Accepted September 7, 2009.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved