In Vitro Cell.Dev.Biol.—Animal DOI 10.1007/s11626-015-9961-4
Augmentation of oxidative stress-induced apoptosis in MCF7 cells by ascorbate–tamoxifen and/or ascorbate–juglone treatments Soraya Sajadimajd 1 & Razieh Yazdanparast 1 & Fariba Roshanzamir 1
Received: 19 May 2015 / Accepted: 16 September 2015 / Editor: Tetsuji Okamoto # The Society for In Vitro Biology 2015
Abstract Since reactive oxygen species (ROS) play diverse roles in cancer, modulating the redox status of cancerous cells seems to be a promising therapeutic approach. Oxidanttargeted therapy appears logical for intervention with the acquired adaptive response to oxidative stress in cancer. In this study, we investigated the cytotoxic effects of juglone (J) and tamoxifen (T) and also the combination of each with ascorbate (A): tamoxifen/ascorbate (TA) and/or juglone/ascorbate (JA) on MCF7 cancerous cells. The results revealed that the growth inhibitory effects of juglone and tamoxifen were each associated with enhanced levels of ROS production and lipid peroxidation. These effects were markedly intensified in tamoxifen/ ascorbate and juglone/ascorbate co-treatments. On the other hand, the intracellular anti-oxidant components such as reduced glutathione (GSH), catalase, superoxide dismutase (SOD), and glutathione peroxidase significantly declined in cells subjected to combination treatments compared to that in cells exposed solely to tamoxifen, juglone, and the untreated control cells. In addition, ascorbate association induced more apoptotic and necrotic or necrotic-like cell death than cells treated with each drug alone. These results were further confirmed by comparing the Bax/Bcl2 ratio in combinationtreated cells. Additionally, ascorbate was able to potentiate the cytotoxic effects of combination therapy via activation of ROS-responsive factors including Foxo family members. Keywords Ascorbate . Chemosensitization . Juglone . MCF7 cells . Oxidative stress . Tamoxifen
* Razieh Yazdanparast
[email protected] 1
Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran, P. O. Box 13145-1384, Tehran, Iran
Introduction Reactive oxygen species (ROS), namely, hydrogen peroxide (H2O2), superoxide anion (O2•−), and hydroxyl radicals (•OH), are defined as oxygen-containing highly reactive biomolecules that are produced even during normal intracellular metabolism (D’Autréaux and Toledano 2007). Oxidative stress arises due to the disturbed physiological balance between ROS and the intracellular anti-oxidant defense systems leading to significant damages to biomolecules such as proteins, lipids, and DNA molecules (Ďuračková 2010). Under stress situation, ROS modulate some of the intracellular signaling cascades leading to a wide variety of cellular responses. One of these is the mammalian forkhead box O (Foxo) pathway which plays essential roles in many cellular processes including cell cycle arrest, programmed cell death, differentiation, and stress resistance. In addition, the presence of a regulated feedback loop between ROS and Foxo proteins is now well established (Vurusaner et al. 2012). Foxo proteins are able to regulate and to be regulated by oxidative stress through posttranslational modification and protein interactions. Furthermore, the outcome of Foxo-ROS pathway is determined via cellular context as well as duration and concentration of ROS insults (Myatt et al. 2011). It has been demonstrated that, in response to stress stimuli, Foxo proteins interact with the tumor suppressor p53 protein, redirecting the cellular fate toward survival or apoptosis (Brunet et al. 2004; Myatt et al. 2011). Similar to Foxo, p53 is a transcription factor involved in several cellular processes including apoptosis, cell cycle arrest, and tumor suppression. Depending on the level and the type of stress-induced ROS, p53 can exhibit either anti-oxidant or pro-oxidant activity through activating the expression of specific molecules involved in redox response (Bensaad and Vousden 2005; Liu and Xu 2011). Regarding the cytotoxic nature of the elevated ROS level
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and the vulnerability of malignant cells to ROS, oxidative stress targeting could be evaluated as an alternative therapeutic strategy for cancer. Therefore, it is reasonable to exploit compounds which selectively induce death through redox modulation of the tumor cells. Ascorbic acid (ascorbate, vitamin C), as an essential nutrient, has long been known for its anti-oxidant capabilities. However, it has also been suggested that, at the pharmacological doses, ascorbate is able to impair the proliferation of some cancer cells through H2O2 production (Chen et al. 2005; 2008; Tareen et al. 2008). Further, synergic effect of ascorbate combination therapy with vitamin K3 and/or other quinones has been reported to induce cell death among various tumor cell lines. This effect is regarded as quinone redox cycling which promotes oxidative stress (Verrax et al. 2005; Tareen et al. 2008). Another natural compound named juglone (5-hydroxy-2methyl-1,4-napthoquinone), a derivative of naphtoquinones, also possesses anti-inflammatory and anti-tumor properties. Anti-cancer effects of juglone, like other naphtoquinones, have been attributed to its ability to modulate oxidative stress and free radical formation through redox cycling and/or reaction with glutathione (Inbaraj and Chignell 2004; Xu et al. 2012). In addition to its ability to induce oxidative stress, juglone also affects some of the regulatory signals such as PI3K and pin1 (Hennig et al. 1998; Chae et al. 2012). Studies have demonstrated that juglone promotes apoptosis in HL60 and gastric SGC-79 cancer cells via mitochondriadependent pathway(s) (Xu et al. 2010; Ji et al. 2011) as well as necrosis in B16F1melanoma cells (Kiran Aithal et al. 2009). It is well documented that certain chemotherapeutic agents also act through induction of oxidative stress. Tamoxifen is a global chemopreventive agent used for treatment of hormonesensitive breast cancer. Its cytotoxic effects have been attributed to ROS generation (Ferlini et al. 1999). Tamoxifen is metabolized to reactive intermediates including quinone derivatives which cause DNA damages (Bolton 2002). However, it should be emphasized that ROS-based therapies might also become associated with resistance. In that case, ROS combination therapies might be operative as alternative treatments. In that respect, it has been found that ascorbate has reinforced the cytotoxic effects of juglone on human bladder carcinoma T24 cells in an oxidative stress-related manner (Kviecinski et al. 2012). In the current study, we aimed to apply the same strategy to enhance or to potentiate the drug efficacy of tamoxifen, compared to juglone, when applied with ascorbate.
Materials and Methods Materials. Human breast cancer MCF-7 cells were purchased from Pasteur Institute of Iran (Tehran, Iran). The cell culture
medium (RPMI1640), fetal bovine serum (FBS), and penicillin–streptomycin were purchased from Gibco BRL (Life Technology, Paisley, Scotland). The culture plates were purchased from Nunc Brand products, Roskilde, Denmark. Dimethyl sulfoxide (DMSO) was obtained from Merck (Darmstadt, Germany). Ethidium Bromide (EtBr), acridine orange (Ao), and Triton X-100 were obtained from Pharmacia LKB Biotechnology AB Uppsala (Bromma, Sweden). MTT [3-(4,5-dimethyl tiazol-2,5-diphenyl tetrazolium bromide], phenylmethylsulphonyl fluoride (PMSF), leupeptin, pepstatin, aprotinin, dithionitrobenzoic acid (DTNB), reduced glutathione (GSH), tamoxifen, juglone, and ascorbate were purchased from Sigma Chem. Co. (Darmstadt, Germany). Nitro blue tetrazolium (NBT), Nacetylcysteine (NAC), nicotinamide adenine dinucleotide reduced form (NADH), phenazine methosulfate (PMS), 2deoxy-2-ribose (2-DR), and thiobarbituric acid (TBA) were obtained from Merck (Darmstadt, Germany). 2′,7′Dichlorofluorescein diacetate (DCFH-DA) was obtained from Molecular Probe (Eugene, OR). Ethylenediaminetetraacetic acid (EDTA) was from Aldrich (Germany). Anti-Bcl-2, antiBax, anti-cleaved caspase-3, and anti-p53 antibodies were purchased from Biosource (Nivelles, Belgium). Anti-Foxo1 and anti-Foxo3a antibodies were obtained from Cell Signaling (Danvers, MA, United States). The chemiluminescent detection system was purchased from AmershamPharmacia (Piscataway, NJ). Cell culture and treatment. MCF-7 cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum and 100 U/ml penicillin in a humidified 5% CO2 atmosphere at 37°C. Viable cells were counted using a hemocytometer, based on their abilities to exclude trypan blue. Drug treatment was usually performed 24 h after seeding the cells. Vitamin C stock solution was prepared fresh in culture medium at darkness and diluted whenever needed with the same medium to the desired concentrations. Juglone and tamoxifen stock solutions (100 mM) were prepared in DMSO (final concentration ≤0.1%) and then diluted with the culture medium to get the desired concentrations. Control groups received DMSO vehicle at a concentration equal to that in drug-treated cells. Cell viability assay. Cytotoxicity of each drug was evaluated by MTT assay as described by Visitica et al. (Vistica et al. 1991). Briefly, MCF-7 cells at a density of 3×104 ml−1 were plated into 96 multiwell plates for 24 h and then treated each with either drug alone (juglone, and tamoxifen) or in combination with ascorbate. Treated cells were incubated at 37°C for the indicated time intervals. After incubation, the media were replaced, and 10 μl MTT (5 mg/ml) was added to each well. Following 4 h of incubation, the media were discarded and DMSO (150 μl) was added to each well for dissolving the formazan crystals. The viability of cells was evaluated by
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measuring the absorbance at 570 nm with the ELISA reader (Exert 96, Asys Hitch, Ec Austria) after 30 min. The values were expressed as mean±SD of four independent experiments carried out in triplicates. In another method, 3×104 cells ml−1 was seeded in triplicate into cell culture plates for 24 h prior to treatment. After treatment with the drugs, at different doses for various lengths of time, cell numbers were established using a hemocytometer and the cell viability was determined by the trypan blue exclusion test. The cells attached to the culture plates were trypsinized with trypsin–EDTA solution. The numbers of attached and unattached cells were determined using a hemocytometer. Isoblogram assay. Isoblogram test is a dose-oriented method for evaluation of the final effects of drug combination using the equation CI=a/A+b/B, where CI is the combination index of drugs, and A and B represent the concentration of each drug alone producing the desired effect (IC50), and a and b are the respective doses of the drugs in combination that induced 50% effects (Chou and Talalay 1984). In this assay, CI>1 represents antagonist effect, CI=1 shows additive effect, and CI
