Effects of astaxanthin on oxidative stress induced by Cu²⁺ in prostate cells

Effects of astaxanthin on oxidative stress induced by Cu 2+ in prostate cells 虾青素对铜离子诱导的前列腺细胞氧化损伤的影响 1 first-author 2 3 2 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:shrshen@zju.edu.cn †E-mail:shrshen@zju.edu.cn shrshen@zju.edu.cn Department of Urology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Urology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, China College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, China Astaxanthin (AST), a carotenoid molecule extensively found in marine organisms and increasingly used as a dietary supplement, has been reported to have beneficial effects against oxidative stress. In the current paper, the effects of AST on viability of prostate cells were investigated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay; cell apoptosis and intracellular reactive oxygen species (ROS) levels were determined by flow cytometry; the mitochondrial membrane potential (MMP) was measured by fluorospectrophotometer; and activities of malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were evaluated by a detection kit. The results show that copper ion (Cu 2+ ) induced apoptosis, along with the accumulation of intracellular ROS and MDA, in both prostate cell lines (RWPE-1 and PC-3). AST treatments could decrease the MDA levels, increase MMP, and keep ROS stable in RWPE-1 cell line. An addition of AST decreased the SOD, GSH-Px, and CAT activities in PC-3 cell line treated with Cu 2+ , but had a contrary reaction in RWPE-1 cell lines. In conclusion, AST could contribute to protecting RWPE-1 cells against Cu 2+ -induced injuries but could cause damage to the antioxidant enzyme system in PC-3 cells. 目的 2 研究虾青素对铜离子诱导的前列腺细胞氧化损伤的影响，并探索其作用机制。 创新点 2 首次研究虾青素对铜离子诱导的前列腺细胞及前列腺癌细胞氧化损伤的影响，并比较其对两种细胞作用的差异。 方法 2 MTT法测定铜离子与虾青素对前列腺细胞（RWPE-1）和前列腺癌细胞（PC-3）生长的影响；采用细胞流式仪测定虾青素对铜离子诱导的RWPE-1和PC-3细胞凋亡的影响；荧光分光光度法测定了虾青素对铜离子诱导的活性氧自由基（ROS）产生的影响；采用罗丹明123（Rh123）染色检测虾青素对铜离子诱导的细胞线粒体膜电位（MMP）变化的影响；采用试剂盒测定了虾青素对铜离子存在下丙二醛（MDA）含量、超氧化物歧化酶（SOD）、过氧化氢酶（CAT）及谷胱甘肽过氧化物酶（GSH-Px）活性变化的影响。 结论 2 结果表明，铜离子能诱导RWPE-1和PC-3细胞凋亡，并伴随细胞内ROS和MDA含量升高；虾青素处理可显著降低RWPE-1细胞中MDA含量，升高线粒体膜电位，并保持ROS含量稳定；虾青素处理可降低PC-3细胞中SOD、GSH-Px和CAT的活性，而对RWPE-1细胞则作用相反。因此，虾青素处理能有效降低铜离子对RWPE-1细胞引起的损伤，而通过降低抗氧化酶活性加剧铜离子对PC-3细胞的损伤。 Oxidative stress PC-3 RWPE-1 Astaxanthin Copper ion 氧化损伤 PC-3细胞 RWPE-1细胞 虾青素 铜离子 Introduction 1 Prostate cancer is the second most frequently diagnosed cancer and the second leading cause of cancer-related death in men; the incidence and mortality of this disease are high in both North America and Western Europe, and currently low, but increasing, in Asia. Considerable evidence indicates that both genetic and environmental factors are primarily involved in its evolution. Copper ion (Cu 2+ ) is an essential trace element for human health. An imbalance in the metabolism of Cu 2+ could be an etiologic factor for prostate cancer development. It participates in a variety of important metabolic pathways in free radical forms, such as superoxide dismutase (SOD) scavenging intracellular free radicals and cytochrome oxidase transmitting respiratory chain electron. Low intracellular Cu 2+ concentrations could influence the activities of these enzymes and the normal metabolisms of the cells. Interestingly, the redox properties of the metal also mediate its toxicity because uncontrolled production of reactive oxygen species (ROS) results in oxidative stress, which does not follow a correct antioxidant response and consequently damages the biological macromolecules such as nucleic acids, proteins, and lipids (Adler et al., 1999 1999 ; Auten and Davis, 2009 2009 ; Maltepe and Saugstad, 2009 2009 ; Linder, 2012 2012 ). Under normal conditions, all processes involved in copper intake, distribution, utilization, and excretion are precisely regulated (Rosenzweig and O'Halloran, 2000 2000 ; Kim et al., 2008 2008 ; de Feo et al., 2009 2009 ; Banci et al., 2010 2010 ; Festa and Thiele, 2011 2011 ; Haas et al., 2011 2011 ). Both exogenous and endogenous sources contributed to the formation of intracellular ROS (Winterbourn, 2008 2008 ). Exogenous sources include radiation and environmental agents. Major endogenous sources of cellular ROS are microsomes, peroxisomes, and mitochondria. Other endogenous sources of ROS include enzymes such as xanthine oxidase, amino-acid oxidases, lipoxygenase, and cyclo-oxygenase. Superoxide release, as a result of the activity of the latter two enzymes, could be especially important in prostate cancer because of prostaglandin biosynthesis (Schewe, 2002 2002 ). In addition, deregulated androgen signaling increases ROS in prostate cancer (Ripple et al., 1997 1997 ; Sun et al., 2001 2001 ; Tam et al., 2003 2003 ; Frohlich et al., 2008 2008 ; Basu et al., 2009 2009 ), which is consistent with the results of other studies that prostate cancer development is associated with oxidative stress (Paschos et al., 2013 2013 ). Antioxidants, especially carotenoids, play an important role in the regulation of the oxidative process. They have strong antioxidant effects due to their double-bonded structures, allowing for their delocalization of impaired electrons. In recent years, the interests in astaxanthin (AST; 3,3'-dihydroxy-β-β'-carotene-4,4'-dione) have been continuously growing. AST is a type of carotenoid, with antioxidant activity that is 100–1000 times greater than that of vitamin E. AST is commonly found in crustaceans such as shrimp and crab, as well as marine organisms such as salmon, krill, and algae (Barros et al., 2014 2014 ). As reported, dietary supplementation with AST has beneficial effects in the treatments of inflammation, cardiovascular disease, and oxidative damages, suggesting that AST is a functional food ingredient (Ohgami et al., 2003 2003 ; Pashkow et al., 2008 2008 ; Fassett and Coombes, 2009 2009 ; Preuss et al., 2009 2009 ). However, there are no reports about the effect of AST on oxidative stress in prostate cell lines, especially in prostate epithelial (RWPE-1) and prostate cancer (PC-3) cell lines treated with Cu 2+ . In this paper, the effects of AST on Cu 2+ -induced oxidative stress in prostate cells and prostate cancer cells are investigated. Materials and methods 1 Materials 2 RWPE-1 and PC-3 cell lines were obtained from the Institute of Biochemistry and Cell Biology, Shanghai Institute for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). Purified preparations of AST and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma (St. Louis, MO, USA). RPMI 1640 was purchased from GIBCO (Grand Island, NY, USA), and Annexin V-fluorescein isothiocyanate (FITC), ROS, SOD, catalase (CAT), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA) detection kits were obtained from Beyotime (Nantong, China). Other reagents used in our research were of analytical grade. Cell culture 2 Both cell lines were cultured in RPMI 1640 supplemented with 10% (0.1 g/ml) fetal bovine serum and 100 U/ml penicillin-streptomycin. The cells were maintained at 37 °C and 5% CO 2 in a humid environment, and stock solutions of AST and CuSO 4 were prepared. Determination of cell viability 2 Cells were harvested and counted, then seeded on a 96-well plate (1×10 5 PC-3 cells/well, 5×10 4 RWPE-1 cells/well) for 12 h. Different concentrations of CuSO 4 and AST were prepared with serial dilutions ranging from 100 to 180 μmol/L and 0.001 to 10 μmol/L, respectively (Shen, 2014 2014 ). AST was dissolved in dimethyl sulphoxide (DMSO) as a stock solution. For cell treatment, the AST was diluted in RPMI 1640 to obtain the final concentration, which was then added in sextuplicate to the cells (DMSO concentration in a dilution was < 0.01%). After a 24-h or 48-h incubation, 20 μl of 5 mg/ml MTT reagent was added to each well. The cells were further incubated for 4 h in the dark, before 150 μl of DMSO was added and fully vibrated. Absorbance was measured at 490 nm ( A 490 ) using a microplate reader (Thermal Lab system, Finland). The relative cell viability (%) was calculated as ( A 490 of treated samples/ A 490 of untreated samples)×100%. Annexin-V/PI double-staining and flow cytometry 2 Apoptosis was double-stained with Annexin V-FITC and propidium iodide (PI) according to the manufacturer’s instructions (Annexin V-FITC apoptosis detection kit) (Saha et al., 2003 2003 ). Live cells were shown as Annexin V-negative and PI-negative (A − /PI − , B3). Since cells with sustained plasma membrane integrity did not take up the PI, cells stained with Annexin V but not with PI were considered to be in the early stages of apoptosis (A + /PI − , B4). In the late apoptosis stage or the necrosis secondary stage to apoptosis, the cell membrane lost its integrity, allowing staining of the cells with both Annexin V and PI (A + /PI + , B2). Cells stained only with PI (A − /PI + , B1) were considered necrotic. Briefly, RWPE-1 (5×10 4 cells/well) and PC-3 (1×10 5 cells/well) were seeded into six-well plates and treated with Cu 2+ and AST for 48 h, washed with an ice-cold phosphate buffer solution (PBS), and subsequently stained with a 100-μl incubation buffer containing 50 μg/ml Annexin V-FITC and 50 μg/ml PI at 37 °C in the dark for 15 min. Treated cells were immediately analyzed in a flow cytometer (FC500, Beckman Coulter, USA). Intracellular ROS levels were measured using the cell permeable substrate 2',7'-dichlorofluorescein diacetate (DCFH-DA), a converted detectable fluorescent product (Rajeshkumar et al., 2015 2015 ). After 48 h incubation, the cells were washed with a cold PBS solution prior to adding 1 ml PBS containing 10 μmol/L DCFH-DA (excitation: 488 nm; emission: 525 nm), and then continually incubated for 20 min in the dark at 37 °C. The treated cells were immediately washed and resuspended in PBS before ROS production was measured by using flow cytometry (FC500, Beckman Coulter, USA). The data were analyzed with CellQuest software. Measurement of mitochondrial membrane potential 2 In order to measure the mitochondrial membrane potential (MMP), the cell suspension was incubated with 10 μmol/L Rh123 (excitation: 488 nm; emission: 534 nm) for 10 min in the dark at 37 °C, then immediately washed and resuspended in PBS for three separate times. The fluorescence was analyzed via a fluorospectrophotometer (Cary Eclipse, Varian, USA) (Brawek et al., 2010 2010 ). Measurements of MDA, SOD, CAT, and GSH-Px 2 RWPE-1 and PC-3 cells were seeded in 6-well plates at a density of 5×10 4 and 1×10 5 cells/well, respectively. Subsequently, the cells were treated with Cu 2+ and AST. After a 48-h incubation, cells were collected for measurements of MDA, SOD, CAT and GSH-Px using a detection kit according to the manufacturer’s instructions. The results were corrected for their protein levels based on the reference manual for a BCA protein assay kit (Changsha Auragene Bioscience, Hunan, China). Statistical analysis 2 All data were reported as the mean±standard deviation (SD). Comparison between groups was made by one-way analysis of variance (ANOVA), using SPSS software (Version 21.0). Each experiment was carried out in triplicate. A P -value of < 0.05 is considered statistically significant. Results 1 Effects of AST and Cu 2+ on viabilities of RWPE-1 and PC-3 cells 2 To investigate the cytotoxicities of AST and Cu 2+ on the viabilities of RWPE-1 and PC-3 cells, concentration-and time-dependent experiments were designed and carried out (Fig. 1 1 ). Cells were incubated with AST and Cu 2+ of increasing initial concentrations (0.01 to 10 μmol/L and 100 to 180 μmol/L, respectively) for either 24 or 48 h and the cell viability was determined by performing MTT assay. AST induced a significant increase of PC-3 cells. Treatment with higher concentrations of AST (more than 0.01 μmol/L) for 48 h resulted in significant increases of RWPE-1 cells. Supplementation of Cu 2+ to PC-3 cells resulted in an increase of cell viability after 24 h, while the growth was significantly suppressed after 48 h. For RWPE-1 cells, Cu 2+ treatment did not lead to significant decreases after 24 h. Moreover, it was shown that Cu 2+ treatment at low concentrations could promote cell growth (up to 129.8% for the viability) after 48 h, while 180 μmol/L Cu 2+ treatment inhibited the growth of cells to 70.6%. Eventually, the effects of different concentrations of AST (0.001, 0.1, and 10 μmol/L as low dose (LD), middle dose (MD), and high dose (HD), respectively) on the oxidative damage of 120 μmol/L Cu 2+ -treated prostate cells were studied. AST promoted PC-3 cell proliferation in 24 and 48 h. AST promoted RWPE-1 cell proliferation in 48 h while inhibited cell proliferation in high concentrations in 24 h. Cu 2+ increased cell viability of PC-3 cells in 24 h, while suppressed cell viability in 48 h. Cu 2+ treatment did not decreased viability of RWPE-1 cells in 24 h. Cu 2+ promoted RWPE-1 cell growth in low concentrations, but inhibited RWPE-1 cell growth in high concentrations in 48 h. Data are expressed as mean±SD ( n =3) Effects of AST on apoptosis of RWPE-1 and PC-3 cells in the presence of Cu 2+ 2 To further confirm effects of AST on apoptosis in both cells induced by Cu 2+ , Annexin V-FITC/PI double staining was performed. Representative dot plot diagrams and specific percentages, obtained through flow cytometry of the RWPE-1 and PC-3 cells treated with different concentrations of AST for 48 h, were shown in Figs. 2 2 and 3 3 and Table 1 1 . In the non-apoptotic, viable control cells, Annexin V-FITC-negative and PI-negative staining was found in B3. The Cu 2+ had a greater effect on the apoptosis of PC-3 cells because the percentage of cells in the late apoptosis stage or the necrosis secondary stage to apoptosis (A + /PI + , B2) increased significantly in the presence of Cu 2+ , which increased the dot numbers of RWPE-1 and PC-3 in B2 from 0.01% to 15.47% and 21.50%, respectively. However, similar results were obtained in AST treatment groups and there were no significant differences when compared with the Cu 2+ treatment group. The population of cells progressed to advanced apoptosis because Cu 2+ could induce cell apoptosis progression and the AST treatment seemed to have no clear indication of protective effects on apoptosis for the RWPE-1 or PC-3 cells. Results were presented as the mean±SD ( n =3). Different lowercase letters in the same column indicated significant differences, P < 0.05. CK: control group; Cu 2+ : 120 μmol/L Cu 2+ group; HD: 10 μmol/L AST+120 μmol/L Cu 2+ group; MD: 0.1 μmol/L AST+120 μmol/L Cu 2+ group; LD: 0.001 μmol/L AST+120 μmol/L Cu 2+ group. B1: necrotic cells; B2: late apoptotic cells; B3: live cells; B4: early apoptotic cells 2 Treatment 2 B1 (%) 2 B2 (%) 2 B3 (%) 2 B4 (%) RWPE-1 PC-3 RWPE-1 PC-3 RWPE-1 PC-3 RWPE-1 PC-3  CK 0.02±0.01 c 0.03±0.01 c 0.01±0.01 b 0.01±0.00 b 99.97±0.01 a 99.96±0.01 a 0.00±0.00 c 0.00±0.00 b  Cu 2+ 0.72±0.04 a 0.23±0.01 a 15.47±0.33 a 21.50±0.42 a 80.98±0.60 b 75.64±0.55 b 2.82±0.31 b 2.64±0.15 a  HD 0.51±0.10 b 0.15±0.01 b 16.28±1.12 a 21.63±2.14 a 79.35±1.43 b 75.57±2.11 b 3.85±0.35 a 2.65±0.16 a  MD 0.43±0.08 b 0.23±0.05 a 14.32±0.63 a 21.02±0.59 a 80.74±0.83 b 76.64±0.69 b 4.50±0.22 a 2.11±0.30 a  LD 0.51±0.04 b 0.23±0.03 a 16.28±0.22 a 23.38±1.75 a 79.15±0.35 b 74.24±1.84 b 4.06±0.19 a 2.15±0.11 a RWPE-1-CK: control group; RWPE-1-Cu 2+ : RWPE-1 cells treated with 120 μmol/L Cu 2+ ; RWPE-1-10 μmol/L: RWPE-1 cells treated with 10 μmol/L AST and 120 μmol/L Cu 2+ ; RWPE-1-0.1 μmol/L: RWPE-1 cells treated with 0.1 μmol/L AST and 120 μmol/L Cu 2+ ; RWPE-1-0.001 μmol/L: RWPE-1 cells treated with 0.001 μmol/L AST and 120 μmol/L Cu 2+ PC-3-CK: control group; PC-3-Cu 2+ : PC-3 cells treated with 120 μmol/L Cu 2+ ; PC-3-10 μmol/L: PC-3 cells treated with 10 μmol/L AST and 120 μmol/L Cu 2+ ; PC-3-0.1 μmol/L: PC-3 cells treated with 0.1 μmol/L AST and 120 μmol/L Cu 2+ ; PC-3-0.001 μmol/L: PC-3 cells treated with 0.001 μmol/L AST and 120 μmol/L Cu 2+ Effects of AST on ROS production in RWPE-1 and PC-3 cells treated with Cu 2+ 2 The cells were stained with DCFH-DA to examine a level of ROS before a fluorospectrophotometer was applied. The data in Fig. 4 4 show that the ROS level in PC-3 cells treated with 120 μmol/L Cu 2+ was higher than that in the untreated groups. The addition of AST significantly impaired Cu 2+ -induced ROS production. The HD-treated groups showed better scavenge ability resulting in a decrease of ROS production as compared with the control groups. In contrast, Cu 2+ induced a significant decrease of ROS accumulation in RWPE-1 cells, implying that Cu 2+ treatment can induce greater damage to PC-3 cells. Values are expressed as mean±SD ( n =3). ** P < 0.01 compared with control; ## P < 0.01 compared with Cu 2+ group Effects of AST on MMP levels in RWPE-1 and PC-3 cells treated with Cu 2+ 2 In order to characterize the changes in the mitochondrial events induced by Cu 2+ treatment, we observed the collapse of MMP in both cells with Rh123. When the MMP level decreased, the mitochondrial cell membrane was partially destroyed, so that Rh123 could be released into a matrix, causing the enhancement of the fluorescence intensity. As shown in Fig. 5 5 , the addition of different concentrations of AST (LD, MD, and HD) on RWPE-1 cells reversely reduced MMP; therefore, the fluorescence intensity decreased from 143.0% to 79.6%, 67.0%, and 26.0% of the level in the control group (100%), respectively. Moreover, the addition of AST resulted in MMP reduction in a concentration-dependent manner in PC-3 cells induced by Cu 2+ treatment. These results suggest that AST protected RWPE-1 cells against apoptosis induced by Cu 2+ by reversing the down-regulation of MMP. Values are expressed as mean±SD ( n =3). ** P < 0.01 compared with control; ## P < 0.01 compared with Cu 2+ group Effects of AST on MDA level and SOD activity in RWPE-1 and PC-3 cells treated with Cu 2+ 2 ROS overproduction could interfere with the intracellular redox homeostasis leading to oxidative stress. To determine whether AST was able to release cell oxidative stress induced by Cu 2+ , the intracellular MDA levels were determined after treatment as described above. As shown in Fig. 6 6 , exposure of both kinds of cells to Cu 2+ resulted in significant increases in the intracellular MDA levels, but the MDA levels in PC-3 cells were lower. However, an addition of LD AST effectively reduced the intracellular MDA generation in RWPE-1 cells, whereas MD and HD AST treatments resulted in a significant increase of MDA in PC-3 cells, indicating that AST accelerated oxidative stress induced by Cu 2+ in PC-3 cells. Values are expressed as mean±SD ( n =3). ** P < 0.01 compared with control; # P < 0.05, ## P < 0.01 compared with Cu 2+ group SOD is an important enzyme that is crucial for the prevention of diseases correlated to oxidative stress. As shown in Fig. 6 6 , the treatment with Cu 2+ resulted in a significant decrease of SOD activity in RWPE-1 and PC-3 cells to 57.1% and 53.8%, respectively. In contrast, HD treatment of AST significantly increased SOD activity, as compared with the control group, while LD treatment resulted in a significant decrease of SOD activity in PC-3 cells, indicating that a low concentration of AST may decrease its ability to scavenge ROS in PC-3 cells. Effects of AST on GSH-Px and CAT in RWPE-1 and PC-3 cells treated with Cu 2+ 2 GSH-Px enzyme, one of the most important antioxidant enzymes in the human body, functions as a detoxification of hydrogen peroxide. As shown in Fig. 6 6 , the treatment with Cu 2+ resulted in a significant decrease of GSH-Px activity in RWPE-1 and PC-3 cells, only reaching to 54.2% and 71.8%, respectively, of the level of the control group; the addition of AST significantly increased GSH-Px activity in RWPE-1 cells and displayed concentration-dependent decreases in both cells, as compared with the cells only treated with Cu 2+ . However, after AST treatment, the GSH-Px activity in PC-3 cells was lower than that after only Cu 2+ treatment in all groups. In summary, AST could increase the activity of GSH-Px in RWPE-1 cells treated with Cu 2+ , resulting in oxidative injury. CAT enzyme could remove hydrogen peroxide in vivo to protect the cells; thus, it is one of the key enzymes in the antioxidant enzyme system. As shown in Fig. 6 6 , treatment with Cu 2+ still resulted in a significant decrease of CAT in both types of cells, and the CAT level in RWPE-1 cells was significantly higher than that in PC-3 cells in all the treated groups. The addition of AST did not reverse CAT reduction in PC-3 induced by Cu 2+ , while the LD concentration of AST increased CAT level significantly in RWPE-1 cells. Discussion 1 The intrinsic balance could be interfered by several environmental stresses. ROS are among the most potent and omnipresent threats faced by any living organism. Intracellular accumulation of ROS such as superoxide anion, hydrogen peroxide, singlet oxygen, hydroxyl radical, and peroxy radical, usually results from toxic reactions or normal metabolic processes. These oxidative modified products might interfere with several functions in cancer cells, such as cell proliferations and genetic mutations. Inherent oxidative stress is a potent factor of angiogenesis and is considered to be involved in pathophysiology of cancers (Kuroki et al., 1996 1996 ). ROS, also termed oxidants, were common by-products of the standard aerobic cellular metabolism, and were continuously generated in cells and synchronously scavenged by an array of antioxidant mechanisms (Dröge, 2002 2002 ). Oxidative stress results from an imbalance between ROS production and the cell antioxidant defense capability, and causes reactions among those oxidative molecules and lipids, proteins and DNA (Montezano and Touyz, 2012 2012 ; Ambati et al., 2014 2014 ). Oxidative stress had been clarified as an interference factor in the prooxidant-antioxidant balance, resulting in potential cell damage (Sies, 1985 1985 ). In our results, Cu 2+ accelerated ROS production in prostate cancer cells. We determined that prostate cancer cells could be more sensitive to oxidative stress induced by Cu 2+ . AST is an orange-pinkish carotenoid extensively found in marine organisms, which have various biological activities such as antioxidant, anti-cancer, and anti-inflammatory activities (Preuss et al., 2009 2009 ; Yang et al., 2013 2013 ; Kimura et al., 2014 2014 ). Some results showed that AST protected neuronal cells against oxidative damage, and thus it has been a potent candidate for brain food (Liu and Osawa, 2009 2009 ), and additionally could protect PC12 cells against glucose toxicity (Zhang et al., 2015 2015 ). On the other hand, Cu 2+ could lead to various cell injuries by inducing oxidative stress and mitochondrial dysfunction (Murphy and Taiz, 1997 1997 ; Mira et al., 2002 2002 ; Ma et al., 2014 2014 ). Cu 2+ -induced production of hydrogen peroxide and hydroxyl radicals has been directly correlated with the damage to proteins and lipids (Wang et al., 2015 2015 ). However, there have been no reports about the effects of AST on oxidative stress induced by Cu 2+ in prostate cells. As an excellent anti-oxidant, AST suppressed ROS production in prostate cancer cells. Also, AST increased the ROS of normal prostate cells treated with Cu 2+ to the same level as the control. Our results confirmed that AST could regulate oxidant status in prostate cells. ROS initiated autocatalytic lipid peroxidation, and generated various potential genotoxic breakdown products, including alkoxyl radicals, peroxyl radicals, and aldehydes such as MDA. MDA has been used for many years as a convenient biomarker for lipid peroxidation because of its facile reaction with thiobarbituric acid to form an intensely colored chromogen (Rodríguez-Sureda et al., 2015 2015 ). In this study, Cu 2+ induced apoptosis along with the accumulation of intracellular ROS and increased MDA levels in both cells. In turn, AST treatment could reduce ROS and MMP accumulation and increase their MDA levels. According to our results, AST could have protective effects against Cu 2+ -induced oxidative damage. There were several enzyme systems (SOD, CAT, and GSH-Px) that catalyzed reactions clearing free radicals and ROS. These form the body’s endogenous defense mechanisms to protect against cell damages induced by free radicals. First, SOD, a major cytoplasmic antioxidant enzyme, catalyzed the dismutation of superoxide radicals to molecular oxygen and hydrogen peroxides which, in turn, were converted by either GSH-Px or CAT to water and oxygen, thus providing a combined enzymatic action against oxygen toxicity. The imbalance of SOD to GSH-Px and CAT resulted in the accumulation of H 2 O 2 which might participate in the Fenton’s reaction and consequently lead to the formation of noxious hydroxyl radicals (de Haan et al., 1996 1996 ). The present study investigated the antioxidant enzyme expression in long-term cultures of prostate cells. Considering the results, Cu 2+ could significantly reduce SOD, GSH-Px, and CAT in both cells as shown in Fig. 6 6 . The addition of AST decreased the SOD, GSH-Px, and CAT activities in PC-3 cells induced by Cu 2+ , while they were up-regulated in RWPE-1 cells. It is speculated that AST could be able to protect RWPE-1 cells against Cu 2+ -induced damage on the cell antioxidant enzyme system, and to a certain extent, promote enzyme activity. Conclusions 1 Our data clearly demonstrated that AST has a potential ability to inhibit Cu 2+ -induced oxidative stress in RWPE-1 cells. The underlying pathway was associated with the properties of AST to scavenge ROS, to restore MMP, and to increase antioxidant enzyme activity. However, AST induced the damage of the antioxidant system induced by Cu 2+ in PC-3 cells, specifically through its regulation on the levels of CAT, GSH-Px, and MMP. 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