Breast Cancer Res Treat (2015) 150:523–534 DOI 10.1007/s10549-015-3329-z

PRECLINICAL STUDY

Differential effects of superoxide dismutase and superoxide dismutase/catalase mimetics on human breast cancer cells Manisha H. Shah1 • Guei-Sheung Liu2,3 • Erik W. Thompson1 • Gregory J. Dusting2,3 Hitesh M. Peshavariya2,3

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Received: 20 October 2014 / Accepted: 3 March 2015 / Published online: 21 March 2015 Ó Springer Science+Business Media New York 2015

Abstract Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H2O2) have been implicated in development and progression of breast cancer. In the present study, we have evaluated the effects of the superoxide dismutase (SOD) mimetic MnTmPyP and the SOD/catalase mimetic EUK 134 on superoxide and H2O2 formation as well as proliferation, adhesion, and migration of MCF-7 and MDAMB-231 cells. Superoxide and H2O2 production was examined using dihydroethidium and Amplex red assays, respectively. Cell viability and adhesion were measured using a tetrazolium-based MTT assay. Cell proliferation was determined using trypan blue assay. Cell cycle progression was analyzed using flow cytometry. Clonal expansion of a single cell was performed using a colony formation assay. Cell migration was measured using transwell migration assay. Dual luciferase assay was used to determine NF-jB reporter activity. EUK 134 effectively reduced both superoxide and H2O2, whereas MnTmPyP removed superoxide but enhanced H2O2 formation. EUK 134 effectively attenuated viability, proliferation, clonal expansion, adhesion, and migration of MCF-7 and MDA-MB-231 cells. In contrast, MnTmPyP only

& Hitesh M. Peshavariya [email protected] Manisha H. Shah [email protected] 1

Victorian Breast Cancer Research, Invasion and Metastasis Unit, St. Vincent’s Institute of Medical Research, 41 Victoria Parade, Fitzroy, VIC 3065, Australia

2

Department of Ophthalmology, Centre for Eye Research Australia, University of Melbourne, Royal Victorian Eye and Ear Hospital, 1/32 Gisborne Street, East Melbourne, VIC 3002, Australia

3

O’Brien Institute, Fitzroy, VIC 3065, Australia

reduced clonal expansion of MCF-7 and MDA-MB-231 cells but had no effect on adhesion and cell cycle progression. Tumor necrosis factor-alpha-induced NF-jB activity was reduced by EUK 134, whereas MnTmPyP enhanced this activity. These data indicate that the SOD mimetic MnTmPyP and the SOD/catalase mimetic EUK 134 exert differential effects on breast cancer cell growth. Inhibition of H2O2 signaling using EUK 134-like compound might be a promising approach to breast cancer therapy. Keywords Breast cancer  Proliferation  Superoxide dismutase  Catalase  Superoxide  H2O2 Abbreviations EUK 134 Eukaration-134; chloro[[2,20 -[1,2ethanediylbis[(nitrilo-jN)methylidyne]] bis[6-methoxyphenolato-jO]]]-manganese H2O2 Hydrogen peroxide HMEC Human dermal microvascular endothelial cells MnTmPyP Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride NF-jB Nuclear factor kappa B ROS Reactive oxygen species SOD Superoxide dismutase TNFa Tumor necrosis factor-alpha

Introduction Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H2O2) are produced by all aerobic cells during metabolism under normal oxygen conditions. Physiologically, ROS can be cleared from the cellular system through either enzymatic or non-enzymatic

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pathways such as glutathione (GSH), thioredoxin (Trx), superoxide dismutase (SOD), catalase, and peroxidases. Thus, superoxide is dismuted into H2O2 by SOD which in turn is converted into water and oxygen by catalase and glutathione peroxidase enzymes [7, 37]. Defined generation of ROS represents the intracellular redox signaling which is important for cell proliferation, migration, and survival [7, 31, 38]. Mounting evidence suggests that cancer cells produce increased levels of ROS compared to normal cells and these overwhelm the antioxidant defense system [33, 36, 37]. Excessive generation of ROS in cancer cells favors tumor growth for the cells, develop resistance to apoptosis, and ROS promote genetic instability, cell proliferation, migration, and metastasis [35, 37, 38]. Inhibition of ROS is a crucial objective in order to reduce the growth and survival of cancer cell. Hydrogen peroxide is well documented as secondary messenger molecule in the intracellular propagation of oncogenic growth signals induced by growth factors and oncogenes [31, 32]. Several investigators have reported that many cancer cell types including breast cancer cells exhibit increased H2O2 levels compared to their normal counterparts [9, 25, 26, 36]. In breast cancer cells, intracellular H2O2 levels are found to be higher due to altered SOD expression and activity, and a decreased expression of catalase [12, 30, 34]. In addition, a recent study showed decreased bioactivity of catalase in human breast cancer tissues compared to their normal counterparts [30], which ultimately lead to increased H2O2 levels and enhanced cell survival pathways such as Akt and ERK1/2 and NF-jB [3, 8, 16, 30]. It would be of great interest to reduce breast carcinogenesis via modulation of intracellular H2O2. To this end, several studies revealed that overexpression of either SOD [39, 40] or catalase [9, 23] inhibits breast cancer cell growth. However, the mechanisms involved by overexpressing SOD and catalase in reduction of breast cancer growth may differ. For instance, SOD overexpression enhances pro-oxidant H2O2 levels beyond the threshold of cancer cells and leads to cytotoxic effects on breast cancer cells [39, 40], whereas overexpression of catalase reduces intracellular H2O2, and blocks several cell survival and proliferation pathways [9, 23, 30]. Although administration of these natural SOD and catalase enzymes reduced cancer progression, there are several limitations to efficacy such as their large size (which limits cell entry and may elicit an immune response), their short circulating half-life, and expensive production which restricts their use therapeutically. To overcome some of these limitations, low molecular weight SOD and SOD/catalase mimetics have been developed [2, 28, 29]. In the present study, we investigated the effects of the low molecular weight, cell permeable SOD mimetic

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MnTmPyP and the SOD/catalase mimetic EUK 134 on proliferation, migration adhesion, and NF-jB signaling in human breast cancer cells.

Materials and methods Cell culture The less invasive human breast cancer epithelial MCF-7 and highly invasive mesodermal MDA-MB-231 cell lines were procured from the American Type Culture Collection (Manassas, VA). Monolayer cultures of MCF-7 and MDAMB-231 cells were maintained at 80 % confluency in RPMI 1640 (GibcoÒ Life Technologies, Victoria, Australia) supplemented with 10 % fetal bovine serum (FBS; GibcoÒ Life Technologies, Victoria, Australia) media. Human microvascular endothelial cells (HMECs) were a kind gift from Centre for Disease Control and Prevention, Atlanta, USA. The cells were cultured in EGM-MV Bullet Kit (hydrocortisone, gentamicin, amphotericin-B, human epidermal growth factor, human fibroblast growth factorbasic, bovine brain extract) with 5 % FBS (Lonza, Victoria, Australia) [19]. All cell lines were maintained at 37 °C in an atmosphere of 95 % air and 5 % CO2. All chemicals were purchased from Sigma-Aldrich otherwise stated. Superoxide measurement using dihydroxyethidium assay Superoxide was measured from cell-free xanthine/xanthine oxidase system as described previously [20]. Briefly, dihydroxyethidium (DHE 25 lM; Molecular Probe Life Technologies, Victoria, Australia) was incubated with xanthine (100 lM) in PBS (0.1 M, pH 7.4) in the absence or presence of either Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin pentachloride (MnTmPyP 25 lM; Cayman Chemical Company, Michigan, USA) or EUK 134 (chloro[[2,20 -[1,2ethanediylbis[(nitrilo-jN)methylidyne]]bis[6-methoxyphenolato-jO]]]-manganese; 25 lM, Cayman Chemical Company, Michigan, USA). After addition of xanthine oxidase (0.03 U/ml), fluorescence intensity was measured for 1 h at 37 °C using a Polarstar microplate reader (BMG laboratories, Germany) at excitation and emission wavelengths 480 ± 10 and 570 ± 10 nm, respectively, under dark adapted conditions as described previously [20]. Superoxide levels in MCF-7 and MBA-MB-231 were detected using DHE assay as described previously [20, 21]. Briefly, cells were trypsinised and re-suspended in KrebsHEPES buffer (in mM: NaCl 98.0, KCl 4.7, NaHCO3 25.0, MgSO4 1.2, KH2PO4 1.2, CaCl2 2.5, D-glucose 11.1, and Hepes-Na 20.0) containing DHE (25 lM). In some cases,

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cells were treated with either MnTmPyP (25 lM) or EUK 134 (25 lM). Fluorescence was then measured at 37 °C with excitation and emission wavelengths at 480 ± 10 and 570 ± 10 nm, respectively, using a Polarstar microplate reader. Hydrogen peroxide measurement by Amplex red assay Hydrogen peroxide (H2O2) levels were measured in cellfree xanthine/xanthine oxidase. Xanthine (100 lM) and xanthine oxidase (0.03 U/ml) in PBS (0.1 M, pH 7.4) were incubated with AmplexÒ Red reagent (10 lM) and horseradish peroxide (HRP; 0.1 U/ml) for 1 h in the presence of either MnTmPyP (25 lM) or EUK 134 (25 lM). Fluorescence was then measured with excitation and emission wavelengths at 550 nm and 590 nm, respectively, using a Polarstar microplate reader at 37 °C under dark adapted conditions. Extracellular H2O2 levels in MCF-7 and MBA-MB-231 were detected using AmplexÒ Red assay according to manufacturer’s instructions (Molecular Probe Life Technologies, Victoria, Australia). Following treatments, trypsinised cells were suspended in Krebs-HEPES buffer containing AmplexÒ Red reagent (10 lM) and HRP (0.1 U/ml). Fluorescence was then measured at excitation and emission wavelengths at 550 and 590 nm, respectively, using a Polarstar microplate reader at 37 °C [21].

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Australia, Australia). Cells were synchronized by being starved overnight in serum-free media. Next day, proliferation was induced with growth medium containing 10 % FBS in the absence or presence of either MnTmPyP (25 and 100 lM) or EUK 134 (25 and 100 lM) for 48 h. Following the incubation, the cells were trypsinised and resuspended in complete growth medium. The cell suspension was then mixed with an equal volume of trypan blue (0.4 %) solution to determine cell numbers using hemocytometer. Analysis of cell cycle distribution The effect of EUK 134 and MnTmPyP on cell cycle distribution was determined by flow cytometry as described previously [14]. Briefly, 5 9 105 cells were seeded in 10-cm dishes (BD FalconTM, New South Wales, Australia, Australia), allowed to adhere by overnight incubation. The cells were subjected to starvation with medium free of FBS for 30 h followed by culturing in medium containing 10 % FCS for 48 h in the presence of a desired concentration of MnTmPyP (25 and 100 lM) or EUK 134 (25 and 100 lM) for 48 h. Both floating and adherent cells were collected, washed with PBS, and fixed with 70 % ethanol. The cells were then treated with RNaseA (0.1 U/ml) and propidium iodide (1 lg/ml) for 30 min as described previously [14]. The stained cells were analyzed using a Coulter Epics XL Flow Cytometer (Beckman Coulter, Miami, FL, USA).

Cell viability assay

Colony formation assay

MCF-7 (5 9 103), MDA-MB-231 (5 9 103), and HMECs (5 9 103) were seeded in 96-well tissue culture plates. After 24 h, cells were replenished with fresh growth medium containing 10 % FBS in the absence or presence of either MnTmPyP (0.5–100 lM) or EUK-134 (0.5–100 lM) for 48 h. MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide], a tetrazolium-based assay was also performed to determine the cell viability. Following 48 h incubation, cells were washed with PBS and incubated with 100 ll of 0.4 mg/ml MTT solution prepared in RPMI for 1 h at 37 °C 5 % CO2. The formation of formazan product was solubilized with dimethyl sulfoxide (DMSO), and the absorbance was then determined at 570 nm using a SpectraMax Microplate reader (Molecular Devices, Australia).

Cells were trypsinised to produce a single cell suspension. MCF-7 (0.25 9 104) and MDA-MB-231 (0.1 9 104) cells were seeded in 6-well tissue culture plates (BD FalconTM, New South Wales, Australia) and allowed to adhere by overnight incubation at 5 % CO2 and 37 °C. Cells were then treated with either MnTmPyP (25 and 100 lM) or EUK 134 (25 and 100 lM) for every 72 h for 9 days. At the end of the experiment, the growth medium was removed from the plates and washed twice with PBS. Cell colonies were then fixed and stained by adding 1.5 ml of solution containing 25 % formalin and 0.5 % crystal violet fixing–staining solution. The plates were incubated for 30 min at room temperature and then washed gently with tap water. The air-dried plates were stored at room temperature until scanned and a colony count performed. The number of colonies was counted from entire well using Image J software.

Cell proliferation assay Breast cancer cell proliferation was measured using trypan blue cell counting assay. MCF-7 (5 9 104), MDA-MB-231 (5 9 104), and HMECs (1 9 105) were seeded in six-well tissue culture plates (BD FalconTM, New South Wales,

Transwell migration assay The transwell migration assay was performed using 24-well TranswellÒ permeable inserts containing polycarbonate

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membranes with 6.5 mm diameter, 8 lm pore size, and 0.3 cm3 bottom area (CLS3422; Sigma-Aldrich). MCF-7 and MDA-MB-231 cells were trypsinised and 1 9 104 live cells/ml re-suspended in the serum-free RPMI medium. While the cell suspensions were prepared, 600 ll of the chemo attractant (RPMI supplemented with 1 % FBS) was dispensed into each well of the 24-well TranswellÒ plates and incubated at 37 °C for 1 h. The TranswellÒ inserts were placed in the bottom wells containing pre-warmed chemo attractant, and 1 9 104 cells (600 ll from the cell suspension) were applied. In some wells, cells were treated with MnTmPyP (25 lM) or EUK 134 (25 lM). The TranswellÒ plates were then incubated at 37 °C for 16 h. The medium in the inserts was then removed, and the membranes were washed twice in PBS. The membranes were then fixed in cold methanol for 1 min. Methanol was then discarded, and the membranes were allowed to air dry. The membrane was washed three times and stained with 0.1 % of Propidium iodide (PI) solution for 1 min, followed by three washes in PBS to remove excess stain. The non-migrated cells on the top side of the membranes were gently wiped off using wet cotton swabs. The membranes were left to air dry and carefully peeled off from the inserts and placed on microscopic slides with the migrated cells facing down, mounted in DEPEX mounting medium. Quantification was done by imaging ten random 910 high-power fields per membrane using Olympus inverted light/fluorescent microscope (Model No. IX81), and the number of migrated cells was counted using Image J software. Adhesion assay Attachment assays were performed as follows. Briefly, the 96-well plates were coated with 100 lg/ml type-1 collagen (PureColÒ; Advanced BioMatrix, California, USA) or 10 lg/ml fibronectin (Merck Millipore, Victoria, Australia) overnight at room temperature. Cells were trypsinised, and single cell suspension was prepared in RPMI medium containing 0.5 % FBS. Cell suspensions were then added in triplicates to the wells at a concentration of 1 9 104 cells/well of the same medium. In some wells, cells were treated with MnTmPyP (25 lM) or EUK 134 (25 lM). Following 1 h incubation at 37 °C, non-adherent cells were removed by three washes of PBS. The colorimetric MTT assay was used to determine the remaining number of adherent cells. NF-jB-binding luciferase reporter assay To test the transcriptional activity of NF-jB, a pGL3 luciferase-based reporter vector (pGL3-NF-jB) containing 6 9 NF-jB-binding site was used to perform the assay

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(a kind gift from Prof Ming-Hong Tai, Institute of Biomedical Science, National Sun Yat-Sen University, Taiwan). HMECs and MDA-MB-231 were seeded in 12-well plates (BD FalconTM, New South Wales, Australia) and transfected using Lipofectamine 2000 (Life Technologies, Victoria, Australia) 400 ng of pGL3-NF-jB and 100 ng of a reference plasmid (pRL-SV40; Promega, Wisconsin, USA) simultaneously. Luciferase assay was performed using dual luciferase kit from Promega (Promega, Wisconsin, USA) as described previously [22].

Statistical analysis Data are expressed as mean ± standard error of the mean (SEM). The mean data were analyzed with Student’s t test or ANOVA followed by post hoc Tukey analysis. A value of P \ 0.05 was regarded as statistically significant.

Results Effect of SOD versus SOD/catalase mimetic on superoxide and H2O2 The effects of the SOD mimetic MnTmPyP and SOD/catalase mimetic EUK 134 on superoxide anion and H2O2 production were examined using DHE and Amplex red assays, respectively, under cell-free and whole cell systems. Superoxide was measured in a cell-free xanthine/ xanthine oxidase system. Incubation of superoxide probe DHE in a solution containing xanthine (100 lM) and xanthine oxidase (0.03 U/ml) caused a substantial increase in fluorescence above background, which was markedly reduced by either MnTmPyP (Fig. 1a) or EUK 134 (Fig. 1a). Similarly, exposure of MCF-7 and MDA-MB231 breast cancer cell lines to DHE increased fluorescence, which was remarkably decreased by the cell permeable MnTmPyP (Fig. 1b) and EUK 134 (Fig. 1b), suggesting that both MnTmPyP and EUK 134 scavenge the superoxide generated by cell-free and cellular systems. Xanthine (100 lM) and xanthine oxidase (0.03 U/ml) reaction lead to spontaneous dismutation of superoxide to H2O2 and enhanced Amplex Red fluorescence. This effect was abolished by SOD/catalase mimetic EUK 134 (Fig. 1c), whereas MnTmPyP (Fig. 1c) substantially enhanced H2O2 production due to its SOD-like activity. Similarly, EUK 134 (Fig. 1d) substantially decreased extracellular production of H2O2 in MCF-7 and MDA-MB-231 cells, whereas MnTmPyP (Fig. 1d) enhanced production of H2O2. Thus, SOD/catalase mimetic EUK 134 reduces both superoxide and H2O2 production, whereas SOD mimetic

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Fig. 1 Effects of MnTmPyP and EUK 134 on superoxide and H2O2 generation in cell-free and cellular systems. DHE (25 lM; a) or Amplex Red (10 lM; c) was incubated with xanthine (100 lM) and xanthine oxidase (0.03 U/ml) prepared in PBS solution (pH 7.4) in the absence or presence of SOD mimetic MnTmPyP (25 lM) or SOD/catalase mimetic EUK 134 (25 lM). Similarly, MCF-7 and MDA-MB-231 cell suspensions prepared in Krebs-HEPES buffer were incubated with either DHE (25 lM; b) or Amplex Red (10 lM; d) and HRP (0.1 U/ml) in the absence or presence of MnTmPyP (25 lM) or EUK 134 (25 lM). Fluorescence was recorded for 1 h, and values (mean ± SEM from 3 to 4 experiments) represent relative fluorescence units (RFU). Asterisk denotes a P B 0.05 value following oneway ANOVA with Tukey– Kramer post hoc analysis

MnTmPyP reduces superoxide but enhances H2O2 production. SOD/catalase mimetic but not SOD mimetic reduces cell proliferation and viability The scavenging of H2O2 has been shown to reduce proliferation and viability of breast cancer and other cells [8, 23]. We examined the effect of the SOD mimetic MnTmPyP and SOD/catalase mimetic EUK 134 on the viability of MCF-7 and MDA-MB-231 cells. Treatment with EUK 134 for 48 h reduced viability of both MCF-7 and MDA-MB-231 cells (Fig. 2b, d) in a concentrationdependent manner with an IC50 of 25 lM. In contrast, MnTmPyP only reduced MCF-7 cells viability by *25 % at 50 and 100 lM (Fig. 2a), whereas no effect was observed on MDA-MB-231 cells viability. The effect of EUK 134 and MnTmPyP on cell proliferation was further confirmed using cell counting assay. As shown in Fig. 2e, MnTmPyP failed to inhibit proliferation of MCF-7 and MDA-MB-231 at concentrations of 25 and 100 lM. Interestingly, MnTmPyP at 25 lM showed small but significantly increased proliferation of MCF-7 cells. In contrast, EUK 134 markedly reduced the

proliferation of both the breast cancer cell types (Fig. 2f). These sets of data suggest that H2O2 is important for breast cancer cell proliferation and survival. SOD/catalase mimetic arrests the cell cycle and induces apoptosis To gain insights into the suppressive effect of EUK 134 on cancer cell proliferation, we determined its effect on cell cycle distribution by flow cytometric analysis. MCF-7 and MDA-MB-231 were synchronized and exposed to two different concentrations (25 and 100 lM) of MnTmPyP and EUK 134 for 48 h. As shown in Fig. 3a, b, MnTmPyP revealed no effect on any phase of the cell cycle. In contrast, exposure of MCF-7 and MDA-MB-231 to 100 lM EUK 134 increased the cells in sub-G0 fraction (Fig. 3c, d), an indicator of DNA fragmentation and apoptosis. Interestingly, treatment of MCF-7 and MDA-MB-231 with 25 lM EUK 134 caused an increase in G2-M fraction compared to their respective controls without affecting sub-G0 fraction (Fig. 3c, d). These results indicate that the EUK-134 mediated inhibition of breast cancer cell proliferation was associated with G2-M phase cell cycle arrest as well as DNA fragmentation and apoptosis.

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Fig. 2 Effects of MnTmPyP and EUK 134 on viability and proliferation of breast cancer cells. MCF-7 or MDA-MB-231 was incubated with various concentrations of SOD mimetic MnTmPyP or SOD/catalase mimetic EUK 134 for 48 h. Cells viability was assessed by MTT (a–d), and cell proliferation was assessed by trypan blue cell counting assay (e, f). Values (mean ± SEM from five experiments) are expressed as percentage of the control (a–d) or viable cell counts (e, f). Asterisk denotes a P B 0.05 value following oneway ANOVA with Tukey– Kramer post hoc analysis

SOD/catalase and SOD mimetics reduce colony formation

SOD/catalase and SOD mimetics reduce cell adhesion and migration

The clonogenic assay assesses the capacity of a single cell to form a colony of cells over a given period of time. We performed a clonogenic assay to evaluate the effects of MnTmPyP and EUK 134 on the colony formation ability of MCF-7 and MDA-MB-231. Both MCF-7 and MDA-MB231 form colonies from a single cell in 9 days. As shown in Fig. 4a, b, treatment with SOD mimetic MnTmPyP (25 and 100 lM) marginally reduced colony formation of breast cancer cells MCF-7 and MDA-MB-231, whereas EUK 134 abolished clonal expansion of these cells at both concentrations (Fig. 4c, d). From these observations, we conclude that EUK 134 inhibits more effectively the clonal expansion of breast cancer cells than MnTmPyP.

Adhesion of breast cancer cells to extracellular matrix and basement membranes are considered to be the initial steps in the process of tissue invasion by metastatic tumor cells. We examined the influence of MnTmPyP and EUK 134 on the adhesion activities of MCF-7 and MDA-MB-231 cells to the substrates pre-coated with collagen type I and fibronectin, which are extracellular matrix components. The adhesion activity of non-metastatic MCF-7 cells on collagen type I and fibronectin remained unchanged with the acute treatment of MnTmPyP or EUK 134 (Fig. 5a). In contrast, the adhesion activity of highly metastatic MDAMB-231 cells was markedly reduced when treated with MnTmPyP and EUK 134 (Fig. 5b).

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Fig. 3 Effects of MnTmPyP and EUK 134 on breast cancer cell cycle progression. MCF-7 or MDA-MB-231 cells were seeded in 10-cm plate and incubated with different concentrations (25 and 100 lM) of MnTmPyP or EUK 134. Representative cell cycle profiles of 48-htreated MnTmPyP (a) or EUK 134 (c) and their control cells. Cells

were acquired by Coulter Epics XL Flow Cytometer software and analyzed with ModFit II software as described in ‘‘Materials and Methods’’ section. Data b, d are percentage of gated total cells (mean ± SEM from three experiments). *P B 0.05 value following a one-way ANOVA with Tukey–Kramer post hoc analysis

Cell migration is a measure of the metastatic potential of breast cancer cells. We examined the migration of human breast cancer cell lines MCF-7 and MDA-MB-231 in a transwell migration assay in the presence or absence of MnTmPyP or EUK 134. MCF-7 cells were unable to migrate in the absence of serum, whereas MDA-MB-231 cells show migration in serum-free medium (Data not shown). Therefore, the effects of MnTmPyP and EUK 134 on MCF-7 cell migration were performed in the presence of 1 % serum. As

shown in Fig. 5c, MCF-7 cells migration was significantly reduced by EUK 134, whereas MnTmPyP-treated cells remained unaffected. Interestingly, the treatment of MnTmPyP significantly enhanced migration of MDA-MB231 cells (Fig. 5d), whereas EUK 134 markedly decreased the cell migration (Fig. 5d). These findings suggest that elevation of intracellular levels of H2O2 potentiated the migration of metastatic breast cancer cells, and removal of H2O2 via EUK 134 inhibits this process.

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Fig. 4 Effects of MnTMPyP and EUK 134 on breast cancer cells colony formation. MCF-7 or MDA-MB-231 cells were seeded in a 6-well plate and incubated with two different concentrations (25 lM and 100 lM) of MnTmPyP (a) or EUK 134 (Fig. 3c). Anchoragedependent colony formation was accomplished as described in ‘‘Materials and Methods’’ section. a and c show representative colony formation after MnTmPyP or EUK 134 treatments and their control cells. The number of colonies formed was counted and results are (mean ± SEM from five experiments) expressed as percentage of the control (b, d). *P B 0.05 value following a one-way ANOVA with Tukey– Kramer post hoc analysis

Fig. 5 Effects of MnTmPyP and EUK 134 on breast cancer cells adhesion and migration. MCF-7 or MDA-MB-231 was allowed to adhere on extracellular matrix proteins such as Collagen-I and fibronectin for 1 h in the absence and presence of MnTmPyP (25 lM; Fig. 4a) or EUK 134 (25 lM; b). MTT assay was performed to determine the adherent cells. Absorbance was measured at 490 nm wavelength and values (mean ± SEM from four experiments) are expressed as percentage of the control. Similarly, MCF-7 or MDAMB-231 migration was determined using TranswellÒ chamber assays.

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In all cases, growth medium containing 1 % fetal bovine serum was placed in the lower chamber. Cancer cells were incubated with MnTmPyP (25 lM; c, d) or EUK 134 (25 lM; c, d) and allowed to migrate into the lower chamber for 16 h. A number of migrated cells were quantified from ten random 109 high-power fields using Image J software (mean ± SEM from three to four experiments) and expressed as percentage of the control. *P B 0.05 value following a one-way ANOVA with Tukey–Kramer post hoc analysis

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SOD/catalase mimetic inhibits endothelial cell proliferation Angiogenesis is a key component of breast cancer growth, invasion, and metastasis. Therefore, inhibition of angiogenesis is an attractive strategy for the treatment of cancer. Endothelial cells are major players in angiogenesis. Previously, we have shown that exogenous addition of catalase reduced endothelial cell proliferation, whereas PEG-SOD and SOD failed to do so [18]. Similarly, the SOD/catalase mimetic EUK 134 decreased proliferation (Fig. 6a) and viability (Fig. 6b) of endothelial cells, whereas the SOD mimetic MnTmPyP showed no effect on cell proliferation and viability compared to their respective controls (Fig. 6a, b). SOD/catalase mimetic inhibits TNFa-induced NF-jB reporter activity Alteration in cellular H2O2 level modulates various intracellular signaling pathways such as nuclear factor kappa B (NF-jB). To examine the effect of MnTmPyP and EUK 134 on tumor necrosis factor-alpha (TNFa)-induced NFjB downstream signaling process, we transfected breast cancer and endothelial cells with human NF-jB-binding sites cloned into pGL3 luciferase reporter vector (pGL3/ NF-jB). As expected, TNFa markedly increased the NFjB reporter activity in MDA-MB-231 (Fig. 7a) and HMECs (Fig. 7b) TNFa-induced NF-jB reporter activity was reduced by EUK 134 in both cell types (Fig. 7a, b). In contrast, MnTmPyP further enhanced TNFa-induced NFjB reporter activity in MDA-MB-231 (Fig. 7a) and

Fig. 6 Effects of SOD mimetic MnTmPyP and SOD/catalase mimetic EUK 134 on endothelial cell proliferation. HMECs were treated with either SOD mimetic MnTmPyP (25 lM) or SOD/catalase mimetic EUK 134 (25 lM) for 48 h. Cell proliferation was assessed by trypan blue cell counting (a) or the MTT cell viability assay (b). Values (mean ± SEM from four experiments) are expressed as cell counts (a) or absorbance at 570 nm (b) expressed as percentage of the control. *P \ 0.05 value following a one-way ANOVA with Tukey– Kramer post hoc analysis

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HMECs (Fig. 7b). These results suggest that the removal of superoxide by SOD mimetic MnTmPyP enhances H2O2 levels and NF-jB activity, whereas the SOD/catalase mimetic EUK 134 effectively reduced both superoxide and H2O2 levels, and ultimately reduced NF-jB activity.

Discussion Given that superoxide is the first species in the cascade of ROS signaling molecules in a number of diseases and SOD is the first line of cellular defense, we investigated the effect of SOD- and SOD/catalase-mimetics on breast cancer growth. EUK 134 effectively reduced both superoxide and H2O2, whereas MnTmPyP decreased superoxide production but enhanced H2O2 production in both cell-free systems and breast cancer cells. Removal of H2O2 by the SOD/catalase mimetic EUK 134 reduced the viability, proliferation, clonal expansion, adhesion, and migration as well as arrested cells in G2-M cell cycle phase. In contrast, SOD mimetic MnTmPyP at a higher concentration had only a minor effect on viability and clonal expansion but no effect on proliferation and adhesion of breast cancer cells. In fact, MnTmPyP enhanced proliferation of MCF-7 and migration of MDA-MB-231 cells. It is well documented that H2O2 has dual role in cell signaling [7, 31, 32]. H2O2 at low concentration enhanced proliferation and migration of mammalian cells, whereas at high concentration it induces cell senescence and apoptosis [5, 7]. In the present study, the SOD mimetic MnTmPyP increased H2O2 levels (Fig. 1d) and affected the viability of cancer cells only at a higher concentration. This may be due to cytotoxic effect of H2O2 generated by SOD-like activity of MnTmPyP. Similar mechanism may imply for clonal expansion assay where initial cancer cells numbers are low and thus both concentrations of MnTmPyP (25 and 100 lM) generate excessive H2O2 to induce cytotoxicity and reduce clonal expansion of breast cancer cells. This accord with recent study which showed the pro-oxidant effects of SOD mimetic in the presence of vitamin C reduce growth and viability of breast cancer cells [6, 42]. Extracellular matrix components such as collagens, fibronectin, and hyaluronan are important in breast cancer cell adhesion, progression, and metastasis [17]. Breast cancer progression and metastasis also required ROS [4, 13]. For instance, it has been shown that antioxidant Nacetyl-L-cysteine (NAC) inhibits radiation-induced adhesion of highly metastatic breast cancer cells MDA-MB-231 [4]. We observed no difference in adhesion of MCF-7 to collagen-1 and fibronectin in the presence of MnTmPyP and EUK-134. In contrast, adhesion of MDA-MB-231 to collagen-1 and fibronectin was significantly reduced by acute treatment with both MnTmPyP and EUK-134. The

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Fig. 7 Effects of SOD mimetic MnTmPyP and SOD/catalase mimetic EUK 134 on NF-jB signaling. TNFa (20 ng/ml)-induced NF-jB-binding activity was measured using reporter-luciferase assay in the absence and presence of SOD mimetic MnTmPyP (25 lM) or SOD/catalase mimetic EUK 134 (25 lM) incubated for 24 h in MDA-MB-231(a) and HMECs (b). Basic pGL3 vector without any

insert was used as a blank control. Luciferase activity is expressed as relative luminescence units (RLU; mean ± SEM from four experiments) and is normalized to control without TNFa stimulation. *P \ 0.05 value following a one-way ANOVA with Tukey–Kramer post hoc analysis

effects of MnTmPyP on reduction in cell adhesion may be via sub-lethal H2O2 production. Indeed, Paquette et al. have shown that H2O2 inhibits cancer cells adhesion to extracellular matrix components in a dose-dependent manner [13]. Redox signaling has been implicated in cancer cell migration and invasion, initial steps of the metastatic cascade [35]. Inhibition of MnSOD and reduction of H2O2 have been shown to block the migration of metastatic breast cancer cell line MDA-MB-231 [12]. In addition, removal of H2O2 by catalase overexpression inhibits migration of MCF-7 suggesting that H2O2 signaling is required for cell migration [9]. Consistent with these findings, we found that removal of H2O2 by SOD/catalase mimetic EUK-134 inhibits migration of MCF-7 and MDA-MB-231. Interestingly, our study also highlights enhanced migration in MnTmPyP-treated MDA-MB-231, and this effect may be due to SOD-like activity (increased H2O2 production) of MnTmPyP. H2O2 is an important mediator for the activation of MAPK-dependent signaling pathways and transcription factors such as NF-jB and AP-1, required for cancer cell survival and progression [16, 31, 32]. More specifically, activation of NF-jB pathway protects cancer cells from apoptosis and has been linked to tumor progression and metastasis [1, 10, 24], therefore NF-jB pathway could be an important target to suppress breast carcinogenesis. We investigated effects of MnTmPyP and EUK-134 on NF-kB signaling and observed differential effects on TNFa-induced NF-jB reporter activity in both breast cancer and endothelial cells. EUK134 abrogated TNFa-induced NFjB gene reporter activity, whereas MnTmPyP enhanced the activity. These results are in accordance with a previous study showing opposite effects of SOD and catalase on

NF-jB activation [27]. As mentioned above, H2O2 regulates multiple signaling pathways. Recently, Sen et al. have shown impaired endogenous catalase activity in breast cancer cells compared to normal mammary epithelial cells [30]. This leads to an enhanced intracellular level of H2O2 and oxidation of cysteine residue of protein phosphatase 2A (PP2A) which, in turn, inactivates this enzyme [30]. Inactivation of PP2A enhances the PI3-Akt signaling pathway involved in proliferation, migration, apoptosis, and clonal expansion of breast cancer cells [11, 15, 30]. Therefore, reducing intracellular levels of H2O2 using EUK 134 may up-regulate PP2A activity and inhibit PI3-Akt signaling that otherwise promotes breast cancer progression. Whether this is the only way EUK 134 affects this complex on molecular pathways requires further investigation. Each cell type maintains different intracellular thresholds of H2O2 for signaling mechanisms. Due to compromised activity and/or expression of catalase in breast cancer cells compared to normal cells, intracellular H2O2 levels are enhanced [9, 23, 30]. To inhibit breast cancer cell growth, it would be appropriate to overexpress catalase or apply a catalase mimetic. Indeed, several studies have shown that catalase overexpression reduced proliferation and metastatic phenotype of breast and other cancer cells [9, 23, 30]. Similarly, the present study also provides evidence that the SOD/catalase mimetic EUK 134 effectively reduced cell proliferation, viability and clonal expansion, adhesion, and migration as described previously [9, 23, 30]. In addition, breast cancer cells overexpressing catalase were more sensitive to chemotherapeutic agents such as paclitaxel, etoposide, and arsenic trioxide [9]. In contrast, Xiao et al. has shown that SOD/catalase mimetic EUK 134 protects against benzyl isothiocyanate (BITC, a dietary

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cancer chemopreventive agent)-induced apoptosis pathway in breast cancer cells [41]. However, the protective effect of EUK 134 was tested acutely (1 h) before the addition of BITC in these cells [41]. Given that chronic treatment of EUK 134 reduced breast cancer cell proliferation, viability, and clonal expansion, it would be interesting to determine the effect of chemotherapeutic agents on cancer cells chronically (24–48 h) pre-treated with EUK 134. In conclusion, this is the first study to our knowledge showing head-to-head comparison of an SOD mimetic MnTmPyP and SOD/catalase mimetic EUK 134 on breast cancer cell growth. EUK 134 showed profound effects on breast cancer cell proliferation, survival, and migration as well as NF-jB signaling as compared to MnTmPyP, and these effects may be due to reduction of intracellular H2O2 levels. Further studies are required to identify the specific pathways that are affected by the SOD/catalase mimetic EUK 134. Acknowledgments MHS was supported by postdoctoral Fellowship (KG080837) from the Susan G. Komen Foundation for the Cure, USA. GJD is the recipient of an National Health and Medical Research Council Research Fellowship (#1003113). Authors are thankful to Dr. Rachana Sainger for her extensive reading and suggestions for this manuscript. The Centre for Eye Research Australia, St. Vincent’s Institute of Medical Research and O’Brien Institute acknowledge the Victorian State Government’s Department of Innovation, Industry and Regional Development’s Operational Infrastructure Support Program. Conflict of interest of interest.

The authors confirm that there are no conflicts

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