Increased expression of heparanase stimulates the progression of various human cancers, including breast cancer. Therefore, a deeper understanding of the mechanisms involved in regulating heparanase is critical in developing effective treatments for heparanase-overexpressing cancers. In this study, we investigated the potential use of extracellular superoxide dismutase (EcSOD) to enhance the inhibitory effects of heparin/low molecular weight heparin (LMWH) in breast cancer cells. EcSOD binds to cell surfaces and the extracellular matrix through heparin-binding domain (HBD). Deleting this HBD rendered the protein a more potent inhibitor of breast cancer growth, survival, and invasion. Among the treatment combinations examined, EcSODΔHBD plus LMWH provided the best tumor suppressive effects in inhibiting breast cancer growth and invasion in vitro. We have further shown that overexpression of EcSOD decreased accumulation of vascular endothelial growth factor in the culture medium and increased the level of intact cell surface-associated heparan sulfate, thus implicating inhibition of heparanase expression as a potential mechanism. Overexpression of EcSOD inhibited steady-state heparanase mRNA levels by >50% as determined by quantitative reverse transcription-PCR. Moreover, heparanase promoter activation was suppressed by EcSOD as indicated by a luciferase reporter assay. These findings reveal a previously unrecognized molecular pathway showing that regulation of heparanase transcription can be mediated by oxidative stress. Our study implies that overexpression of EcSOD is a promising strategy to enhance the efficacy of heparin/LMWH by inhibiting heparanase as a novel treatment for breast cancer. [Cancer Res 2009;69(15):6355–63]

Heparanase has been widely implicated as an important regulator of proliferation, invasion, and metastasis, as well as a significant promoter of malignancy-associated angiogenesis in breast cancer (1, 2). Heparanase is an endoglucuronidase that is involved in the degradation of heparan sulfate (HS), a linear sulfated glycosaminoglycan (3). As a key component of cell surface proteoglycans and extracellular matrix (ECM), HS participates in the self-assembly, insolubility, and barrier properties of the basement membrane (4). HS also serves as a storage depot for various members of the heparin-binding family of growth factors, sequestering them in inactive forms (5). Cleavage of HS by heparanase releases these signaling molecules, which can then be activated through binding to their corresponding receptors, thereby promoting cancer growth, angiogenesis, and invasion (6). Furthermore, by degrading the constituents of basement membranes and ECM, heparanase participates in invasion of cancer cells into the underlying stroma and metastasis to distal sites via vascular and lymphatic routes (7). In addition, heparanase has been shown to simulate angiogenesis by inducing vascular endothelial growth factor (VEGF).

The findings that heparanase is elevated in a wide variety of human cancer cells, including breast cancer (8), has led to an explosion of therapeutic strategies to inhibit its enzymatic activity. These inhibitors include neutralizing antibodies, small interfering RNAs (siRNA), HS mimetics, such as polyanionic molecules, as well as the anticoagulants heparin and low molecular weight heparin (LMWH; refs. 7, 9). Due to its highly sulfated structure, heparin is the most negatively charged biological molecule, rendering it a potent inhibitor of heparanase as a HS mimic. LMWHs, in general, are considered more effective antiangiogenic and antimetastatic agents than the unfractionated heparin due to their smaller variation of fragment sizes, longer half lives, and greater bioavailabilities (10). In addition to heparanase, HS degradation has also been shown to be mediated by reactive oxygen species (ROS; ref. 11). Superoxide dismutases (SOD) are cellular antioxidant enzymes that provide the primary defense against these ROS by converting superoxide radicals (O2·) into hydrogen peroxide (H2O2), which is subsequently removed by cellular catalase and peroxidases (12). This suggests that scavenging ROS with SOD may help in protecting the integrity of HS to inhibit heparanase-mediated cancer progression, as illustrated in our proposed model in Fig. 6D.

As the name implies, extracellular SOD (EcSOD) is the predominant superoxide scavenger in extracellular space. An important and unique feature of this enzyme is that it contains a heparin-binding domain (HBD; ref. 13). Once produced and secreted into the extracellular space, the full-length EcSOD binds to cell surfaces and ECM via this HBD. Removal of this domain occurs naturally by posttranslational proteolysis, producing a truncated form of the protein (EcSODΔHBD) that remains in circulation but has lost the ability to associate with HS (1416). Because full-length EcSOD has a stronger affinity for heparin/LMWH than HS, addition of heparin/LMWH will result in the redistribution of this EcSOD from cell surfaces and ECM into the circulation system (17). These circulating EcSODs will modulate the oxidative stress in the tumor microenvironment. In addition to increasing the circulating level of EcSOD, heparin has also been shown to induce the expression of this antioxidant enzyme (18). It is, therefore, logical to speculate that the antitumor effect of heparin and LMWH may extend to their effects in increasing the circulating level of EcSOD.

We investigated whether overexpression of EcSOD could enhance the inhibitory effect of heparin/LMWH in breast cancer cells. Our data showed that overexpression of EcSOD, particularly the EcSODΔHBD, inhibited the in vitro growth and invasion of two aggressive breast cancer cell lines. The inhibitory effects of either form of EcSOD were greatly enhanced with the addition of heparin/LMWH. We have further shown that EcSOD caused down-regulation of heparanase transcription, hence protecting the cleavage of cell surface HS proteoglycan and decreasing VEGF accumulation in the medium.

Cell culture. Immortalized, nonmalignant breast epithelial MCF-10A cells and human mammary adenocarcinoma cell lines, MCF-7 cells, MDA-MB231 cells, and MDA-MB435 cells, were purchased from American Type Culture Collection. These cells were cultured as described previously (19).

Adenovirus transduction. Overexpression of EcSODs was achieved by adenovirus infection, as previously described with a multiplicity of infection (MOI) of 50 for 72 h (20). The adenovirus constructs used were generated to overexpress a human full-length wild-type EcSOD gene, the EcSOD gene with a deletion in the HBD, EcSODΔHBD, or the mutated EcSOD gene with a R213G mutation in the HBD, EcSOD/R213G, as described (2023).

Heparin and LMWH treatment. After 24 h of adenovirus infection in serum-free media, cells were incubated in complete media supplemented with heparin (0.5 mg/mL, Sigma Aldrich) and LMWH (50 IU/mL, Pharmion) for 48 h.

RNA interference. Predesigned double-stranded siRNA against heparanase (siHPSE) was purchased from Ambion, Inc. with the sequence of 5′-GCAAUUGCUACUCCGAGAAtt-3′. As a negative control, a siNegative siRNA was obtained from Ambion. Transfection of siRNAs was performed, as previously described (20).

Real time reverse transcription-PCR analysis. Quantitative real-time reverse transcription-PCR (RT-PCR) assay for heparanase mRNA expression was performed with gene-specific heparanase primer/probe mix (Assays-on-Demand, Applied Biosystems, Inc.) according to the manufacturer's instructions.

Western blot analysis. Protein expression of EcSOD was determined by Western blot analysis, as previously described (20).

Antioxidant enzyme activity gels. Conditioned media were harvested from cells transduced with adenovirus vectors for 72 h. Activity of EcSOD was evaluated by native SOD activity gel assay by the method of Beauchamp and Fridovich (24), as previously described (20).

Cell growth. After 72 h of adenoviral infection or siRNA transfection, cells were seeded at a density of 5 × 103 in 24-well plates in complete media or media supplemented with heparin/LMWH. For the growth analysis, cells were counted daily for 10 d using a Coulter counter. Cell population doubling times in hours were determined, as previously described (25).

Clonogenic assay. After 72 h of adenoviral vector infection or siRNA transfection, cells were plated in triplicate into 60-mm dishes in complete media or media supplemented with heparin/LMWH. The dishes were maintained in the incubator for 12 to 14 d. The colonies containing >50 cells were scored.

Invasion assay. The in vitro invasive properties of the aggressive breast cancer cells were performed using the BD Bio-Coat Matrigel invasion assay system (BD Biosciences). Briefly, 2 × 105 of the cells were suspended in serum-free medium and seeded into the Matrigel chambers consisting with 8-μm pores. The transwell chambers were then placed into 24-well plates, into which we added complete medium or medium containing heparin/LMWH. After incubating cells for 18 h, the upper surface of the transwell chambers was removed with a cotton swab and the invading cells were fixed and stained with Giemsa stain. Photographs were taken under a light microscope, and the numbers of cells invaded were counted in five random microscopic fields.

Determination of extracellular superoxide level. Conditioned media were incubated at room temperature with 500 μmol/L hypoxanthine (Sigma) and 40 units/mL xanthine oxidase (Sigma) in the presence of 50 mmol/L DMPO (Dojindo Labs.). The mixed samples were then placed into a high sensitivity cavity using an AquaX (4-bore) sample cell (Bruker Instruments). Electron spin resonance spectra were obtained with a Bruker EMX spectrometer. Signal heights were measured using the second downfield peak of the four-line DMPO spin adduct and reported in arbitrary units.

VEGF assay. The effects of EcSOD on VEGF levels in the media were determined by a Quantikine Human VEGF Immunoassay kit (R&D Systems). After 72 h of adenovirus infection or siRNA transfection, cells were harvested and seeded at 1 × 104 in 24-well in complete media or media supplemented with heparin/LMWH. After 24 h of incubation, the cell culture supernatant was collected for the VEGF analysis, as described by the manufacturer.

Flow cytometry analysis of intact HS. Cells were trypsinized, washed with PBS containing 0.1% bovine serum albumin, and incubated with anti-HS antibody (10E4, Seikagaku) for 30 min. They were then washed and labeled with FITC-conjugated goat anti-mouse immunoglobulin (Becton Dickinson). A suspension of stained cells was subjected to FACSCalibur flow cytometry (BD Biosciences) to evaluate the fluorescence intensity.

Heparanase activity. The ability of endogenous heparanase to digest exogeneously added HS was determined with Heparanase Assay kit using a homogeneous time resolve fluorescence (HTRF) technology (Cisbio). In brief, cell lysates were added to a 384-well assay plate followed by an addition of biotinylated HS labeled with europium cryptate. After incubation at 37°C for 60 min, fluorescent-labeled streptavidin was then added. The HTRF value in each well was measured using a SpectraMax M5e (Molecular Devices) plate reader.

Heparanase promoter activity assay. EcSODs were overexpressed by adenovirus infection as described above with or without coinfection with an adenovirus vector constructed to express the luciferase gene driven by a heparanase promoter (AdHPSE.luc) or a cytomegalovirus (CMV) promoter (AdCMV.luc; ref. 26) at an MOI of 50 for each virus for 48 h. Luciferase activity of the infected cell lysate (20 μg total protein) was evaluated with Luciferase Assay System with Reporter Lysis Buffer (Promega) using the Tecan SpectraFluor plate reader.

Statistical analysis. Statistical analyses were performed using SYSTAT. For some experiments, a single-factor ANOVA followed by post-hoc Tukey test was used to determine statistical differences between means. Statistical analyses were assessed using a two-tailed Student's t test. Results shown are representative of at least three separate experiments each performed in triplicate.

Heparanase expression is up-regulated in breast cancer cell lines. In comparison to the nonmalignant mammary epithelial cell MCF-10A, breast cancer cell lines showed elevated heparanase mRNA expression as determined by quantitative real-time RT-PCR (Fig. 1A). The highest expression was detected in MDA-MB231 cells followed by MDA-MB435 cells, which are both aggressive and invasive breast cancer cells. In an attempt to show that heparanase is the main contributing factor in stimulating breast cancer growth and invasion, we have identified a siRNA against human heparanase, siHPSE, which efficiently inhibited the heparanase expression in MDA-MB231 cells, as shown in Fig. 1B.

Overexpression of EcSODs in MDA-MB231 cells. Western blot analysis in Fig. 1C shows that MDA-MB231 cells do not express EcSOD, but high levels of both forms of EcSOD were detected from the cell lysates after 72 hours of AdEcSOD and AdΔHBD infection. Note that, although this cell line does not express this antioxidant enzyme by nature, it is able to process the ∼31-kDa full-length protein into a ∼27-kDa truncated form, which is presumably the EcSODΔHBD. The bottom panel of the Western blot analysis indicates that this breast cancer cell line is able to secrete both forms of EcSOD into the conditioned media.

The overexpressed EcSODs are catalytically active.Figure 1D clearly indicates that both the full-length and the truncated form of EcSODs are catalytically active in the conditioned media harvested from the AdEcSOD- and AdΔHBD-infected MDA-MB231 cells. Addition of heparin and LMWH shifted the mobility of the full-length EcSOD in the gel, indicating that the HBD of this protein is intact and functional in associating with these negatively charged compounds. It is noteworthy that the heparin has an average molecular weight of 12 to 15 kDa, whereas the LMWH used in this study ranges from 5.5 to 7.5 kDa in size. The mobility of the truncated EcSOD, on the other hand, was not affected by heparin/LMWH, consistent with it lacking HBD.

EcSOD plus heparin/LMWH inhibited the growth of breast cancer cells. To investigate the effect of EcSODs with or without heparin/LMWH on breast cancer cell growth, we determined the cell doubling time from growth rate analysis. Overexpression of the full-length EcSOD moderately prolonged the cell doubling time of MDA-MB231 cells (Fig. 2A) and MDA-MB435 cells (Fig. 2C). Interestingly, overexpression of the truncated EcSOD dramatically slowed the growth of both aggressive breast cancer cell lines by >2-fold compared with control or AdEmpty-infected cells. Heparin and LMWH slightly increased the cell doubling time in both cell lines, and they provided additive effects in cells overexpressing either forms of the EcSODs. To show that the reduction in cell growth is due to inhibition of heparanase, we down-regulated the expression of heparanase with RNA interference and showed that siHPSE transfection resulted in an increase in cell doubling time of both cell lines.

EcSODs plus heparin/LMWH additively reduced clonogenic survival. Consistent with growth inhibition, Fig. 2 shows that EcSOD overexpression greatly reduced the clonogenic survival of both MDA-MB231 cells (Fig. 2B) and MDA-MB435 cells (Fig. 2D), with a more dramatic effect seen with EcSODΔHBD overexpression. Again, EcSODΔHBD plus LMWH was the best combination treatment in inhibiting the clonogenic survival of breast cancer cells.

EcSODs enhanced the inhibitory effect of heparin/LMWH on the invasion of breast cancer cells. The photomicrographs in Fig. 3A show representative fields of adenovirus vector-transduced MDA-MB231 cells, which have invaded through the Matrigel invasion chamber pores (round 8 μm circles). This invasion assay convincingly illustrates the potent effect of EcSODs plus heparin/LMWH in suppressing the invasion of MDA-MB231 cells. The results of this invasion assay were quantified as shown in Fig. 3B. Cells overexpressing full-length and truncated EcSODs showed >5.2-fold and 13.5-fold reduction (versus AdEmpty-infected cells) in invasion potential, respectively. Heparin and LMWH treatment alone also reduced the invasion, but most importantly, dramatic inhibitory effects were observed when cells simultaneously overexpress either the full-length EcSOD or EcSODΔHBD. Similar abrogation of invasion was also observed in MDA-MB435 cells (Fig. 3C).

Secreted EcSODs (in conditioned media) suppressed breast cancer invasion. Because EcSOD is an extracellular protein, we investigated whether the secreted EcSOD in the conditioned media would affect the invasion of breast cancer cells. Indeed, our data showed that breast cancer cells are less invasive in conditioned media containing full-length and truncated EcSODs compared with the conditioned media obtained from control and AdEmpty-infected cells (Fig. 4A). Quantitative analyses of the invasion assay are shown in Fig. 4B for MDA-MB231 cells and Fig. 4C for MDA-MB435 cells. In the presence of soluble full-length and truncated EcSODs in the media, suppressing heparanase expression with siHPSE provided a further inhibition of the invasion to 10 ± 1.2% and 5 ± 0.8%, respectively, relative to the siNegative-transfected cells in the AdEmpty-conditioned media (Fig. 4D).

EcSOD decreased extracellular superoxide levels. We then performed a DMPO adduct spin trapping electron paramagnetic resonance (EPR) assay to further confirm and quantify the specificity of the superoxide scavenging ability of the EcSODs in the conditioned media. The relative rates of superoxide formation correspond to the intensities of the DMPO-OH spin adduct as shown in Fig. 5A. EPR spectra from control and AdEmpty-treated conditioned media showed typical peak patterns specific to superoxide generation in MDA-MB231 cells. The intensities of the peak heights were significantly diminished in conditioned media containing either the full-length EcSOD or EcSODΔHBD. The relative extracellular superoxide levels were quantified from the peak heights and are shown in Fig. 5B.

EcSOD and heparanase inhibitors decreased levels of VEGF in the media. Because VEGF is one of the effecter molecules affected by heparanase, we determined whether EcSOD will inhibit the bioavailability of this angiogenic factor. As shown in Fig. 5C, cells that overexpressed either the long form of EcSOD or EcSODΔHBD significantly decreased VEGF protein levels in the media. Addition of LMWH to these EcSOD-overexpressing cells resulted in a further reduction in VEGF levels.

EcSOD and LMWH increased intact HS on breast cancer cell surface. To investigate whether scavenging extracellular superoxide radicals with EcSOD overexpression would prevent heparanase-mediated HS chain degradation, we assessed the immunoreactivity of breast cancer cell surface HS by flow cytometry with monoclonal antibody 10E4. This antibody reacts with epitopes containing N-sulfated glucosamine residues on intact HS side chains of proteoglycans but not with the heparanase-digested HS proteoglycan (27, 28). Figure 5D shows that cells overexpressing either form of the EcSOD have higher content of intact cell surface HS than the control and AdEmpty-infected cells. This protective effect against HS degradation was enhanced with the addition of LMWH.

Overexpression of EcSOD inhibited heparanase activity. We then examined whether EcSOD prevents HS degradation by inhibiting the activity of endogenous heparanase. Figure 6A shows that EcSOD overexpression inhibited heparanase activity by >50%. This inhibitory effect is enhanced in the presence of LMWH. The ability of heparin and LMWH to block the activity of heparanase is well documented (3), but the effect of EcSOD on heparanase activity has not been reported.

EcSOD down-regulated heparanase transcription in breast cancer cells. We then determined whether EcSOD inhibited the heparanase activity by affecting its gene expression. Results from quantitative real-time RT-PCR analysis in Fig. 6B (left) showed that full-length EcSOD-overexpressing cells had less than half the steady-state levels of heparanase mRNA compared with AdEmpty-infected MDA-MB231 cells. AdΔHBD infection also resulted in a reduction of heparanase mRNA expression. We have also observed similar down-regulation of heparanase gene expression by these EcSODs in MDA-MB435 cells (Fig. 6B,, right), indicating that this inhibitory effect is not cell line specific. These results imply that the inhibition of heparanase activity seen in EcSOD/EcSODΔHBD–overexpressing cells (Fig. 6A) is likely due to the decreased expression level of heparanase mRNA; that is, overexpression of these antioxidant enzymes is unlikely to have changed the specific activity of heparanase.

EcSOD inhibited heparanase promoter activity. To address the mechanism underlying decreased heparanase gene expression by EcSODs, we used a human heparanase promoter-luciferase reporter construct. When the heparanase promoter activity was determined, we found that endogenous overexpression of EcSODs via adenovirus vectors, as well as exogenous EcSODs in conditioned media, decreased the expression of luciferase by >50% compared with both the control and the AdEmpty infection in both breast cancer cell lines (Fig. 6C). These data clearly show that EcSOD impairs the expression of heparanase, thus limiting the activity of this HS degrading enzyme to provide an antitumor effect in breast cancer cells.

A critical event in the process of cancer invasion and metastasis is the degradation of various components of ECM, including HS proteoglycan. Heparanase is the only known endoglycosidase involved in HS cleavage, and it has been shown to play an important role in sustaining the pathology of malignant tumors (8). Recent experimental and clinical evidence shows that inhibiting heparanase with heparin/LMWH significantly reduced the growth and metastasis of various human cancers, including breast cancer (2, 29). In this report, we have shown that overexpression of EcSOD significantly inhibited heparanase expression and total activity and enhanced the inhibitory effects of heparin/LMWH in two aggressive and invasive breast cancer cell lines.

Inhibiting heparanase expression with either the pharmaceutical drugs (heparin and LMWH) or by genetic manipulation (siHPSE) leads to a slower growth, reduced clonogenic capacity, and invasive potential of aggressive breast cancer cell lines (Figs. 24), which is in agreement with numerous reports. However, this is the first investigation showing that overexpression of the antioxidant enzyme EcSOD has such a profound antitumor effect in breast cancer cells. Consistent with our previous findings in pancreatic cancer cells (20), AdEcSOD transduction resulted in a slower growth and a decreased clonogenic capacity in breast carcinoma cells (Fig. 2). We further showed that overexpression of full-length EcSOD interfered with the invasion potential of these cells (Figs. 3 and 4). Furthermore, exogeneous EcSOD in the conditioned media had the ability to inhibit invasion of breast cancer cells. This exciting observation suggests that systemic application of EcSOD may have promise in the therapeutic intervention for breast cancer treatment. In support of this, Wheeler and colleagues (30) have shown that i.m. injection of an adenoviral vector with EcSOD construct before tumor implantation blunted melanoma growth in mice, suggesting that secreted EcSOD has tumor-suppressive properties.

It is intriguing yet puzzling that removal of the HBD from EcSOD rendered the truncated antioxidant enzyme a better inhibitor of growth and invasion in breast cancer cells. These findings do not parallel the evidence provided by other reports showing that deleting HBD from EcSOD resulted in a loss of its protective effects in reducing arterial pressure during hypertension and preventing vascular dysfunction (21, 23, 31), pointing to the importance of physical interaction between EcSOD and cell surfaces for its cardiovascular functions. Having said that, considering the fact that cells naturally produce this truncated EcSOD devoid of the HBD, EcSODΔHBD must serve some biological roles that have yet to be elucidated. In this report, we have shown that liberating EcSOD from HS interaction resulted in a greater inhibition of growth, clonogenic survival, and invasion of breast cancer cells (Figs. 24). The mechanisms involved in these suppressive effects of EcSODΔHBD are unclear, but we speculate that deleting this domain imposes less physical restriction compared with the full-length EcSOD, which are mainly sequestered on cell surfaces. In other words, the free, non–HS-bound EcSODΔHBD may possess greater suppressive effects than its full-length counterpart due to a greater bioavailability. It has also been suggested that the HBD of this antioxidant enzyme participates in the internalization of the protein by endocytosis and subsequent degradation of EcSOD by lysosomal proteases (32, 33). Therefore, removal of this domain may increase the half life of the truncated protein. However, considering the variety of the binding partners for the HBD of EcSOD, which include but are not limited to heparin (34), HS (35), hyaluronan (36), collagen type I (37), and fibulin 5 (38), further investigations are needed to understand the biological significance of this domain of EcSOD. Moreover, identification of the protease involved in the processing of this domain will accelerate our understanding of the role played by this antioxidant enzyme.

Interestingly, a natural mutation, Arg213 to Gly (R213G), located in the HBD has been described for human EcSOD. This polymorphism, which has been found in 4% of Swedish, 3% of Australian, and 6% of Japanese people, resulted in an increase in the concentration of plasma EcSOD by 10-fold to 30-fold due to a reduced heparin affinity of this mutant (39, 40). Although the consequences of this polymorphism are unclear, it has been shown to be associated with the poor outcome in diabetic patients who require hemodialysis and an increased risk factor–associated with cardiovascular disease (41, 42). However, this R213G variant has also been shown to confer resistance to the development of chronic obstructive pulmonary disease in some smokers, which was suggested to be linked to a greater availability of the circulating EcSOD-R213G, thereby providing a better antioxidant and inflammatory response (43). These rather conflicting results imply that the R213G variant or an increased circulating EcSOD level may contribute to different outcomes under varying pathophysiologic conditions, and our study indeed provides evidence that increased circulating EcSOD may be therapeutically beneficial in breast cancer treatment. In an attempt to further characterize the role of the HBD or heparin affinity of EcSOD, we have examined the effect of EcSOD-R213G in breast cancer tumorigenesis via adenovirus-mediated gene transduction (AdR213G). Overexpression of this variant EcSOD resulted in an intermediate tumor phenotype in inhibiting growth, clonogenic survival, and invasion compared with AdEcSOD- and AdΔHBD-infected MDA-MB231 cells (Supplementary Fig. S1). This study provides further evidence that association with cell surface HS is not essential in the growth and invasion-suppressive effects of EcSOD.

Furthermore, we have illustrated that in the presence of heparin/LMWH, the inhibitory effects of EcSOD overexpression on growth, clonogenic capacity, and invasion is greatly enhanced in breast cancer cells. However, this additive effect cannot be attributed to the interaction between heparin/LMWH and EcSOD because deleting the HBD rendered the truncated form of this antioxidant enzyme a more potent inhibitor of malignant behavior in breast carcinoma cells in the presence of heparin/LMWH (Figs. 24). These data strongly suggest that increased circulating EcSOD, in a truncated form or in combination with heparin/LMWH, may be therapeutically beneficial for breast cancer.

In addition to scavenging extracellular superoxide radicals (Fig. 6), we have provided evidence that EcSOD enhanced the antitumor effects of heparin/LMWH in part by inhibiting heparanase mRNA expression in breast cancer cells (Fig. 6). This down-regulation of heparanase transcription likely led to the observed decrease in the bioavailability of the angiogenic VEGF, as well as higher content of intact cell surface HS (Fig. 5). Previous reports have shown that EcSOD can prevent oxidative fragmentation of HS (44) and the negatively charged extracellular matrix component hyaluronan (36) in the lung. However, this is the first report illustrating the ability of EcSOD to regulate the expression of heparanase, thus providing a potential mechanism for the observed antitumor effects of EcSOD. Although further work is needed to show how this occurs, we suggest that EcSOD may exert its effect on heparanase expression by blocking activation of redox-sensitive transcription factors that participate in regulating heparanase transcription, such as Egr1, nuclear factor-κB (NF-κB), and Ets1/2 (45). These transcription factors are regulated by oxidative stress. It has been reported that EcSOD inhibited the expression of VEGF in an in vivo melanoma study by preventing oxidative activation of NF-κB (30) and activation of this transcription factor has been shown to correlate with heparanase expression in various carcinomas (46). In addition, ROS and the renin-angiotensin system have been shown to up-regulate glomerular heparanase expression in rats with Adriamycin nephropathy, further supporting that modulation of oxidative stress with an antioxidant enzyme, such as EcSOD, can inhibit the expression of heparanase. We also showed that EcSOD inhibited the expression of heparanase independent of its HBD because deleting this domain provided similar inhibitory effect on heparanase transcription and promoter activity (Fig. 6). Although this study showed the promising use of EcSOD expression to amplify the therapeutic efficacy of heparin/LMWH in breast cancer cells, these combination treatments did not completely abolish the clonogenic survival of the cells, suggesting that there are limitations to these strategies and that multiple strategies will likely be needed.

In conclusion, this study indicates that scavenging ROS with EcSOD down-regulated heparanase expression and activity and prevented loss of cell surface HS in breast cancer cells. This property of EcSOD enhanced the inhibitory effects of heparin/LMWH in breast cancer cells, indicating that EcSOD plus heparin/LMWH combination could be used as promising therapeutic agents in breast cancer treatment by targeting heparanase-mediated pathways as represented in Fig. 6D. Most significantly, this report provides evidence showing that the HBD of EcSOD is not essential for its tumor-suppressive effects, implying a biological role for the truncated EcSODΔHBD or the circulating EcSOD released by heparin/LMWH.

No potential conflicts of interest were disclosed.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

We dedicate this work to the late Dr. Larry Oberley, in honor of his pioneering studies on SOD in cancer.

Grant support: Komen for the Cure Postdoctoral Fellowship Award KG080437 (M. Teoh) and NIH grants CA115438 and CA073612 (F.E. Domann).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. James Crapo (National Jewish Medical and Research Center) for providing antibodies to EcSOD and Brett Wagner (University of Iowa) for EPR studies supported by grant P30CA086862.

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Supplementary data