Epithelial ovarian cancer (EOC) is the fourth leading cause of death due to cancer in women and comprises distinct histologic subtypes, which vary widely in their genetic profiles and tissues of origin. It is therefore imperative to understand the etiology of these distinct diseases. Ovarian clear cell carcinoma (OCCC), a very aggressive subtype, comprises >10% of EOCs. In the present study, we show that mitochondrial superoxide dismutase (Sod2) is highly expressed in OCCC compared with other EOC subtypes. Sod2 is an antioxidant enzyme that converts highly reactive superoxide (O2•−) to hydrogen peroxide (H2O2) and oxygen (O2), and our data demonstrate that Sod2 is protumorigenic and prometastatic in OCCC. Inhibiting Sod2 expression reduces OCCC ES-2 cell tumor growth and metastasis in a chorioallantoic membrane (CAM) model. Similarly, cell proliferation, migration, spheroid attachment and outgrowth on collagen, and Akt phosphorylation are significantly decreased with reduced expression of Sod2. Mechanistically, we show that Sod2 has a dual function in supporting OCCC tumorigenicity and metastatic spread. First, Sod2 maintains highly functional mitochondria, by scavenging O2•−, to support the high metabolic activity of OCCC. Second, Sod2 alters the steady-state ROS balance to drive H2O2-mediated migration. While this higher steady-state H2O2 drives prometastatic behavior, it also presents a doubled-edged sword for OCCC, as it pushed the intracellular H2O2 threshold to enable more rapid killing by exogenous sources of H2O2. Understanding the complex interaction of antioxidants and ROS may provide novel therapeutic strategies to pursue for the treatment of this histologic EOC subtype. Cancer Res; 75(22); 4973–84. ©2015 AACR.

Ovarian clear cell carcinomas (OCCC) represent approximately 10% to 25% of all epithelial ovarian cancer (EOC), depending on ethnic background (1). It is now evident that OCCC differs widely from the more common high-grade serous adenocarcinoma. While the primary tumor mass of OCCC is found on the ovary, its origin is not thought to be the ovary or fallopian tube but rather to stem from endometrioid tissue and endometriosis. Because of the reactive oxygen species (ROS) stress associated with endometrioisis, OCCC has been characterized as a stress-responsive cancer (2, 3). Gene expression studies have shown increased expression of a number of stress-related and metabolic genes, in particular those related to hypoxic insult, glycolysis, and antioxidant defense mechanisms (4). The Nrf2 stress response pathway has been implicated in driving some of these changes, including the enhanced expression of mitochondrial manganese-containing superoxide dismutase (Sod2; ref. 5).

Sod2 is a nuclear-expressed mitochondria-targeted antioxidant enzyme, which catalyzes the conversion of two molecules of superoxide anion (O2•−) to hydrogen peroxide (H2O2) and oxygen (O2). Because a small amount of O2•− leakage occurs during normal oxidative phosphorylation from the mitochondrial electron transport chain, Sod2 is of importance in preventing redox-mediated damage of mitochondrial proteins and preserving mitochondrial function. Enhanced O2•− production can occur in response to stress, such as hypoxia, and has been linked to a number of cancer types. For this reason, Sod2 was initially characterized as a tumor suppressor gene (6). However, recent data now point to a dichotomous role of Sod2 during tumor progression. Evidence suggests that Sod2 expression is often increased during metastatic progression, potentially as an adaptation to enhanced levels of intra- and extracellular ROS (7). While Sod2 may initially prevent ROS-mediated DNA damage to facilitate tumor initiation, increased Sod2 expression appears to conversely contribute to the metastatic phenotype by altering redox signaling pathways (8). We and others have observed that increased Sod2 expression correlates with a shift to higher intracellular H2O2 levels, contributing to prometastatic behavior (7, 9, 10).

In the present study, we set out to interrogate the role of Sod2 in OCCC and found a dual function for this mitochondrial antioxidant in OCCC tumorigenicity. Sod2 not only provides a protective role in scavenging mitochondrial O2•−, thereby maintaining high mitochondrial function and proliferation, but also alters the steady-state ROS balance to drive H2O2-mediated migration and metastasis of OCCC cells.

Oncomine data and ovarian cancer cell line microarray data

Oncomine.org was used to screen Sod2 expression in ovarian cancer histologic subtypes (Supplementary Fig. S1). Two representative datasets are shown in Fig. 1A and B (GEO accession nos. GSE2109 and GSE6008). Microarray data of the following ovarian cancer cell lines were obtained using the GeneChip Human Genome U133A 2.0 Array (Affymetrix; GEO accession no.: GSE25428; refs. 4, 11). Data represent expression of Sod2 probe 215223_s_at (log2 RMA normalized). OCCC: JHOC-5, JHOC-7, JHOC-8, JHOC-9, KOC-5C, KOC-7C, OVISE, OVTOKO, RMG-1, RMG-2, RMG-5, TAYA, TOV-21-G. Serous adenocarcinoma: CAOV3, Fuov1, HEY, Hey-A8, Hey-Ce, JHOS-2, JHOS-3, JHOS-4, M41, M41-cisR, OV90, OVARY1847, OVCA420, OVCA429, OVCA432, OVCAR3, PEO1, PEO4, SKOV3. Mucinous: JHOM-1, JHOM-2B, MCAS, OMC-3. Endometrioid: OVK-18, TOV-112D. Adenocarcinoma: A2780 (A2780-J), A2780J-cisR, DOV13, OVCAR2, OVCAR5, OVCAR8. Teratocarcinoma: CH1, PA1. Undifferentiated: TYK-nu, TYK-nu cisR. Prior to microarray analysis, cell lines were authenticated by STR analysis at the Fragment Analysis Facility, Johns Hopkins University (Baltimore, MD; PowerPlex 1.2 System; Promega) or at the University of Colorado Cancer Center (Aurora, CO; AmpFlSTR Identifier Plus PCR Kit, Applied Biosystems; ref. 11).

Figure 1.

Increased Sod2 mRNA expression is observed in OCCC compared with other ovarian carcinoma histologic subtypes. Two representative datasets from ovarian cancer microarray studies are displayed as box and whisker plots (data obtained through Oncomine.org). A, clear cell (n = 16), serous (n = 24), mucinous (n = 8), endometrioid (n = 29), serous surface papillary (n = 107); one-way ANOVA (P < 0.0002), Tukey posttest. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01 (GSE2109). B, clear cell (n = 8), serous (n = 41), mucinous (n = 13), endometrioid (n = 37); one-way ANOVA (P < 0.0001), Tukey posttest. ***, P < 0.001 (GSE6008). C, Sod2 mRNA expression was significantly increased in a panel of OCCC lines, listed in Materials and Methods, compared with serous adenocarcinoma cell lines (Affymetrix array; ANOVA Tukey posttest: *, P < 0.05). D, semiquantitative real-time RT-PCR was performed to assess Sod2 mRNA expression in serous ovarian cancer cell lines OVCA433, OVCA429, and OVCAR3 and OCCC cell lines ES-2 and TOV-21-G (data expressed relative to OVCA433, which displayed lowest Sod2 expression). E, immunoblot analysis and densitometric quantification of Sod2 protein expression in ovarian cancer cell lines (for D and E: mean ± SEM, n = 3, ANOVA; Tukey posttest. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05).

Figure 1.

Increased Sod2 mRNA expression is observed in OCCC compared with other ovarian carcinoma histologic subtypes. Two representative datasets from ovarian cancer microarray studies are displayed as box and whisker plots (data obtained through Oncomine.org). A, clear cell (n = 16), serous (n = 24), mucinous (n = 8), endometrioid (n = 29), serous surface papillary (n = 107); one-way ANOVA (P < 0.0002), Tukey posttest. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01 (GSE2109). B, clear cell (n = 8), serous (n = 41), mucinous (n = 13), endometrioid (n = 37); one-way ANOVA (P < 0.0001), Tukey posttest. ***, P < 0.001 (GSE6008). C, Sod2 mRNA expression was significantly increased in a panel of OCCC lines, listed in Materials and Methods, compared with serous adenocarcinoma cell lines (Affymetrix array; ANOVA Tukey posttest: *, P < 0.05). D, semiquantitative real-time RT-PCR was performed to assess Sod2 mRNA expression in serous ovarian cancer cell lines OVCA433, OVCA429, and OVCAR3 and OCCC cell lines ES-2 and TOV-21-G (data expressed relative to OVCA433, which displayed lowest Sod2 expression). E, immunoblot analysis and densitometric quantification of Sod2 protein expression in ovarian cancer cell lines (for D and E: mean ± SEM, n = 3, ANOVA; Tukey posttest. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05).

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Cell lines and cell culture conditions

At commencement of this study ES-2 and TOV-21-G cells were newly obtained from ATCC. Authenticity was verified by ATCC using STR analysis. ES-2 cells were maintained in McCoy 5A media + 10% FBS and TOV-21-G cells in 40% Media199/40% MCBD supplemented with 20% FBS and sodium bicarbonate. Cells were maintained at 37°C with 5% CO2.

Sod2 knockdown using RNA interference

Scramble nontargeting control and Sod2-specific siRNA oligonucleotides were synthesized by Life Technologies/Dharmacon. 5′- CAACAGGCCUUAUUCCACU-3′ and 5′- AAGUAAACCACGAUCGUUA-3′ sequences were used as siSod2_#1 and siSod2_#2, respectively (Supplementary Fig. S2) and 10 pmol transfected into cells using Lipofectamine RNAiMax (Invitrogen). shRNA with nontargeting scramble sequence or targeting Sod2 (shSod2_#1: 5′-CTGACGGCTGCATCTGTTGGTGTCCAAGG-3′, and shSod2_#2: 5′-ACCTGAACGTCACCGAGGAGAAGTACCAG-3′) in pGFP-V-RS vector (Origene; TG309190) were used to stably transfect ES-2 cells (Fig. 2; Supplementary Fig. S3). The clone expressing shSod2_#1 was used in Figs. 2–7.

Figure 2.

Inhibition of Sod2 expression abrogates OCCC tumorigenicity A, Sod2 expression and activity were reduced using RNA interference in OCCC cell lines ES-2 (shSod2_#1) and TOV-21-G (siRNASod2_#1). Sod2 expression was analyzed using Western blotting (top) and activity determined using in-gel zymography (bottom). B, cell proliferation rate was decreased with reduced Sod2 expression (shSod2_#1), analyzed by cell counting using trypan blue exclusion assay (mean ± SEM. ANOVA Tukey posttest; ****, P < 0.0001). C, Sod2 RNA interference decreases single-cell survival of ES-2 cells in clonogenicity assays. D, reduced Sod2 levels significantly inhibited the tumor size and weight of ES-2 tumors grown in the ex ovo CAM model. Fertilized chicken eggs were incubated at 37°C for 7 days after inoculating the cells (shSod2_#1 or scramble transfected ES-2) onto the CAM, after which tumors were collected and measured (control, n = 12; shSod2, n = 10; mean ± SD; Student t test; **, P < 0.01).

Figure 2.

Inhibition of Sod2 expression abrogates OCCC tumorigenicity A, Sod2 expression and activity were reduced using RNA interference in OCCC cell lines ES-2 (shSod2_#1) and TOV-21-G (siRNASod2_#1). Sod2 expression was analyzed using Western blotting (top) and activity determined using in-gel zymography (bottom). B, cell proliferation rate was decreased with reduced Sod2 expression (shSod2_#1), analyzed by cell counting using trypan blue exclusion assay (mean ± SEM. ANOVA Tukey posttest; ****, P < 0.0001). C, Sod2 RNA interference decreases single-cell survival of ES-2 cells in clonogenicity assays. D, reduced Sod2 levels significantly inhibited the tumor size and weight of ES-2 tumors grown in the ex ovo CAM model. Fertilized chicken eggs were incubated at 37°C for 7 days after inoculating the cells (shSod2_#1 or scramble transfected ES-2) onto the CAM, after which tumors were collected and measured (control, n = 12; shSod2, n = 10; mean ± SD; Student t test; **, P < 0.01).

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Figure 3.

Sod2 preserves OCCC mitochondrial respiration. A, reduced Sod2 levels significantly attenuated the mitochondrial oxidative phosphorylation in stably transfected ES-2 cells (shSod2_#1). OCR was measured using extracellular flux analysis and pharmacologic manipulation of mitochondrial activity to interrogate bioenergetics parameters. Oligomycin A (O) was used to derive ATP-linked OCR, FCCP (F) to stimulate maximal OCR, and antimycin A (A) to inhibit all mitochondrial OCR. B, basal OCR, ATP-linked OCR, maximum OCR, and respiratory reserve capacity (max OCR − basal OCR) were significantly decreased with stable shRNA-mediated Sod2 knockdown in ES-2 (one representative of three experiments is shown, mean ± SEM; control, n = 8; shSod2, n = 9. ANOVA, Tukey posttest; ***, P < 0.001; ****, P < 0.0001). C, transiently transfected siRNA targeting Sod2 in TOV-21-G cells showed significantly reduced mitochondrial oxidative phosphorylation (siRNA construct #1). D, basal OCR, maximum OCR, and respiratory reserve capacity (max OCR − basal OCR) were significantly decreased with Sod2 knockdown in TOV-21-G cells. (one representative of three experiments is shown; mean ± SEM; control, n = 4; siSod2, n = 5; ANOVA, Tukey posttest; *, P < 0.05). E, superoxide-mediated oxidation of MitoSox redox–sensitive dye was measured in live cells. Increased MitoSox fluorescence was observed in ES-2 cells stably transfected with shRNA-Sod2_#1 compared with scramble-transected control cells, suggesting that the amount of mitochondrial superoxide accumulation is higher in cells with reduced Sod2 expression. MitoSox florescence was abrogated by addition of 10 μmol/L of the porphyrin superoxide scavenger ortho tetrakis(N-n-butoxyethylpyridinium-2-yl) porphyrin (MnTnBuOE-2-PyP5+), representative of three experiments is shown; mean ± SEM, n = 4; ANOVA, ANOVA Tukey posttest; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

Sod2 preserves OCCC mitochondrial respiration. A, reduced Sod2 levels significantly attenuated the mitochondrial oxidative phosphorylation in stably transfected ES-2 cells (shSod2_#1). OCR was measured using extracellular flux analysis and pharmacologic manipulation of mitochondrial activity to interrogate bioenergetics parameters. Oligomycin A (O) was used to derive ATP-linked OCR, FCCP (F) to stimulate maximal OCR, and antimycin A (A) to inhibit all mitochondrial OCR. B, basal OCR, ATP-linked OCR, maximum OCR, and respiratory reserve capacity (max OCR − basal OCR) were significantly decreased with stable shRNA-mediated Sod2 knockdown in ES-2 (one representative of three experiments is shown, mean ± SEM; control, n = 8; shSod2, n = 9. ANOVA, Tukey posttest; ***, P < 0.001; ****, P < 0.0001). C, transiently transfected siRNA targeting Sod2 in TOV-21-G cells showed significantly reduced mitochondrial oxidative phosphorylation (siRNA construct #1). D, basal OCR, maximum OCR, and respiratory reserve capacity (max OCR − basal OCR) were significantly decreased with Sod2 knockdown in TOV-21-G cells. (one representative of three experiments is shown; mean ± SEM; control, n = 4; siSod2, n = 5; ANOVA, Tukey posttest; *, P < 0.05). E, superoxide-mediated oxidation of MitoSox redox–sensitive dye was measured in live cells. Increased MitoSox fluorescence was observed in ES-2 cells stably transfected with shRNA-Sod2_#1 compared with scramble-transected control cells, suggesting that the amount of mitochondrial superoxide accumulation is higher in cells with reduced Sod2 expression. MitoSox florescence was abrogated by addition of 10 μmol/L of the porphyrin superoxide scavenger ortho tetrakis(N-n-butoxyethylpyridinium-2-yl) porphyrin (MnTnBuOE-2-PyP5+), representative of three experiments is shown; mean ± SEM, n = 4; ANOVA, ANOVA Tukey posttest; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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Figure 4.

Metastatic spread of ES-2 cells is attenuated with reduced Sod2 expression in the ex ovo CAM tumor model. A, representative images show the location of the primary tumors and micrometastasis of GFP-labeled cells in the membrane (scale bar, 1 mm; CAM tumor study carried out as in Fig. 2). B, reduced Sod2 expression inhibited tumor metastasis into the chick embryo and CAM (shSod2_#1). Quantification of percentage metastasis of tumor cells into CAM, liver, and lung of chick embryo. Representative images show the percentage of tumors with metastatic cancer cells present in the chicken embryo liver and lung (control, n = 12; shSod2, n = 10).

Figure 4.

Metastatic spread of ES-2 cells is attenuated with reduced Sod2 expression in the ex ovo CAM tumor model. A, representative images show the location of the primary tumors and micrometastasis of GFP-labeled cells in the membrane (scale bar, 1 mm; CAM tumor study carried out as in Fig. 2). B, reduced Sod2 expression inhibited tumor metastasis into the chick embryo and CAM (shSod2_#1). Quantification of percentage metastasis of tumor cells into CAM, liver, and lung of chick embryo. Representative images show the percentage of tumors with metastatic cancer cells present in the chicken embryo liver and lung (control, n = 12; shSod2, n = 10).

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Figure 5.

A, cell migration is significantly inhibited with Sod2 knockdown. A, migration was analyzed by wound-healing assays using Ibidi inserts and the area covered by cells quantified following 72 hours of migration (normalized to day 0). Data from one representative experiment are shown (n = 6, mean ± SEM; t test; **, P < 0.01; scale bar, 500 μm). B, representative images of attachment and outgrowth of ES-2 spheroids on uncoated and collagen I–coated surfaces (scale bar, 1 mm). Reduced Sod2 levels significantly inhibited the outgrowth of spheroids in both uncoated and collagen I–coated surfaces. Data from one representative experiment are shown (n = 4, mean ± SEM; ANOVA, Tukey posttest; **, P < 0.01). C, Akt phosphorylation is dependent on Sod2 expression. Protein expression of phosphorylated and total levels of Akt, p130cas, and FAK were analyzed by Western blotting. Data from one representative experiment are shown. Levels of phosphorylated proteins were quantified by densitometry and normalized to total levels (n = 3, mean ± SEM).

Figure 5.

A, cell migration is significantly inhibited with Sod2 knockdown. A, migration was analyzed by wound-healing assays using Ibidi inserts and the area covered by cells quantified following 72 hours of migration (normalized to day 0). Data from one representative experiment are shown (n = 6, mean ± SEM; t test; **, P < 0.01; scale bar, 500 μm). B, representative images of attachment and outgrowth of ES-2 spheroids on uncoated and collagen I–coated surfaces (scale bar, 1 mm). Reduced Sod2 levels significantly inhibited the outgrowth of spheroids in both uncoated and collagen I–coated surfaces. Data from one representative experiment are shown (n = 4, mean ± SEM; ANOVA, Tukey posttest; **, P < 0.01). C, Akt phosphorylation is dependent on Sod2 expression. Protein expression of phosphorylated and total levels of Akt, p130cas, and FAK were analyzed by Western blotting. Data from one representative experiment are shown. Levels of phosphorylated proteins were quantified by densitometry and normalized to total levels (n = 3, mean ± SEM).

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Figure 6.

H2O2 contributes to OCCC migration. A, steady-state H2O2 levels decrease with reduced Sod2 expression. Steady-state H2O2 levels were derived in ES-2 cells stably transfected with either scramble shRNA control (closed circles) or shRNA targeting Sod2 (open circles), using aminotriazole inhibition of catalase kinetics assays, as described in Materials and Methods. First-order decay curves for catalase inhibition by aminotrizaole were used to determine rate constants of inactivation (k; one representative decay curve from three replicate experiments shown). H2O2 concentrations were determined using the equation [H2O2] = k/k1, where k1 is the rate of catalase compound I formation [1.7 × 107 (mol/L)−1 s−1]. H2O2 data represent an average of three experiments ± SD. B, there was no change in catalase activity and catalase protein expression between control and shRNA-Sod2 cells. C, H2O2 scavenging by catalase abrogates migration of ES-2 cells, whereas H2O2 can stimulate migration in cells with low Sod2 expression. Migration was analyzed by wound-healing assays using Ibidi inserts and the area covered by cells quantified following 72 hours of migration (normalized to day 0). Scavenging of endogenous H2O2 by catalase significantly reduced the migration of ES-2 scramble–transfected control cells. Recombinant catalase protein (500 units/mL) was added in serum-free media at time 0 and left on cells for 72 hours. Conversely, migration of shSod2 cells was significantly increased with treatment of 5 μmol/L H2O2, whereas this level of H2O2 was toxic to control cells and effects on migration could therefore not be determined (N/D). Data represent one of two independent cellular migration experiments (n = 5, mean ± SEM; ANOVA, Tukey posttest; *, P < 0.05). D, H2O2 can stimulate Akt signaling in OCCC. Dose-dependent increases in phospho-Akt S473 levels were observed by Western blotting following 2 hours of H2O2 treatment with indicated doses in serum-free media.

Figure 6.

H2O2 contributes to OCCC migration. A, steady-state H2O2 levels decrease with reduced Sod2 expression. Steady-state H2O2 levels were derived in ES-2 cells stably transfected with either scramble shRNA control (closed circles) or shRNA targeting Sod2 (open circles), using aminotriazole inhibition of catalase kinetics assays, as described in Materials and Methods. First-order decay curves for catalase inhibition by aminotrizaole were used to determine rate constants of inactivation (k; one representative decay curve from three replicate experiments shown). H2O2 concentrations were determined using the equation [H2O2] = k/k1, where k1 is the rate of catalase compound I formation [1.7 × 107 (mol/L)−1 s−1]. H2O2 data represent an average of three experiments ± SD. B, there was no change in catalase activity and catalase protein expression between control and shRNA-Sod2 cells. C, H2O2 scavenging by catalase abrogates migration of ES-2 cells, whereas H2O2 can stimulate migration in cells with low Sod2 expression. Migration was analyzed by wound-healing assays using Ibidi inserts and the area covered by cells quantified following 72 hours of migration (normalized to day 0). Scavenging of endogenous H2O2 by catalase significantly reduced the migration of ES-2 scramble–transfected control cells. Recombinant catalase protein (500 units/mL) was added in serum-free media at time 0 and left on cells for 72 hours. Conversely, migration of shSod2 cells was significantly increased with treatment of 5 μmol/L H2O2, whereas this level of H2O2 was toxic to control cells and effects on migration could therefore not be determined (N/D). Data represent one of two independent cellular migration experiments (n = 5, mean ± SEM; ANOVA, Tukey posttest; *, P < 0.05). D, H2O2 can stimulate Akt signaling in OCCC. Dose-dependent increases in phospho-Akt S473 levels were observed by Western blotting following 2 hours of H2O2 treatment with indicated doses in serum-free media.

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Figure 7.

Exogenous H2O2 treatment significantly decreases viability of cells with higher steady-state H2O2 levels. A, ES-2 scramble control cells show significantly lower viability than shSod2 ES-2 cells using crystal violet assays, in response to increasing concentrations of H2O2 for 72 hours. Each data point represents an average of 6 replicates ± SEM. Tukey posttest; *, P < 0.05. B, proposed mechanisms of Sod2-mediated OCCC tumorigenesis and metastasis. OCCC has high Sod2 expression, which provides efficient superoxide scavenging and maintenance of high mitochondrial function to drive increased cell proliferation. In addition, Sod2 shifts the intracellular ROS balance from O2•− to H2O2, which drives tumor cell migration and metastasis. This concomitantly enhances the intracellular H2O2 threshold, enabling more rapid killing by exogenous H2O2.

Figure 7.

Exogenous H2O2 treatment significantly decreases viability of cells with higher steady-state H2O2 levels. A, ES-2 scramble control cells show significantly lower viability than shSod2 ES-2 cells using crystal violet assays, in response to increasing concentrations of H2O2 for 72 hours. Each data point represents an average of 6 replicates ± SEM. Tukey posttest; *, P < 0.05. B, proposed mechanisms of Sod2-mediated OCCC tumorigenesis and metastasis. OCCC has high Sod2 expression, which provides efficient superoxide scavenging and maintenance of high mitochondrial function to drive increased cell proliferation. In addition, Sod2 shifts the intracellular ROS balance from O2•− to H2O2, which drives tumor cell migration and metastasis. This concomitantly enhances the intracellular H2O2 threshold, enabling more rapid killing by exogenous H2O2.

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Immunoblotting

Protein expression was analyzed by standard Western blotting using antibodies from Cell Signaling Technology (pAkt-s473, Akt, pFAK-Y397, FAK, p-p130cas-Y165, p130cas) or Abcam (Sod2). Primary antibodies were diluted in blocking solution (5% nonfat milk in TBS with 0.1% Tween-20, 1:1,000) and incubated overnight at 4°C. Blots were visualized using Femto and Pico ECL chemiluminescence substrate (Thermo scientific) and imaged using a ChemiDoc MP system (BioRad). Densitometric analysis was performed using ImageJ software (NIH). Each protein band was normalized to the respective GAPDH or β-actin loading control band.

Sod2 zymography

Sod2 activity was analyzed using Sod2 in-gel zymography as previously described (12). Briefly, cell lysates were loaded on nondenaturing acrylamide gels, followed by electrophoresis. Sod2 activity is visualized by the inhibition of nitroblue tetrazolium reduction.

Clonogenicity and cell viability

Single-cell survival clonogenicity assays were performed as previously described (13). Briefly, 100 cells were plated in each well of 6-well plate colonies visualized after 10 days using crystal violet. Viability was assessed by cell counting using trypan blue (1%) staining or crystal violet uptake assays (13).

Chorioallantoic membrane assay

Each chorioallantoic membrane (CAM) was inoculated with 5 × 105 ES-2 cells stably expressing either scramble-shRNA-GFP or Sod2-shRNA_#1-GFP that were suspended in 50 μL PBS (with 1 mmol/L MgCl2, 0.5 mmol/L CaCl2, 100 U/mL penicillin, and 100 μg/mL streptomycin), essentially as previously described (14). Tumors were allowed to form for 7 days prior to termination of the experiments by sacrificing the chick embryo. Tumors on the CAM were removed and measured. CAM and chick embryo organs (liver and lung) were collected for tumor metastasis analysis by surveying for GFP-labeled cells.

Seahorse XF24 extracellular flux analysis

Oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and mitochondria stress tests were measured using the Seahorse XF243 Extracellular Flux Analyzer (Seahorse Bioscience), as described previously (13). Cells were plated at a density of 40,000 cells per well and media replaced with XF media the following day 1 hour prior to the assay. Three measurements of OCR and ECAR were taken at baseline and after each injection of the following mitochondrial stress test compounds: oligomycin (1 μmol/L; complex V inhibitor); FCCP (0.75 μmol/L; proton gradient uncoupler); and antimycin A (1 μmol/L; complex III inhibitor). Basal and maximal respiration were normalized by subtracting nonmitochondrial OCR (i.e., after antimycin A addition). Respiratory reserve capacity was calculated as the difference between maximal and basal OCR. ATP-linked OCR was derived as the difference between basal and oligomycin A–inhibited OCR. Data were normalized to total protein content in each well.

Wound-healing assay

Cell migration was assessed in serum-free media by wound-healing assays using Ibidi inserts (Martinsried) and quantified after 72 hours. Ibidi inserts were removed from a monolayer of GFP-labeled cells to expose the cell-free wound area. Fluorescent images were taken after 72 hours of migration and overlayed with corresponding images at time 0 hour. Pixels representing GFP-labeled cells were quantified within the wound area using ImageJ and corrected by subtracting any GFP-detected cells in the same area at time 0.

Spheroid attachment assay

Cells were plated at a density of 1,000 cells per well in ultra-low attachment 96-well plates (Corning) and incubated for 5 days. Spheroids were transferred to 24-well plates with or without Collagen I coating. Percentage outgrowth was calculated by subtracting the area covered by migrating cells onto the collagen matrix from the area of the spheroid at time 0 hour.

Live cell imaging of MitoSox oxidation

As an indicator of mitochondrial O2•−, the oxidation and fluorescence of the redox-sensitive MitoSox Red dye (Life Technologies) were monitored by live cell imaging according to manufacturer's instructions. Cells were imaged to detect oxidation and fluorescence of MitoSox using a Leica SP5 II AOBS confocal microscope following incubation with dye in HBSS for 30 minutes at 37°C. The Manganese Porphyrin O2•− scavenger ortho tetrakis(N-n-butoxyethylpyridinium-2-yl) porphyrin (MnTnBuOE-2-PyP5+) was generously provided by Dr. Ines Batinic Haberle (Duke University, Durham, NC).

Measurement of cellular H2O2 using catalase activity assay

Concentration of cellular H2O2 was determined by measuring the rate of inactivation of catalase with amino1,2,4-triazol (ATZ), according to Yusa and colleagues (15). ATZ irreversibly inactivates catalase by covalently binding with intermediate compound I, formed following oxidation of catalase by H2O2. Briefly, cells were treated with 20 mmol/L ATZ for different time intervals (15 and 30 minutes). Cells were washed with PBS and protein lysates were prepared in 50 mmol/L phosphate buffer (pH, 7.4) with protease inhibitors. Decomposition of H2O2 by catalase in protein lysates was analyzed using ultraviolet spectroscopy at 240 nm wavelength. H2O2 concentration was determined using the following equation: |$\rm [H_2O_2] = \it k/k_1$|⁠, where k is the empirically determined pseudo first-order rate constant of catalase inactivation in the cells by ATZ (Fig. 6A), whereas k1 is the rate of compound I formation [1.7 × 107 (mol/L)−1s−1].

Statistical analysis

All data presented are representative of at least three independent experiments and expressed as mean ± SEM, unless otherwise stated. Statistical data analysis (ANOVA with Tukey post-test or Student t test) was performed using GraphPad Prism Software v6. P < 0.05 was considered to be significant.

Sod2 expression is significantly increased in OCCC compared with other EOC histologic subtypes

Mining publicly available expression data (Oncomine.org) revealed that Sod2 mRNA levels are elevated in OCCC compared with other ovarian cancer histologic subtypes (Supplementary Fig. S1). Two representative datasets displayed in Fig. 1A and B show that levels of this antioxidant enzyme are statistically higher than in any other ovarian cancer subtype. Similarly, significantly higher Sod2 mRNA expression was observed by microarray analysis in a panel of OCCC cell lines when compared with cell lines of high-grade serous adenocarcinoma origin (Fig. 1C). This observation was further verified by assessing Sod2 expression in cultured ovarian cancer cell lines, using semiquantitative real-time RT-PCR and Western blotting (Fig. 1D and E).

Reduced Sod2 levels significantly inhibit OCCC cell proliferation in vitro and tumor formation in the CAM model

To further investigate the role of Sod2, we used two OCCC cell lines, ES-2 and TOV-21-G. Sod2 expression was inhibited by shRNA and siRNA transfection, which was demonstrated to lead to a concomitant decrease in Sod2 enzyme activity (Fig. 2A; Supplementary Figs. S2 and S3A). Following Sod2 expression knockdown, ES-2 cell proliferation rate (Fig. 2B; Supplementary Fig. S3B) and clonogenicity (Fig. 2C; Supplementary Fig. S3C) were significantly attenuated. This appeared to be Sod2 concentration-dependent. A 30% reduction in Sod2 levels mediated by stable shRNA transfection reduced clonogenicity by approximately 50% (shSod2_#1), whereas a 70% Sod2 decrease almost completely abrogated the cells' ability to survive in this assay (shSod2_#2; Supplementary Fig. S3C). Analysis of PARP cleavage and Annexin V staining suggested that the decrease in cell viability observed in cells with 30% Sod2 knockdown is not related to a significant increase in apoptosis (Supplementary Fig. S4). This cell line, referred to as shSod2 in the following figures, was chosen for subsequent studies to achieve pathophysiologically relevant changes in Sod2 expression (rather than complete loss), which also closely reflects Sod2 levels observed in non-OCCC cell lines OVCA433 and OVCA429 (Fig. 1E). The CAM ex ovo model was used to further test the role of Sod2 on ES-2 tumorigenicity. A significant decrease in tumor size and weight was observed in tumors grown from ES-2 cells with reduced Sod2 expression (shSod2; Fig. 2D). In addition, the shRNA-Sod2 tumors exhibited less vascularization than the control groups (Fig. 2D), suggesting that Sod2 may contribute to both proliferation and the recruitment of blood vessels to the tumor.

Sod2 maintains OCCC mitochondrial respiration

We previously demonstrated that OCCC cell lines are highly energetic and depend on both oxidative phosphorylation and glycolysis for their energy needs (13). Given that Sod2 has a primary role in protecting mitochondria from excess O2•−, the effect of Sod2 knockdown on mitochondrial respiration was assessed. OCR, representing mitochondrial oxidative phosphorylation, and ECAR, correlating with glycolytic activity, were measured using extracellular flux analysis in both ES-2 and TOV-21-G OCCC cell lines following Sod2 knockdown (13, 16). Stable shRNA-Sod2 decreases in ES-2 and transient siRNA-mediated knockdown of Sod2 in TOV-21-G cells resulted in significant reduction in basal OCR compared with control scramble RNAi–transfected cells (Fig. 3A–D; Supplementary Fig. S5A). Furthermore, respiratory reserve capacity, a measure of the ability of cells to enhance respiration in response to physiologic cues and stress, was significantly inhibited with reduced Sod2 levels (Fig. 3B and D), suggesting that Sod2 plays a major role in maintaining mitochondrial health to support maximal respiration. Although slight, a consistent negative effect on OCR was observed in stable shRNA-Sod2 cells following FCCP treatment (Fig. 3B). A wide range of FCCP concentrations was tested on these cells, but none were able to enhance OCR with Sod2 loss. FCCP can be inhibitory at high concentrations, and it has been speculated that this may be due to a loss in the ability of mitochondria to accumulate respiratory substrates (16). While not tested here, it is possible that sustained Sod2 expression decreases, and concomitant increases in mitochondrial O2•-− levels, may exacerbate this FCCP-dependent OCR inhibition, thereby influencing mitochondrial membrane integrity and substrate transport. No significant increases in ECAR were observed between control and shRNA- or siRNA-transfected cells, suggesting that decreases in Sod2 expression do not influence a compensatory shift toward glycolysis (Supplementary Fig. S5B–S5D). The above observations show that both siRNA- and shRNA-mediated decreases in Sod2 reduced basal OCR and respiratory reserve capacity, indicating that Sod2 is important in maintaining mitochondrial respiration in OCCC.

To assess whether a decrease in Sod2 expression results in compromised O2•− scavenging, which may be one of the causes of compromised mitochondrial function, the presence of mitochondrial O2•− was evaluated using the mitochondria-targeted redox-sensitive dye MitoSox. As expected, increased oxidation and consequential enhanced fluorescence of MitoSox were observed in the Sod2-knockdown cells compared with controls (Fig. 3E and F). Addition of the Sod2 mimetic porphyrin (MnTnBuOE-2-PyP5+), which acts as a O2•− scavenger, reduced MitoSox oxidation in both control and shRNA-Sod2 groups (Fig. 3E and F).

Sod2 knockdown attenuates metastasis of cancer cells in the CAM model

We have previously demonstrated that enhanced Sod2 expression is implicated with metastatic progression (7, 8, 17). To investigate the role of Sod2 during OCCC metastasis, the appearance of metastatic lesions of GFP-labeled ES-2 cells was investigated in the CAM tumor model. Single cells and micrometer-sized cellular clusters were highly abundant throughout the membrane in the control group, which could be observed 2 to 3 cm from the tumor (Fig. 4A). In contrast, metastatic spread from shRNA-Sod2 knockdown tumors was limited to the appearance of single cells in the membrane confined to an approximate 1- to 1.5-cm radius from the tumor (Fig. 4A). Furthermore, lung metastases in the chick embryo were observed in 11 of the 12 controls compared with only 5 of 10 embryos in the shRNA-Sod2 group (Fig. 4B). Furthermore, 10 of 12 control tumors metastasized into the liver, whereas only 4 of 10 liver metastases were observed in the shRNA-Sod2 group (Fig. 4B). While clusters of five or more cells were found in the lungs and livers of control groups, only single cells were detected in the Sod2-knockdown group (Fig. 4B).

Sod2 levels modulate cell migration and tumor spheroid outgrowth

Because of the significant abrogation of metastatic spread in response to Sod2 expression decreases, the role of Sod2 on cell migration was further investigated. Cell migration, assessed by wound-healing assays, was significantly inhibited with reduced Sod2 expression in ES-2 cells (Fig. 5A; Supplementary Fig. S6). Furthermore, the ability of cellular spheroid clusters to attach and cells to migrate from the spheroid onto collagen I and uncoated surfaces was also compromised with reduced Sod2 expression (Fig. 5B). Anchorage-independent spheroid formation is a commonly observed phenotype of ovarian cancer cells metastasizing via the transcoelomic route through the intraperitoneal cavity, and these have shown the ability to attach on the peritoneum to form metastatic lesions. These data suggest that Sod2 plays an important role in tumor spheroid metastasis (Fig. 5B). While spheroids of equal size were chosen for this assay, it should be noted that Sod2 knockdown also decreased ES-2 spheroid growth in anchorage independence (data not shown).

To gain mechanistic insights into the signaling pathways that may be altered by Sod2-mediated metastasis, phosphorylation profiles of Akt, p130cas, and focal adhesion kinase (FAK) were investigated. These were chosen on the basis of previous observations of their redox regulation and involvement in tumor cell migration (8, 9, 18, 19). shRNA-Sod2 cells exhibited a 50% decrease in phospho-Akt levels compared with scramble control cells, whereas no appreciable change was observed in phosphorylation of FAK or the focal adhesion adapter protein p130cas (Fig. 5C). The effects on Akt phosphorylation were also dependent on Sod2 concentration, where cells with lower Sod2 expression demonstrated a more striking decrease in Akt phosphorylation (Supplementary Fig. S6). These data suggest that Akt signaling may be important in driving Sod2-mediated tumorigenicity and metastasis of OCCC.

OCCC cell migration is H2O2-dependent

Sod2 is the primary enzyme involved in converting O2•− to H2O2 within the mitochondria. While it serves as a protective mechanism to maintain mitochondrial function (Fig. 3) by removal of damaging O2•−, a shift toward increasing levels of H2O2 has also been observed in response to enhanced Sod2 expression (8, 10, 18, 20, 21). Because of its relative stability and ease in traversing cellular membranes, H2O2 can mediate redox signaling, including events that drive migration (9). To test whether Sod2 changes the steady-state H2O2 levels in OCCC, we assessed intracellular H2O2 status in control and shRNA-Sod2 ES-2 cells using a biochemical assay on the basis of the irreversible inhibition of catalase by aminotriazole (15, 17). Steady-state levels of H2O2 were reduced approximately by 50% in Sod2-knockdown cells compared with controls (Fig. 6A), whereas baseline catalase activity and protein expression were comparable (Fig. 6B).

To test whether OCCC cell migration is H2O2-dependent, wound-healing assays were carried out in the presence of catalase, which catalyzes the conversion of H2O2 to H2O and O2. As previously demonstrated, exogenous application of recombinant catalase resulted in accumulation of catalase within ES-2 cells (Supplementary Fig. S7; ref. 9). Catalase significantly reduced the migration of both control and shRNA-Sod2 cells, suggesting that H2O2 is a promoter of ES-2 cell migration (Fig. 6C). Conversely, treatment with low levels of H2O2 (5 μmol/L) significantly reversed the slow migration of shRNA-Sod2 cells (Fig. 6C). Furthermore, 5- and 50-μmol/L H2O2 treatment was able to increase phospho-Akt levels (Fig. 6D), while catalase expression abrogated Akt phosphorylation (Supplementary Fig. S8), suggesting that this may be an important redox-dependent signaling pathway in OCCC.

It was noted that ES-2 control cells were not able to tolerate long-term exposure to low-dose H2O2 during migration assays (Fig. 6C). To examine this further, cell viability was assessed in response to H2O2. A significant reduction in cell survival in response to H2O2 was observed in the control group compared with cells with decreased Sod2 expression (Fig. 7A). This suggests that a higher intracellular steady-state H2O2 milieu in OCCC predisposes cells to enhanced killing by additional exposure to low-level exogenous H2O2. The above data imply that high Sod2 expression provides several advantages to OCCC by protecting mitochondrial function through scavenging of O2•− and driving H2O2-dependent migration. While these attributes are advantageous for OCCC survival and metastatic progression, an enhanced intracellular steady-state H2O2 level presents a double-edged sword, as these cells are consequentially more susceptible to H2O2 toxicity (Fig. 7B).

Although the five different EOC histologic subtypes share the same primary tumor location on the ovaries, it is now evident that these are distinct diseases with vastly different tissue origins and genetic and epigenetic profiles (2, 11). In the present study, we show that Sod2 is highly expressed in OCCC compared with other EOC histologic subtypes and that this mitochondrial antioxidant plays a significant role in OCCC tumorigenicity and metastasis.

Intracellular ROS are maintained within a narrow range tightly regulated by the balance of the rate of ROS production and ROS scavenging/detoxifying by antioxidant enzymes. This balance is often disrupted in the context of cancer, due to high ROS production as a consequence of changes in metabolism or the tumor environment (e.g., hypoxia) and the resulting changes in antioxidant expression. Because the mitochondrial respiratory chain is the major site of O2•− generation within cells, Sod2 plays an important role in maintaining cellular ROS balance. On the basis of the above findings, Sod2 appears to play a dual role in enhancing OCCC tumorigenicity, first, by protecting cells from mitochondrial O2•− damage, and second, by shifting the steady-state ROS balance toward H2O2.

We recently demonstrated that a distinguishing feature of OCCC is their unique metabolic phenotype. Compared with serous adenocarcinoma cells, OCCC cell lines were significantly more energetic, displaying both very high levels of mitochondrial oxidative phosphorylation and glycolytic flux (13). Our data suggest that Sod2 is intricately involved in maintaining this high rate of oxygen consumption, potentially by preserving mitochondrial function as a consequence of O2•− scavenging (Fig. 3). By preventing mitochondrial electron transport chain complex damage mediated by O2•− or secondary products, Sod2 likely supports the high rate of OCCC proliferation, clonogenicity, and tumor growth. The results of the present study suggest that inhibiting mitochondrial antioxidant defenses may provide an alternative strategy to therapeutically target OCCC.

In addition to scavenging O2•− and maintaining mitochondrial health for optimal cell proliferation, we believe that Sod2 has another role in promoting the aggressiveness of OCCC, by shifting steady-state H2O2 levels and driving prometastatic behavior (Figs. 4–6). It has been previously shown that high expression of Sod2 is associated with metastatic progression (8, 10, 17, 20–23) and that dependence of cancer cell migration is related to cellular H2O2 production (9, 19, 24, 25). For example, we have shown that steady-state increases in H2O2 can lead to induction of the FAK pathway and migration of metastatic bladder cancer cells and cells with enforced Sod2 expression (9, 19). This effect was mediated by oxidation-dependent inhibition of the phosphatase PTPN12, leading to enhanced phosphorylation of p130cas and Rac1 activation. In addition, work from the Melendez group has shown that Sod2 expression significantly contributes to the expression of the matrix-degrading enzyme MMP-1 in an H2O2-dependent manner (21) and that the Sod2/H2O2-dependent inhibition of the dual-lipid protein tyrosine phosphatase PTEN enhances Akt/GSK3β/VEGF-dependent angiogenesis (18), both processes contributing significantly to metastasis. Our present data suggest that Sod2 may similarly contribute to metastatic progression of OCCC by activating Akt signaling (Figs. 5 and 6). In addition to its prosurvival function, Akt has been shown to influence metastasis and cell migration by regulating cytoskeletal rearrangement, prometastatic cell signaling, and gene transcription (26). These results are of specific importance to OCCC, which, unlike other ovarian cancer histologic subtypes, has been characterized by high-frequency Akt pathway activation. Seventy percent of early- and 68% of late-stage OCCC cases have been shown to display phospho-Akt (S473) staining (27). About 38% of OCCC cases show PTEN loss (28) and 40% of cases PI3K activating mutations (29). Our data imply that Sod2-dependent Akt phosphorylation may also contribute to high activation of Akt signaling in OCCC. Because Akt phosphorylation was highly susceptible to H2O2 treatment, it suggests that this signaling pathway is redox-regulated in OCCC, with a plausible mechanism for this being the oxidation of PTEN (18).

Although an increase in Sod2 should theoretically not result in higher levels of H2O2 production based on the enzyme's kinetic properties (30), a number of studies have demonstrated increases in H2O2 levels that correlate with Sod2 expression (18, 31–33). While the reason for this observation in OCCC has not been investigated, there are plausible explanations for this increase in steady-state H2O2 as a consequence of Sod expression. These explanations primarily relate to changes in the reaction rates within the mitochondrial electron transport chain (ETC). For example, it has been proposed that Sod2 in the mitochondria may alter the flux of O2•− from some quinone/semiquinone/hydroquinone triads, such as coenzyme Q, thereby driving the reaction into the direction of O2•− production, potentially leading to enhanced localized dismutation to H2O2 by Sod2 (31, 34). Alternatively, inhibition of cytochrome c oxidase by nitric oxide, arising as a consequence of Sod2 expression, may influence the reduction state of the ETC and drive O2•- and H2O2 production (35). Our observation that Sod2 knockdown also decreases H2O2 levels suggests that Sod2 is involved in regulating H2O2 balance within cells, and this may contribute to H2O2-mediated redox signaling.

While Sod2 appears to contribute to H2O2-mediated metastatic progression, an enhanced steady-state H2O2 milieu may also present a disadvantage to OCCC cells. Our data suggest that cells with high Sod2 levels and concomitant increases in intracellular H2O2 are more susceptible to exogenous sources of redox stress (Fig. 7A). This likely puts cells closer to the cytotoxic threshold of H2O2, which is reached once cells are further challenged by exogenous ROS. Interestingly, OCCC cells do not appear to have enhanced expression of catalase to provide additional scavenging of excess H2O2 (Fig. 6A). While sublethal levels of H2O2 have been shown to contribute to redox signaling, high levels of H2O2 can elicit tumor cell death by a number of pathways, including apoptosis, protein/DNA damage, and mitochondrial dysfunction (36–38). Furthermore, it was recently reported that H2O2 exposure of tumor cells with enforced Sod2 expression can result in Sod2 peroxidase activity, leading to mitochondrial damage and dysfunction (39). An increased H2O2 steady-state has been observed in a number of cancer cells (17, 40–42) and lends credence to the idea that this higher H2O2 threshold may be exploited therapeutically. In this regard, the use of high-dose ascorbic acid, which is oxidized within tumor cells to produce H2O2, has recently been revisited for use in cancer treatment (43–45) and has shown promise in early clinical trials in advanced-stage cancers (46, 47). Ascorbate and concomitant H2O2-mediated DNA damage and apoptosis were shown to enhance ovarian cancer cell death and increase chemosensitivity (48). While that study was not focused on OCCC, this type of treatment may be of particular benefit to this histologic subtype given the high expression of Sod2. It is important to highlight that cancer cells with enhanced Sod2 expression may respond differently to ROS-producing agents, depending on both the type and cellular location of the ROS/reactive nitrogen species generated. For instance, Sod2 may enhance scavenging of O2•− and therefore provide chemoresistance benefits to the tumor cells in response to these ROS. Conversely, while an increase in steady-state H2O2 facilitates redox signaling beneficial to the cancer cells, this higher threshold may facilitate H2O2-mediated OCCC cell death in response to further insult by exogenous sources of H2O2. Understanding the complex interaction of antioxidants and ROS in OCCC is therefore of importance and may provide novel therapeutic avenues to pursue for this histologic subtype of ovarian cancer.

No potential conflicts of interest were disclosed.

Conception and design: L.P.M.P. Hemachandra, D.-H. Shin, L.M. Uusitalo, N. Hempel

Development of methodology: L.P.M.P. Hemachandra, N. Hempel

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): L.P.M.P. Hemachandra, U. Dier, J.N. Iuliano, L.M. Uusitalo, S.K. Murphy

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): L.P.M.P. Hemachandra, D.-H. Shin, N. Hempel

Writing, review, and/or revision of the manuscript: L.P.M.P. Hemachandra, D.-H. Shin, U. Dier, S.A. Engelberth, L.M. Uusitalo, S.K. Murphy, N. Hempel

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): D.-H. Shin, S.A. Engelberth

Study supervision: N. Hempel

The ortho tetrakis(N-n-butoxyethylpyridinium-2-yl) porphyrin (MnTnBuOE-2-PyP5+) was generously provided by Dr. Ines Batinic-Haberle (Duke University). The authors thank Dr. J. Andres Melendez (SUNY Poly) for helpful discussions and Dr. Nate Cady for use of his microscope. They appreciate the help of Jeffrey Richards for assistance with spheroid assays.

This work was supported by NIH/NCI grant R00CA143229.

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.

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