Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2009 Aug 1.
Published in final edited form as: Cancer Biol Ther. 2008 Aug 13;7(8):1271–1279. doi: 10.4161/cbt.7.8.6300

Mitochondria-targeted antioxidant enzyme activity regulates radioresistance in human pancreatic cancer cells

Carolyn J Fisher 1, Prabhat C Goswami 1
PMCID: PMC2581876  NIHMSID: NIHMS77296  PMID: 18497575

Abstract

In recent years, cellular redox environment gained significant attention as a critical regulator of cellular responses to oxidative stress. Cellular redox environment is a balance between production of reactive oxygen species and their removal by antioxidant enzymes. We investigated the hypothesis that mitochondrial antioxidant enzyme activity regulates radioresistance in human pancreatic cancer cells. Vector-control and manganese superoxide dismutase (MnSOD) overexpressing human pancreatic cancer cells were irradiated and assayed for cell survival and activation of the G2-checkpoint pathway. Increased MnSOD activity significantly increased cell survival following irradiation with 6 Gy of gamma-radiation (p < 0.05). The MnSOD overexpressing irradiated cells also revealed 3–4 folds increase in the percentage of G2 cells compared to irradiated vector-control. Furthermore, MnSOD overexpressing irradiated cells exhibited increased loss of phosphorylated histone H2AX protein levels. The radiation-induced increase in cyclin B1 protein levels in irradiated vector-control cells was suppressed in irradiated MnSOD overexpressing cells. Mitochondria-targeted catalase overexpression increased the survival of irradiated cells. These results support the hypothesis that mitochondrial antioxidant enzyme activity and mitochondria-generated reactive oxygen species-signaling (superoxide and hydrogen peroxide) could regulate radiation-induced G2 checkpoint activation and radioresistance in human pancreatic cancer cells.

Keywords: MnSOD, ROS, G2 delay, radioresistance, mitochondrial catalase, cyclin B1

INTRODUCTION

Cancer is a multifaceted disease with various treatment modalities that include ionizing radiation (IR). IR exposures have been shown to produce reactive oxygen species (ROS) in living cells.1 ROS (superoxide and hydrogen peroxide) are ameliorated by cellular defense systems such as endogenous antioxidant enzymes (AOE). The AOE include superoxide dismutase (SOD), which converts superoxide to hydrogen peroxide and catalase (CAT) that neutralizes hydrogen peroxide to water. There are three types of SODs in mammalian cells, extracellular (EC-SOD), copper-zinc (CuZnSOD), found in the cytoplasm and nucleus, and manganese (MnSOD), found in the mitochondrion.

A nuclear encoded mitochondria localized protein, MnSOD, is essential and biologically significant to aerobic cells. It has been reported that total MnSOD gene knockout is lethal in mice2 and heterozygous mice with decreased MnSOD activity were more susceptible to oxidative injury.3 These and numerous other studies have shown that MnSOD protects against oxidant stress including radiation injury.4–8 Recently, it has been reported that ectopic expression of MnSOD altered the intracellular oxidation-reduction (redox) state9 and also affected radiosensitivity in tumor cells.10–12 Although the mechanisms associated with MnSOD overexpression and radiosensitivity in tumor cells are not completely understood, it is possible that mitochondria-generated ROS could regulate radiation-induced cell cycle checkpoint pathways, which could impact upon cancer cells’ responses to radiation exposures.

The crucial role of cell cycle checkpoints13 is to maintain genomic stability in response to internal (oxidative) and external (IR) cellular stressors by controlling the order of cell cycle events and halting at discrete points of surveillance identified as the G1, S, and G2 checkpoints. Tumor cells that exhibit the mutant p53 phenotype have defective G1 checkpoint activation, and cells possessing this type of genetic deficiency rely heavily on their G2 checkpoint to protect against cellular stressors. The molecular mechanisms associated with the DNA damage response and the G2 checkpoint activation in mammalian cells have identified the ataxia telangiectasia mutated (ATM) protein kinase, which is auto-phosphorylated (activated) following IR as an initiating event.14

The ATM kinase regulation of biological responses to DNA damage is coordinated by its phosphorylation of a wide range of targets including checkpoint kinases 1 and 2, (Chk 1/2) leading to inhibition of the downstream effector, cyclin B1 resulting in a delay in the activation of cyclin B1/Cdk1 kinase activity at the G2/M border. Cyclin B1 regulates progression from G2 to M and has been found downregulated following IR signifying its role in the G2 delay.15

Another ATM target phosphorylated following IR is the histone H2A variant, H2AX. In response to DNA double strand breaks, H2AX, which represents 2–25% of the H2A pool in nucleosomes, is rapidly phosphorylated on a carboxy terminal SQE motif to form the γH2AX foci along mega base domains of chromatin.16 It has been suggested that this modification in chromatin could affect recruitment of DNA repair proteins and would also be a factor in the G2 delay. Evidence suggests that mice lacking H2AX were deficient in the IR-induced G2 checkpoint.17

This study was initiated to investigate the hypothesis that mitochondrial antioxidant enzyme activity regulates radiosensitivity in human pancreatic cancer cells. Our results show that human pancreatic cancer cells overexpressing MnSOD were radioresistant, which was accompanied with 3–4 folds increase in the percentage of G2 cells. The MnSOD overexpressing irradiated cells showed increased loss of γH2AX protein levels. The radiation-induced increase in cyclin B1 protein levels in irradiated vector-control cells was suppressed in irradiated MnSOD overexpressing cells. Overexpression of catalase in the mitochondria significantly increased the survival of vector-control and MnSOD overexpressing cells following irradiation with 6 Gy.

MATERIALS AND METHODS

Cell culture and irradiation

Human pancreatic cancer cells (MIA PaCa-2) stably transfected with human MnSOD cDNA were kindly provided by Dr. Joseph Cullen (University of Iowa). MnSOD overexpressing cells (Mn1 and Mn7) and an expression vector-control (V2B) were used in this study. MnSOD overexpression was confirmed with western blotting and by the indirect nitroblue tetrazolium (NBT) biochemical assay for enzymatic activity.18 Total glutathione (GSH) and glutathione disulfide (GSSG) were assayed by following the previously published method of Griffith.19 All measurements were normalized to the protein content of whole cell homogenate. Cells were exposed to irradiation using a 137Cs source (J. L. Shepherd model 81-16A) at a dose rate of 0.89 Gy/min.

Cell survival assay

Following irradiation, cells were plated in triplicate into 60 mm tissue culture dishes at limiting dilutions and were incubated for 2 weeks to allow colony formation. The colonies were then fixed in 70% ethanol and stained with 0.1% Coomassie blue. A population of 50 cells per colony was scored.20 Plating efficiencies (PE) were calculated using the following formula: PE = (number of colonies counted/number of cells seeded) × 100. Survival fractions (SF) were calculated using the following formula: SF = (number of colonies counted)/(number of cells seeded × PE).

Flow cytometry assays: Propidium iodide (PI) staining for DNA content

Cells were harvested at predetermined intervals following irradiation and fixed in 70% ethanol. Approximately 1 × 106 cells were incubated with 100 μL of RNase A (1 mg/mL) for 30 min followed by incubation with 35 μg/mL of PI for 1 h.21

BrdU pulse-labeling

Cells were pulse-labeled with 10 μM 5-bromo-2′-deoxy-uridine (BrdU) for 30 min at 37°C and fixed in 70% ethanol. Nuclei were isolated and incubated with monoclonal primary antibody to BrdU (Becton Dickinson) and fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Becton Dickinson). Cells were counter-stained with PI and fluorescence measured following our previously published protocol.22

Mitotic Index (MI)

The MI was determined by measuring histone H3 phosphorylation levels following the previously published protocol reported by Xu and Kastan.23 Cells were irradiated with 6 Gy and fixed in 70% ethanol at indicated times. Fixed cells were stained with an antibody against phospho-histone H3 (Ser-10) (Upstate Technologies), followed by a FITC-conjugated secondary antibody, and counter stained with PI.

Immunofluorescence of γ-H2AX

Phosphorylated form of histone H2AX (γ-H2AX) was assessed using a DNA damage kit (Upstate Technologies) per the manufacturer’s instructions. Irradiated cells were fixed for 20 min over ice, permeabilized and were either stained with anti-phospho-Histone H2AX-FITC (Ser-139) conjugate or the negative control mouse IgG-FITC conjugate.

Immunofluorescence of ATM

Irradiated cells were fixed in 70% ethanol, rehydrated in 0.25% Triton X-100 in PBS and stained with anti phospho-ATM (Ser-1981) antibody (Upstate Technologies). Cells were counterstained with FITC-conjugated secondary antibody.24

Immunofluorescence of cyclin B1

Un-irradiated control and irradiated monolayer cells were harvested by trypsinization and fixed in 70% cold ethanol. Ethanol fixed cells were washed and incubated overnight at 4°C with anti-cyclin B1 antibody (BD Pharmingen) followed by 1 h incubation with FITC-conjugated secondary antibody. Cells were counter-stained with PI for DNA content measurement.

Fluorescence measurements and quantitation

The fluorescence of PI and/or FITC stained cells were measured using a FACScan (Becton Dickinson, San Jose, CA) instrument equipped with a 488 nm blue light laser excitation source. For detection, the FL1 detector at 530 nm emission filters (FITC), and FL-2 detector at 585 nm emission filters (PI) were used. Data was analyzed using CellQuest Pro analyses software (Becton Dickinson). The DNA content was evaluated with ModFit deconvulation software (Verity Software).

Measurements of reactive oxygen species (ROS) steady state levels

Live cells were labeled with the fluorescent dye, dihydroethidium (DHE, 10 μM; in 1% DMSO) (Molecular Probes, Eugene, Oregon) for 40 min at 37°C and harvested on ice using phenol free trypsin-EDTA followed by phenol free EMEM containing 10% FBS.25 Centrifuged cells were re-suspended in PBS and analyzed using a FACScan flow cytometer. The mean fluorescence intensity of 10,000 cells were analyzed and corrected for autofluorescence from unlabeled cells.

Antioxidant enzyme activity assays

SOD enzymatic activity was measured using an indirect competition assay between SOD and nitroblue tetrazolium (NBT) following the method of Spitz and Oberley.18 Specific activity was reported as Units per mg protein. Catalase (CAT) enzymatic activity was assayed using the method of Aebi26 where the disappearance of H2O2 was measured spectrophotometrically at 240 nm. Specific activity was reported as mkU per mg protein.

Western blotting assay

Cells were harvested, washed, and either lysed by sonic disruption on ice at low power, or with 100 μL of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5% deoxycholate, 0.1% SDS, 0.1% Triton-X-100, 5 mM EDTA) supplemented with protease inhibitors (Sigma). Equal amounts of protein were separated by SDS-PAGE (12.5%) and transferred to nitrocellulose membranes. Blots were incubated with antibodies directed against either MnSOD (generously donated by Dr. Larry W. Oberley, University of Iowa), cyclin B1 (BD Pharmingen), or actin (Chemicon), followed by a horseradish peroxidase-conjugated secondary antibody (Chemicon). The enhanced chemiluminescence (ECL) plus Western Blotting solution (GE Healthcare) was used for visualization of immunoreactive bands.

Adenoviral infections

Cells were incubated with a replication-defective adenovirus containing either the human catalase cDNA containing a mitochondrial targeting sequence (AdmCAT) 27 or the empty vector (AdBgl II) (DNA Vector Core, University of Iowa) for 24 h in serum-free media. Media was replaced with complete media for an additional 24 h before cells were harvested for analyses. Cells were infected with adenovirus containing the green fluorescence protein cDNA (AdGFP) and transduction efficiency was determined by flow cytometry. The transduction efficiency for 25 MOI infected cells was calculated to be 77% (data not shown).

Small interfering (si) RNA Transfection

Small interfering RNA (siRNA) induced gene silencing in mammalian cells is a tool to suppress the expression of specific genes.28 The MnSOD siRNA corresponding to sequence CUUUUAGAUAAUCAGGCC (Silencer pre-designed siRNA: ID 9052) was obtained from Ambion (Austin, TX). The negative control siRNA (Silencer negative control #1, Ambion) is a 19 bp scrambled sequence with no sequence homology to any known gene sequences from mouse, rat, or human. MIA PaCa-2 wild type cells (105) were transfected with 10 μl of Lipofectamine™ 2000 (Invitrogen Co. Carlsbad, CA) and 8 ng of siRNA μM stock) for 96 h. MnSOD protein levels were verified by western blot.

Statistical analyses

The comparison between groups was determined by ANOVA with Dunnett’s post hoc evaluations using GraphPad Prism version 4. Probability (p) values less than 0.05 were considered significant. All experiments were performed a minimum of three times. Error bars represent the experimental average of 3 separate experiments (n=3) reported as standard error of the mean (SEM).

RESULTS

MnSOD activity regulates radioresistance in human pancreatic cancer cells

Initially, we determined if MnSOD activity regulates cellular redox environment and cell growth. Exponentially growing asynchronous cultures of vector-control and MnSOD overexpressing (Mn1 and Mn7) human pancreatic cancer cells (MIA PaCa-2) were harvested and analyzed for MnSOD protein levels and activity. MnSOD overexpression increased MnSOD protein levels approximately 1.5-fold in Mn1 and 2-fold in Mn7 cells compared to vector-control cells (Figure 1A). MnSOD activity was approximately 20 U per mg protein in vector-control cells, 30 U in Mn1 and 60 U per mg protein in Mn7 cells (Figure 1B). Cells harvested from replicate plates were assayed for glutathione (GSH) and glutathione disulfide (GSSG) levels. GSH levels were significantly (p <0.001) decreased in MnSOD overexpressing cells compared to vector-control cells (Figure 1C). The percent GSSG in all three cell types were less than 1% (data not shown).

Figure 1.

Figure 1

Open in a new tab

The effect of MnSOD activity on exponentially growing cells. (A) MnSOD protein levels. MIA PaCa-2 cells overexpressing MnSOD (Mn1 and Mn7) and vector control (V2B) were lysed and whole cell lysates were subjected to SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with antibodies against MnSOD and actin (loading control). (B) MnSOD enzymatic activity. Cell pellets were homogenized in ice-cold DETAPAC buffer and enzymatic activity measured using the indirect NBT assay. Data was normalized to mg of protein of whole cell homogenate. (C) Glutathione levels. Exponentially growing cultures were plated (1×105 per 60 mm dish), continued in culture for 48 h, and scrape harvested in cold PBS. Total glutathione (GSH) and glutathione disulfide (GSSG) were distinguished by the addition of 2-vinylpyridine. Results are reported as nmoles/mg protein. (D) Cell cycle phase distribution. Cells were pulse-labeled with 10 μM of BrdU for 30 min, harvested, and fixed in 70% ethanol. The DNA content and BrdU incorporation was measured using PI staining and anti-BrdU antibody, respectively. The percent of G1, S, and G2 cells was determined relative to total population (G1+S+G2). Asterisks represent statistical significance compared to vector-control cells (V2B).

To determine if MnSOD overexpression affects cellular growth, monolayer cultures were pulse-labeled with BrdU for 30 min and harvested for flow cytometry analysis of cell cycle phase distributions (Figure 1D). The G1 percentages of cells overexpressing MnSOD were slightly higher compared to vector-control cells (Mn1 36 ± 1; Mn7 38 ± 1; V2B 33 ± 1). There was a decrease in the percent S-phase in Mn1 (47 ± 1) and Mn7 (46 ± 2) cells compared to vector-control (50 ± 2).

In order to determine the effect of MnSOD activity on radiosensitivity, monolayer cultures of exponentially growing asynchronous cells were irradiated (2–6 Gy) and surviving fractions (SF) determined using the clonogenic assay. Results presented in Figure 2A showed MnSOD overexpression increased cell survival that was significantly different at the 4 and 6 Gy doses compared to vector-control (p <0.01). A direct correlation was observed between MnSOD activity and SF (Figure 2B, r2 = 0.99). Additional experiments were performed to measure DHE-fluorescence in irradiated cells. The DHE-fluorescence increased in irradiated V2B cells at 12 h compared to 0 h post-irradiation. The DHE-fluorescence was similar in irradiated Mn1 and Mn7 cells at 12 compared to 0 h post-irradiation. Subsequently, the DHE fluorescence increased further at 24 h post-irradiation compared to 0 h in each cell line. However, this increase was slightly more in V2B compared to Mn1 and Mn7 (Figure 2C).

Figure 2.

Figure 2

Open in a new tab

MnSOD overexpression increases cell survival. (A) Survival fraction (SF). Cells were irradiated (0–6 Gy γ rays), seeded into 60 mm tissue culture plates at limiting dilutions, and incubated for 2 weeks to allow colony formation. The colonies were then fixed in 70% ethanol and stained with 0.1% coomassie blue. A population of 50 cells per colony was scored. Asterisks represent statistical significance compared to vector-control cells. (B) Correlation plot of normalized survival fraction (NSF) at 6 Gy and relative MnSOD activity. The SF and MnSOD activity were normalized to the vector control values. C) DHE Fluorescence. Cells were irradiated (0 or 6 Gy), incubated with 10 μM DHE, and harvested over ice at 0, 12, 24, and 48 h following radiation. Fluorescence was measured using flow cytometry.

MnSOD overexpression alters radiation-induced activation of the G2-checkpoint pathways

To determine if MnSOD overexpression affects radiation-induced G2 checkpoint pathway, irradiated V2B, Mn1, and Mn7 cells were harvested at various hours post-irradiation and assayed for phosphorylated ATM and H2AX protein levels. Since phosphorylation of ATM at serine 1981 (p-ATM) correlates to its kinase activity, a flow cytometry based assay was used to measure p-ATM levels in irradiated cells. Results showed p-ATM levels increased in all cell lines at 0.5 h post-irradiation (Figure 3A). However, the fold-change was lower in irradiated Mn1 and Mn7 compared to V2B cells at 2 h post-irradiation (Figure 3A). An inverse correlation (Figure 3B) was observed between p-ATM and MnSOD activity (r2 = 0.97). Activated ATM is known to phosphorylate H2AX (γH2AX) within minutes of irradiation. Cells from replicate dishes were analyzed for γH2AX protein levels using a flow-cytometry based assay. There were increased changes in γH2AX protein levels in unirradiated Mn1 and Mn7 compared to V2B cells (Figure 4A). As expected irradiation increased γH2AX protein levels in all three cell types (Figure 4B). However, the fold-increase in irradiated Mn1 and Mn7 cells was significantly (a = p< 0.05) lower at 1 and 2 h post-irradiation compared to irradiated V2B cells. An inverse correlation was observed between surviving fraction and γH2AX protein levels (Figure 4C, r2 = 0.86).

Figure 3.

Figure 3

Open in a new tab

MnSOD overexpression alters ATM phosphorylation. (A) Phosphorylated ATM levels (p-ATM): cells (1 × 105 per 60 mm dish) were irradiated with 6 Gy, harvested at 0.5 and 2 h post-irradiation and fixed in 70% ethanol. Ethanol-fixed cells were incubated with anti-phospho-ATM antibody (Ser 1981) followed by FITC-conjugated secondary antibody. The fluorescence of FITC stained cells was measured using flow cytometry. (B) Correlation plot of relative p-ATM levels (2 h) and relative MnSOD activity.

Figure 4.

Figure 4

Open in a new tab

MnSOD activity and phosphorylated H2AX. (A) Exponentially growing cultures of vector and MnSOD overexpressing cells were fixed in ethanol and stained with either FITC-conjugated anti-phospho H2AX (γH2AX) antibody (Ser 139) or control IgG-FITC-conjugate and fluorescence measured using flow cytometry. Cells positive for γH2AX were determined relative to control (IgG-FITC). Data represents basal (unirradiated) levels. (B) Monolayer cultures (1 × 105 per 60 mm dish) were irradiated with 6 Gy, harvested at 0.5, 1, and 2 h following irradiation, and analyzed for γH2AX levels. Fold changes were determined relative to un-irradiated cultures (a = p< 0.05). (C) Correlation plot of normalized survival fraction (NSF) and relative γH2AX protein levels 1 h following radiation.

The possible effect of MnSOD activity regulating the radiation-induced G2-checkpoint pathways was further evident from the results presented in Figures 5A & 5B. Total cellular protein extracts were analyzed for cyclin B1 protein levels by immunoblotting. Cyclin B1 protein levels increased in irradiated V2B cells at 12 and 24 h post-irradiation. Cyclin B1 protein levels in irradiated Mn1 cells showed a small increase at 24 h post-irradiation, while irradiated Mn7 cells did not show any change in cyclin B1 protein levels at 12 and 24 h post-irradiation compared to 0h (Figure 5A). These results were consistent with results obtained from a flow cytometry assay determining cyclin B1 protein levels in irradiated cells (Figure 5B). Irradiated V2B, Mn1, and Mn7 cells were harvested at 12 h post-irradiation and fixed in ethanol. Ethanol-fixed cells were assayed for cyclin B1 protein levels using flow cytometry. Cyclin B1 protein levels were significantly (p < 0.05) suppressed 60–70 percent in Mn1 and Mn7 cells compared to V2B cells (Figure 5B).

Figure 5.

Figure 5

Open in a new tab

MnSOD overexpression alters radiation-induced G2 checkpoint activation. (A) Cyclin B1 protein levels. Monolayer cultures were irradiated with 6 Gy and harvested 12 and 24 h following radiation. Cells were lysed with 100 μL of lysis buffer and equal amounts of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with antibodies against cyclin B1 and actin (loading control). (B) Flow cytometric analysis of cyclin B1. Ethanol fixed cells were incubated overnight (4°C) with anti-cyclin B1 antibody followed by 1 h incubation with FITC-conjugated secondary antibody. Cells were counter-stained with PI for DNA content measurement. The fluorescence of PI and/or FITC stained cells was measured by flow cytometry. The percentage of FITC-positive cells with G2 DNA content was calculated. Data represents values relative to V2B. (C) Mitotic index (MI). Monolayer cultures were irradiated with 6 Gy and harvested 0, 0.5, and 2 h post-irradiation. Ethanol fixed cells were incubated with phosphorylated histone H3 (Ser-10) primary antibody followed by FITC-conjugated secondary antibody. Cells were counter-stained with PI for measurements of DNA content. The fluorescence of PI and/or FITC stained cells was measured by flow cytometry. The percentage of FITC-positive cells with G2 DNA content, representative of mitotic cells, was calculated. The MI was determined relative to un-irradiated cells. (D) G2 accumulation: 6 Gy-irradiated monolayer cultures were harvested 0, 12, 24, and 48 h post-irradiation. Ethanol fixed cells were incubated with RNase A, stained with PI and the fluorescence measured by flow cytometry. Data represents the fold-change in irradiated cells normalized to un-irradiated cells (a = p< 0.05, b = p< 0.01).

Consistent with the above results, MnSOD activity altered radiation-induced G2-checkpoint activation. Initially, the mitotic index (MI) was determined using a flow cytometry based assay that measures the phosphorylated form of histone H3 (p-H3). Phosphorylated histone H3 is a specific marker for mitosis and the decrease in mitotic cells following radiation indicates activation of the G2 checkpoint. MnSOD overexpressing cells showed a small increase in MI at 0.5 h post-irradiation compared to V2B cells (Figure 5C). All three cell types showed a decrease in MI at 2 h post-irradiation suggesting that the G2-checkpoint is active in V2B as well as Mn1 and Mn7 cells. The MI at 6 h post-irradiation was comparable to 0 h indicating a 6 h delay in cell division in 6 Gy irradiated cells (data not shown). The percentage of cells in G2 was determined using the BrdU/PI staining and flow cytometry. Exponential cultures were irradiated with 6 Gy and pulse-labeled with BrdU 30 min prior to harvesting at indicated hours post-irradiation. The fold-change in the percentage of cells in G2 increased approximately 2-fold at 12 h post-irradiation in irradiated V2B cells compared to 0h (Figure 5D). Irradiated Mn1 and Mn7 cells showed a significant (a = p < 0.05, b = p< 0.01) increase in percent G2-cells at 12 and 24 h post-irradiation compared to V2B. The percentage of G2-cells in all three cell types was comparable at 48 h post-irradiation (Figure 5D) suggesting that while the G2-duration is not significantly altered among the three cell types MnSOD overexpression did increase the percentage of cells in G2 following irradiation.

Mitochondrial ROS regulate radioresistance

MnSOD is a nuclear encoded and mitochondrial localized antioxidant enzyme known to convert superoxide to hydrogen peroxide. The above results suggest MnSOD activity and therefore mitochondria-derived superoxide-signaling could regulate radiosensitivity in human pancreatic cancer cells. To further determine the role of mitochondria-derived ROS-signaling regulating cancer cells’ response to radiation exposure, cells were infected with adenovirus carrying the mitochondria-targeted human catalase cDNA (AdmCAT). The catalase activity was approximately 10 mkU/mg in un-irradiated V2B cells compared to 4 mkU/mg in Mn1 and Mn7 cells (Figure 6A). Cells infected with 25 MOI of AdmCAT showed 4–5 folds increase in catalase activity at 48 h post-infection (Figure 6B). Increase in catalase activity enhanced cell survival in irradiated V2B and Mn1 cells while Mn7 cells with highest MnSOD activity did not show any significant change in the fraction of surviving cells (Figure 6C). To further assess the effects of MnSOD on radioresistance in MIA PaCa-2 cells, gene knock-down experiments using siRNA were performed. Wild-type cells transfected with siRNA directed against MnSOD show a decrease in MnSOD protein levels compared to cells transfected with control siRNA. The decrease in MnSOD protein levels resulted in a significant decrease in cell survival following 3 Gy irradiation (Figure 6D).

Figure 6.

Figure 6

Open in a new tab

Mitochondria ROS regulate radiosensitivity. (A) Catalase enzymatic activity. Exponentially growing cultures were plated (1×105 per 60 mm dish), continued in culture for 48 h, and scrape harvested. Catalase enzymatic activity was assayed where the disappearance of H2O2 was measured spectrophotometrically at 240 nm. (B) Catalase activity in cells infected with adenovirus containing a mitochondria-targeted human catalase cDNA (AdmCAT). Cells were incubated with 25 MOI of AdmCAT for 24 h in serum-free media. Cells were harvested at 48 h post-infection and analyzed for catalase enzymatic activity. (C) Survival Fraction. Control and AdmCAT infected cells were irradiated with 6 Gy at 48 h post-infection and the fraction of surviving cells assayed by clonogenic assay. Asterisks represent significance compared to un-infected irradiated V2B cells. (D) SiRNA Transfection: Wild-type MIA PACa-2 cells were transfected with siRNA against MnSOD or control siRNA, irradiated with 3 Gy. Equal amounts of protein were analyzed by SDS-PAGE and transferred to nitrocellulose membranes. Blots were probed with antibodies against MnSOD and actin (loading control). Cell survival was measured using the clonogenic assay. Asterisks represent significance compared to control siRNA infected irradiated cells.

DISCUSSION

Manganese superoxide dismutase (MnSOD) is a mitochondria-localized antioxidant enzyme that regulates the intracellular oxidation-reduction (redox) state by converting superoxide to hydrogen peroxide. Hydrogen peroxide is then neutralized by catalase and glutathione peroxidase. In recent years, the intracellular redox state and cell cycle checkpoint pathways have drawn significant attention as modulators of radioresistance. The mechanisms associated with MnSOD activity, the intracellular redox state, and radioresistance are not well understood, but could be related to cell cycle checkpoints, and in particular, the G2 checkpoint. The G2 checkpoint, which prevents cells from entering mitosis when DNA is damaged, is initiated in response to endogenous (reactive oxygen species) and exogenous (ionizing radiation) stressors. In tumor cells, the G1 checkpoint is mostly regulated by the p53 tumor suppressor gene that is mutated in most cancers. Tumor cells therefore, are more likely to undergo a G2 checkpoint activation following ionizing radiation. The G2 checkpoint activation has been demonstrated in virtually all eukaryotic cells examined to date in response to ionizing radiation.29

Ectopic expression of MnSOD in human pancreatic cell line, MIA PaCa-2 increased protein and enzymatic activities above basal levels (Figures 1A & B). The fold changes although modest, more appropriately represent physiological conditions. The increased MnSOD activity showed minimal changes in the percentage of G1 and S-phase cells, which correlated with small changes in cell growth rate (data not shown). Consistent with previously published results12, MnSOD overexpressing cells showed lower levels of glutathione indicating that these cells are adapted to a different redox-state (Figure 1C). Increased MnSOD activity rendered cells resistant to ionizing radiation relative to vector-control cells (Figure 2A). These results are similar to earlier reports where MnSOD overexpressing human neuroblastoma tumor cells, human ovarian cancer cells, hepatocellular carcinoma cells, and oral squamous carcinoma cells were reported to be more resistant to radiation-induced cell killing compared to control cells.10–12, 30 In further support of the role of MnSOD activity and radioresistance, a direct correlation was observed between MnSOD activity and the fraction of surviving cells (Figure 2B). Furthermore, MnSOD overexpression suppressed radiation-induced increase in DHE-fluorescence (Figure 2C), supporting the hypothesis that MnSOD activity regulates mitochondria-generated ROS (presumably superoxide) in irradiated cells. Our results and earlier reports reviewed by Kinnula and Crapo31 support the hypothesis that MnSOD activity could be used as a prognostic marker of tumor cell radiosensitivity.

The increased MnSOD activity altered the radiation-induced G2 checkpoint pathway. Radiation-induced increase in p-ATM and γH2AX protein levels inversely correlated with MnSOD activity (Figures 3 & 4). Likewise, radiation-induced increase in cyclin B1 protein levels was suppressed in MnSOD overexpressing cells (Figure 5). Furthermore, MnSOD overexpression significantly increased the percentage of G2 cells at 12 and 24 h post-irradiation compared to 48 h post-irradiation (Figure 5D). Initial observations on the variations of responses of cancer cells to irradiation during the cell cycle were reported by Terasima and Tolmach.32 More recently, it has been suggested that two molecularly distinct G2-M checkpoints could be identified.33 The first of these G2-M checkpoints represents the failure of cells that were in G2 at the time of radiation to progress to M. It occurs early following the radiation exposure, and is suggested to be dependent upon ATM and independent of dose. The second of the G2-M checkpoints represents the accumulation of cells that were in other phases of the cell cycle at the time of radiation that progress to and are delayed in exiting G2. It occurs late following the radiation exposure, and is suggested to be independent of ATM and dependent of dose. Previous studies have demonstrated a close relationship between radiation-induced G2+M delay and radiosensitivity where increased cell survival was correlated to an increase in the percentage of G2-cells.34 Our results demonstrate that MnSOD activity regulates both of these phenomenon (Figure 5C & 5D). However, distinct differences exist, accelerating M-transit in the early phase and enhancing G2-accumulation at later times. Such a difference in cellular responses could be attributed to MnSOD activity that varies during the cell cycle.35 MnSOD activity could exert its pronounced effects on cellular radioresistance via modulation of the G2-checkpoint pathway.

MnSOD is a nuclear-encoded and mitochondria localized antioxidant enzyme known to convert mitochondria-derived superoxide to hydrogen peroxide. Therefore, our results also suggested mitochondrial generated ROS-signaling could regulate radiation-induced G2-checkpoint pathway and radiosensitivity in human pancreatic cancer cells. This hypothesis is further supported by results presented in Figure 6. Mitochondrial overexpression of catalase significantly increased the survival of irradiated V2B and Mn1 cells compared to un-infected irradiated cells (Figure 6C). Catalase overexpression in Mn7 cells with highest MnSOD activity did not show any significant change in the fraction of surviving cells suggesting that the catalase overexpression might not be enough to decrease the steady state levels of hydrogen peroxide in irradiated Mn7 cells. Furthermore, MnSOD siRNA mediated decrease in MnSOD protein levels significantly decreased surviving fraction compared to control siRNA treated cells (Figure 6D). Overall, our results support the hypothesis that mitochondria-derived superoxide and hydrogen peroxide-signaling could regulate cell cycle checkpoint pathways and cellular responses to ionizing radiation.

A relationship of cell cycle checkpoints and intracellular redox state has been suggested in a current review article36 stating that a redox cycle within the cell cycle could regulate progression from one cell cycle phase to the next. In this study, we have provided evidence linking cellular radioresistance with MnSOD-activity and radiation-induced activation of the G2-checkpoint pathway. Although the exact mechanisms of ROS-signaling regulating cell cycle checkpoint proteins are not completely understood, it is possible that thiol-disulfide exchange reactions could modify protein function. Redox modifications of proteins could occur by reduction (-SH) and/or oxidation (-S-S-) of critical cysteines in cell cycle regulatory proteins. A previous report showed redox-modifications of cysteines in the active site of Cdc25C resulted in a decrease in phosphatase activity and subsequent degradation.37 Because Cdc25C regulates cyclin/Cdk kinase activity; its redox modification could affect cyclin/Cdk kinase activity thereby modifying the oxidative stress-induced activation of cell cycle checkpoint pathways.

In summary, results presented in this study showed that human pancreatic cancer cells (MIA PaCa-2) overexpressing MnSOD were radioresistant, which was accompanied with a significant increase in the percentage of cells in the G2-phase. The MnSOD overexpressing irradiated cells also showed decrease in p-ATM, γH2AX, and cyclin B1 protein levels. Mitochondria-targeted overexpression of catalase enhanced cell survival in irradiated cells, and a decrease in MnSOD protein levels sensitized cells to radiation-induced cell death. These results support the hypothesis that mitochondria-localized antioxidant enzyme activity and ROS (superoxide and hydrogen peroxide)-signaling could regulate mitochondria to nuclear communication influencing nuclear processes which subsequently lead to the activation of the cell cycle checkpoint pathways and cellular responses to radiation exposures (Figure 7). We propose that combinatorial cancer therapy protocols targeted to cell cycle checkpoint proteins and intracellular antioxidant enzymes could provide innovative mechanistic based treatment for human pancreatic cancer.

Figure 7.

Figure 7

Open in a new tab

A schematic representation of radiation-induced ROS-signaling communicating between mitochondria and nuclear processes.

Acknowledgments

We would like to thank the University of Iowa Core facilities, and Dr. Larry W. Oberley, University of Iowa for his constant encouragement during this study. This work was funded in part by NIH CA111365, University of Iowa Carver Trust Fund, and NIH Training Grant -CA078586-0651.

ABBREVIATIONS

MnSOD

manganese superoxide dismutase

ROS

reactive oxygen species

CAT

catalase

AOE

antioxidant enzymes

AdmCAT

adenovirus containing mitochondria-targeted human catalase cDNA

DHE

dihydroethidium

Footnotes

There are no conflict of interest

References

  • 1.Riley PA. Free radicals in biology: oxidative stress and the effects of ionizing radiation. Int J Radiat Biol. 1994;65:27–33. doi: 10.1080/09553009414550041. [DOI] [PubMed] [Google Scholar]
  • 2.Williams MD, Van Remmen H, Conrad CC, Huang TT, Epstein CJ, Richardson A. Increased oxidative damage is correlated to altered mitochondrial function in heterozygous manganese superoxide dismutase knockout mice. J Biol Chem. 1998;273:28510–5. doi: 10.1074/jbc.273.43.28510. [DOI] [PubMed] [Google Scholar]
  • 3.Tsan MF. Superoxide dismutase and pulmonary oxygen toxicity: lessons from transgenic and knockout mice (Review) Int J Mol Med. 2001;7:13–9. doi: 10.3892/ijmm.7.1.13. [DOI] [PubMed] [Google Scholar]
  • 4.Petkau A, Chelack WS, Pleskach SD. Letter: Protection of post-irradiated mice by superoxide dismutase. Int J Radiat Biol Relat Stud Phys Chem Med. 1976;29:297–9. doi: 10.1080/09553007614550341. [DOI] [PubMed] [Google Scholar]
  • 5.Oberley LW, Lindgren LA, Baker SA, Stevens RH. Superoxide lon as the cause of the oxygen effect. Radiat Res. 1976;68:320–8. [PubMed] [Google Scholar]
  • 6.St Clair DK, Wan XS, Oberley TD, Muse KE, St Clair WH. Suppression of radiation-induced neoplastic transformation by overexpression of mitochondrial superoxide dismutase. Mol Carcinog. 1992;6:238–42. doi: 10.1002/mc.2940060404. [DOI] [PubMed] [Google Scholar]
  • 7.Hirose K, Longo DL, Oppenheim JJ, Matsushima K. Overexpression of mitochondrial manganese superoxide dismutase promotes the survival of tumor cells exposed to interleukin-1, tumor necrosis factor, selected anticancer drugs, and ionizing radiation. FASEB J. 1993;7:361–8. doi: 10.1096/fasebj.7.2.8440412. [DOI] [PubMed] [Google Scholar]
  • 8.Yamaguchi S, Sakurada S, Nagumo M. Role of intracellular SOD in protecting human leukemic and cancer cells against superoxide and radiation. Free Radic Biol Med. 1994;17:389–95. doi: 10.1016/0891-5849(94)90165-1. [DOI] [PubMed] [Google Scholar]
  • 9.Oberley TD. Mitochondria, manganese superoxide dismutase, and cancer. Antioxid Redox Signal. 2004;6:483–7. doi: 10.1089/152308604773934242. [DOI] [PubMed] [Google Scholar]
  • 10.Takada Y, Hachiya M, Park SH, Osawa Y, Ozawa T, Akashi M. Role of reactive oxygen species in cells overexpressing manganese superoxide dismutase: mechanism for induction of radioresistance. Mol Cancer Res. 2002;1:137–46. [PubMed] [Google Scholar]
  • 11.Motoori S, Majima HJ, Ebara M, Kato H, Hirai F, Kakinuma S, Yamaguchi C, Ozawa T, Nagano T, Tsujii H, Saisho H. Overexpression of mitochondrial manganese superoxide dismutase protects against radiation-induced cell death in the human hepatocellular carcinoma cell line HLE. Cancer Res. 2001;61:5382–8. [PubMed] [Google Scholar]
  • 12.Kalen AL, Sarsour EH, Venkataraman S, Goswami PC. Mn-superoxide dismutase overexpression enhances G2 accumulation and radioresistance in human oral squamous carcinoma cells. Antioxid Redox Signal. 2006;8:1273–81. doi: 10.1089/ars.2006.8.1273. [DOI] [PubMed] [Google Scholar]
  • 13.Hartwell LH, Weinert TA. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989;246:629–34. doi: 10.1126/science.2683079. [DOI] [PubMed] [Google Scholar]
  • 14.Bakkenist CJ, Kastan MB. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature. 2003;421:499–506. doi: 10.1038/nature01368. [DOI] [PubMed] [Google Scholar]
  • 15.Muschel RJ, Zhang HB, Iliakis G, McKenna WG. Cyclin B expression in HeLa cells during the G2 block induced by ionizing radiation. Cancer Res. 1991;51:5113–7. [PubMed] [Google Scholar]
  • 16.Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998;273:5858–68. doi: 10.1074/jbc.273.10.5858. [DOI] [PubMed] [Google Scholar]
  • 17.Celeste A, Petersen S, Romanienko PJ, Fernandez-Capetillo O, Chen HT, Sedelnikova OA, Reina-San-Martin B, Coppola V, Meffre E, Difilippantonio MJ, Redon C, Pilch DR, Olaru A, Eckhaus M, Camerini-Otero RD, Tessarollo L, Livak F, Manova K, Bonner WM, Nussenzweig MC, Nussenzweig A. Genomic instability in mice lacking histone H2AX. Science. 2002;296:922–7. doi: 10.1126/science.1069398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Spitz DR, Oberley LW. An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal Biochem. 1989;179:8–18. doi: 10.1016/0003-2697(89)90192-9. [DOI] [PubMed] [Google Scholar]
  • 19.Griffith OW. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem. 1980;106:207–12. doi: 10.1016/0003-2697(80)90139-6. [DOI] [PubMed] [Google Scholar]
  • 20.Puck TT, Marcus PI. Action of x-rays on mammalian cells. J Exp Med. 1956;103:653–66. doi: 10.1084/jem.103.5.653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Krishan A. Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining. The Journal of cell biology. 1975;66:188–93. doi: 10.1083/jcb.66.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Menon SG, Sarsour EH, Spitz DR, Higashikubo R, Sturm M, Zhang H, Goswami PC. Redox regulation of the G1 to S phase transition in the mouse embryo fibroblast cell cycle. Cancer Res. 2003;63:2109–17. [PubMed] [Google Scholar]
  • 23.Xu B, Kastan MB. Analyzing cell cycle checkpoints after ionizing radiation. Methods Mol Biol. 2004;281:283–92. doi: 10.1385/1-59259-811-0:283. [DOI] [PubMed] [Google Scholar]
  • 24.Huang X, Kurose A, Tanaka T, Traganos F, Dai W, Darzynkiewicz Z. Sequential phosphorylation of Ser-10 on histone H3 and ser-139 on histone H2AX and ATM activation during premature chromosome condensation: relationship to cell-cycle phase and apoptosis. Cytometry A. 2006;69:222–9. doi: 10.1002/cyto.a.20257. [DOI] [PubMed] [Google Scholar]
  • 25.Miller FJ, Jr, Griendling KK. Functional evaluation of nonphagocytic NAD(P)H oxidases. Methods in enzymology. 2002;353:220–33. doi: 10.1016/s0076-6879(02)53050-0. [DOI] [PubMed] [Google Scholar]
  • 26.Aebi H. Catalase in vitro. Methods Enzymol. 1984;105:121–6. doi: 10.1016/s0076-6879(84)05016-3. [DOI] [PubMed] [Google Scholar]
  • 27.Bai J, Rodriguez AM, Melendez JA, Cederbaum AI. Overexpression of catalase in cytosolic or mitochondrial compartment protects HepG2 cells against oxidative injury. J Biol Chem. 1999;274:26217–24. doi: 10.1074/jbc.274.37.26217. [DOI] [PubMed] [Google Scholar]
  • 28.Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 2001;411:494–8. doi: 10.1038/35078107. [DOI] [PubMed] [Google Scholar]
  • 29.Bernhard EJ, Maity A, Muschel RJ, McKenna WG. Effects of ionizing radiation on cell cycle progression. A review Radiat Environ Biophys. 1995;34:79–83. doi: 10.1007/BF01275210. [DOI] [PubMed] [Google Scholar]
  • 30.Wong GH. Protective roles of cytokines against radiation: induction of mitochondrial MnSOD. Biochim Biophys Acta. 1995;1271:205–9. doi: 10.1016/0925-4439(95)00029-4. [DOI] [PubMed] [Google Scholar]
  • 31.Kinnula VL, Crapo JD. Superoxide dismutases in malignant cells and human tumors. Free Radic Biol Med. 2004;36:718–44. doi: 10.1016/j.freeradbiomed.2003.12.010. [DOI] [PubMed] [Google Scholar]
  • 32.Terasima T, Tolmach LJ. Variations in several responses of HeLa cells to x-irradiation during the division cycle. Biophys J. 1963;3:11–33. doi: 10.1016/s0006-3495(63)86801-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Xu B, Kim ST, Lim DS, Kastan MB. Two molecularly distinct G(2)/M checkpoints are induced by ionizing irradiation. Mol Cell Biol. 2002;22:1049–59. doi: 10.1128/MCB.22.4.1049-1059.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Nagasawa H, Keng P, Harley R, Dahlberg W, Little JB. Relationship between gamma-ray-induced G2/M delay and cellular radiosensitivity. Int J Radiat Biol. 1994;66:373–9. doi: 10.1080/09553009414551311. [DOI] [PubMed] [Google Scholar]
  • 35.Li N, Oberley TD. Modulation of antioxidant enzymes, reactive oxygen species, and glutathione levels in manganese superoxide dismutase-overexpressing NIH/3T3 fibroblasts during the cell cycle. J Cell Physiol. 1998;177:148–60. doi: 10.1002/(SICI)1097-4652(199810)177:1<148::AID-JCP16>3.0.CO;2-9. [DOI] [PubMed] [Google Scholar]
  • 36.Menon SG, Goswami PC. A redox cycle within the cell cycle: ring in the old with the new. Oncogene. 2007;26:1101–9. doi: 10.1038/sj.onc.1209895. [DOI] [PubMed] [Google Scholar]
  • 37.Savitsky PA, Finkel T. Redox regulation of Cdc25C. J Biol Chem. 2002;277:20535–40. doi: 10.1074/jbc.M201589200. [DOI] [PubMed] [Google Scholar]

RESOURCES