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

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 G(2)-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 G(2) 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 G(2) checkpoint activation and radioresistance in human pancreatic cancer cells.

Figure 1

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).

Figure 2

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.

Figure 3

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

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.

Figure 5

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).

Figure 6

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.

Figure 7

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