Decreasing peroxiredoxin II expression decreases glutathione, alters cell cycle distribution, and sensitizes glioma cells to ionizing radiation and H2O2

doi:10.1016/j.freeradbiomed.2008.07.015

Free Radical Biology and Medicine

Volume 45, Issue 8, 15 October 2008, Pages 1178-1189

https://doi.org/10.1016/j.freeradbiomed.2008.07.015 Get rights and content

Abstract

Glioblastomas are notorious for their resistance to ionizing radiation and chemotherapy. We hypothesize that this resistance to ionizing radiation is due, in part, to alterations in antioxidant enzymes. Here, we show that rat and human glioma cells overexpress the antioxidant enzyme peroxiredoxin II (Prx II). Glioma cells in which Prx II is decreased using shRNA exhibit increased hyperoxidation of the remaining cellular Prxs, suggesting that the redox environment is more oxidizing. Of interest, decreasing Prx II does not alter other antioxidant enzymes (i.e., catalase, GPx, Prx I, Prx III, CuZnSOD, and MnSOD). Analysis of the redox environment revealed that decreasing Prx II increased intracellular reactive oxygen species in 36B10 cells; extracellular levels of H₂O₂ were also increased in both C6 and 36B10 cells. Treatment with H₂O₂ led to a further elevation in intracellular reactive oxygen species in cells where Prx II was decreased. Decreasing Prx II expression in glioma cells also reduced clonogenic cell survival following exposure to ionizing radiation and H₂O₂. Furthermore, lowering Prx II expression decreased intracellular glutathione and resulted in a significant decline in glutathione reductase activity, suggesting a possible mechanism for the observed increased sensitivity to oxidative insults. Additionally, decreasing Prx II expression increased cell cycle doubling times, with fewer cells distributed to S phase in C6 glioma cells and more cells redistributed to the most radiosensitive phase of the cell cycle, G2/M, in 36B10 glioma cells. These findings support the hypothesis that inhibiting Prx II sensitizes glioma cells to oxidative stress, presenting Prxs as potential therapeutic targets.

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Materials

All chemicals and reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA) unless stated otherwise. Medium and medium components were purchased from Invitrogen (Carlsbad, CA, USA).

Cell cultures

Primary rat astrocytes were isolated from the cerebral cortex of 2- to 3-day-old Sprague–Dawley rat pups as described previously [6]. Type II astrocytes were maintained in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum, 4 mM L-glutamine, and 38.9 mM glucose. Medium was

Prx II is overexpressed in human and rat glioma cell lines

As astrocytes, the normal counterpart of glioma cells, express low levels of Prx II, overexpression of Prx II may be important for protecting glioma cells from endogenous and exogenous sources of oxidative stress. Therefore, several rat and human glioma cell lines were examined for expression of Prx II (Figs. 1A and B). When compared with primary rat astrocytes, Prx II levels were at least 4-fold higher in the rat glioma cell lines (Fig. 1A). Similarly, when human glioma cells were compared

Discussion

Gliomas are extremely refractory to IR and chemotherapy. Despite significant advances, median patient survival remains only 14.6 months with optimal therapy [1], [2], [61]. We hypothesized that this resistance is due, in part, to altered levels of antioxidant enzymes. We have previously shown that catalase is overexpressed in glioma cells and that this overexpression contributes to their radioresistance; inhibiting catalase expression sensitized glioma cells to oxidative stress [6]. We now

Acknowledgments

This work was supported by NIH Grants CA110274 (P.S.S.), CA112593 (M.E.R.), GM050389 (L.B.P.), andCA086862 and CA100045 (D.R.S.). We extend special thanks to Mitchell C. Coleman (University of Iowa) for analysis of cellular glutathione content and for technical assistance.