Abstract
Phospholipid hydroperoxide glutathione peroxidase (PhGPx) is an antioxidant enzyme that reduces cellular phospholipid hydroperoxides (PLOOHs) to alcohols. Cellular peroxide tone has been implicated in cell growth and differentiation. By reducing the PLOOH level in the cell membrane, PhGPx regulates the peroxide tone and thereby might be involved in cell growth. We hypothesized that overexpression of PhGPx in human breast cancer cells would decrease their growth rate. We stably transfected MCF-7 cells (Wt) with L-PhGPx and measured cell doubling time, plating efficiency, and cell cycle phase transit times. P-4 cells (8-fold increase in PhGPx activity) showed a 2-fold increase in doubling time; doubling time increased directly with PhGPx activity (r=0.95). The higher the PhGPx activity, the lower the plating efficiency (r=-0.86). The profile of other antioxidant enzymes was unchanged. Overexpression of PhGPx lowered the steady-state level of PLOOH (by >60%). Results from bromodeoxyuridine pulse-chase experiments and flow cytometry indicate that PhGPx induced a delay in MCF-7 proliferation that was primarily due to a slower progression from G1 to S. These results support the hypothesis that PhGPx plays a regulatory role in the progression of MCF-7 cells from G1 to S possibly by regulating the steady-state levels of PLOOH. These data suggest that PhGPx can lower the peroxide tone, which might change the cellular redox environment resulting in a delay in G1 transit. Thus, PhGPx could be an important factor in cell growth.
- Glutathione peroxidase
- Lipid hydroperoxide
- Cell cycle
- Antioxidant enzymes
- BrdU, bromodeoxyuridine
- CAT, catalase
- CuZnSOD, copper zinc superoxide dismutase
- EDTA, disodium ethylenediaminetetraacetic acid
- EtOH, ethanol
- GSH, glutathione
- GSSG, glutathione disulfide
- GPx-1, cytosolic glutathione peroxidase
- GR, glutathione reductase
- HO, hydrogen peroxide
- MCF-7, human breast carcinoma cells
- MnSOD, manganese superoxide dismutase
- Neo, MCF-7 cells with empty vector
- L-PhGPx, mitochondrial phospholipid hydroperoxide glutathione peroxidase
- LOH, lipid alcohol
- LOOHs, lipid hydroperoxides
- P-1, P-2, P-3, P-4, are MCF-7 clones stably transfected with PhGPx
- PBS, phosphate buffered saline
- PE, plating efficiency
- PLOOH, phosphatidylcholine hydroperoxide
- PI, propidium iodide
- RM, relative movement parameter
- Se-PhGPx, represents the reduced enzyme
- Se-O-PhGPx, represents the oxidized enzyme
- SOD, superoxide dismutase
- TEMED, N,N,N′,N′-tetramethylethylene diamine
- Wt, parental MCF-7 cells