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. 2013 Jul 11;8(7):e69030.
doi: 10.1371/journal.pone.0069030. Print 2013.

Mitochondrial function in human neuroblastoma cells is up-regulated and protected by NQO1, a plasma membrane redox enzyme

Jiyeong Kim  1 , Su-Kyung Kim, Hwa-Kyung Kim, Mark P Mattson, Dong-Hoon Hyun
Affiliations

Mitochondrial function in human neuroblastoma cells is up-regulated and protected by NQO1, a plasma membrane redox enzyme

Jiyeong Kim et al. PLoS One. .

Abstract

Background: Recent findings suggest that NADH-dependent enzymes of the plasma membrane redox system (PMRS) play roles in the maintenance of cell bioenergetics and oxidative state. Neurons and tumor cells exhibit differential vulnerability to oxidative and metabolic stress, with important implications for the development of therapeutic interventions that promote either cell survival (neurons) or death (cancer cells).

Methods and findings: Here we used human neuroblastoma cells with low or high levels of the PMRS enzyme NADH-quinone oxidoreductase 1 (NQO1) to investigate how the PMRS modulates mitochondrial functions and cell survival. Cells with elevated NQO1 levels exhibited higher levels of oxygen consumption and ATP production, and lower production of reactive oxygen species. Cells overexpressing NQO1 were more resistant to being damaged by the mitochondrial toxins rotenone and antimycin A, and exhibited less oxidative/nitrative damage and less apoptotic cell death. Cells with basal levels of NQO1 resulted in increased oxidative damage to proteins and cellular vulnerability to mitochondrial toxins. Thus, mitochondrial functions are enhanced and oxidative stress is reduced as a result of elevated PMRS activity, enabling cells to maintain redox homeostasis under conditions of metabolic and energetic stress.

Conclusion: These findings suggest that NQO1 is a potential target for the development of therapeutic agents for either preventing neuronal degeneration or promoting the death of neural tumor cells.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. ATP production and oxygen consumption are increased by overexpression of NQO1.
Intact mitochondria were prepared and ATP production and oxygen consumption were assessed in the presence of electron donors. (A–C) ATP production rates in the presence of NADH (A), glutamate/malate (B) and succinate (C). (D–F) Oxygen consumption rates in the presence of NADH (D), glutamate/malate (E) and succinate (F). Values are the mean ± SEM (n = 6). *p<0.01 compared with the value for untransfected control cells.
Figure 2
Figure 2. Activities of mitochondrial complexes I and II are elevated and production of ROS are reduced in human neuroblastoma cells overexpressing NQO1.
Cells were cultured and then mitochondrial fractions were isolated by centrifugal fractionation. Mitochondrial complex activities were measured using appropriate electron donors. Complex I activity in the presence of NADH (A) and glutamate/malate (B). Complex II activity in the presence of succinate (C). ROS production in the presence of NADH (D), glutamate/malate (E) and succinate (F). Values are the mean ± SEM (n = 6). *p<0.01, **p<0.05 compared with the value for untransfected control cells.
Figure 3
Figure 3. The cytoplasmic NAD+/NADH ratio and levels of p53 are elevated in cells overexpressing NQO1.
(A) The NAD+/NADH ratio was measured using a NAD/NADH Quantitation Kit. Values are the mean ± SEM (n = 6). *p<0.01 compared with the value for untransfected control cells under normal culture conditions. # p<0.01 compared with the values in the mitochondrial fractions between untransfected control and NQO1-transfected cells. (B) p53 levels were assessed following treatment with cycloheximide for 6, 12, 18 and 24 hr.
Figure 4
Figure 4. Neuroblastoma cells overexpressing NQO1 exhibit reduced vulnerability to mitochondrial toxins.
Cells were exposed to the indicated concentrations (micromolar) of rotenone (A and C) or antimycin A (B and D) for the indicated time periods (A and B) or for 24 hr (C and D) and cell viability was measured by trypan blue exclusion (A and B, ○: control, •: NQO1) and MTT-reduction assay (C and D). Values are the mean ± SEM (n = 6). *p<0.01 compared to untransfected cells exposed to the same concentration of the toxin.
Figure 5
Figure 5. Complex I activity is increased in cells with elevated NQO1 levels and is decreased by rotenone.
Cells were cultured in the presence of 50 and 100 µM rotenone for 6 hr and the mitochondrial fractions were isolated and used for the complex I assay using 5 mM NADH (A) or 5 mM glutamate/malate (B). The mitochondrial fractions were separated from cells cultured under normal culture conditions and the complex I assay was performed in the presence of 10 and 100 nM rotenone using 5 mM NADH (C) or 5 mM glutamate/malate (D). Values are the mean ± SEM (n = 6). *p<0.01 compared with the value for untransfected cells and to NQO1-overexpressing cells not exposed to rotentone.
Figure 6
Figure 6. Levels of oxidative damage to lipids and proteins are attenuated in cells with elevated NQO1 levels compared with control cells.
Cell extracts were used to measure levels of protein carbonyls (A and B) and nitrotyrosine (C and D) following exposure to 50 µM rotenone (A and C) or 100 µM antimycin A (B and D) for 24 hr. Values are the mean ± SEM (n = 6). *p<0.01 compared with the value for untransfected cells exposed to rotenone or antimycin A.
Figure 7
Figure 7. Neuroblastoma cells with elevated NQO1 levels exhibit resistance to apoptisis induced by mitochondrial toxins.
Following exposure to rotenone (A) or antimycin A (B), cells were lysed and the levels of the cleaved form of PARP were determined by immunoblot analysis. Three independent experiments were performed and representative blots are shown. Levels of cell membrane permeabilization (propidium iodide-positive cells) (C and D) and chromatin condensation (E and F) were also measured after the addition of 50 µM rotenone (C and E) or 100 µM antimycin A (D and F) for 24 hr. Values are the mean ± SEM (n = 6). *p<0.01 compared with the value for untransfected control cells exposed to rotenone or antimycin A.
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Grants and funding

This study was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF-2010-003064), South Korea. This work was also funded, in part, by the Intramural Research Program of the National Institute on Aging. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.