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. 2018 Jun 21;7(7):64.
doi: 10.3390/cells7070064.

Hypoxia and IF₁ Expression Promote ROS Decrease in Cancer Cells

Gianluca Sgarbi  1 , Giulia Gorini  2   3 , Francesca Liuzzi  4 , Giancarlo Solaini  1 , Alessandra Baracca  5
Affiliations

Hypoxia and IF₁ Expression Promote ROS Decrease in Cancer Cells

Gianluca Sgarbi et al. Cells. .

Abstract

The role of reactive oxygen species (ROS) in the metabolic reprogramming of cells adapted to hypoxia and the interplay between ROS and hypoxia in malignancy is under debate. Here, we examined how ROS levels are modulated by hypoxia in human cancer compared to untransformed cells. Short time exposure (20 min) of either fibroblasts or 143B osteosarcoma cells to low oxygen tension down to 0.5% induced a significant decrease of the cellular ROS level, as detected by the CellROX fluorescent probe (−70%). Prolonging the cells’ exposure to hypoxia for 24 h, ROS decreased further, reaching nearly 20% of the normoxic value. In this regard, due to the debated role of the endogenous inhibitor protein (IF₁) of the ATP synthase complex in cancer cell bioenergetics, we investigated whether IF₁ is involved in the control of ROS generation under severe hypoxic conditions. A significant ROS content decrease was observed in hypoxia in both IF₁-expressing and IF₁- silenced cells compared to normoxia. However, IF₁-silenced cells showed higher ROS levels compared to IF1-containing cells. In addition, the MitoSOX Red-measured superoxide level of all the hypoxic cells was significantly lower compared to normoxia; however, the decrease was milder than the marked drop of ROS content. Accordingly, the difference between IF₁-expressing and IF₁-silenced cells was smaller but significant in both normoxia and hypoxia. In conclusion, the interplay between ROS and hypoxia and its modulation by IF₁ have to be taken into account to develop therapeutic strategies against cancer.

Keywords: F1F0-ATPase; IF1; ROS; cancer cells; hypoxia; osteosarcoma; superoxide radical.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Validation of ROS detection by CellROX in human fibroblasts. (A) Typical top right quadrant (green-framed) analysis of cell fluorescence distribution as an index of ROS level. CellROX-loaded fibroblasts were analyzed following the exposure to 1 mM NAC or 200 μM Luperox, under both normoxia and hypoxia (6 h). (B) Quantitation of high fluorescent cells as an index of ROS content. (C,D) Fluorescence of CellROX-loaded control cells exposed to 4 h hypoxia followed by 4 h re-oxygenation. Data are means ± SD of three independent experiments, each carried out on four different cell lines. * p ≤ 0.05 and ** p ≤ 0.01 indicate the statistical significance of data compared to basal conditions.
Figure 2
Figure 2
ROS level in human fibroblasts grown under hypoxia. (A) HIF-1α level determined upon 6 h exposure of fibroblasts to either normoxia or hypoxia. (B) Representative top right quadrant analysis of CellROX-loaded cells maintained in either normoxia or hypoxia (0.5% O2) up to 24 h. (C) Scatter graph showing the time-dependence of cellular ROS level decrease during 24 h hypoxic exposure. Data are means ± SD of three independent experiments, each carried out on four different cell lines. ** p ≤ 0.01 indicates the statistical significance of data compared to normoxia.
Figure 3
Figure 3
ROS level in osteosarcoma cells grown under hypoxia. (A) HIF-1α level determined upon 6 h exposure of osteosarcoma cells to either normoxia or hypoxia. (B) Representative top right quadrant analysis of CellROX-loaded cells maintained in either normoxia or hypoxia (0.5% O2 ) up to 24 h. (C) Scatter graph showing the time-dependence of cellular ROS level decrease during 24 h hypoxic exposure. Data are means ± SD of three independent experiments. ** p ≤ 0.01 indicates the statistical significance of data compared to normoxia.
Figure 4
Figure 4
IF1 affects ROS level in osteosarcoma cells grown under either normoxia or hypoxia. (A) Immunoblot analysis of IF1 protein level in parental cells , scrambled and IF1-silenced clones (E9 and G3). (B) Representative top right quadrant analysis and (C) bars graph of ROS levels measured in all types of CellROX-loaded cells cultured in either normoxia or hypoxia (0.5% O2 ) for 24 h. Data are means ± SD of four independent experiments. ** p ≤ 0.01 and ## p ≤ 0.01 indicate the statistical significance of data compared to normoxia and to controls, respectively.
Figure 5
Figure 5
IF1 affects superoxide anion production in osteosarcoma cells grown under either normoxia or hypoxia. (A) Representative top right quadrant analysis and (B) bars graph of superoxide anion levels measured in MitoSOX Red-loaded parental (143B), scrambled (Scr) and IF1-silenced (E9 and G3) cells cultured in either normoxia or hypoxia (0.5% O2) for 24 h. Data are means ± SD of four independent experiments. ** p ≤ 0.01, indicates the statistical significance of data compared to normoxia; # p ≤ 0.05 and ## p ≤ 0.01, indicate the statistical significance of data compared to controls.
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References

    1. Ray P.D., Huang B.W., Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell. Signal. 2012;24:981–990. doi: 10.1016/j.cellsig.2012.01.008. - DOI - PMC - PubMed
    1. Valko M., Rhodes C.J., Moncol J., Izakovic M., Mazur M. Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem. Biol. Interact. 2006;160:1–40. doi: 10.1016/j.cbi.2005.12.009. - DOI - PubMed
    1. Boveris A., Chance B. The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem. J. 1973;134:707–716. doi: 10.1042/bj1340707. - DOI - PMC - PubMed
    1. Solaini G., Landi L., Pasquali P., Rossi C.A. Protective effect of endogenous coenzyme q on both lipid peroxidation and respiratory chain inactivation induced by an adriamycin-iron complex. Biochem. Biophys. Res. Commun. 1987;147:572–580. doi: 10.1016/0006-291X(87)90969-7. - DOI - PubMed
    1. Wu M.Y., Yiang G.T., Liao W.T., Tsai A.P., Cheng Y.L., Cheng P.W., Li C.Y., Li C.J. Current mechanistic concepts in ischemia and reperfusion injury. Cell. Physiol. Biochem. 2018;46:1650–1667. doi: 10.1159/000489241. - DOI - PubMed

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