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RESEARCH ARTICLE

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Relationship between Optical Redox Status and Reactive Oxygen Species in Cancer Cells

Allison Podsednik, Annemarie Jacob, Lin Z. Li, and He N. Xu 

Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 

Correspondence: hexu2@pennmedicine.upenn.edu (H.N.X.) 

Podsednik A et al. Reactive Oxygen Species 9(26):95–108, 2020; ©2020 Cell Med Press

http://dx.doi.org/10.20455/ros.2020.815

(Received: November 22, 2019; Revised: December 18, 2019; Accepted: December 19, 2019) 

ABSTRACT | Shifted NAD(H) redox status and enhanced reactive oxygen species (ROS) scavenging systems have been observed in cancers. However, how such redox shift is related to the ROS level in cancer cells is less clear. Based on collecting the intrinsic fluorescence of oxidized flavoproteins (Fp containing flavin adenine dinucleotide) and reduced nicotinamide adenine dinucleotide (NADH), optical redox imaging (ORI) provides a quantitative measure of the mitochondrial redox state by the optical redox ratio, Fp/(NADH+Fp), a surrogate marker of the NAD+-coupled redox state NAD+/NADH. Our study aims to explore the relationship between NAD(H) redox status and ROS by imaging NADH, Fp, and ROS levels using cultured breast cancer cell models. By manipulating either ROS levels via application of exogenous H2O2 or redox status via metabolic perturbation compounds, we found that: (1) oxidation of NAD(H) redox status correlates with ROS levels at lower H2O2 concentrations (up to ~700 µM), but not necessarily at higher concentrations; (2) an elevated ROS level diminishes NADH and reduces redox ratio plasticity; (3) either more oxidized or more reduced status can correlate to an increased ROS level; and (4) sometimes, a more oxidized status can correlate to a decreased ROS level depending on cell lines. These observations indicated that cellular NAD(H) redox state and ROS are intricately related but can also change separately. This study can benefit cancer research as both NAD(H) redox status and ROS have been implicated in cancer transformation and progression. 

KEYWORDS | Breast cancer; Intrinsic fluorescence; Flavoproteins; NADH; Optical redox imaging; Reactive oxygen species; Redox ratio 

ABBREVIATIONS | AA, antimycin A;DHE, dihydroethidium; DMSO, dimethyl sulfoxide; DPBS+, Dulbecco’s phosphate-bufferedsaline with calcium and magnesium;FCCP, trifluoromethoxy carbonyl cyanide phenylhydrazone;Fp, flavoprotein; NADH, reduced nicotinamide adenine dinucleotide;Oligo, oligomycin;ORI, optical redox imaging;ROS, reactive oxygen species;ROT, rotenone;ROTAA, rotenone and antimycin A; TNBC, triple negative breast cancer 

CONTENTS 

  1. Introduction
  2. Materials and Methods

2.1. Cell Culture

2.2. Treatment with Chemical Agents

2.3. Live Cell Optical Redox Imaging

2.4. Data Analysis and Statistics

  1. Results

3.1. Confirmation of the Redox Response from Intrinsic Fp and NADH Fluorescence Signals

3.2. Redox Changes Correlate with Exogenous H2O2 Concentrations

3.3. Elevated Exogenous ROS Results in Reduced Redox Plasticity

3.4. A Change of Redox Status toward either Oxidation or Reduction is Associated with an Increase of Endogenous ROS

3.5. A Change of Redox State Correlates to a Change in Mitochondrial ROS

3.6. No Change in ROS Levels Correlates to No Change of Redox Status under Mitochondrial Metabolic Perturbation

  1. Discussion

4.1. Concomitant and Non-Concomitant Relationship between the Redox Status and ROS Levels

4.2. Both Redox Extremes Correlate to Increased Cytoplasmic ROS Levels

4.3. Differential Changes of Mitochondrial ROS Levels between Redox Extremes

4.4. Cell Line-Dependent Relationship between Redox Status and Mitochondrial ROS

4.5. Increased ROS Decreases Redox Plasticity

4.6. Limitations of This Study

  1. Conclusion

1. INTRODUCTION 

Reactive Oxygen Species (ROS) are formed intracellularly as a byproduct of metabolism and include oxygen free radicals, such as superoxide anion radicals (O2˙ˉ) and hydroxyl radicals (OH˙) resulting from the non-radical oxidant hydrogen peroxide (H2O2) [1]. It has been shown that up to 90% of ROS are of mitochondrial origin in the brain [2] and that mitochondria are the main source of ROS in triple negative breast cancer(TNBC) cells [3] although they can be generated in other cellular compartments. The homeostatic balance between antioxidant scavenging systems and ROS production systems determines the ROS level within the cell [4]. Studies of the relationship between cellular redox status and ROS found that either oxidized or reduced state leads to ROS overflow in intact cardiomyocyte cells and that oxidative stress altered mitochondrial bioenergetics and modified pancreatic acinar cell death [4, 5]. 

ROS also play a role in promoting cancer transformation and progression to metastasis [3, 6]. Cancer cells rely on metabolic reprograming to support rapid proliferation requiring quick adenosine triphosphate (ATP) generation and increased biosynthesis as well as maintenance of appropriate redox environment [7]. As a result, there is an elevation of ROS generation from mitochondria which is balanced by enhanced ROS-scavenging antioxidant systems [8, 9]. 

NAD+/NADH is an important cellular redox pair, among many others, and may regulate ROS generation and scavenging [10, 11]. However, not much is known about the exact relationship between NAD(H) redox status and ROS levels in cancer models. In this paper, we aim to explore the relationship between cellular NAD(H)-coupled redox status and ROS levels in cancer using TNBC cell lines as model systems. We employ optical redox imaging (ORI), a technique used to detect intrinsic fluorescence signals of oxidized flavoproteins (Fp containing flavin adenine dinucleotide) and NADH, to measure cellular redox status. Pioneered by Chance et al. [12–14], this technique has been widely applied to study energetics, metabolism, disease diagnosis/prognosis, and treatment response [15–23]. To quantify both cytoplasmic and mitochondrial ROS, we used two separate probes: dihydroethidium (DHE) and MitoSOX, respectively [24–29]. 

Our findings indicate that deviations from baseline cellular redox status correlate to ROS imbalances, yet the relationship between NAD(H) redox state and ROS levels is complicated and may vary depending on TNBC subgroups.

2. MATERIALS AND METHODS 

2.1. Cell Culture 

Breast cancer MDA-MB-231 and HCC1806 cells were maintained in T-25 flasks with phenol-red containing RPMI 1640 Medium (Cat#11875085, Gibco, Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS). The cells were incubated at 37°C with 5% CO2 and passaged at approximately 80% confluency using 0.25% trypsin-EDTA. The cells were seeded on glass-bottom dishes coated with poly-d-lysine at a density of 40,000 cells/200 µl (two-day treatment) or 50,000 cells/200 µl (one-day treatment) or on non-coated 35-mm glass-bottom dishes at a density of 50,000 cells/1 ml (two-day treatment) or 100,000 cells/1 ml (one-day treatment). After 4 h in incubation, 1ml medium was added to coated dishes. Experiments with successive addition of H2O2 were performed with 4-chamber glass-bottom dishes (Cat# D35C4-20-1.5-N, Cellvis, Mountain View, CA, USA). Approximately 45 min before imaging, dishes were rinsed twice with DPBS+ (Dulbecco’s phosphate-bufferedsaline with calcium and magnesium) and 1 ml Live Cell Imaging Solution (LCIS, Molecular Probes/Thermo Fisher) supplemented with glucose (11.5 mM) and glutamine (2 mM) was added. Since endogenous fluorescence of NADH and Fp are relatively weak, supplemented LCIS was used during imaging to minimize fluorescence background that interferes with the imaging results. 

2.2. Treatment with Chemical Agents 

All chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) except that CB-839 and MitoSOX red were purchased from Cayman Chemical (An Arbor, MI, USA) and Thermo Fisher, respectively. These chemical reagents (except H2O2, 3% aqueous solution) were first reconstituted in 100% dimethyl sulfoxide (DMSO) and aliquoted for storage in either a –80°C or –20°C freezer until use. 

H2O2 experiments were performed by treating cell dishes with H2O2 to achieve the following final concentrations: 176, 704, and 1408 µM. Dishes were imaged approximately 2–3 min post H2O2 addition. DPBS+ was added to control dishes in a volume equal to the volume of H2O2 added to achieve 1408 µM (1.6 µl). 

FX11 experiments were performed by treating dishes with FX11 to achieve a final concentration of either 3 µM or 5 µM. Dishes were imaged approximately 1 h post FX11 addition. 0.5 µl DMSO (same as in 5 µM FX11) was added to control dishes. Since DHE detects cytoplasmic O2•− not specific to mitochondrial O2•−, ROS levels were measured by staining cells with either DHE or its mitochondrion-targeted form MitoSOX (red) [24–29]. To stain with DHE, immediately after ORI, 2 mM DHE was added to cell dishes to achieve a final concentration of 2 µM. Dishes were wrapped in aluminum foil and incubated at 37°C for 40 min. After incubation, cell dishes were rinsed twice with DPBS+ and fresh supplemented LCIS was added to dishes for imaging. To stain with MitoSOX, immediately after ORI, 5 mM MitoSOX was added to each dish to give a final concentration of 2.5 µM. Dishes were wrapped in aluminum foil and incubated at 37°C for 10 min. Cell dishes were rinsed twice with DPBS+ and fresh supplemented LCIS was added to dishes for imaging. 

Redox plasticity measurements were achieved by sequentially adding a mitochondrial oxidative phosphorylation uncoupler, trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP), and a mixture of mitochondrial inhibitors rotenone and antimycin A (ROTAA). In some experiments, oligomycin (Oligo) was also used to inhibit mitochondrial complex V. Chemical agents were added to cell dishes at the following concentrations: Oligo 2 µg/ml, FCCP 0.5 µM, ROTAA (ROT 1 µM + AA 1.25 µg/ml). Images were taken approximately 3–5 min post addition of each agent. In some experiments, to evaluate separate effects of these chemical agents, either Oligo, FCCP, ROT, or AA was added to the dish in the previously specified concentration and imaged 3–5 min after. 

2.3. Live Cell Optical Redox Imaging 

A Zeiss wide-field microscope (Axio Observer 7, Zeiss, Oberkochen, Germany) set at 37°C was used for imaging. Images were taken using a 20× lens (NA = 0.8). The NADH signals were collected through the following optical bandpass filters: Ex 370–400 nm and Em 414–450 nm. The Fp signals were acquired at Ex 450–488 nm and Em 500–530 nm. The DHE signals were acquired at Ex 540–570 nm and Em 580–610 nm. The MitoSOX signals were acquired at Ex 370–400 nm and Em 580–610 nm. To avoid photo-bleaching, transmitting light was used to locate and focus on regions of interest. Three to five randomly selected, non-overlapping fields of view per dish were imaged. 

2.4. Data Analysis and Statistics 

The NIH ImageJ was used to split the files into separate channels. A custom MATLAB program was employed to evaluate various parameters including the intensities of NADH, Fp, and ROS. The redox ratio (Fp/[NADH+Fp]) images were generated pixel-by-pixel from NADH and Fp images. The custom program analyzed the images through a series of steps: image flattening using a third-degree polynomial surface fit function, background removal by drawing a ROI in a cell-free region and computing the mean and standard deviation of the field of view, and thresholding to set pixel limits of signal-to-noise ratio ≥ 3, as described in detail previously [18]. 

Fields of view from each dish were averaged, which were further averaged across dishes to obtain the group mean. Bar graphs grouped by treatment are displayed as the mean ± standard deviation (SD). To compare three or more groups, significance was evaluated using Prism 8 (San Diego, CA, USA) to run one-way ANOVA tests followed by Tukey tests to correct for multiple comparisons with p = 0.05 as the threshold of significance. The mean of each column (control and all treatment conditions) was compared to the mean of every other column. For comparison between two groups, unpaired t-tests with unequal variance were used. Significant differences are shown as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001.

3. RESULTS 

3.1. Confirmation of the Redox Response from Intrinsic Fp and NADH Fluorescence Signals 

To confirm that ORI is sensitive to redox changes, we imaged cells under metabolic perturbations. Typical images of MDA-MB-231 cells acquired by the ORI technique are shown in Figure 1. Upon addition of FCCP, a mitochondrial oxidative phosphorylation uncoupler [30], to MDA-MB-231 cells ORI detected a decrease in NADH levels and an increase in the redox ratio. A decrease in NADH was expected due to uncoupled mitochondrial oxidative phosphorylation leading to an exhaustion of NADH and dissipation of the proton gradient. Upon addition of ROTAA, mitochondrial inhibitors, to MDA-MB-231 cells ORI detected a significant spike in NADH levels and a decrease in the redox ratio. An increase in NADH signal was the expected outcome of ROT blocking the activity of complex I, and to a greater extent, the outcome of AA blocking complex III. Figure 2 quantifies the imaging results and demonstrates that ORI-detected Fp and NADH signals of cells in culture under the experimental setup are sensitive to metabolic/redox modulations.

 

FIGURE 1. Typical pseudo-color images of MDA-MB-231 cells in culture produced using MATLAB. Images display Fp, NADH, and Fp/(NADH+Fp) intensity levels of the control group. The color bars of Fp and NADH images represent the signal intensities in arbitrary units and the color bar of the redox ratio image indicates the redox ratio ranging from 0 to 1.

 

FIGURE 2. (a) Fp, NADH, and the redox ratio of MDA-MB-231 cells under 0.5 µM FCCP modulation and (b) Fp, NADH, and the redox ratio of MDA-MB-231 cells under 1 µM ROT + 1.25 µg/ml AA modulation. Data represent the mean ± SD (n = 4). *, p < 0.05; ****, p < 0.0001. 

3.2. Redox Changes Correlate with Exogenous H2O2 Concentrations 

To evaluate whether and how the redox status responds to exogenous ROS, we added various concentrations of H2O2 to MDA-MB-231 cell dishes. Upon successive addition of H2O2, NADH intensity decreased in a concentration-dependent manner with the 704 and 1408 µM concentrations resulting in significant decreases relative to the control dishes, as illustrated in Figure 3. In accordance, increased redox ratio correlated with larger H2O2 concentrations in a concentration-dependent manner. All H2O2 concentrations used for experimentation (176, 704, and 1408 µM) produced a significant oxidative shift in the redox ratio from the control and preceding concentration. Additionally, Figure 4confirms that increasing concentrations of exogenous H2O2 correlate to increasing levels of intracellular ROS measured by DHE intensity. Quantitatively, the 176 µM H2O2 condition resulted in an 87.2% ROS level increase relative to control, and the 704 µM and 1408 µM H2O2 conditions resulted in similar ROS level increases from the control, 138.0% and 132.8%, respectively. The difference of intracellular ROS levels between the latter two concentration conditions was insignificant.

 

FIGURE 3. Fp and NADH intensities and the redox ratios of MDA-MB-231 cell dishes following successive H2O2 addition of various concentrations ranging from 176 µM to 1408 µM. The 0 µM condition represents the control dishes with DPBS+ added. Data represent the mean ± SD (n = 4). *, p < 0.05; **, p < 0.01.

 

FIGURE 4. (a) Typical ROS (DHE) images of MDA-MB-231 cells under various exogenous H2O2 concentrations and (b) quantification of ROS measured by DHE intensity corresponding to various H2O2 treatments. In panel (a), all images have the color bar scaled in the same range (0‒4000 in arbitrary unit). The warmer colors represent higher ROS levels. In panel (b), data represent the mean ± SD (n = 4). *, p < 0.05; ***, p < 0.001. 

3.3. Elevated Exogenous ROS Results in Reduced Redox Plasticity 

Redox plasticity herein refers to the changes of NADH, Fp, or redox ratio and is another useful parameter reflecting metabolic changes in addition to the redox baseline [19]. To explore the effect of elevated exogenous ROS levels on redox plasticity, we added 3 different concentrations of H2O2 (0, 176, and 704mM) to MDA-MB-231 cells followed by sequential redox status manipulations by chemical agents: first FCCP, then ROTAA. Changes of the redox indices, i.e., redox plasticity, DFp, DNADH, and DFp/(NADH+Fp) were obtained from the differences between FCCP and ROTAA treatments. We found a concentration-dependent downward trend in DNADH and a reduction in redox ratio plasticity due to H2O2 treatment (Figure 5).

 

FIGURE 5. Redox plasticity measurements achieved by sequential addition of mitochondrial uncoupler FCCP and inhibitors ROTAA to MDA-MB-231 cells. Plasticity measurements were taken after 10 min pre-incubation of various concentrations of H2O2 (or DPBS+ in the case of the 0 µM condition). Data represent the mean ± SD (n = 3). **, p < 0.01. 

3.4. A Change of Redox Status toward either Oxidation or Reduction is Associated with an Increase of Endogenous ROS 

To investigate whether oxidized or reduced redox state is associated with endogenous ROS generation, we quantified the ROS generation (Figure 6) resultingfromredoxstatusshiftsduetoFCCPorROTAA perturbation. There are significant ROS increases in response to the redox changes. Relative to the control, cells treated with FCCP experienced a 68.4% ROS increase and cells treated with ROTAA experienced a 146.4% ROS increase detected by DHE staining. Corresponding redox changes for these treatments can be seen in Figure 6a or 2. Thus, we observed both more oxidized and more reduced redox states are associated with ROS increase.

 

FIGURE 6. ROS levels corresponding to redox changes in MDA-MB-231 cells. (a) Redox changes due to FCCP or ROTAA perturbation (separated charts were presented in Figure 2). (b) The corresponding ROS levels. Data represent the mean ± SD (n = 4). *, p < 0.05; **, p < 0.01; ****, p < 0.0001. 

We also manipulated the redox status in MDA-MB-231 cells with FX11. FX11 is a lactate dehydrogenase A (LDHA) inhibitor which inhibits the interconversion between lactate and pyruvate and was reported to be present in both mitochondria and cytosol [31]. Quantitative analysis revealed that both the 3 µM and 5 µM FX11 additions bear significant increases in NADH levels, 194.4% and 258.3%, respectively,relativetothecontrol(Figure7a).In accordance, both concentrations of FX11 produced a significant reductive shift in the redox ratios of the cells, whereas, staining cells with DHE revealed increased generation of ROS in dishes treated with FX11 (Figure 7b). The 3 µM and 5 µM treatment conditions resulted in a 154.7% and 179.5% increase in ROS, respectively, relative to the control, but no significant difference in ROS levels between each other.

 

FIGURE 7. (a) Quantification of the redox effects of FX11 on MDA-MB-231 cells, and (b) the corresponding ROS levels. Data represent the mean ± SD (n = 3). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. 

3.5. A Change of Redox State Correlates to a Change in Mitochondrial ROS 

Since DHE is not specific to mitochondrial O2˙ˉ[24, 25], and NADH and Fp signals detected by ORI are mainly from mitochondria, we employed a mitochondrial ROS dye, MitoSOX red. We first tested MitoSOX in MDA-MB-231 cells. When treated with 1.25 µg/ml AA, as expected mitochondrial ROS generation increased corresponding to the reduced redox status induced by AA (Figure 8). However, when cells were treated with 0.5 µM FCCP we detected a decrease in mitochondrial ROS, which was opposite to the intracellular ROS level change detected by DHE (Figure 6).

 

FIGURE 8. Altered redox status by FCCP (0.5 µM) or AA (1.25 µg/ml) corresponds to a decrease or an increase of mitochondrial ROS, respectively, in MDA-MB-231 cells. Data represent the mean ± SD (n = 6). *, p < 0.05. 

To investigate whether the relationship between redox state and ROS levels holds for other TNBC cells, we performed experiments on another TNBC cell line HCC1806 (Figure 9). Similarly, AA addition to HCC1806 cells caused expected redox changes, namely increased NADH and a decreased redox ratio. As a result of redox changes, ROS level increased. Upon FCCP treatment, NADH levels decreased and the redox ratio increased, similar to the redox response of MDA-MB-231 cells; however, FCCP treatment increased mitochondrial ROS generation which is opposite to the response observed in MDA-MB-231 cells. In HCC1806 cells, a shift toward either oxidation or reduction both resulted in increased ROS generation.

 

FIGURE 9. (a) Altered redox status by FCCP (0.5 µM) and AA (1.25 µg/ml) corresponds to (b) an increase of mitochondrial ROS in HCC1806 cells. Data represent the mean ± SD (n = 3). *, p < 0.05; **, p < 0.01; ****, p < 0.0001. 

3.6. No Change in ROS Levels Correlates to No Change of Redox Status under Mitochondrial Metabolic Perturbation 

In some cases, the chemical agents used for metabolic perturbation had no significant effect on redox state or ROS levels. As seen in Figures 10 and11, respectively, when Oligo (complex V inhibitor, 2 µg/ml) or ROT (1mM) was administered to HCC1806 cells there was no significant change in redox state or mitochondrial ROS level. Similarly, when MDA-MB-231 cells were treated with ROT(1mM) there was no change in redox state or mitochondrial ROS level (Figure 12).

 

FIGURE 10. (a) ORI results and (b) mitochondrial ROS levels in HCC1806 cells due to2 µg/ml Oligo treatment. Data represent the mean ± SD (n = 3).

 

FIGURE 11. (a) ORI results and (b) mitochondrial ROS levels in HCC1806 cells due to1 µM ROT treatment. Data represent the mean ± SD (n = 3).

 

FIGURE 12. (a) ORI results and (b) mitochondrial ROS levels in MDA-MB-231 cells due to 1 µM ROT treatment. Data represent the mean ± SD (n = 6).

4. DISCUSSION 

4.1. Concomitant and Non-Concomitant Relationship between the Redox Status and ROS Levels 

We observed concomitant redox changes and ROS changes with exogenous H2O2, FCCP, ROTAA, AA, and FX11 treatments in MDA-MB-231 cells and with FCCP and AA treatments in HCC1806 cells. We also observed, in some experiments, no changes to cellular redox status upon ROT and Oligo modulation and no change in mitochondrial ROS level, either, which may indicate insufficient concentrations of chemical agents. Overall, redox and ROS changes correlate well. 

However, a non-concomitant change between redox status and ROS levels was also observed. We observed changes in both redox state and cytoplasmic ROS levels due to addition of exogeneous H2O2 up to 704 µM, after which we saw a change in redox statebutnofurtherincreaseincytoplasmicROS (Figures 3 and 4). One might question if the detection of further increases in cytoplasmic ROS was possible or if the concentration of dye (DHE) was insufficient, which could cause signal saturation and make higher levels of ROS undetectable. However, at the highest measured ROS level in the H2O2 addition experiment (704 µM) we observed a 138.0% increase from control ROS values. Referring to the FX11 addition experiment using the same DHE concentration, we observed a 179.5% increase in ROS levels in the 5 µM condition from the control, which is a larger percentage increase than was illustrated in the H2O2 addition experiment. This confirms that the amount of DHE used would have been able to detect further increases in ROS during the H2O2 addition if they were to exist. This halted ROS generation above 704 µM H2O2 is supported by the study of Armstrongetal.[5],whichcites500µMH2O2tobethe concentration at which the peak effect on ROS generation is observed for pancreatic acinar cells. No further increases in intracellular ROS levels under high concentrations of exogenous H2O2 were presumably due to enhanced ROS scavenging in cells, which was consistent with the decrease in NADH we observed. 

With 1 µM ROT or 2 µg/ml Oligo treatment, we did not detect significant redox changes or ROS changes (Figures 1012).These concentrations were probably too low to induce changes in these cell lines under the experimental conditions of this study. 

4.2. Both Redox Extremes Correlate to Increased Cytoplasmic ROS Levels 

It is interesting that a redox shift in MDA-MB-231 cells, whether toward reduction or oxidation, caused an increase in cytoplasmic ROS. This finding could imply that general redox stress (either oxidative or reductive) to the cell is linked to a cytoplasmic ROS overflow. These results are supported by the redox-optimized ROS balance hypothesis, which theorizes that ROS are minimum in an intermediate range of redox potential and that ROS overflow occurs at either extreme, i.e., more oxidized or more reduced compared to the intermediate range of redox potential [4]. When redox state is reduced, ROS overflow is suggested to occur due to free radical production outweighing scavenging capacity. On the other hand, in a more oxidized redox state, ROS overflow is suggested to occur due to depletion of the ROS scavenging molecules such as NADPH and reduced glutathione. Though more studies are needed to attribute an exact mechanism, we observed the phenomenon of intracellular ROS overflow at both ends of the cellular redox state spectrum in MDA-MB-231 cells. 

4.3. Differential Changes of Mitochondrial ROS Levels between Redox Extremes 

Under reductive stress caused by AA or ROTAA, we found both mitochondrial and cytoplasmic ROS increases in MDA-MB-231 cells as detected by MitoSOX and DHE, respectively. However, upon uncoupling with 0.5 µM FCCP, which resulted in a more oxidized state (a higher redox ratio), we observed decreased mitochondrial ROS despite observing increased cytoplasmic ROS. It has been reported that mild uncoupling can increase or decrease mitochondrial ROS depending on the redox environment [4, 32, 33]. Uncoupling by low concentrations of FCCP resulting in lower ROS level has been reported to contribute to the survival advantage of cancer cells [34]. 

4.4. Cell Line-Dependent Relationship between Redox Status and Mitochondrial ROS 

Under FCCP treatment, MDA-MB-231 cells experienced decreased mitochondrial ROS production but HCC1806 cells experienced increased mitochondrial ROS production. This suggests that the mitochondria of the two different cell lines are operating within different redox environments. MDA-MB-231 and HCC1806 were classified as two different TNBC subgroups, i.e., mesenchymal and basal-like, respectively [35]. It has also been shown that TNBC subgroups can have different mitochondrial ROS baselines and heterogeneous responses to metabolic modulation or antioxidants [3]. It is possible that the homeostatic redox state of MDA-MB-231 cells is toward the more reduced side of their minimum ROS production range.In comparison, HCC1806 cellsmight fall on their minimum ROS production range. More studies are needed to better understand these observations. 

4.5. Increased ROS Decreases Redox Plasticity 

We found that increasingly higher cytoplasmic ROS levels correlated to a decreased redox plasticity of NADH and redox ratio in MDA-MB-231 cells. This is an important finding that suggests a reduced ability of the cell to respond to stressors when experiencing increased cytoplasmic ROS levels. 

4.6. Limitations of This Study 

NADPH is an important ROS scavenger. However, ORI cannot differentiate between NADH and NADPH since both have the same absorption and fluorescence spectrum. Therefore, changes observed in the NADH channel may reflect changes of either or both species. However, it has been estimated that NADPH contributes much less than NADH to the total NADPH signals [36, 37]. Therefore, the signal of the NADH channel is expected to be dominated by NADH. 

Neither DHE nor MitoSOX is as specific as was intended for cytoplasmic O2˙ˉor mitochondrial O2˙ˉ, respectively, but they serve well as probes for detecting intracellular oxidant formation [24, 38]. DHE as a dye for staining cytoplasmic O2˙ˉcan form two fluorescence products with overlapping fluorescence spectra upon entering cells: ethidium and 2-hydroxyethidium via a non-specific redox reaction and a specific adduct of O2˙ˉ, respectively. Additionally, the ethidium can enter the nucleus and bond to double-stranded DNA, generating an enhanced fluorescence. All contribute to the total fluorescence, resulting in an over-estimation of cytoplasmic ROS levels. However, this should not significantly affect our conclusions since we are interested in the relative intracellular ROS change. MitoSOX used in high concentration (> 2 µM) can stain non-mitochondrial ROS when accumulated in cytoplasm [24]. While using 2.5 µM MitoSOX with 2-hydroxyethidium-specific 385 nm excitation [27–29], we observed more mitochondria-localized staining for the baseline level of the two TNBC cell lines; however, for higher ROS levels, the staining was less mitochondria specific.

5. CONCLUSION 

Employing optical redox imaging, we found that both the NAD(H) redox status and ROS level change in response to exogenous H2O2 or perturbation of metabolic activity in TNBC cell cultures; however, ROS increase can be associated with either a more oxidized or a more reduced redox status. Overall, this study serves as a starting point to broaden the knowledge of the relationship between NAD(H) redox state and ROS in cancer cells. 

ACKNOWLEDGMENTS 

This work was supported by McCabe Pilot Award (to H.N.X.) and the U.S. NIH Grant R01CA191207 (to L.Z.L.). The authors thank the Cell and Developmental Biology (CDB) Microscopy Core in the Perelman School of Medicine, University of Pennsylvania, PA, USA.The authors declare no conflicts of interest. 

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