Inhibition of ATP-synthase potentiates cytotoxicity of combination drug menadione/ascorbate in leukaemia lymphocytes

Abstract The combination drug menadione/ascorbate (M/A) manifests synergistic dose-dependent antiproliferative and cytotoxic effects towards cancer cells, but not towards normal cells of the same origin especially at concentrations that can be achieved in vivo by its oral and parenteral administration. It is assumed that M/A alters selectively dysfunctional cancerous mitochondria. However, the exact molecular mechanism is not clear yet. The aim of the present study was to elucidate the role of adenosine triphosphate (ATP) synthase activity and its suppression by oligomycin-A on M/A-induced cytotoxicity, mitochondrial superoxide and ATP level in leukaemic lymphocytes. Cells were treated with different concentrations of M/A in the absence and presence of oligomycin-A (100 ng/mL) for 24 h and 48 h. The cell growth and viability, steady-state ATP level and mitochondrial superoxide were analysed using conventional analytical tests. The results showed that suppression of ATP synthase activity by oligomycin-A decreased the cell growth and viability and increased the production of mitochondrial superoxide and depletion of ATP in cells treated with low/tolerable doses of M/A (up to 5/500 µM/µM), compared to the cells treated with M/A only. Oligomycin-A did not affect these parameters in cells treated with high doses of M/A (10/1000 and 20/2000 µM/µM). The inhibition of ATP synthase potentiated the cytotoxicity of M/A, particularly in leukaemic lymphocytes treated with low/tolerable doses. We assume that the cytotoxicity of M/A is tightly connected to impairment of oxidative phosphorylation, and mitochondrial ATP depletion is a crucial factor for cell death.


Introduction
Oxidative phosphorylation (OXPHOS) is the main source of energy (adenosine triphosphate (ATP)) in normal cells. Unlike them, most cancer cells are characterized by a unique shift in bioenergetics -reduction of OXPHOS and increased glycolysis, the so-called 'Warburg effect' [1]. This compensatory switch is accompanied by some advantages: (i) maintenance of biosynthetic pathways during rapid cell proliferation (carbons from glucose and the Krebs cycle are used for amino acid, fatty acid and nucleic acid synthesis) and (ii) acidification of microenvironment due to increased lactate levels from glucose metabolism [2]. Thus, via bioenergetics adaptation, cancer cells revive and sustain their survival and growth, which promotes their invasion and metastasis. Moreover, through metabolic 'rearrangements' , cancer cells become more protected against conventional anticancer therapies. On the other hand, combined anticancer treatments, targeting both mitochondrial respiration and glycolysis, could be lethal to cancer cells [3][4][5].
Until recently, it was generally accepted that cancer cells use glycolysis as a main source of ATP and do not rely on mitochondrial respiration for ATP production. However, recent studies demonstrated that the majority of ATP in cancer cells is produced by mitochondria, and some tumours show heavy dependence on OXPHOS [6][7][8]. Therefore, blocking mitochondrial respiration should induce cell death in these tumours. These studies provide strong evidence that dysfunctional mitochondria are priority target to successful anticancer therapy. We believe that it is more correct to call cancerous mitochondria 'altered' instead of 'dysfunctional' , because in fact they are functional.
Menadione (pro-vitamin K3) and ascorbate (vitamin C) interfere directly with the mitochondrial electron transport chain (eTC). Studies have demonstrated that naphthoquinones and benzoquinones affect mitochondrial respiration, bypassing Complex-I deficiency [9][10][11]. Pharmacological ascorbate and menadione are applied as adjuvants in the treatment of mitochondrial diseases [11,12]. The combination of ascorbate and menadione has been used clinically to bypass Complex-III deficiency of the eTC [11,12]. Both substances have been applied as a dietary supplement in combination with coenzyme q10 (Coq10), niacin, riboflavin and thiamin to bypass Complex-I and Complex-III of the eTC [13]. Menadione is also a common supplement in animal feed (including diets for laboratory animals).
Menadione and ascorbate are redox-cyclers and administered alone, or in combination, they induce intracellular production of reactive oxygen species (ROS) by interaction with molecular oxygen [14,15]. The redox-active combination menadione/ascorbate (the ratio of 1/100 mol/mol menadione to ascorbate; M/A) has attracted the attention of researchers for more than 20 years due to its unusual ability to kill cancer cells without affecting the viability of normal cells, as well as with the synergistic anticancer effects of both molecules [16][17][18][19][20][21][22][23][24][25]. This targeted cytotoxicity of M/A is accompanied by overproduction of mitochondrial superoxide and impairment of mitochondrial functionality in cancer cells only, assessed by a decrease in mitochondrial potential, NADH, NAD + and oncometabolite succinate [26,27]. We assume that M/A-mediated suppression of cancer cell growth is a result of a specific mitochondrial redox-cycling between both substances, leading to severe oxidative stress and ATP depletion. We hypothesized that this specific redox-cycling of M/A occurs in the altered cancerous mitochondria only due to their overcharged and unbalanced 'Coq-pools' , overloading with metabolites (such as NADH and succinate), and 'reversed' Krebs cycle [27,28]. However, the exact molecular mechanism of M/A-mediated targeting and influencing OXPHOS in cancerous mitochondria is not clear yet.
Oligomycin-A is a mitochondrial ATP synthase inhibitor, widely used to suppress OXPHOS in living cells. It is considered that oligomycin-A blocks the proton channel, which significantly reduces the electron flow through the eTC, but it is not completely stopped due to a process known as 'proton leak' or 'mitochondrial uncoupling' -the protons return to the mitochondrial matrix independently of ATP synthase activity [29]. In addition, a strong relationship between proton and electron leakage was demonstrated, which is explained by the sensitivity of superoxide production to reduced proton mobility due to its leakage [30]. It was reported that treatment of cancer cells with oligomycin-A results in a decreased generation of ATP, which shifts their bioenergetics to glycolysis [31]. We consider this as a suitable model to clarify whether the antiproliferative and cytotoxic effect of M/A is mediated by ATP synthase activity and OXPHOS.
The aim of the present study was to elucidate the role of ATP synthase activity and its suppression by oligomycin-A in M/A-induced cytotoxicity, mitochondrial superoxide and ATP level in leukaemic lymphocytes, as well as to clarify the role of mitochondrial ATP depletion in the antiproliferative and cytotoxic effects of this combination drug.

Chemicals
Oligomycin-A was purchased from Selleck Chemicals (USA); menadione and ascorbate were purchased from Sigma-Aldrich (Weinheim, Germany). Other chemicals and kits were purchased from various suppliers. All reagents used in the experiments were analytical or HPLC grade.
Before each experiment, the cells were sedimented by centrifugation (1000 × g/10 min) and placed in fresh medium without antibiotics. The lymphocytes were incubated with oligomycin-A (100 ng/mL) alone or in combination with menadione/ascorbate (M/A) for different time intervals, and at each time interval, aliquots were used for biochemical analyses. Ascorbate was dissolved in phosphate-buffered saline (PBS) (10 mM, pH 7.4). Oligomycin-A and menadione were dissolved in dimethyl sulfoxide (DMSO) (to 5 mM and 10 mM stock solutions, respectively) and then several working solutions in PBS were prepared. To avoid influence on cell viability, the final concentration of DMSO in the cell suspension was below 1%.

ATP assay
CellTiter-Glo™ Luminescent Cell Viability Assay (Promega, Madison, WI, USA) was used to analyse ATP levels in cell suspensions. The assay is based on the generation of a luminescent signal from a luciferin/ luciferase reaction, which is proportional to the production of ATP in live cells [32]. Shortly, aliquots of cell suspension (180 μL containing 1 × 10 6 cells/mL) were placed in 96-well transparent plates and incubated with 10 µL of oligomycin-A (final concentration 100 ng/mL) and 10 μL of M/A (at different concentrations − 2/200, 3/300, 5/500, 10/1000 and 20/2000 µM/ µM) for 24 and 48 h, in a humidified atmosphere (at 37 °C, 5% CO 2 ). A hundred microlitres of the cell suspension from each well were transferred to 96-well white plates, and 100 µL of the CellTiter-Glo reagent, containing luciferin and luciferase, were added to each well and incubated according to the manufacturer's protocol. Luminescence was detected using a microplate reader (TeCAN Infinite® M1000, Vienna, Austria), working in chemiluminescent mode.

Cell proliferation and viability assay
Cell viability and proliferation were analysed by trypan blue staining and automated cell counting, using Countess™ Automated Cell Counter (Invitrogen, Oregon, USA). Briefly, 10 μL of 0.4% trypan blue solution was added to 10 μL of cells suspension, incubated for 30 s, and 10 μL of cell suspension was placed in a Countess™ (Invitrogen) glass chamber. The number of live and dead cells was counted automatically. The linear range to operate with the automated cell counter was 1 × 10 4 -5 × 10 6 cells/mL, and the optimal cell size was in the range of 5-60 μm.

Analysis of mitochondrial superoxide
A fluorogenic probe -MitoSOX™ Red Mitochondrial Superoxide Indicator (MitoSOX; Molecular Probes, Invitrogen, eugene, Oregon, USA) was used for highly selective detection of superoxide in the mitochondria of live cells. The probe is cell-penetrating and localizes in the mitochondria. MitoSOX exhibits red fluorescence after oxidation by mitochondrial superoxide [33].
Briefly, on the day of the experiment, MitoSOX™ Red was dissolved in DMSO to 5 mM stock solution, which was diluted with Hank's Balanced Salt Solution (HBSS, containing Ca 2+ and Mg 2+ ) to prepare final 3 μM working solution. One-millilitre aliquots of cells (1 × 10 6 cells/mL) were collected via centrifugation, and the pellet was re-suspended in 1 mL of working MitoSOX solution. After 30 min at room temperature and protection from light, all samples were washed three times with PBS using centrifugation and finally re-suspended in 1 mL of PBS. The fluorescence intensity was detected immediately at λ ex = 510 nm and λ em = 605 nm, using a microplate reader (TeCAN Infinite® M1000, Vienna, Austria).

Statistical analysis
All data are expressed as mean values with standard deviation (±SD). Comparisons between the groups were performed using Student's t-test. Differences were considered statistically significant at the level of p < 0.05.  doses of M/A (≥5/500 µM/µM) possessed a strong antiproliferative and even cytotoxic effect. The initial cell number in all suspensions was 1 × 10 6 cells/mL. In Figure 1, the values below the red dashed line indicate a cytotoxic effect, while the values above the red dashed line indicate an antiproliferative effect (decreasing, but not stopping proliferation). Oligomycin-A (100 ng/mL) applied alone also suppressed the growth of leukaemic lymphocytes, but this effect was relatively slightly expressed. In the combination, oligomycin-A affected the effect of M/A on cell growth and viability in two ways: (i) it potentiated the antiproliferative effect of low/tolerable doses of M/A (<5/500 µM/µM); (ii) but it did not affect the antiproliferative and cytotoxic effect of high doses of M/A (Figure 1 -grey columns versus black columns).

Results and discussion
The effect of M/A on the steady-state level of ATP in the cancer cells also was dose-dependent and correlated with the number of cells in the respective sample and their viability (Figures 2 and 3). Data from ATP analysis were processed in two ways: (i) as absolute values of ATP-based luminescence in treated samples and untreated controls and (ii) as ATP-based luminescence normalized to the same number of living cells in each sample, since only living cells synthesize ATP. This approach for data processing was described in details in our previous study [27]. M/A provoked a significant decrease in ATP-based luminescence in leukaemic lymphocytes, especially at high doses (≥5/500 µM/µM of M/A) (Figure 2). Oligomycin-A (applied alone) also decreased the absolute steady-state level of ATP in the cells, but this effect was slightly expressed. In the combination, oligomycin-A further increased the ATP depletion in   Data are means ± SD from three independent experiments with four parallel measurements for each experiment. *p < 0.05, **p < 0.01, p < 0.001: oligomycin/m/a-treated versus the respective m/a-treated cells or oligomycin-treated sample versus the untreated cells (control). atp-based luminescence in the untreated cells was considered 100%.
Studies have suggested that a menadione concentration of 5 µM is crucial for its mitochondrial redox-cycling [11,34,35]. This concentration is considered a threshold level, since Complex-I bypass and ATP recovery in menadione-treated cells occurs only at concentrations below 5 µM [11]. In our study, we observed that M/A at concentrations ≥5/500 µM/µM induces irreversible ATP depletion (Figures 2 and 3 black columns), which could be a result of suppression of mitochondrial respiration due to irreversible eTC damage. However, at low/tolerable concentrations of M/A, the effect on ATP depletion and mitochondrial respiration is reversible. Inhibition of ATP synthase by oligomycin-A potentiates the M/A-mediated ATP depletion in leukaemic lymphocytes, which is evidence for mitochondrial mechanism of its antiproliferative and cytotoxic effects.
To investigate the effect of M/A and/or oligomycin-A on mitochondrial superoxide, we used fluorogenic probe MitoSOX Red Superoxide Indicator. The sensor easily crosses the cell membrane and localizes mainly in the mitochondria. In the absence of superoxide, MitoSOX has no fluorescence, but fluorescence appears after oxidation of MitoSOX by superoxide. The results are shown in Figure 4. In M/A-treated leukaemic lymphocytes, the superoxide level increased in a dose-dependent and time-dependent manner: from ~2 times for low/tolerable concentrations of M/A to ~10 times for high concentrations of M/A within 48 h of incubation. Incubation of cells with oligomycin-A also induced production of mitochondrial superoxide (~3 times over the control level). The combination oligomycin/M/A induced an additive increase in mitochondrial superoxide at M/A concentrations below 5/500 µM/µM. Oligomycin-A did not affect the level of mitochondrial superoxide in cells treated with high doses of M/A (≥5/500 µM/µM).
Our data suggest that inhibition of ATP synthase by oligomycin-A potentiated the antiproliferative and cytotoxic effect of M/A, particularly in leukaemic lymphocytes treated with low/tolerable doses, which was accompanied by ATP depletion and overproduction of mitochondrial superoxide. We assume that the antiproliferative and cytotoxic effects of M/A towards cancer cells are mediated by mitochondria-dependent mechanism(s), in particular by mitochondrial ATP synthesis.
It is interesting to note that a slight relapse was observed in cell proliferation and viability of M/A-treated cells in the absence and presence of oligomycin-A within the incubation time (48 h versus 24 h of incubation) (Figure 1). This observation raises the question of whether leukaemic lymphocytes can reorganize and adapt their bioenergetics after M/A-treatment, shifting from OXPHOS to other pathways for ATP synthesis, when ATP synthase activity is inhibited by oligomycin-A.
Hao et al. [31] have demonstrated that cancer cell lines with higher respiration rate show larger gains in glycolytic flux upon oligomycin-A treatment (100 ng/mL), which is an indicator for the compensatory role of glycolysis in energy supply [31]. Inhibition of ATP synthase by oligomycin-A was also found to trigger fatty acid oxidation (FAO) as another alternative mechanism for ATP synthesis in cancer cells [36]. Based on these observations, we assume that proliferation recover y af ter M/A-treatment of leukaemic lymphocytes could be due to activation of regulatory mechanism(s) related to reorganization and adaptation of their bioenergetics to glycolysis and/or FAO after oligomycin-A application. The increased generation of mitochondrial superoxide in oligomycin/M/A-treated cells compared to M/A-treated, especially at low/tolerable doses of M/A, is a factor disrupting the eTC, leading to additional decrease in proliferation activity and cell survival.
The findings from this study support our assumption [27] that the possible reasons for the specific overproduction of mitochondrial superoxide in M/A-treated cancer cells are as follows: (i) a direct impairment of mitochondrial eTC by compromising its functionality, mainly Complex-I and Complex-III that are known to produce superoxide [37,38] and (ii) a specific mitochondrial redox-cycling of both substances, mediated by dysfunctional (altered) mitochondria, but not by the mitochondria of non-transformed cells [11,27]. Our hypothesis is that cancerous mitochondria could be discharged and offloaded by redox-cycling between menadione and ascorbate in them [27]. Once superoxide exceeds a certain threshold, vital functional mitochondrial units (such as eTC, ATP synthase and others) can be destroyed, which causes inhibition of mitochondrial ATP synthesis, release of caspase-independent apoptosis-inducing factor (AIF) and cell death most likely via autoschizis [39][40][41]. Inhibition of ATP synthase by oligomycin-A may exacerbate these effects, especially at low/tolerable M/A concentrations, potentiating the impairment of mitochondrial functionality in cancer cells.

Conclusions
Suppression of ATP synthase activity by oligomycin-A decreased the proliferation and viability and increased the production of mitochondrial superoxide and depletion of ATP in leukaemic lymphocytes treated with low/tolerable concentrations of M/A (up to 5/500 µM/µM), compared to the cells treated with M/A only. We assume that the cytotoxicity of M/A towards cancer cells is tightly connected to alteration of vital functional mitochondrial units (such as eTC, ATP synthase and others), which is accompanied by impairment of OXPHOS, and mitochondrial ATP depletion is one of the crucial factors for cell death.

Disclosure statement
No potential conflict of interest was reported by the authors.

Authors' contributions
SS, ZZ and RB produced the first draft of manuscript. SS, DI, BN, GZ, RB and ZZ conducted the experiments. IA was involved in the critical review of the drafts and final version. All authors read and approved the final version of the manuscript.

Data availability statement
The data that support the findings of this study are available on request from the corresponding authors (ZZ and BN).

Funding
This study was partially supported by the IC-MedTech Co. (USA) (ICM/qST grant, granted to R.B.) and the Japanese Agency for Medical Research and Development (AMeD) (Project for Cancer Research and Therapeutic evolution, P-CReATe, No. 16 cm0106202h0001).