Investigating the In Vitro Mitochondria-Mediated Anticancer Activity of the Plant Metabolite Ursolic Acid

TL;DR

This study shows that ursolic acid, a plant compound, kills breast cancer cells by targeting mitochondria and works better when combined with glycolysis inhibitors.

Contribution

The study reveals a direct mitochondrial inhibitory effect of ursolic acid and proposes a novel dual treatment strategy for breast cancer.

Findings

Ursolic acid induces cytotoxicity and antiproliferative effects in breast cancer cells.

Ursolic acid depolarizes mitochondrial membrane potential and inhibits mitochondrial enzyme activity.

Combining ursolic acid with glycolytic inhibitors enhances cytotoxicity in breast cancer cells.

Abstract

This study investigated the cellular and mitochondrial toxicities of the pentacyclic triterpenoid and plant-specialized metabolite ursolic acid (UA) in human breast adenocarcinoma cell lines. Cell viability and clonogenic assays showed that UA induced potent cytotoxic and antiproliferative effects in MDA-MB-231 and MCF7 cells. Confocal images of living cells showed that UA caused a depolarization of the mitochondrial membrane potential and spectrophotometric measurement of electron transport chain enzyme activity in isolated organelles showed that UA induced a dose-dependent decrease in mitochondrial succinate-cytochrome reductase activity. These results demonstrate a direct, site-specific inhibitory effect of UA on mitochondrial bioenergetic function. Furthermore, the efficacy of a drug combination aimed concurrently at both major pathways of ATP production in breast cancer cells was investigated. The data show that when MDA-MB-231 and MCF7 cells were treated with UA in combination with either 2-deoxy-D-glucose or 3-bromopyruvate, two inhibitors of glycolysis, the resulting cytotoxicity was greater than that induced by any of the compounds used independently. The results of this study are important in that they demonstrate direct mitochondrial targets of UA and suggest the possibility of using this natural, plant-derived metabolite in combination with glycolytic inhibitors as a novel and effective dual treatment strategy for breast cancer cell killing.

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1. Introduction

According to the World Health Organization, breast cancer is the most commonly diagnosed malignancy and the most common cause of cancer death in women worldwide [1]. There are currently a variety of treatments used against breast cancer, including surgery, chemotherapy, and hormonal and radiation therapies [2]. However, despite the numerous discoveries and achievements made by the pharmaceutical industry during the past several decades, drug resistance, adverse side effects, and limited efficacy continue to present major challenges in the treatment of breast cancer. Compounding these challenges is the fact that different breast cancer subtypes, which are characterized by their hormone or growth factor receptor status, also respond very differently to various forms of treatment.

The limitations and adverse side effects of conventional therapies, along with their high development and production costs, have sparked interest in the use of plant-based “natural” medicines as alternatives or complements in the treatment and prevention of a variety of diseases, including cancer. One such compound, ursolic acid (UA), is a pentacyclic triterpenoid found in a variety of fruits, herbs, berries, and teas (Figure 1) [3]. Ursolic acid has demonstrated a number of health benefits, including anti-inflammatory [4], antioxidant [5], anti-diabetic [6], cardioprotective [7], and neuroprotective [8] effects. Several studies also show that UA exerts potent in vitro and in vivo anticancer activity through multiple mechanisms, including inhibition of cell proliferation and induction of apoptosis [9,10]. For example, in human colon cancer cells, UA was shown to inhibit cell proliferation by suppressing the phosphorylation of EGFR and to induce apoptosis through down-regulation of Bcl-2 and Bcl-xL and activation of caspase-3 and caspase-9 [11]. In cervical cancer cells, the apoptotic mechanism induced by UA treatment was determined to occur through the mitochondrial intrinsic pathway and to be closely associated with the suppression of the ERK1/2 signaling pathway [12]. In breast cancer specifically, UA was shown to inhibit tumor growth and induce apoptosis via modulation of PI3K/Akt/mTOR pathway signaling [13] and to suppress tumor migration and metastasis via modulation of c-Jun N-terminal kinase (JNK), Akt, and mTOR signaling [14]. More recently, UA has been shown to dramatically impair both glycolytic metabolism and mitochondrial respiratory function in breast cancer cells [15,16].

The purpose of this study was to investigate the in vitro cellular and mitochondrial toxicity of UA in MDA-MB-231 and MCF7 breast cancer cells, and to assess the possibility that the anticancer activity of this plant metabolite might be enhanced by dual treatment with glycolytic inhibitors. The data obtained show that UA has potent cytotoxic and antiproliferative effects on both breast adenocarcinoma cell lines, that it decreases the mitochondrial membrane potential in intact breast cancer cells, and that it directly inhibits electron transport activity in isolated mitochondria. Furthermore, when MDA-MB-231 and MCF7 cells were treated with UA in combination with either 2-deoxy-D-glucose (2DG) or 3-bromopyruvate (3BP), two inhibitors of the glycolytic pathway, the resulting cytotoxicity was shown to be greater than that induced by any of the compounds used independently.

2. Results

2.1. The Effect of UA on the Viability and Proliferative Capacity of Breast Cancer Cells

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay is a common laboratory-based technique that is used to monitor the metabolic activity and viability of cultured cells. It is based on the principle that living cells with active metabolism convert MTT into a purple formazan product, while dead cells are unable to do so. In this study, the MTT assay was used to assess the cytotoxic effect of UA on MDA-MB-231 and MCF7 cells after exposure to varying concentrations of the compound for 24 h. The results in Figure 2 show that UA induced a concentration-dependent cytotoxicity in both breast cancer cell lines. The effect of UA on cell viability was similar in both cell lines, with the concentration of compound required to kill half of the exposed cell population (LC_50_) observed at approximately 50 μM.

The clonogenic (or colony formation) assay is an important in vitro technique that evaluates the ability of a single cell to grow into a colony of cells. It differs from the MTT assay, which monitors metabolic activity and cell viability over a very short time period, in that is assesses the long-term survival and proliferative capacity of cells. In essence, the clonogenic assay reveals whether cells maintain the ability to reproduce and contribute to tumor regrowth after exposure to cytotoxic agents or treatment conditions. In this study, the clonogenic assay was used to determine the long-term, anti-proliferative effect of UA on MDA-MB-231 and MCF7 breast cancer cell lines. This was accomplished by measuring colony formation after 7–10 days in both cell populations after 24 h exposure to varying concentrations of the compound. The results in Figure 3 show that UA induced a concentration-dependent inhibition of colony formation in both cell lines. However, MDA-MB-231 cells appear to be less sensitive than MCF7 cells to the effects of UA, with the LC_50_ observed at approximately 15 μM and approximately 2 μM, respectively. As expected, the results also show that the concentration of UA required to induce anti-proliferative effects in both cell lines was much lower than that required to induce cytotoxic effects.

2.2. Effect of UA on Mitochondrial Function

The mitochondrial membrane potential is a key indicator of mitochondrial health and the cell’s ability to generate ATP through oxidative phosphorylation. Therefore, its measurement in living cells can be used to assess how a therapeutic compound or toxin impacts overall mitochondrial function in real-time. In this study, the effect of UA on mitochondrial membrane potential was monitored in cultured MDA-MB-231 and MCF7 cells. This was accomplished using confocal microscopy to observe the fluorescence intensity of tetramethyl rhodamine ethyl ester (TMRE), a cell-permeant, cationic dye that is accumulated in the mitochondria matrix of living cells in response to a negative inside membrane potential. Figure 4 shows that 10 min of exposure to UA caused a significant and concentration-dependent decrease in TMRE fluorescence in MDA-MB-231 and MCF7 cells compared to the control condition (t = 0). Specifically, exposure to 150 μM UA induced no obvious change in TMRE fluorescence intensity in either cell line, indicating no effect on the mitochondrial membrane potential. Exposure to a higher 250 μM concentration of UA caused a partial decrease in TMRE staining, indicating a partial loss of mitochondrial membrane potential, in MDA-MB-231 cells. However, the same concentration of UA caused no observable effect in MCF7 cells. Exposure to 350 μM UA induced a total dissipation of TMRE staining in both cell lines. This loss was similar to that observed after exposure of the cells to the proton ionophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), indicating a complete depolarization of the mitochondrial membrane potential at the highest concentration of UA tested.

To determine whether UA might have a direct effect on the mitochondrial electron transport chain (ETC), freeze-thawed preparations of isolated mitochondria were exposed to varying concentrations of UA and spectrophotometric measurement of electron transport enzyme activities was subsequently performed. The mechanisms of ETC activity are common among mammalian mitochondria; therefore, rat liver was used as a convenient and cost-effective source of mitochondria in these experiments. To discern any potential site-specific action of UA, three individual assays that monitored electron transfer activity in limited segments of the ETC were chosen. The NADH-ubiquinone oxidoreductase assay was used to measure the rate of electron transfer from the oxidizable substrate NADH through complex I. The succinate-cytochrome c reductase assay was used to measure the rate of electron transfer from succinate, through complex II and III, and then on to cytochrome c. The cytochrome c oxidase assay was used to measure the rate of complex IV directly by monitoring its rate of oxidation of reduced cytochrome c.

The data presented in Figure 5 indicate that UA had no significant effect on either NADH-ubiquinone oxidoreductase (5A) or cytochrome c oxidase (5C) activities over the concentration range tested. However, as shown in Figure 5B, UA induced a dose-dependent inhibition of mitochondrial succinate-cytochrome reductase activity at a concentration range between 0 and 1.0 mM, with a half-maximal inhibitory concentration achieved at approximately 0.2 mM. This indicates that UA has a direct, site-specific inhibitory effect on electron transport activity at or between complexes II and III of the mitochondrial electron transport chain. Interestingly, the inhibitory effect of UA on succinate-cytochrome c reductase activity occurred within the same range of concentrations of the compound that was found to induce membrane potential loss in live cells. This suggests that ETC inhibition by UA may be a causal or contributing factor to mitochondrial membrane potential depolarization and subsequent cytotoxicity.

2.3. The Effect of UA in Combination with 2DG or 3BP on the Viability of Breast Cancer Cells

A recent study conducted in this laboratory investigated the cytotoxic and antiproliferative effects of a dual treatment strategy aimed simultaneously at both mitochondrial and glycolytic pathways of ATP production in human breast cancer cells [17]. That study involved concurrent exposure of MDA-MB-231 and MCF7 cells to the synthetic mitochondria-targeted chemotherapeutic agent, elesclomol, in combination with either of two glycolytic inhibitors, 2DG or 3BP. The results showed a significantly greater in vitro cytotoxic effect against both breast cancer cell lines when the combination of mitochondrial-targeted and glycolytic inhibitor compounds was used than when any of the compounds were used as a single agent.

In the current study, once the mitochondrial toxicity of UA was established, it was of interest to determine whether a similar enhancement of cytotoxicity could be achieved when breast cancer cells were exposed to UA in combination with the same glycolytic inhibitors. First, the effective cytotoxic concentration range of 2DG and 3BP, as single agents, was established in MDA-MB-231 and MCF7 cells using the MTT colorimetric assay. The results presented in Figure 6 show that each glycolytic inhibitor induced a concentration-dependent decrease in cell viability in both breast cancer cell lines. 3BP appeared to be the more potent of the two glycolytic inhibitors, with the LC_50_ observed at approximately 60–70 μM and complete cell death observed by 200 μM for both cell lines. For 2DG, higher concentrations of the compound were required to achieve the LC_50_, which was approximately 100 mM for MDA-MB-231 cells and approximately 175 mM for MCF7 cells. Interestingly, there was no further increase in toxicity demonstrated in either cell line even at the highest concentration of 2DG tested.

Next, the MTT assay was used to measure and compare the cytotoxic effect of the UA plus 3BP and UA plus 2DG combination treatments versus single-agent treatments with each compound in each of the cell lines. The data presented in Figure 7 are from a series of MTT assays using test concentrations of 31 μM UA, 50 μM 3BP, and either 25 mM or 100 mM 2DG for the MDAMB231 or MCF7 cell lines, respectively. These specific concentrations were chosen based on the results from the dose–response curves in Figure 2 and Figure 6, reflecting concentrations at which moderate cytotoxic effects with single agents and potential enhancements with dual treatment could be observed. The results show that when the mitochondria-targeted plant metabolite UA was combined with either glycolytic inhibitor, the cytotoxic effect in both MDA-MB-231 and MCF7 cell lines was enhanced compared to that observed for any of the compounds used as single agents. Ordinary one-way ANOVA analyses of the data for MCF7 cells showed statistically significant differences in the cytotoxic effect induced by UA, 3BP, or 2DG as single agents compared to that induced by UA in combination with 3BP or UA in combination with 2DG. Analysis of the data for MDA-MB-231 cells showed statistically significant differences in the cytotoxic effect for the 3BP versus UA plus 3BP comparison and for the UA versus UA plus 2DG comparison. However, the calculated p values for the UA versus UA plus 3BP comparison and for the 2DG versus UA plus 2DG comparison indicate that these differences did not achieve statistical significance. Table 1 presents the data obtained from these experiments in numerical form as percent control cell number for each treatment condition and comparison with calculated p values.

3. Discussion

This study investigated the in vitro cellular and mitochondrial toxicity of UA in MDA-MB-231 and MCF7 breast cancer cells. The results show that UA has potent cytotoxic and antiproliferative effects on both breast adenocarcinoma cell lines, that it decreases the mitochondrial membrane potential in intact breast cancer cells, and that it has a direct inhibitory effect on mitochondrial electron transport activity in isolated organelles. These results support and extend those of previously mentioned studies [15,16] showing UA caused depolarization of the mitochondrial membrane potential and impairment of basal and maximal mitochondrial respiratory function in the same human breast cell lines. Taken together, these findings suggest that at least some anticancer effects induced by UA in breast adenocarcinoma are mediated through specific mitochondrial targets. Future studies are warranted to elucidate more fully the underlying molecular mechanisms of the mitochondrial and cellular toxicity induced by UA. Additionally, it will be of interest to explore the potential efficacy of other plant-based mitochondria ETC inhibitors for in vitro and in vivo anticancer activity.

Breast cancer cells are known to display a high degree of metabolic plasticity. This is a fundamental property of cancer cells that allows them to shift between mitochondrial oxidative phosphorylation and glycolysis in order to generate enough ATP to survive in a variety of growth conditions. Unfortunately, this property also allows breast cancer cells to bypass many traditional monotherapies aimed at only one or the other major metabolic pathway. In this study, we explored the effectiveness of a drug combination aimed simultaneously at both metabolic pathways by combining the mitochondria-targeted UA with certain compounds that are known to inhibit glycolysis. One of these compounds, 2DG, is a synthetic glucose analog that competes with the natural substrate for binding and phosphorylation at the first hexokinase-catalyzed reaction in glycolysis. Unlike glucose, phosphorylated 2DG cannot be further metabolized via glycolysis. Instead, it accumulates within the cell, causing allosteric and competitive inhibition of hexokinase, decreasing ATP production [18] and eventually leading to cell death [19]. The other compound, 3BP, is a structural analog of pyruvate and lactate. It inhibits glyceraldehyde-3-phosphate dehydrogenase, the enzyme that catalyzes the sixth step in glycolysis [20]. This inhibition causes a disruption in ATP production and oxidative stress, eventually leading to cell death [21,22]. The results of this study show that when MDA-MB-231 and MCF7 cells were treated with the mitochondria-targeted natural plant metabolite UA in combination with either of two glycolytic inhibitors, 2DG or 3BP, the resulting cytotoxicity was shown to be greater than that induced by any of the compounds used independently.

In this study, the combination treatment of UA plus 3BP or UA plus 2DG was tested in two different human breast cancer subtypes with different metabolic profiles [23]. The MCF7 cells are an estrogen and progesterone receptor-positive subtype [24] that is more dependent on oxidative phosphorylation than on glycolysis for its energy supply [25,26]. In contrast, MDA-MB-231 is a triple-negative breast cancer (TNBC) subtype that lacks the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 [27,28] and relies more heavily on glycolysis than on oxidative phosphorylation for its energy supply [29,30]. The TNBC subtype is more aggressive and invasive than the receptor-positive subtype and is generally more resistant to chemotherapeutic agents [31]. Our data show that both MDA-MB-231 and MCF7 cell lines are equally sensitive to the cytotoxic effects of UA when used as a single agent, and that both cell lines show at least some enhancement in cytotoxicity when UA is combined with either 2DG or 3BP. These results have positive implications for the therapeutic potential of UA as a single agent and in combination with glycolytic inhibitors for both breast cancer subtypes.

It is noteworthy that the enhancement of cytotoxicity demonstrated in this study when UA was combined with 2DG or 3BP was similar to that shown in our previously published study when elesclomol was combined with either of the same glycolytic inhibitors [20]. Elesclomol is a synthetic bis(thiohydrazide) amide that inhibits mitochondrial ETC activity at complex I, while UA is a natural pentacyclic triterpenoid that inhibits mitochondrial ETC activity between complex II and III. The comparable enhancement of cytotoxic effects achieved when two very distinct mitochondria-targeted compounds are used in combination with glycolytic inhibitors suggests the potential for more widespread applicability of a dual treatment strategy that simultaneously targets both major pathways of ATP production in human breast cancer cells.

The choice of UA as the test compound in this study was important for several reasons. Ursolic acid is a plant specialized metabolite, one of a wide array of species-specific organic compounds that are produced by plants to help attract pollinators, to defend against pests and pathogens, and to mediate interactions with environmental stressors. Specialized metabolites have long been used by humans as spices, aromatics, and insect repellants, and for their health benefits, which include antioxidant, anti-inflammatory, and antimicrobial effects. As a natural plant metabolite, UA is an abundant, inexpensive, and relatively safe compound. It is found in a variety of common plant sources, most notably in fruits such as apples (especially the peels) and raisins, in berries such as cranberries and blueberries, in herbs such as basil and rosemary, and in green tea [32]. Ursolic acid can be extracted easily and is already available as a dietary supplement in various forms. Clinical trials have suggested that UA supplementation is well-tolerated with manageable toxicity [32,33,34].

Furthermore, UA as a single agent has already been shown to induce potent in vitro and in vivo anticancer effects against numerous different cancer cell types, including prostate, colon, pancreatic and liver cancers, leukemia, melanoma, glioma and others [3,9,10]. Interestingly, a modified diamine derivative of UA in combination with 2DG was previously shown to synergistically inhibit cancer cell growth in vitro in HEPG2 hepatoma and A-375 melanoma cell lines and in vivo in a H22 hepatoma-bearing mouse model [35]. The authors proposed that this synergy occurred via dual targeting of caspase-mediated apoptosis and glycolysis. These findings, taken together with those of our study, warrant future investigations to assess the efficacy of the UA plus 3BP/2DG dual treatment strategy against a wide variety of cancer types.

4. Materials and Methods

4.1. Materials and Reagents

Ursolic acid (UA; catalog no. 158253), 2-deoxy-D-glucose (2DG; catalog no. AC111980050), and dimethyl sulfoxide (DMSO) (catalog no. BP231-100) were obtained from Fisher Scientific. (Waltham, MA, USA) UA was made fresh on the day of experimentation at a concentration of 50 mM in DMSO and 2DG was made fresh on the day of experimentation at a concentration of 1 M in cell culture medium. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (catalog no. 475989-1GM), FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone) (catalog no. SML2959), and 3-bromopyruvate (3BP; catalog no. 376817-M) were obtained from Millipore Sigma (Burlington, MA, USA). 3BP was made fresh on the day of experimentation at a concentration of 50 mM in dH_2_O. Tetramethylrhodamine ethyl ester, perchlorate (TMRE) (Cat # 70016) was obtained from Biotium (Fremont, CA USA), resuspended in DMSO at a concentration of 5 mM, and stored in single-use aliquots at −20 °C. NucBlue was obtained from Invitrogen (Carlsbad, CA, USA; Winooski, VT, USA); catalog no. R37605)

4.2. Cell Cultures

The human breast adenocarcinoma cell lines MDA-MB-231 and MCF7 were obtained from ATCC and grown in Dulbecco’s Modified Eagles Medium (DMEM; ATCC catalog no. 30-2002) supplemented with 10% Fetal bovine serum (FBS; catalog no. 30-2020). Cells were incubated at 37 °C in a 5% CO_2_ atmosphere.

4.3. Cell Viability Assays

The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay was used to assess the effect of UA, 3BP and 2DG, alone and in combination, on MDA-MB-231 and MCF7 cell viability. On the day of the experiment, cells were harvested, counted, and diluted to a concentration of 5 × 10^4^ cells/mL. Sample wells of a 96-well plate were seeded with 100 μL of the cell suspension (0.5 × 10^3^ cells) and plates were incubated at 37 °C. After 24 h., varying concentrations and combinations of the test compound(s) were added to the wells and the plates were returned to the incubator for another 24 h. After this exposure period, MTT was added to the sample wells and the plates were incubated for 4 h. A solubilization buffer was then added to each well and the plates were returned to the incubator for 24 h. A BioTek EL800 Microplate Reader (BioTek Instruments, Inc.; Winooski, VT, USA) was used to measure the absorbance at 540 nm of each sample against a blank well and a graph of the absorbance (y-axis) against the concentration of drug (x-axis) was plotted. Each treatment condition was tested in triplicate for each experiment and each experiment was repeated at least three times on cultures from different platings in order to obtain averaged results.

4.4. Cell Proliferation Assays

The colony formation assay, or clonogenic assay, was used to evaluate the proliferative capacity of each cell type in the absence and presence of each compound or combination of compounds. Cells were harvested, counted, and diluted to a concentration of 1 × 10^3^ cells/mL. Using 6-well plates, 2 mL/well of the cell suspension (i.e., 2000 cells/well) was seeded in each well and the cells were returned to the incubator. The following day, the media was aspirated and 2 mL/well of media containing varying concentrations of the test compound(s) was added. After exposure to the compound(s) for 24 h, the media was aspirated and the wells were rinsed with PBS. Each well then received 2 mL of compound-free media, and the plates were returned to their growth chambers. When the colonies in the control wells reached sufficient size for counting (approximately 7–10 days later), the plates were stained with crystal violet and the colonies in each well were counted. Each treatment sample and an appropriate blank was performed in triplicate for each experiment. Experiments were repeated three times on cultures from different platings.

4.5. Mitochondrial Membrane Potential in Whole Cells

The cell-permeant, positively charged, fluorescent dye TMRE was used to monitor mitochondrial membrane potential in live cultured MDA-MB-231 and MCF7 cells. Cells were seeded and grown to 90% confluency in glass-bottom 35 mm plates (MatTek, Ashland, MA, USA). On the day of the experiment, cells were washed twice with sterile PBS and incubated with 100 nM TMRE in phenol red-free Eagle’s minimum essential medium and two drops/mL of the nuclear stain NucBlue Live for 10 min. Using a Zeiss 800 confocal microscope (Carl Zeiss, Oberkochen, Germany), cells were observed and photographed immediately before the addition of UA (t = 0), and then again after 10 min of exposure to the compound. FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone; 100 μM), a proton ionophore and known uncoupler of oxidative phosphorylation, was used as a positive control for dissipation of mitochondrial membrane potential.

4.6. Isolation of Mitochondria

Mitochondria were isolated from rat liver by a cell fractionation process using differential centrifugation as previously described [36]. Briefly, 5–6 g of rat liver was minced and homogenized in an isotonic STE buffer (250 mM sucrose, 1 mM Tris- HCI, and 1 mM EDTA (ethylenediaminetetraacetic acid, pH 7.4)), and centrifuged at a low speed of 600× g for 10 min. The supernatant was then collected and centrifuged for 10 min at a higher speed of 8000× g. The resulting pellet, which contained the mitochondria, was resuspended and washed twice by centrifugation for 10 min at 8000× g in STE. This was followed by an additional wash in ST buffer (250 mM sucrose and 1 mM Tris-HC1 (pH 7.4)), and the final pellet was re-suspended in ST.

4.7. Mitochondrial Electron Transport Enzyme Assays

The activity of mitochondrial electron transport enzymes was measured using a GENESYS 10S UV-VIS spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) essentially as described [36,37]. NADH-ubiquinone oxidoreductase activity, which is a measure of electron transport through respiratory complex I, was determined by monitoring the rate of decrease in absorbance at 340 nm due to the oxidation of NADH. First, the isolated mitochondria fraction was subjected to three cycles of freeze-thawing in liquid nitrogen. A freeze-thawed sample (300 μg) was then added to a cuvette containing 50 mM potassium phosphate, 3 mg/mL fatty acid-free BSA, 300 μM KCN, and 100 μM NADH in the absence or presence of varying concentrations of UA (0–0.5 mM) and the reaction was initiated by adding 10 μM ubiquinone^1^ to the cuvette. The change in absorbance was recorded over time in the presence and absence of 10 μM rotenone, and the rotenone-sensitive activity of ETC complex I was determined by subtracting the rate measured in the presence of rotenone from the rate obtained in the absence of rotenone.

The succinate-cytochrome c reductase assay, which measures electron transport activity through respiratory complexes II and III, was determined by monitoring the rate of increase in absorbance at 550 nm due to the reduction in oxidized cytochrome c. An isolated mitochondria preparation was freeze-thawed at −20 °C and a 300 μg sample was added to a cuvette containing dH_2_O, 50 mM potassium phosphate (KH_2_P0_4_/K_2_HP0_4_; pH 7.4), 30 μM KCN, in the absence or presence of varying concentrations of UA. In order to fully activate the enzyme, the sample was pre-incubated with substrate (20 mM succinate) for 10 min and the reaction was initiated by adding 50 μM oxidized cytochrome c to the cuvette. The change in the absorbance was recorded over time.

Cytochrome c oxidase activity, which is a measure of electron transport through complex IV, was determined by monitoring the decrease in absorbance at 550 nm due to the oxidation of reduced cytochrome c. An isolated mitochondria preparation was freeze-thawed at −20 °C and a 300 μg sample was added to a cuvette containing dH_2_O, 40 mM potassium phosphate (KH_2_PO_4_/K_2_HPO_4_; pH 7.0), in the absence or presence of varying concentrations of UA. The reaction was initiated by adding 50 μM reduced cytochrome c, and the change in the absorbance was recorded over time.

4.8. Statistical Analysis

An ordinary one-way ANOVA analysis was performed followed by Sidak’s multiple comparison test using GraphPad Prism Version 10.6.1 for Mac (Graph-Pad Software, Inc., San Diego, CA, USA) in order to identify statistical differences in data generated from the control and treatment conditions. A minimum of three biological and three technical replicates were used for all experiments. The threshold for statistical significance was set at * p < 0.05, ** p < 0 01., *** p < 0.001

5. Conclusions

This study investigated the in vitro cellular and mitochondrial toxicity of UA in MDA-MB-231 and MCF7 breast cancer cells. The data obtained show that UA has a potent cytotoxic and antiproliferative effect on both breast adenocarcinoma cell lines, that it decreases the mitochondrial membrane potential in intact breast cancer cells, and that it has a direct inhibitory effect on mitochondrial electron transport activity in isolated organelles. Furthermore, the effectiveness of a dual treatment strategy aimed simultaneously at both mitochondrial and glycolytic pathways of ATP production in breast cancer cells was also explored. The data show that when MDA-MB-231 and MCF7 cells are treated with the mitochondria-targeted natural plant metabolite UA in combination with either of two glycolytic inhibitors, 2DG or 3BP, the resulting cytotoxicity is greater than that induced by any of the compounds used independently. The results of this study are important in that they reveal direct mitochondrial targets of UA and suggest the possibility of using this plant-derived metabolite in combination with glycolytic inhibitors as a novel and effective dual treatment strategy for breast cancer cell killing.