Oxidative phosphorylation inhibitors inhibit proliferation of endometriosis cells

All reagents were obtained from ThermoFisher or Sigma Aldrich, unless specified otherwise. All reagents and antibodies were strictly maintained at optimal storage conditions and tested for activity using appropriate quality control assays. The 12Z cells were kindly provided to us by Dr. Asgi Fazleabas (Michigan State University) with permission from Dr. Anna Starzinski-Powitz (Goethe University Frankfurt) whose lab first developed this cell line. The 12Z cell line was developed through the SV40 transformation of epithelial-like cells from peritoneal endometriosis lesions and are considered to be an excellent model for endometriosis ( Chelariu-Raicu, et al. 2016 , Ruiz, et al. 2018 , Zeitvogel, et al. 2001 ). The cells were maintained in DMEM-F12 media and tested for mycoplasma contamination each month. Effect of plumbagin, curcumin and atovaquone on the viability of 12Z cells was monitored using the MTT assay ( Kumar, et al. 2018 ). In this assay, NADPH dependent oxidoreductase enzymes reduce the tetrazolium dye, MTT, to insoluble formazan crystals which have purple color. The intensity of the color is a measure of cell viability. Briefly, 5X10 3 cells /well were plated in 96 well tissue culture plates, allowed to grow for 24 h and then treated with the test compounds. Following 72 h treatment, 20 μl MTT (stock 5 mg/ml) was added to each well and the plate was incubated in a tissue culture incubator for 3 h. Subsequently, the media was removed from all wells and formazan crystals formed in each well were dissolved in 100 μL DMSO. The optical density was read at 570 nm using the Spectramax M3 microplate reader. Apoptosis was measured by monitoring the expression of phosphatidylserine on the cell surface using FITC-conjugated Annexin V ( Vermes, et al. 1995 ). The untreated and treated cells were stained with Annexin V–FITC and propidium iodide (PI) according to manufacturer’s instructions and analyzed by flow cytometry on a Attune NXT (ThermoFisher) spectral flow cytometer. The data was analyzed using FlowJo software. The 12Z cells were loaded with the oxygen radical sensor, H2DCFDA which gains fluorescence when cleaved by reactive oxygen species ( Eruslanov and Kusmartsev 2010 ). The H2DCFDA dye loaded cells were treated with compounds for 15 min, washed, harvested and fluorescence in the untreated and compound-treated groups was measured on the Attune NXT spectral flow cytometer. The data was analyzed using FlowJo software. Oxygen consumption rate (OCR) in 12Z cells was measured using a Seahorse XFe96 Extracellular Flux Analyzer (Seahorse Bioscience, Billerica, MA, USA). 12Z were seeded in XFe96 cell culture plates at a density of 2.5X10 4 cells/well in their culture medium and incubated overnight at 37°C. The cells were then treated with either DMSO (vehicle control) or 15 μM Atovaquone, 2.5 μM plumbagin, 7.5 μM curcumin for 30 min. Following incubation, cells were washed with a pre-warmed XF buffer and were kept in a non-CO 2 incubator for 1h to allow temperature and pH equilibration. The OCR was measured according to the manufacturer’s instruction using pre-optimized concentration of FCCP. The data was analyzed using the Wave software as described previously by our group ( Kapur, et al. 2018 ). Effect of the test compounds on the mitochondria electron transport complexes was measured using the high-resolution respirometer Oxygraph-2K (Oroboros Instruments, Innsbruck, Austria). Complex activities were measured using the substrate inhibitor titration protocol using the Mitochondrial Respiration medium MIR-05 kit from Oroboros ( Makrecka-Kuka, et al. 2015 ). Briefly, 2.5X10 6 cells/ml of mitochondrial respiration medium from MIR-05 kit were used in each chamber of the oxygraph. The baseline oxygen consumption of the cells was measured, followed by addition of 16 μM (previously optimized) digitonin to permeabilize the cells. Once the oxygen consumption stabilized, DMSO (vehicle) was added to chamber A (control) and the test compounds were added to Chamber B. At 5 min post drug treatment Complex I substrates, malate (2 mM) and glutamate (10 mM) were added to initiate respiration followed by the addition of 5 mM ADP to achieve maximum respiration. Activated Complex I was then inhibited by adding 1.25 μM rotenone. Complex II/III respiration was stimulated by adding 10 mM succinate (complex II substrate) followed by inhibition with 2.5 μM antimycin A (Complex III inhibitor). Lastly, complex IV was activated by addition of 1 mM tetramethyl -p-phenylene-diamine (TMPD) dissolved in 0.8 M ascorbate and complex IV was inhibited by 500 mM sodium azide. Data analysis was performed following normalization to the number of cells using the DatLab software 5.1. The microfluidic device fabrication is described in ( Ayuso, et al. 2018 , Jimenez-Torres, et al. 2016 ). Briefly, microdevices were fabricated by soft lithography: a template containing the microdevice geometry was fabricated using SU-8 100 (Y131273, MICROCHEM), then polydimethylsiloxane (PMDS) (Dow Corning) was poured on top and polymerized for 4 hours at 80 °C. PDMS microdevices were detached from the SU-8 wafer and assembled. PDMS rods were fabricated by injecting liquid PDMS through 23-gauge needles (BD Precision Glide). After PDMS polymerization, rods were removed from the needles and placed into the microdevices. Finally, the assembled microdevices and 60 mm glass-bottom Petri dishes (P50G-1.5–30-F, MatTek) were treated with oxygen plasma and bonded together to create a permanent bonding. The microdevices were sterilized by UV light exposure for 20 minutes. To improve the attachment of the collagen hydrogel, microdevices were pretreated with 2% aqueous poly(ethyleneimine) (Sigma-Aldrich, 03880) for 10 minutes and 0.4% glutaraldehyde (Sigma-Aldrich, G6257) diluted in water for 30 minutes. Microdevices were washed three times with sterile distilled water to remove potential residues. The collagen hydrogel was prepared by mixing 10.6 μl of 10X PBS (79382, Sigma), 2.34 μl of 1N NaOH (221465, Sigma), 93.65 μl of 9.61 mg/ml collagen type I (354249, Corning) and finally 93.4 μl of 12Z culture medium. 10 μl of this hydrogel mixture was injected through the gel loading port in each microdevice. Microdevices were placed into the incubator at 37° C and 5% CO 2 for 20 min to polymerize the collagen hydrogel. Then, PDMS rods were removed using sterile tweezers, generating a lumen through the collagen hydrogel. 1.5 μl of 20×10 6 12Z cells/ml was injected through the lumen and microdevices were immediately placed upside-down in the incubator for 15 min to allow the cells to attach to the top side of the lumen. Then the microdevices were flipped upside-up again and left in the incubator for 2 hours to allow the remaining cells to attach to the bottom of the lumen. Finally, microdevices were covered with 4 ml of growth medium and placed in the incubator. After 24 hours, media was supplemented with curcumin or plumbagin at different concentrations and cell viability was evaluated 3 days later. Stock solutions of 5 mg/ml calcein acetoxymethyl ester (CAM) and 2 mg/ml propidium iodide (PI) were dissolved in DMSO and distilled water respectively. To test cell viability within microfluidic devices, stock solutions of CAM and PI were diluted to 5 and 4 μg/ml, respectively, in PBS. The CAM/PI solution was perfused through the lumen and cells were incubated for 15 min to label viable and dead cells in green and red respectively. Cell viability was evaluated using 20X magnification in a Leica SP8 3X STED Super-resolution microscope. Z-stacks were generated by acquiring images every 10 microns, and images were analyzed with Fiji software ( https://fiji.sc/ ). Briefly, red and green images were binarized and the area occupied by live and dead cells was quantified. A custom-built inverted multiphoton microscope (Bruker Fluorescence Microscopy, Middleton, WI, USA), was used to acquire fluorescence intensity and lifetime images. The equipment consists of an ultrafast laser (Spectra Physics, Insight DSDual), an inverted microscope (Nikon, Eclipse Ti), and a 40X water immersion (1.15 NA, Nikon) objective. NAD(P)H and FAD images were obtained for the same field of view. FAD fluorescence was isolated using an emission bandpass filter of 550/100 nm and excitation wavelength of 890 nm. NAD(P)H fluorescence was isolated using an emission bandpass filter of 440/80 nm and an excitation wavelength of 750 nm. Fluorescence lifetime images were collected using time-correlated single-photon counting electronics (SPC-150, Becker and Hickl, Brookline, MA, USA) and a GaAsP photomultiplier tube (H7422P-40, Hamamatsu, Bridgewater, NJ, USA). 512×512 pixel images were obtained using a pixel dwell time of 4.8 μs over 60 s total integration time. To guarantee adequate photon observations for lifetime decay fits and no photobleaching, the photon count rates were maintained at 1–2 × 10 5 photons/s. The instrument response function was calculated from the second harmonic generation of urea crystals excited at 900 nm. A Fluoresbrite YG microsphere (Polysciences Inc., Warrington, PA, USA) was imaged as a daily standard for fluorescence lifetime. The lifetime decay curves for the YG microsphere standard were fit to a single exponential decay and the fluorescence lifetime was measured to be 2.1 ns (n = 7), which is consistent with published values. Optical redox ratio values for all treatment conditions were normalized to the control condition. NAD(P)H and FAD intensity and lifetime images were analyzed using SPCImage software (Becker & Hickl, Berlin, Germany) as described previously ( Sharick, et al. 2020 ). The fluorescence lifetime decay curve was deconvolved with the instrument response function and fit to a two-component exponential decay model at each pixel, I(t) = α 1* e(−t/τ 1 ) + α 2 *e(−t/τ 2 ) + C, where I(t) represents the fluorescence intensity at time t after the laser excitation pulse, α accounts for the fractional contribution from each component, C represents the background light, and τ is the fluorescence lifetime of each component. Since both NAD(P)H and FAD can exist in two conformational states, bound or unbound to enzymes, a two-component model was used. For NAD(P)H, the short (τ 1 ) and long (τ 2 ) lifetime components correspond with the unbound and bound conformations, respectively. The mean lifetime (τ m ) was calculated using τ m = α 1* τ 1 + α 2* τ 2 , for both NAD(P)H and FAD. The optical redox ratio was determined from the NAD(P)H and FAD intensity images. For each pixel, the intensity of NAD(P)H was then divided by the intensity of FAD. Cell cytoplasms were isolated using Cell Profiler ( Walsh and Skala 2014 ). Values for NAD(P)H τ m , FAD τ m , NAD(P)H intensity, FAD intensity, and the optical redox ratio (NAD(P)H/FAD intensity) were averaged for all pixels within each cell cytoplasm. A minimum of 100 cells were analyzed to obtain statistically robust results. The results from all experiments were plotted in GraphPad Prizm software 6.04 for Windows (GraphPad Software La Jolla CA, USA, www.graphpad.com ). Statistical analysis was conducted using the features available through this software package. Typically, we used the two-tailed unpaired t-test or ANOVA to determine statistical significance and calculate the p-values.

Our group and others have demonstrated the chemotoxic effects of curcumin and plumbagin ( Fig. 1A ) in cancer cells. Here, we demonstrate that both these compounds can also inhibit the proliferation of the endometriosis cell line, 12Z. By employing MTT assays we demonstrate that curcumin and plumbagin inhibit the proliferation of 12Z cells with an IC 50 of 2.5 and 7.5 μM, respectively ( Fig. 1B ). Curcumin and plumbagin belong to a class of compounds that contain the unsaturated carbonyl functional group ( Fig. 1 A ). In our on-going works we are observing that several other small molecule agents that contain the unsaturated carbonyl group are also able to inhibit the viability of cancer cells. A prominent example is atovaquone ( Fig. 1A ), an FDA-approved antimalarial agent that is widely used against the plasmodium parasite ( Mather, et al. 2005 , Nixon, et al. 2013 , Srivastava, et al. 1997 ). We demonstrate for the first time that atovaquone is also a potent agent against the 12Z cells and mediates its effects at an IC 50 of 20 μM ( Fig. 1A ). The 12Z cells were seeded in the 3D lumen of the microdevice that was suspended in a collagen hydrogel ( Fig. 2A ). After 24 hours, the cells were completely attached and formed a lumen that was lined continuously with a single layer of 12Z cells. Atovaquone, plumbagin, or curcumin were added to the lumen to evaluate their effect on cell viability. After 3-day incubation with the drugs, the 12Z cells in the microfluidic devices were labeled with CAM and propidium iodide to stain for viable and dead cells. The viable (labeled with green CAM fluorescence) and dead (PI labeled red cells) cells were qualitatively and quantitatively evaluated by confocal microscopy. In the absence of drugs, 12Z cells exhibited low cell death and high cell viability (>95%) ( Fig. 2B – D ). Treatment with curcumin and plumbagin (2.5 and 10 μM) resulted in an increased number of dead cells. Based on the MTT assays, it was clear that atovaquone was toxic to the 12Z cells at higher concentrations (a feature we have also observed in unpublished experiments conducted with ovarian and endometrial cancer cell lines and is likely due to the lower solubility of atovaquone in aqueous media). In 3D environments, higher concentration of the test compounds is often required to observe chemotoxicity. We therefore tested atovaquone at 25 and 100 μM concentrations. At both concentrations, we observed a statistically significant increase in the number of dead cells as compared to untreated controls ( Fig. 2B and C ). When the experiments were conducted with curcumin, plumbagin (10 μM each) and atovaquone (25 and 100 μM) we observed a corresponding statistically significant decrease in live cells ( Fig. 2D ). Our microfluidic device also allows tracking of the cells away from the lumen. This parameter has been successfully used by our group to monitor cell migration in response to specific stimuli. Here, we used this parameter to determine the effect of the drug candidates on the migration of 12Z cells ( Fig. 2E ). Higher degree of invasion of the 12Z cells from the lumen was evident in the control experiments. The extent of invasion significantly decreased when curcumin (2.5 and 10 μM), plumbagin (10 μM) and atovaquone (25 and 100 μM) were placed in the lumen ( Fig. 2E ). To determine if the inhibition of cell viability was the result of cell death, we monitored the 12Z cells for apoptosis after they had been exposed to curcumin, plumbagin and atovaquone for 24h. Treatment of the 12Z cells with any of the three agents resulted in a significant increase in the binding of Annexin V to the cells indicating significant apoptosis ( Fig. 3 A ). The results from the annexin V binding experiments complimented the observation that exposure to curcumin, plumbagin and atovaquone increased expression of cleaved caspase 3 ( Fig 3B ). We also observed that these three agents increased the expression and enzymatic activity of cleaved caspase 9 ( Fig 3C ). We did not observe any change in the expression of cleaved caspase 8 in the curcumin, plumbagin or atovaquone treated 12Z cells ( Fig. 3D ). These data indicated that all three of the agents were inducing cell death via the intrinsic apoptotic pathway. In our previous studies we demonstrated that curcumin, plumbagin and atovaquone induce an intracellular oxygen radical flux. We therefore asked if the cell death observed in 12Z cells was also associated with an increase in oxygen radicals upon exposure to curcumin, plumbagin and atovaquone. The H2DCFDA-labeled 12Z cells were treated with the three compounds and analyzed by flow cytometry for an increase in intracellular oxygen radicals. We observe a 2–3 fold increase in oxygen radicals when the12Z cells were treated with each of the three agents ( Fig. 4A ). A common consequence of oxidative stress is DNA damage. We determined that the increase in intracellular oxygen radicals was associated with a 10-fold increase in γ-H2Ax ( Fig. 4B ). Next, we employed imaging cytometry to identify the cellular location of the oxygen radical flux in 12Z cells treated with each of the three drugs. The 12Z cells were labeled with fluorescent markers to stain the nucleus and mitochondria in addition to the mitochondria-specific oxygen radical sensing dye, MitoSox. When the curcumin, plumbagin and atovaquone-treated 12Z cells were examined by imaging cytometry, a major surge in oxygen radicals was observed in the mitochondria. These data suggested that the oxidative stress in the drug-treated 12Z cells was originating in the mitochondria. The mitochondria are the major producers of oxygen radicals. In OXPHOS, high energy electrons from NADH and FADH 2 are gradually transferred to molecular oxygen. This transfer occurs through multiprotein complexes present in the inner mitochondrial membrane. Inefficient transfer of the electrons results in generation of oxygen radicals. Previously, we have demonstrated that plumbagin inhibits OXPHOS in cancer cells and in the process, generates intracellular oxygen radicals. It is also confirmed that atovaquone produces its therapeutic effects by blocking electron transport in the malarial parasite. Here, we confirm that even in the endometriosis cell line, 12Z, curcumin, plumbagin and atovaquone inhibit oxygen consumption ( Fig. 5 A – F ). With either drug, the basal and maximal oxygen consumption of 12Z cells was decreased by 4–8 fold ( Fig. 5 B , D and F ). When the cells were treated with FCCP to remove the mitochondrial proton gradient, we observed that 12Z cells treated with any of the three drugs continued to consume less oxygen as compared to controls indicating that the spare respiratory capacity of the cells was regulated by curcumin, plumbagin and atovaquone ( Fig. 5 A , C and E ). The decreased oxygen consumption is an indicator of compromised OXPHOS. This inference was supported by the results demonstrating that the 12Z cells when treated with the three agents had decreased levels of ATP as compared to the controls ( Fig. 5 B , D and F ). We conducted additional experiments to identify the stages of the electron transport pathway that are blocked by curcumin, plumbagin and atovaquone. In each experiment, the 12Z cells were exposed to either of the three compounds (curcumin, plumbagin and atovaquone) followed by addition of substrates and inhibitors of the protein complexes required for mitochondrial electron transport. During the course of the experiment, the cell media was sequentially supplemented with substrates of complex I (glutamate and malate) or complex II (succinate) and the oxygen consumption rate was measured ( Fig. 6 ). These experiments indicated that supplementation with the complex I substrates significantly reversed the inhibitory effects of curcumin and plumbagin ( Fig. 6 A , B ). Similarly, succinate, the substrate for complex II, also blocked the OXPHOS inhibitory potential of curcumin and plumbagin ( Fig. 6 A , B ). Additionally, when the cells were provided the substrates for Complex IV, the inhibitory effects of curcumin and plumbagin were fully reversed. These data indicate that curcumin and plumbagin likely produce their effects by blocking Complex I. On the other hand, atovaquone significantly inhibited oxygen consumption even when the 12Z cells were supplemented with the substrates for complex I and II ( Fig. 6 C ). To the best of our knowledge, there is no specific substrate or inhibitor of complex III. Therefore, we were not able to specifically test the effect of atovaquone on complex III. However, when cells were supplemented with the substrate for complex IV (TMPD), the ability of atovaquone to block oxygen consumption was fully reversed. These data suggest two possibilities. Atovaquone either inhibits at Complex III (as shown in experiments with the malarial parasites) or can inhibit electron transport at Complexes I, II and III. Based on the observations that curcumin, atovaquone and plumbagin inhibit the mitochondrial complexes that mediate electron transport, we expected that there would be metabolic changes occurring in the 12Z cells. Our group has successfully used the ratio of the autofluorescence of NAD(P)H to FAD, termed as the redox ratio, as an early marker of cellular stress. The microfluidic culture device allowed us to conduct real-time assessment of NAD(P)H and FAD autofluorescence. The fluorescence of curcumin interferes with that of NAD(P)H and FAD and therefore this compound could not be tested in these autofluorescence experiments. Optical redox ratio is sensitive to shifts in metabolic pathways that oxidize/reduce NAD(P)H and FAD and reflects the overall redox state of the cell ( Chance, et al. 1979 , Franklin, et al. 2020 , Meleshina, et al. 2017 , Walsh, et al. 2012 , Walsh, et al. 2013 ). We employed the microfluidic device to conduct real-time monitoring of the redox ratio in the 12Z cells in response to plumbagin and atovaquone. The inherent fluorescence of curcumin interferes with the autofluorescence spectra of NADH and FAD and hence, this agent could not be tested in these experiments. The 12Z cells were implanted in the microdevice followed by treatment with atovaquone and plumbagin for 24 h. We first monitored the autofluorescence of NAD(P)H and FAD of the cells in the microfluidic devices. Optical redox ratio (ratio of the fluorescence of NAD(P)H/FAD), a metabolic indicator of cellular stress was significantly lower in the 12Z cells treated with atovaquone and plumbagin as compared to the matching controls ( Fig. 7 A and B ). Next, we determined the fluorescence lifetime (τ m ) of NAD(P)H to monitor the cellular fate of this cofactor ( Pasch, et al. 2019 , Yu, et al. 2017 ). We consistently observe a statistically significant decrease in the τ m of NAD(P)H suggesting an increase in cellular levels of NAD(P)H that was not conjugated with protein ( Fig. 7 C and D ). This increase in free cellular NAD(P)H is consistent with the hypothesis that inhibition of OXPHOS by atovaquone and plumbagin would result in accumulation of free NADH in the mitochondria.

In this study, we demonstrate that curcumin, plumbagin and atovaquone can cause apoptotic cell death of the prototypic endometriotic cell line, 12Z. We further show that all three of these agents inhibit mitochondrial electron transport in 12Z cells. This inhibition results in inefficient transfer of electrons to molecular oxygen resulting in increase in oxygen radicals in the cells which trigger DNA damage and cell death. Based on these studies, we propose that cytotoxic therapy using curcumin, plumbagin or atovaquone can serve as a novel strategy for the treatment of endometriosis. In addition to developing a novel therapeutic strategy for treatment of endometriosis, we also set the goal of developing a device to model the endometriosis tissue microenvironment. Conventional studies have either grown human endometriosis lesions under tissue culture conditions or have transplanted human tissues in rodents to study endometriosis. The in vitro culture model typically does not allow manipulations for selective monitoring of the various cell types from the endometriosis tissue microenvironment. The rodent models are also challenging because the xenograft experiments cannot be carried out in immunocompetent rodents. We therefore employed an approach that our team has successfully used to investigate tumor microenvironments. The microfluidic device that we have employed, tracks cell viability of endometriotic cells that line the lumen. In our experiments, the OXPHOS inhibitors, curcumin, plumbagin and atovaquone were introduced in the lumen and cellular assays were used to monitor their effects on the viability, migratory potential and the cellular metabolic status of the 12Z cells. The collagen matrix surrounding the lumen, mimics the extracellular matrix of the lesion. The prototype microfluidic device that we report in this study can be further modified to include stromal, immune and endothelial cells around the lumen to study the behavior of the aberrant endometriotic epithelial cells in response to other cellular and acellular components from the tissue microenvironment ( Ayuso, et al. 2021 , Lugo-Cintrón, et al. 2021 ). In our on-going work, we plan to use this device to seed endometriosis tissues from patients to develop a high throughput method to investigate novel therapies against this gynecologic disorder. We also demonstrated that the oxidative stress induced by plumbagin and atovaquone triggers a cascade of events that collectively contribute to cell death. A major observation we recently reported was that plumbagin-induced oxidative stress results in activation of the tumor suppressor, p53. Pifithrin-α, a p53 inhibitor, attenuates apoptosis in plumbagin-treated cells ( Kapur, et al. 2018 ). We have made similar observations with atovaquone ( Kapur, et al. 2022 ). Additionally, we have also demonstrated that plumbagin and atovaquone-induced oxidative stress results in downregulation of Na + /K + -ATPase ( Alharbi, et al. 2020 , Alharbi, et al. 2019 ). Consequently, the transport of Na + and K + across its plasma membrane makes the cell unable to maintain its membrane potential. Our result showing curcumin as an inducer of oxidative stress may seem to be inconsistent with the established observation that this molecule is an antioxidant ( Deguchi 2015 , Patel, et al. 2020 , Weber, et al. 2005 ). However, the two paradoxical results can be reconciled when the cellular responses of curcumin are considered on a temporal scale. In our experiments we monitor oxygen radical formation and OXPHOS inhibition within 15 min to 1 h after exposing the cells to curcumin. We have observed that the antioxidant effect is a delayed response by the cell to compensate and overcome the oxidative stress caused by curcumin. Similar observations were also made by our group when we tested plumbagin and atovaquone in cellular assays (Kapur et al unpublished observations). The cytotoxic effects of curcumin, plumbagin and atovaquone are the result of the oxidative stress induced by these agents in the cells. OXPHOS is a major metabolic pathway that is essential for energy production in normal tissues. Proliferating cells (cancer cells and activated immune cells, for example) show a significant shift in their metabolic pathways by relying on aerobic glycolysis, also known as the Warburg effect ( Potter, et al. 2016 , Schwartz, et al. 2017 ). Several studies have now confirmed that while proliferative cells employ the Warburg effect, their mitochondria continue to be functional ( Zong, et al. 2016 ). Therefore, targeting aberrantly proliferative cells such as those in endometriosis lesions with OXPHOS inhibitors is a valid strategy for development of novel therapeutics against this disorder. Endometriosis lesions experience chronic oxidative stress due to increased exposure to oxygen radicals ( Kajiyama, et al. 2019 ). While some oxidative stress can promote cell growth, the oxygen radicals can also be deleterious to the functioning of a cell. Based on the results presented in this study, we propose that when OXPHOS inhibitors such as atovaquone are administered, the endometriotic lesions will encounter an oxygen radical flux that is more harmful to the lesions and cannot be neutralized by the aberrant cells using endogenous antioxidant mechanisms (Nrf-2 signaling or via neutralization of the radicals by glutathione, catalase or superoxide dismutases. This oxygen radical flux in the endometriosis tissues will result in significant damage to DNA as we have demonstrated in Fig. 4B . Therefore, atovaquone and other OXPHOS inhibitors will serve as chemotoxic agents that eliminate the lesion. This discussion identifies two important factors that contribute to the efficacy of OXPHOS inhibitors as chemotoxic agents. First, the amount of oxygen radicals generated will depend on the concentration of the OXPHOS inhibitors reaching the lesions. At lower concentrations of the OXPHOS inhibitors, the oxidative stress generated in the endometriotic lesions may not be of sufficient amplitude to produce cell death. To the contrary, lower levels of oxygen radicals may induce a proliferative response in the endometriotic cells. The low amplitude of oxygen radicals created following treatment with lower concentration of the OXPHOS inhibitors will also trigger anti-oxidant mechanisms through increased expression and activity of Nrf-2, catalase, MnSOD, glutathione synthesizing enzymes and other enzymes that have been identified as vitagenes that are triggered by vitagens (small organic molecules that are not synthesized by humans and mammals but play essential biological roles in protecting against oxidative damage, for example acting as neuroprotectants) ( Cornelius, et al. 2013 , Trovato Salinaro, et al. 2014 ). Chemotoxicity of the OXPHOS inhibitors may be realized only at sufficiently high concentrations of the OXPHOS inhibitors when the oxygen radical flux is so severe that (a) the cellular damage caused is significant and (b) is beyond the cells capacity to neutralize via its endogenous antioxidant mechanisms. In other words, the OXPHOS inhibitors will likely exhibit a hormetic response, similar to that reported for curcumin and plumbagin in previous studies ( Concetta Scuto, et al. 2019 , Son, et al. 2010 ). Such hermetic responses may provide protection against the chemotoxic effects of the OXPHOS inhibitors up to a threshold concentration beyond which the endometriosis lesions will not be able to protect themselves from the deleterious effects of the inhibitors. Identifying the optimal dose of the OXPHOS inhibitors will therefore be important for efficient control of endometriosis. The approach we are proposing with the OXPHOS inhibitors is fundamentally distinct from what is currently used to manage endometriosis. Endometriotic lesions experience cyclical proliferative and quiescent states in response to the steroid hormones ( Lebovic, et al. 2000 ). In the follicular phase, high levels of estrogen induce proliferation in the lesions whereas high levels of progesterone in the luteal phase decreases cell proliferation. Therefore, the current standard of care for treatment of endometriosis is hormonal treatment. Progestins are used to counter the proliferative effects of estrogen on endometriotic lesions. In comparison, the OXPHOS inhibitors will induce direct chemotoxic effects in the endometriosis lesions and hence will be a curative rather than a maintenance strategy. With atovaquone already an FDA approved agent, it will be convenient to repurpose this drug to conduct a novel clinical trial for the treatment of endometriosis.

Endometriosis affects over 180 million reproductive age women worldwide and is a major cause for infertility and pelvic pain ( de Ziegler, et al. 2010 , Zondervan, et al. 2018 ). The negative impact of endometriosis results in loss of productivity in addition to the mental stress and physical pain for patients as well as their families ( Facchin, et al. 2015 ). Endometriosis is associated with a 3-fold higher risk for developing clear cell and endometrioid ovarian cancers ( Pearce, et al. 2012 , Ruderman and Pavone 2017 ) highlighting the need to develop novel therapeutic strategies to cure this benign disorder. Early studies conducted by Samson ( Sampson 1927 , Ulukus, et al. 2006 ) indicated that retrograde menstruation implants endometrial tissues on the reproductive and peritoneal organs in the abdominal cavity. In women of reproductive age, the proliferation of endometriotic lesions increases in response to high estrogen in the follicular phase whereas high progesterone in the luteal phase inhibits their growth. These observations have resulted in the development of progestins and GnRH antagonists (to block the estrogen surge) to manage endometriosis ( Vercellini, et al. 2016 , Vercellini, et al. 2014 ). However, these therapies are not curative and often cause hot flashes and other menopause-related symptoms. Additionally, treatment with progestins leads to blocking of fertility and hence are a barrier in women planning for pregnancies. Here, we investigate a novel approach to controlling endometriosis. In our prior work in ovarian cancer models, we demonstrated that agents such as plumbagin, atovaquone and curcumin (Kapur and Patankar et al, unpublished reports) induce oxidative stress and thereby induce apoptosis in cancer cells ( Alharbi, et al. 2020 , Alharbi, et al. 2019 , Kapur, et al. 2018 , Kapur, et al. 2016 , Liu, et al. 2012 , Nayak, et al. 2018 , Zeng, et al. 2015 ). These small molecule agents interfere with electron transport in the mitochondria and cause an increase in intracellular oxygen radical flux. The sudden increase in oxygen radicals damages DNA and other biomolecules triggering cell death. Based on these preliminary data, we hypothesized that curcumin, plumbagin and atovaquone may also be able to induce oxidative stress-mediated cell death in endometriosis and can serve as novel therapeutics for this benign reproductive disorder. The current study was undertaken to prove this hypothesis. Using an established cell line model for endometriosis, the 12Z cells, and a novel microfluidic device that has the potential to recapitulate the microenvironment of endometriosis lesions, we demonstrate chemotoxic effects of curcumin, plumbagin and atovaquone. We further propose that because atovaquone is already FDA-approved as a prophylactic for malaria, this drug is a prime candidate for repurposing for the treatment of endometriosis.