Stigmasterol Causes Ovarian Cancer Cell Apoptosis by Inducing Endoplasmic Reticulum and Mitochondrial Dysfunction

pharmaceutics

Article Stigmasterol Causes Ovarian Cancer Cell Apoptosis by Inducing Endoplasmic Reticulum and Mitochondrial Dysfunction

Hyocheol Bae 1 , Gwonhwa Song 1,* and Whasun Lim 2,* 1 Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea; [email protected] 2 Department of Food and Nutrition, College of Science and Technology, Kookmin University, Seoul 02707, Korea * Correspondence: [email protected] (G.S.); [email protected] (W.L.); Tel.: +82-2-3290-3012 (G.S.); +82-2-910-4773 (W.L.); Fax: +82-2-3290-4994 (G.S.) Received: 27 April 2020; Accepted: 25 May 2020; Published: 28 May 2020

Abstract: Background: Phytosterols have physiological effects and are used as medicines or food supplements. Stigmasterol has shown anticancer effects against various cancers such as hepatoma, cholangiocarcinoma, gall bladder carcinoma, endometrial adenocarcinoma and skin, gastric, breast, prostate, and cervical cancer. However, there are no reports on stigmasterol’s effects on ovarian cancer. Methods: We investigated the effects of stigmasterol on proapoptotic signals, mitochondrial function, reactive oxygen species production, and the cytosolic and mitochondrial calcium levels in human ovarian cancer cells, to understand the mechanisms underlying the effects of stigmasterol on ovarian cancer cells. We also conducted migration assay to confirm whether that stigmasterol inhibits ovarian cancer cell migration. Results: Stigmasterol inhibited development of human ovarian cancer cells. However, it induced cell apoptosis, ROS production, and calcium overload in ES2 and OV90 cells. In addition, stigmasterol stimulated cell death by activating the ER-mitochondrial axis. We confirmed that stigmasterol suppressed cell migration and angiogenesis genes in human ovarian cancer cells. Conclusions: Our findings suggest that stigmasterol can be used as a new treatment for ovarian cancer.

Keywords: stigmasterol; ovarian cancer; apoptosis; endoplasmic reticulum; mitochondria; migration

1. Introduction Over the years, phytosterols have received special attention because of their ability to lower cholesterol and prevent cardiovascular disease [1,2]. In addition, various pharmacological properties of phytosterols have been reported, including anti-inﬂammatory, antidiabetic, and anticancer properties [3,4]. Stigmasterol is a plant sterol and is a component of the cell membrane that is responsible for its function [5]. Stigmasterol is an unsaturated phytosterol and is found in many vegetables, including legumes, nuts, seeds, herbs, and edible oils. It is also found in unpasteurized milk; however, stigmasterol is weakened by mild heat; thus, it is destroyed during pasteurization at a low temperature [6]. Stigmasterol is used as an intermediate in the biosynthesis of various hormones, including progesterone, androgens, estrogens, and corticoids; it also serves as a precursor to vitamin D3 [4,7,8]. Constant consumption of stigmasterol can reduce blood LDL cholesterol by 8%–10%, which lowers the risk of cardiovascular disease [9]. Further, stigmasterol inhibits the accumulation of fatty acids [10]. In plant seeds, stigmasterol promotes cell proliferation, maturation, and germination [11]. Stigmasterol inhibits the survival of human umbilical vein endothelial cells (HUVECs) and iPSC-derived cardiomyocytes [12]. Notably, stigmasterol suppresses the development

Pharmaceutics 2020, 12, 488; doi:10.3390/pharmaceutics12060488 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 488 2 of 16 of various cancers, including hepatoma [13], cholangiocarcinoma [14], gall bladder carcinoma [15], skin cancer [16], gastric cancer [17], breast cancer [18,19], endometrial adenocarcinoma [18], prostate cancer [20], and cervical cancer [19]. Among gynecological cancers, ovarian cancer is the fifth-most common cause of death due to difficulty in its diagnosis, high mortality rate, and frequent relapses [21,22]. Therefore, there is a need for the development of new anticancer drugs. In this study, we investigated the effects of stigmasterol on ES2 and OV90 cells. First, we identified whether stigmasterol induces the activity of apoptosis-related factors in ovarian cancer cells. Next, the effect of stigmasterol on cell death, free radical production, and mitochondrial function of ovarian cancer cells was observed. We also investigated the inhibitory effect of stigmasterol on cell proliferation and development of ovarian cancer. The effects of stigmasterol on intracellular signaling pathways were also studied.

2. Materials and Methods

2.1. Reagents Stigmasterol (cat number: S2424) was purchased from Sigma-Aldrich (St Louis, MO, USA). It was diluted in dimethyl sulfoxide (DMSO) before treatment.

2.2. Cell Culture ES2 and OV90 cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). ES2 (type—clear cell carcinoma) and OV90 (type—serous adenocarcinoma) cell lines were grown in McCoy’s 5A medium supplemented with 10% fetal bovine albumin (FBS). Cells were incubated at 37 ◦C with 5% carbon dioxide (CO2). To conﬁrm the eﬀects of stigmasterol, all cells, including those in the control group, were starved with serum-free medium for 24 h prior to treatment.

2.3. Western Blotting The antibodies used in the Western blotting analyses are listed in Table1. The ovarian cancer cells were cultured on cell culture dishes and incubated with stigmasterol (0, 5, 10, and 20 µg/mL) for 24 h. The cells were rinsed twice with PBS to remove the treatment medium before protein extraction. The cells were lysed, and protein concentration was calculated via the Bradford assay. Equal amounts of each protein were mixed with loading buﬀer. The protein loading mixture was denatured at 95 ◦C for 5 min using a heating block. Denatured proteins were separated based on size using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred onto nitrocellulose membranes for immunoblotting. The membranes were sequentially incubated with primary and secondary antibodies, and chemiluminescence was detected using the ChemiDoc EQ system (Bio-Rad, Hercules, CA, USA).

Table 1. List of antibodies.

Primary Antibodies Dilution Supplier Catalog Number Cleaved caspase-3 1:1000 Cell Signaling 9664 Cleaved caspase-9 1:1000 Cell Signaling 9501 Cytochrome c 1:1000 Cell Signaling 11940 BAK 1:1000 Cell Signaling 12105 BAX 1:1000 Cell Signaling 2774 Phospho-PERK (Thr981) 1:1000 Santa Cruz sc-32577 Phospho-eIF2α (Ser51) 1:1000 Cell Signaling 3398 IRE1α 1:1000 Cell Signaling 3294 Pharmaceutics 2020, 12, 488 3 of 16

Table 1. Cont.

Primary Antibodies Dilution Supplier Catalog Number GADD153 1:1000 Santa Cruz sc-7351 ATF6α 1:1000 Santa Cruz sc-166659 GRP78 1:1000 Santa Cruz sc-13968 TUBA 1:1000 Santa Cruz sc-5286 VDAC 1:1000 Cell Signaling 4661 IP3R1 1:1000 Invitrogen PA1-901 IP3R2 1:1000 Santacruz sc-398434 VAPB 1:1000 Invitrogen PA5-53023 FAM82A2 1:1000 Abcam ab182105 Phospho-ULK1 (SER555) 1:1000 Cell Signaling 5869 ULK1 1:1000 Cell Signaling 8054 BECN1 1:1000 Cell Signaling 3495 ATG5 1:1000 Cell Signaling 12994 Phospho-AKT (SER473) 1:1000 Cell Signaling 4060 AKT 1:1000 Cell Signaling 9272 Phospho-P70S6K 1:1000 Cell Signaling 9204 (Thr421/Ser424) P70S6K 1:1000 Cell Signaling 9202 Phospho-S6 (Ser235/236) 1:1000 Cell Signaling 2211 S6 1:1000 Cell Signaling 2217 Phospho-ERK1/2(Thr202/Tyr204) 1:1000 Cell Signaling 9101 ERK1/2 1:1000 Cell Signaling 4695 Phospho-JNK (Thr183/Tyr185) 1:1000 Cell Signaling 4668 JNK 1:1000 Cell Signaling 9252 Phospho-P38 (Thr180/Tyr182) 1:1000 Cell Signaling 4511 P38 1:1000 Cell Signaling 9212

2.4. Spheroid Assay ES2 and OV90 cells were dropped onto the cover of a culture dish (3 103 cells). Vehicle-treated × and stigmasterol-treated cells were cultured for 72 h. Cancer morphology was observed with a DM3000 microscope (Leica, Wetzlar, Germany). Tumor area was calculated using ImageJ software (http://rsb.info.nih.gov/ij/docs/index.html). 3D density was estimated using ReViSP software (https: //sourceforge.net/projects/revisp/).

2.5. Annexin V and PI Staining ES2 and OV90 cells grown to 50% conﬂuence in 6-well plates. FBS-starved cells were incubated with stigmasterol (0, 5, 10, and 20 µg/mL) for 48 h. The cells were washed twice with PBS before being treated with 0.25% trypsin-EDTA. The suspended cells were collected via centrifugation and washed twice using PBS to remove the 0.25% trypsin-EDTA. The collected cells were stained with FITC Annexin V (5 µL) and propidium iodide (PI; 5 µL) for 15 min. Fluorescence was detected using a ﬂow cytometer (BD Bioscience, Franklin Lakes, NJ, USA). All experimental results were estimated by comparing them to vehicle-treated controls. Pharmaceutics 2020, 12, 488 4 of 16

2.6. Cell Cycle Assay ES2 and OV90 cells were grown to 50% conﬂuence in 6-well plates. FBS-starved cells were incubated with stigmasterol (0, 5, 10, and 20 µg/mL) for 48 h. The cells were washed twice to remove the stigmasterol before 0.25% trypsin-EDTA treatment. The suspended cells were collected via centrifugation and washed twice using PBS to remove the remaining 0.25% trypsin-EDTA. The collected cells were incubated with RNase A and PI for 30 min. Fluorescence was detected using a ﬂow cytometer (BD Bioscience). All experimental results were estimated by comparing them to those of the vehicle-treated control cells.

2.7. JC-1 Staining ES2 and OV90 cells were grown to 50% confluence in 6-well plates. FBS-starved cells were incubated with stigmasterol (0, 5, 10, and 20 µg/mL) for 48 h. The cells were washed twice to remove the stigmasterol before trypsinization. The suspended cells were collected via centrifugation and washed twice with PBS to remove the 0.25% trypsin-EDTA. The cells were then incubated with JC-1 (Sigma-Aldrich, St Louis, MO, USA) for 20 min at 37 ◦C. JC-1-stained cells were washed twice using 1 JC-1 staining buffer. JC-1 staining was observed using a flow cytometer (BD Bioscience). × All experiments results were calculated by comparing them to those of the cells from the control group.

2.8. Measurement of Reactive Oxygen Species (ROS) Production ES2 and OV90 cells were grown in 6-well plates. The cells were detached with 0.25% trypsin-EDTA, and the suspended cells were collected via centrifugation and rinsed twice with PBS. The cells were treated with 10 µM 20,70-dichloroﬂuorescein diacetate (DCFH-DA; Sigma) for 30 min. The stained cells were washed twice with PBS and treated with stigmasterol (0, 5, 10, and 20 µg/mL) for 1 h. After stigmasterol treatment, the cells were rinsed twice with PBS. ROS production was detected using a ﬂow cytometer (BD Bioscience).

2.9. Cytosolic Ca2+ Level Analysis ES2 and OV90 cells were grown to 50% confluence in 6-well plates. FBS-starved cells were incubated with stigmasterol (0, 5, 10, and 20 µg/mL) for 48 h. The cells were washed twice to remove the stigmasterol before 0.25% trypsin-EDTA was added to trypsinize the cells. The suspended cells were collected via centrifugation and washed twice with PBS. The cells were incubated with 3 µM fluo-4 acetoxymethyl ester (AM; Invitrogen) for 20 min at 37 ◦C. The stained cells were rinsed twice with PBS. Fluo-4 intensity was detected using a flow cytometer (BD Bioscience). All experimental results were calculated by comparing them to those of the cells from the control group.

2.10. Mitochondrial Ca2+ Level Analysis ES2 and OV90 cells were grown to 50% confluence in 6-well plates. FBS-starved cells were incubated with stigmasterol (0, 5, 10, and 20 µg/mL) for 48 h. The cells were washed twice to remove the stigmasterol before adding 0.25% trypsin-EDTA. The suspended cells were collected via centrifugation and washed twice with PBS to remove any remaining 0.25% trypsin-EDTA. The collected cells were incubated with Rhod-2 for 20 min at 37 ◦C and subsequently washed twice using PBS. Rhod-2 fluorescence was detected using a flow cytometer (BD Bioscience). All experimental results were calculated by comparing them to those of the cells from the control group.

2.11. Proliferation Assay The proliferation of ovarian cancer cells was observed using ELISA, a BrdU kit (Roche, Basel, Switzerland), and a microplate reader (BioTek, Winooski, VT, USA). Cells at 50% conﬂuence were treated with various concentrations of stigmasterol in a 96-well plate for 48 h. The cells were then incubated with BrdU for an additional 2 h. BrdU was labeled with anti-BrdU-POD for 90 min. Pharmaceutics 2020, 12, 488 5 of 16

The samples in each well were washed three times with PBS, and a color reaction was induced using a chromogenic substrate. All experiments were calculated by comparison of the results to those of the cells from the control group.

2.12. Migration Assay Cell migration was observed using a Transwell migration assay. Ovarian cancer cells were seeded on Transwell inserts and incubated for 12 h with stigmasterol or vehicle. The Transwell membranes were fixed with methanol for 10 min and stained with hematoxylin for 30 min. The membranes were washed and covered using mounting media. Cell migration was observed with a DM3000 microscope (Leica).

2.13. Quantitative Real-Time PCR Gene expression was measured using quantitative RT-PCR using SYBR green, as previously described [23] (see in Table2).

Table 2. List of primers.

Primer Sequence Size Forward 5 -TTGTACAAGATCCGCAGACG-3 VEGFA 0 0 100 bp Reverse 50-TCACATCTGCAAGTACGTTCG-30 Forward 5 -CAACTGCCCAAAGAAATTCG-3 PLAU 0 0 148 bp Reverse 50-AAGGACAGTGGCAGAGTTCC-30 Forward 5 -GGCATTCAGGAGCTCTATGG-3 MMP2 0 0 137 bp Reverse 50-ATCTCACCACGGATCTGAGC-30 Forward 5 -TTGACAGCGACAAGAAGTGG-3 MMP9 0 0 145 bp Reverse 50-TCAGTGAAGCGGTACATAGGG-30 Forward 5 -GCAGAAGTTTTACGGCTTGC-3 MMP14 0 0 117 bp Reverse 50-ACATTGGCCTTGATCTCAGC-30

2.14. Statistical Analysis The significance of all experiments was identified through the appropriate error terms according to the expectation of the mean square of the error. P-values less than 0.05 were considered significant. Data are shown as the least-square means (LSMs) with standard errors [24]. (*** = p < 0.001, ** = p < 0.01, and * = p < 0.05)

3. Results

3.1. Induction of Cell Apoptosis and Inhibition of Cell Aggregation by Stigmasterol in ES2 and OV90 Cells Western blotting showed that stigmasterol activated proapoptotic signals in ES2 and OV90 cells. Stigmasterol (0, 5, 10, and 20 µg/mL) stimulated cleavage of caspase 3 and caspase 9 in a dose-dependent manner in each cell type. In addition, stigmasterol activated cytochrome c, BAK, and BAX in both cell types. The levels of alpha-tubulin (TUBA) did not show changes following stigmasterol treatment (Figure1A,B). Stigmasterol increased the tumor area by 150.9% and 146.9% in the case of ES2 and OV90 cells, respectively. However, tumor volume was reduced by 72.8% and 60.1% in ES2 and OV90 cells, respectively, by administration of stigmasterol (20 µg/mL). We identiﬁed that ovarian cancer cells cannot aggregate in the presence of stigmasterol. In the vehicle-treated control, the 3D volume of ovarian cancer cells increased, but the 2D area decreased because of cell aggregation. However, as ovarian cancer cells did not aggregate in response to stigmasterol treatment, the 3D volume decreased, and the cells spread laterally to increase the 2D area (Figure1C,D). We investigated the cell states to conﬁrm the occurrence of programmed cell apoptosis by stigmasterol based on the dot Pharmaceutics 2020, 12, 488 6 of 16

population of the upper-right quadrant. In the case of ES2 cells, the proportion of cells showing late apoptosis was increased by 1.9%, 7.8%, and 29.8% following treatment with 5, 10, and 20 µg/mL of stigmasterol, respectively, compared to the vehicle-treated controls, which showed a 1.1% increase in the proportion of cells showing late apoptosis (Figure1E). In the case of OV90 cells, the proportion of cells in the upper-right quadrant was increased by 0.6%, 2.5%, and 8.5% following treatments with 5, 10, and 20 µg/mL of stigmasterol, respectively, compared to the vehicle-treated control, which showed a 0.1% increase in the proportion of cells in the upper-right quadrant (Figure1F). In the evaluation of cell cycle progression, in the case of ES2 cells, the proportion of cells in the subG1 phase was increased by 11.1% following treatment with 20 µg/mL of stigmasterol (vehicle-treated controls showed a 0.8% increase) (Figure1G). In the case of OV90 cells, the proportion of cells in the subG1 phase increased by 5.6% following treatment with 20 µg/mL of stigmasterol (vehicle-treated controls showed a 0.8% increase)Pharmaceutics (Figure 20201H)., 12, x FOR PEER REVIEW 6 of 17

184 Figure 1. Stigmasterol aﬀects ovarian cancer cell apoptosis and tumor formation in ES2 and OV90 185 Figure 1. Stigmasterol affects ovarian cancer cell apoptosis and tumor formation in ES2 and OV90 186 cells.cells. (A,B (A,B)) Western Western blot blot bands bands showed showed the the expression expression of of proapoptotic proapoptotic signalingsignaling molecules in in both both cell 187 typescell following types following stigmasterol stigmasterol treatments treatments (0, 5, (0, 10, 5, 10, and and 20 20µg μ/mL).g/mL). Alpha-tubulin Alpha-tubulin (TUBA) (TUBA) was was used used as 188 a control.as a control. (C,D) Spheroid(C,D) Spheroid formation formation of ovarian of ovarian cancer cancer cells ce waslls was compared compared between between vehicle-treated vehicle-treated cells 189 and stigmasterol-treatedcells and stigmasterol-treated cells. (E ,cells.F) Annexin (E,F) Annexin V and propidiumV and propidium iodide (PI)iodide staining (PI) staining were performedwere 190 to determineperformed cell to determine death in ES2cell death and OV90in ES2 cells.and OV90 The ce quadrantlls. The quadrant of the dotof the blot dot representsblot represents the the state of 191 apoptosisstate of in apoptosis ES2 and in OV90 ES2 and cells. OV90 The cells. comparative The comparative graph graph represents represents changes changes in in late late apoptosisapoptosis due 192 to stigmasteroldue to stigmasterol treatment treatment (0, 5, 10,(0, 5, and 10, 20andµ 20g/mL) μg/mL) compared compared to to the the vehicle-treated vehicle-treated control control (100%) (100%) in 193 ES2 andin ES2 OV90 and cells.OV90 (cells.G,H )(G,H) Histogram Histogram presents presents cell cell cycle cycle progression progressionin in stigmasterol-treatedstigmasterol-treated (0, (0, 5, 5, 10, 194 10, and 20 μg/mL) ovarian cancer cells. Comparative graph represents the % of cells in the subG1, and 20 µg/mL) ovarian cancer cells. Comparative graph represents the % of cells in the subG1, G0/G1, 195 G0/G1, S, and G2/M phases in stigmasterol-treated (0, 5, 10, and 20 μg/mL) ovarian cancer cells. S, and G2/M phases in stigmasterol-treated (0, 5, 10, and 20 µg/mL) ovarian cancer cells. 196 3.2. Changes in Mitochondrial Function and ROS Levels by Stigmasterol in ES2 and OV90 Cells 197 Mitochondrial function was dramatically altered by stigmasterol (0, 5, 10, and 20 μg/mL) in ES2 198 cells. Mitochondrial depolarization was increased to 700%, 1433%, and 3100% by 5, 10, and 20 μg/mL 199 of stigmasterol, respectively, compared to that in the case of the vehicle-treated control (100%) in ES2 200 cells (Figure 2A). Similarly, the mitochondrial depolarization in OV90 cells was increased to 292%, 201 400%, and 492% by 5, 10, and 20 μg/mL of stigmasterol, respectively, compared to that in the case of 202 the vehicle-treated control (100%) (Figure 2B). ROS production in ES2 cells was dose-dependently 203 increased by 5.5%, 6.9%, and 10.4% by 5, 10, and 20 μg/mL of stigmasterol, respectively, compared to 204 that in the case of the vehicle-treated control (5.1%) (Figure 2C). ROS generation in OV90 cells also 205 increased by 3.8% and 4.1% by 10 and 20 μg/mL of stigmasterol, respectively, compared to that in the 206 case of the vehicle-treated control (2.8%) (Figure 2D).

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3.2. Changes in Mitochondrial Function and ROS Levels by Stigmasterol in ES2 and OV90 Cells Mitochondrial function was dramatically altered by stigmasterol (0, 5, 10, and 20 µg/mL) in ES2 cells. Mitochondrial depolarization was increased to 700%, 1433%, and 3100% by 5, 10, and 20 µg/mL of stigmasterol, respectively, compared to that in the case of the vehicle-treated control (100%) in ES2 cells (Figure2A). Similarly, the mitochondrial depolarization in OV90 cells was increased to 292%, 400%, and 492% by 5, 10, and 20 µg/mL of stigmasterol, respectively, compared to that in the case of the vehicle-treated control (100%) (Figure2B). ROS production in ES2 cells was dose-dependently increased by 5.5%, 6.9%, and 10.4% by 5, 10, and 20 µg/mL of stigmasterol, respectively, compared to that in the case of the vehicle-treated control (5.1%) (Figure2C). ROS generation in OV90 cells also increased by 3.8% and 4.1% by 10 and 20 µg/mL of stigmasterol, respectively, compared to that in the Pharmaceuticscase of the 2020 vehicle-treated, 12, x FOR PEER control REVIEW (2.8%) (Figure2D). 7 of 17

207 208 FigureFigure 2. 2.AlterationAlteration of mitochondrial of mitochondrial membrane membrane potential potential and ROS and generation ROS generation by stigmasterol by stigmasterol in both 209 cells.in both (A,B) cells. JC-1 (dyeA,B )was JC-1 used dye to was investigate used to the investigate alteration the of alterationmitochondrial of mitochondrial membrane potential membrane by 210 stigmasterolpotential by (0, stigmasterol 5, 10, and 20 (0, μg/mL). 5, 10, andQuadrant 20 µg /ofmL). the Quadrantdot blot represented of the dot the blot state represented of mitochondrial the state 211 membraneof mitochondrial potential membrane in ES2 potentialand OV90 in cells. ES2 andComparative OV90 cells. graph Comparative represented graph the represented loss of the the 212 mitochondrialloss of the mitochondrial membrane potential membrane compared potential to comparedthe control to group the control (100%) group in ES2 (100%) and OV90 in ES2 cells. and 213 (C,D)OV90 DCF cells. fluorescence (C,D) DCF ﬂuorescenceintensity was intensity observed was to observedinvestigate to the investigate change by the stigmasterol change by stigmasterol treatment 214 (0,treatment 5, 10, and (0, 20 5, μ 10,g/mL). and The 20 µ histogramg/mL). The represented histogram representedthe state of ROS the stateproduction of ROS in production ES2 (Figure in 2C, ES2 215 left)(Figure and2 OV90C, left) (Figure and OV90 2D, left) (Figure cells.2D, Comparative left) cells. Comparative graph represen graphted ROS represented production ROS compared production to 216 vehicle-treatedcompared to vehicle-treated control (100%) control in ES2 (100%) (Figure in ES22C, (Figureright) 2andC, right) OV90 and (Figure OV90 2D (Figure, right)2D, cells. right) DCF: cells. 217 dichlorofluorescein.DCF: dichloroﬂuorescein.

218 3.3. Upregulation of Cytosolic and Mitochondrial Calcium Levels by Stigmasterol in ES2 and OV90 Cells 219 To determine the regulation of calcium concentrations in the cytosol and mitochondria, we 220 incubated ES2 and OV90 cells with 0, 5, 10, and 20 μg/mL of stigmasterol for 48 h. After this, the 221 calcium levels of the cytosol and mitochondria were observed using Fluo-4-AM and Rhod-2 staining, 222 respectively. The cytosolic calcium level of ES2 cells was increased by 4.1%, 5.8%, and 25.2% by 5, 10, 223 and 20 μg/mL of stigmasterol, respectively, compared to that in the case of the vehicle-treated 224 controls (3.9%) (Figure 3A). The cytosolic calcium level in OV90 cells was increased by 3.6%, 5.8%, 225 and 6.6% by 5, 10, and 20 μg/mL of stigmasterol, respectively, compared to that in the case of the 226 vehicle-treated control (3.5%) (Figure 3B). In addition, the mitochondrial calcium levels in ES2 cells 227 were increased by 1.5%, 3.5%, and 4.0% by 5, 10, and 20 μg/mL of stigmasterol, respectively, 228 compared to that in the case of the vehicle-treated control (1.1%) (Figure 3C). In OV90 cells, the 229 mitochondrial calcium was upregulated by 5.8%, 8.9%, and 13.2% by 5, 10, and 20 μg/mL of 230 stigmasterol, respectively, compared to that in the case of the vehicle-treated control (5.0%) (Figure 231 3D). 232

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3.3. Upregulation of Cytosolic and Mitochondrial Calcium Levels by Stigmasterol in ES2 and OV90 Cells To determine the regulation of calcium concentrations in the cytosol and mitochondria, we incubated ES2 and OV90 cells with 0, 5, 10, and 20 µg/mL of stigmasterol for 48 h. After this, the calcium levels of the cytosol and mitochondria were observed using Fluo-4-AM and Rhod-2 staining, respectively. The cytosolic calcium level of ES2 cells was increased by 4.1%, 5.8%, and 25.2% by 5, 10, and 20 µg/mL of stigmasterol, respectively, compared to that in the case of the vehicle-treated controls (3.9%) (Figure3A). The cytosolic calcium level in OV90 cells was increased by 3.6%, 5.8%, and 6.6% by 5, 10, and 20 µg/mL of stigmasterol, respectively, compared to that in the case of the vehicle-treated control (3.5%) (Figure3B). In addition, the mitochondrial calcium levels in ES2 cells were increased by 1.5%, 3.5%, and 4.0% by 5, 10, and 20 µg/mL of stigmasterol, respectively, compared to that in the case of the vehicle-treated control (1.1%) (Figure3C). In OV90 cells, the mitochondrial calcium was upregulated by 5.8%, 8.9%, and 13.2% by 5, 10, and 20 µg/mL of stigmasterol, respectively, comparedPharmaceutics to that 2020 in, 12 the, x FOR case PEER of the REVIEW vehicle-treated control (5.0%) (Figure3D). 8 of 17

Figure 3. Calcium concentrations in the cytosol and mitochondria in stigmasterol-treated ovarian cancer 233 cells. (A,B) Fluo-4 dye was used to determine the intracellular calcium levels. The histogram represents 234 the changeFigure of 3. cytosolicCalcium concentrations calcium levels in following the cytosol stigmasterol and mitochondria treatments in stigmasterol-treated (0, 5, 10, and 20 ovarianµg/mL) in 235 cancer cells. (A,B) Fluo-4 dye was used to determine the intracellular calcium levels. The histogram ES2 (Figure3A, left) and OV90 (Figure3B, left) cells. The comparative graph represents changes 236 represents the change of cytosolic calcium levels following stigmasterol treatments (0, 5, 10, and 20 in intracellular calcium levels following stigmasterol treatments compared to the vehicle-treated 237 μg/mL) in ES2 (Figure 3A, left) and OV90 (Figure 3B, left) cells. The comparative graph represents 238 controlschanges (100%) in inintracellular ES2 (Figure calcium3A, right) levels and following OV90 (Figurestigmasterol3B, right) treatments cells. (comparedC,D) Rhod-2 to the ﬂuorescence vehicle- 239 was usedtreated to determinecontrols (100%) the mitochondrialin ES2 (Figure 3A, calcium right) levels and OV90 in stigmasterol-treated (Figure 3B, right) cells. ovarian (C,D)cancer Rhod-2 cells. 240 The histogramfluorescence represents was used changes to determine in mitochondrial the mitochondria calciuml calcium levels levels following in stigmasterol-treated stigmasterol treatments ovarian (0, 241 5, 10, andcancer 20 cells.µg/mL) The histogram in ES2 (Figure represents3C, left) changes and in OV90 mitochondrial (Figure3D, calcium left) cells.levels Thefollowing comparative stigmasterol graph 242 representstreatments changes (0, in5, 10, mitochondrial and 20 μg/mL) calcium in ES2 levels (Figure following 3C, left) stigmasterol and OV90 (Figure treatments 3D, left) compared cells. The to the 243 vehicle-treatedcomparative control graph (100%) represents in ES2 changes (Figure in3C, mitoch right)ondrial and OV90 calcium (Figure levels3D, following right) cells. stigmasterol 244 treatments compared to the vehicle-treated control (100%) in ES2 (Figure 3C, right) and OV90 (Figure 245 3D, right) cells.

246 3.4. Regulation of Endoplasmic Reticulum Stress, the Endoplasmic Reticulum-Mitochondria Axis, and 247 Autophagy by Stigmasterol in ES2 and OV90 cells 248 Endoplasmic reticulum (ER) stress and the ER-mitochondria axis were investigated using 249 Western blotting analysis. Activation of representative unfolded protein response (UPR) proteins was 250 observed. Phosphorylated PKR-like ER resident kinase (p-PERK), phosphorylated eukaryotic 251 translation-initiation factor 2α (p-eIF2α), inositol-requiring enzyme 1α (IRE1α), growth arrest and 252 DNA damage-induced-153 (GADD153), activating transcription factor 6α (ATF6α), and glucose- 253 regulated protein 78 (GRP78) were activated by stigmasterol (0, 5, 10, and 20 μg/mL) treatments, 254 compared to the case for TUBA, in ES2 and OV90 cells. p-PERK level increased four point three-fold 255 and two point six-fold in response to stigmasterol (20 μg/mL) treatment relative to that in vehicle-

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3.4. Regulation of Endoplasmic Reticulum Stress, the Endoplasmic Reticulum-Mitochondria Axis, and Autophagy by Stigmasterol in ES2 and OV90 Cells Endoplasmic reticulum (ER) stress and the ER-mitochondria axis were investigated using Western blotting analysis. Activation of representative unfolded protein response (UPR) proteins was observed. Phosphorylated PKR-like ER resident kinase (p-PERK), phosphorylated eukaryotic translation-initiation factor 2α (p-eIF2α), inositol-requiring enzyme 1α (IRE1α), growth arrest and DNA damage-induced-153 (GADD153), activating transcription factor 6α (ATF6α), and glucose-regulated protein 78 (GRP78) were activated by stigmasterol (0, 5, 10, and 20 µg/mL) treatments, compared to the case for TUBA, in ES2 and OV90 cells. p-PERK level increased four point three-fold and two point six-fold in response to stigmasterol (20 µg/mL) treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively. p-eIF2α level increased six-fold and four point six-fold in response to stigmasterol treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively. IRE1α levels increased nine point nine-fold and seven point eight-fold in response to stigmasterol treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively. GADD153 expression increased three point nine-fold and six point two-fold in response to stigmasterol treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively. ATF6α expression increased three point six-fold and three point seven-fold in response to stigmasterol treatment relative to that in controls in ES2 and OV90 cells, respectively. GRP78 expression was upregulated two-fold and three-fold in response to stigmasterol treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively (Figure4A,B). In addition, voltage-dependent anion channel (VDAC), inositol 1,4,5-triphosphate receptor 1 (IP3R1), IP3R2, vesicle-associated membrane protein-associated protein B/C (VAPB), family with sequence similarity 82, member a2 (FAM82A2), beclin-1 (BECN1), phosphorylated UNC-51-like kinase 1 (p-ULK1), and autophagy-related 5 (ATG5) were activated in a dose-dependent manner by stigmasterol (0, 5, 10, and 20 µg/mL), compared to the case for TUBA, in ES2 and OV90 cells. VDAC expression increased two point six-fold and three point seven-fold in response to stigmasterol (20 µg/mL) treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively. IP3RI expression was upregulated three point seven-fold and three-fold upon stigmasterol treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively. IP3R2 expression increased three point eight-fold and one point six-fold upon stigmasterol treatment in ES2 and OV90 cells, respectively. VAPB expression increased three point seven-fold and one point eight-fold upon stigmasterol treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively. FAM82A2 expression increased ﬁve point two-fold and three point four-fold upon stigmasterol treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively. p-ULK1 level increased two point four-fold and two point eight-fold upon stigmasterol treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively. BECN1 was upregulated four point two-fold and three point four-fold upon stigmasterol treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively. ATG5 expression increased two point four-fold and two point three-fold upon stigmasterol treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively (Figure4C–F).

3.5. Restriction of Growth and Inactivation of Intracellular Signals by Stigmasterol in ES2 and OV90 Cells Stigmasterol inhibited cell growth in both ES2 and OV90 cells. The cell growth of ES2 cells was decreased by up to 50.0% when treated with 20 µg/mL of stigmasterol (Figure5A). The cell proliferation of OV90 cells was restricted by up to 54.3% when treated with 20 µg/mL of stigmasterol (Figure5B). Additionally, the phosphorylation of AKT, P70S6K, S6, ERK1 /2, JNK, and P38 was inhibited by stigmasterol in a dose-dependent manner (Figure5C,D). Next, we used inhibitors to verify the correlation of stigmasterol with the intracellular signaling mechanism. We used the PI3K inhibitor (LY294002; 20 µM), ERK1/2 inhibitor (U0126; 20 µM), JNK inhibitor (SP600125; 20 µM), and P38 inhibitor (SB203580; 20 µM) with stigmasterol. ES2 and OV90 cells were treated with these inhibitors prior to stigmasterol treatment (Figure6). Phosphorylation of AKT in ES2 cells was almost completely Pharmaceutics 2020, 12, x FOR PEER REVIEW 9 of 17

256 treated controls in ES2 and OV90 cells, respectively. p-eIF2α level increased six-fold and four point 257 six-fold in response to stigmasterol treatment relative to that in vehicle-treated controls in ES2 and 258 OV90 cells, respectively. IRE1α levels increased nine point nine-fold and seven point eight-fold in 259 response to stigmasterol treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, 260 respectively. GADD153 expression increased three point nine-fold and six point two-fold in response 261 to stigmasterol treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, 262 respectively. ATF6α expression increased three point six-fold and three point seven-fold in response 263 to stigmasterol treatment relative to that in controls in ES2 and OV90 cells, respectively. GRP78 264 expression was upregulated two-fold and three-fold in response to stigmasterol treatment relative to 265 that in vehicle-treated controls in ES2 and OV90 cells, respectively (Figure 4A,B). In addition, voltage- 266 dependent anion channel (VDAC), inositol 1,4,5-triphosphate receptor 1 (IP3R1), IP3R2, vesicle- 267 associated membrane protein-associated protein B/C (VAPB), family with sequence similarity 82, 268 member a2 (FAM82A2), beclin-1 (BECN1), phosphorylated UNC-51-like kinase 1 (p-ULK1), and 269 autophagy-related 5 (ATG5) were activated in a dose-dependent manner by stigmasterol (0, 5, 10, 270 and 20 μg/mL), compared to the case for TUBA, in ES2 and OV90 cells. VDAC expression increased 271 two point six-fold and three point seven-fold in response to stigmasterol (20 μg/mL) treatment 272 relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively. IP3RI expression was 273 upregulated three point seven-fold and three-fold upon stigmasterol treatment relative to that in 274 vehicle-treated controls in ES2 and OV90 cells, respectively. IP3R2 expression increased three point 275Pharmaceutics eight-fold2020 and, 12, 488one point six-fold upon stigmasterol treatment in ES2 and OV90 cells, respectively.10 of 16 276 VAPB expression increased three point seven-fold and one point eight-fold upon stigmasterol 277 treatment relative to that in vehicle-treated controls in ES2 and OV90 cells, respectively. FAM82A2 278inhibited expression by all increased these inhibitors five point but two-fold was only and inhibitedthree point by four-fold LY294002 upon and stigmasterol SB203580 intreatment OV90 cells. 279The phosphorylationrelative to that in vehicle-treated of P70S6K was controls almost in completely ES2 and OV90 inactivated cells, respectively. by LY294002, p-ULK1 U0126, level increased and SB203580 280in ES2two cells point and four-fold by LY294002, and two SP600125, point eight-fold and SB203580 upon stigmasterol in OV90 cells. treatment The phosphorylation relative to that in ofvehicle- S6 protein 281was almosttreated completelycontrols in ES2 blocked and OV90 by all cells, inhibitors respective exceptly. BECN1 U0126 was in upregulated ES2 cells and four was point completely two-fold and blocked 282by allthree inhibitors point four-fold except upon SP600125 stigmasterol in OV90 treatment cells. Therelative phosphorylation to that in vehicle-treated of ERK1 controls/2 was completelyin ES2 283blocked and byOV90 U0126 cells, and respectively. SB203580 ATG5 in both expression cell types increased and only two slightly point four-fold blocked byand LY294002 two point in three- ES2 cells. 284JNK phosphorylationfold upon stigmasterol was treatment completely relative blocked to that by allin vehicle-treated inhibitors in both controls cells. in TheES2 and phosphorylation OV90 cells, of 285 P38 wasrespectively completely (Figure blocked 4C–F). by all inhibitors except SP600125 in both cell lines. 286

287 Figure 4. Increased expression of endoplasmic reticulum (ER) stress sensor proteins and 288 ER-mitochondrialFigure 4. Increased axis proteins expression due toof stigmasterol endoplasmic treatment reticulum in (ER) ovarian stress cancer sensor cells. proteins (A,B) Immunoblotsand ER- 289 representingmitochondrial the expressionaxis proteins of due unfolded to stigmast proteinerol treatment response in ovarian (UPR) cancer proteins cells. following (A,B) Immunoblots stigmasterol

treatments (0, 5, 10, and 20 µg/mL). TUBA was used as a control and is shown at the bottom of the figure. (C,D) Immunoblots representing the expression of ER-mitochondrial axis proteins following stigmasterol treatments (0, 5, 10, and 20 µg/mL). TUBA was used as a control and is shown at the bottom of the figure. (E,F) Immunoblots representing the expression of autophagy proteins following stigmasterol treatment. TUBA was used as a control and is shown at the bottom of the figure.

3.6. Calcium Regulation by Stigmasterol in the Death of ES2 and OV90 Cells Ruthenium red, an inhibitor of calcium overload, inhibited cell apoptosis induced by stigmasterol in ES2 and OV90 cells. Additional treatment of ruthenium red to stigmasterol decreased cell apoptosis from 9.3% (stigmasterol alone) to 3.6% and from 7.7% to 3.4% in ES2 and OV90 cells, respectively (Figure7A,B). Further, the stigmasterol-increased mitochondrial depolarization was decreased from 7.0% to 2.1% and from 5.9% to 2.6% in ES2 and OV90 cells, respectively, following cotreatment with stigmasterol and ruthenium red (Figure7C,D). Mitochondrial calcium levels were increased by stigmasterol by up to 8.9% and 6.9% in ES2 and OV90 cells, respectively. However, treatment with stigmasterol and ruthenium red reduced the mitochondrial calcium from 8.9% to 3.8% and from 6.9% to 3.7% in ES2 and OV90 cells, respectively (Figure7E,F). Pharmaceutics 2020, 12, x FOR PEER REVIEW 10 of 17

290 representing the expression of unfolded protein response (UPR) proteins following stigmasterol 291 treatments (0, 5, 10, and 20 μg/mL). TUBA was used as a control and is shown at the bottom of the 292 figure. (C,D) Immunoblots representing the expression of ER-mitochondrial axis proteins following 293 stigmasterol treatments (0, 5, 10, and 20 μg/mL). TUBA was used as a control and is shown at the 294 bottom of the figure. (E,F) Immunoblots representing the expression of autophagy proteins following 295 stigmasterol treatment. TUBA was used as a control and is shown at the bottom of the figure.

296 3.5. Restriction of Growth and Inactivation of Intracellular Signals by Stigmasterol in ES2 and OV90 Cells 297 Stigmasterol inhibited cell growth in both ES2 and OV90 cells. The cell growth of ES2 cells was 298 decreased by up to 50.0% when treated with 20 μg/mL of stigmasterol (Figure 5A). The cell 299 proliferation of OV90 cells was restricted by up to 54.3% when treated with 20 μg/mL of stigmasterol 300 (Figure 5B). Additionally, the phosphorylation of AKT, P70S6K, S6, ERK1/2, JNK, and P38 was 301 inhibited by stigmasterol in a dose-dependent manner (Figure 5C,D). Next, we used inhibitors to 302 verify the correlation of stigmasterol with the intracellular signaling mechanism. We used the PI3K 303 inhibitor (LY294002; 20 μM), ERK1/2 inhibitor (U0126; 20 μM), JNK inhibitor (SP600125; 20 μM), and 304 P38 inhibitor (SB203580; 20 μM) with stigmasterol. ES2 and OV90 cells were treated with these 305 inhibitors prior to stigmasterol treatment (Figure 6). Phosphorylation of AKT in ES2 cells was almost 306 completely inhibited by all these inhibitors but was only inhibited by LY294002 and SB203580 in 307 OV90 cells. The phosphorylation of P70S6K was almost completely inactivated by LY294002, U0126, 308 and SB203580 in ES2 cells and by LY294002, SP600125, and SB203580 in OV90 cells. The 309 phosphorylation of S6 protein was almost completely blocked by all inhibitors except U0126 in ES2 310 cells and was completely blocked by all inhibitors except SP600125 in OV90 cells. The 311 phosphorylation of ERK1/2 was completely blocked by U0126 and SB203580 in both cell types and 312 only slightly blocked by LY294002 in ES2 cells. JNK phosphorylation was completely blocked by all 313 inhibitors in both cells. The phosphorylation of P38 was completely blocked by all inhibitors except Pharmaceutics314 2020SP600125, 12, 488 in both cell lines. 11 of 16 315 316

317 Figure318 5. RestrictionFigure 5. Restriction of cell growthof cell growth and and cell cell growth-related growth-related signals signals in ovarian in ovarian cancer cells cancer following cells following stigmasterol319 Pharmaceutics treatments.stigmasterol 2020, 12treatments., (xA FOR,B) PEER Cell (A,B) REVIEW proliferation Cell proliferation data data shows shows the the cell cell viability viability (%)(%) following the 11the of 17 treatment 320 treatment of ovarian cancer cells with stigmasterol. The comparative graph represents the relative of ovarian cancer cells with stigmasterol. The comparative graph represents the relative proliferation of 321 proliferation of each cell type following treatments with stigmasterol, compared to vehicle-treated

each322 cell typecontrol following cells (100%). treatments (C,D) Western with blots stigmasterol, present the changes compared of PI3K toand vehicle-treated MAPK expression following control cells (100%). (C,D323) Westernthe blots treatments present of ovarian the changescancer cells ofwith PI3K stigmasterol and MAPK (0, 5, 10, and expression 20 μg/mL). following the treatments of ovarian cancer cells with stigmasterol (0, 5, 10, and 20 µg/mL). 324

325 Figure326 6. CotreatmentFigure 6. Cotreatment of stigmasterol of stigmasterol and inhibitors and inhibitors in ovarian in ovarian cancer cancer cells.cells. (A,B) (A,B Western) Western blots blots showed 327 showed changes of PI3K and MAPK by cotreatment with stigmasterol or each inhibitor in ovarian changes of PI3K and MAPK by cotreatment with stigmasterol or each inhibitor in ovarian cancer cells. 328 cancer cells. Comparative graph represented the change of phosphorylation compared to the vehicle- Comparative329 treated graph control represented (100%) in theES2 and change OV90 ofcells. phosphorylation compared to the vehicle-treated control (100%) in ES2 and OV90 cells. 330 3.7. Calcium Regulation by Stigmasterol in the Death of ES2 and OV90 Cells 331 Ruthenium red, an inhibitor of calcium overload, inhibited cell apoptosis induced by 332 stigmasterol in ES2 and OV90 cells. Additional treatment of ruthenium red to stigmasterol decreased 333 cell apoptosis from 9.3% (stigmasterol alone) to 3.6% and from 7.7% to 3.4% in ES2 and OV90 cells, 334 respectively (Figure 7A,B). Further, the stigmasterol-increased mitochondrial depolarization was 335 decreased from 7.0% to 2.1% and from 5.9% to 2.6% in ES2 and OV90 cells, respectively, following 336 cotreatment with stigmasterol and ruthenium red (Figure 7C,D). Mitochondrial calcium levels were 337 increased by stigmasterol by up to 8.9% and 6.9% in ES2 and OV90 cells, respectively. However, 338 treatment with stigmasterol and ruthenium red reduced the mitochondrial calcium from 8.9% to 3.8% 339 and from 6.9% to 3.7% in ES2 and OV90 cells, respectively (Figure 7E,F).

Pharmaceutics 2020, 12, x FOR PEER REVIEW 12 of 17 Pharmaceutics 2020, 12, 488 12 of 16 340

341 Figure 7. A B 342 Figure 7. CalciumCalcium regulation regulation and and cell cell apoptosis apoptosis by by stigmasterol stigmasterol in in ovarian ovarian cancer. cancer. (A,B) ( , Annexin) Annexin V V and PI were observed to investigate the cell death of ES2 and OV90 cells. Quadrant of the dot 343 and PI were observed to investigate the cell death of ES2 and OV90 cells. Quadrant of the dot blot blot represented the state of cell apoptosis in ES2 and OV90 cells. Comparative graph represented 344 represented the state of cell apoptosis in ES2 and OV90 cells. Comparative graph represented the the change of late apoptosis by stigmasterol or a combination with ruthenium red compared to the 345 change of late apoptosis by stigmasterol or a combination with ruthenium red compared to the control control (100%) in ES2 and OV90 cells. (C,D) JC-1 dye was used to investigate the alteration of the 346 (100%) in ES2 and OV90 cells. (C,D) JC-1 dye was used to investigate the alteration of the mitochondrial membrane potential by stigmasterol or a combination with ruthenium red. Quadrant 347 mitochondrial membrane potential by stigmasterol or a combination with ruthenium red. Quadrant of the dot blot represented the state of the mitochondrial membrane potential. Comparative graph 348 of the dot blot represented the state of the mitochondrial membrane potential. Comparative graph represented the loss of the mitochondrial membrane potential compared to the control group (100%). 349 represented the loss of the mitochondrial membrane potential compared to the control group (100%). (E,F) Rhod-2 ﬂuorescence was observed to investigate the mitochondrial calcium levels in stigmasterol 350 (E,F) Rhod-2 fluorescence was observed to investigate the mitochondrial calcium levels in or a combination with ruthenium red. The histogram represented a change of the mitochondrial 351 stigmasterol or a combination with ruthenium red. The histogram represented a change of the calcium. Comparative graph represented the change of the mitochondrial calcium levels. 352 mitochondrial calcium. Comparative graph represented the change of the mitochondrial calcium 353 levels.

354 3.7. Decreased Migration Activity of ES2 and OV90 Cells Following Stigmasterol Treatment

Pharmaceutics 2020, 12, 488 13 of 16

3.7. DecreasedPharmaceutics Migration 2020, 12, x FOR Activity PEER REVIEW of ES2 and OV90 Cells Following Stigmasterol Treatment 13 of 17 355 Cell migrationCell migration was was inhibited inhibited by by stigmasterol stigmasterol treatment, decreasing decreasing to 28.2% to 28.2% in ES2 in cells ES2 and cells to and to 35627.4% 27.4% in OV90 in OV90 cells cells compared compared to to the the respective respective vehicle-treatedvehicle-treated control control cells cells (Figure (Figure 8A,B).8A,B). Expression Expression 357 of genes that played a pivotal role in cancer cell migration, including VEGFA, PLAU, MMP2, MMP9, of genes that played a pivotal role in cancer cell migration, including VEGFA, PLAU, MMP2, MMP9, 358 and MMP14, were reduced in dose-dependent manner of stigmasterol in both cell lines (Figure 8C,D). and MMP14, were reduced in dose-dependent manner of stigmasterol in both cell lines (Figure8C,D). 359

360 Figure 8. Reduced cell migration by stigmasterol in ovarian cancer cells. (A,B) Cell migration was 361 observedFigure using 8. Reduced Transwell cell inserts. migration Migrated by stigmasterol cells through in ovarian the cancer membrane cells. were(A,B) Cell measured migration on imageswas of 362 five nonoverlappingobserved using Transwell locations. inserts. (C,D )Migrated Gene expression cells through of migration-relatedthe membrane were genesmeasured were on investigatedimages of by 363 quantitativeﬁve nonoverlapping real-time (RT)-PCR. locations. All (C,D) experiments Gene expression were conducted of migration-related in triplicate. genes Scale were bar represents investigated 100 µm. 364 by quantitative real-time (RT)-PCR. All experiments were conducted in triplicate. Scale bar represents 3654. Discussion100 μm.

366 The4. Discussion pharmacological functions of phytosterol, a plant-derived natural product, have been highly studied. Stigmasterol, a type of phytosterol, has been reported to have various physiological 367 The pharmacological functions of phytosterol, a plant-derived natural product, have been highly effects [4]. Recently, stigmasterol has been shown to have anticancer effects against various cancers. 368 studied. Stigmasterol, a type of phytosterol, has been reported to have various physiological effects 369However, [4]. Recently, the anticancer stigmasterol effects has ofbeen stigmasterol shown to have on ovariananticancer cancer effects have against not various yet been cancers. reported. 370Stigmasterol However, suppresses the anticancer skin effects cancer of stigmasterol by increasing on ovarian the lipid cancer peroxide have not levels yet been and causingreported. DNA 371damage Stigmasterol [16] and inhibits suppresses the skin development cancer by increasing of cholangiocarcinoma the lipid peroxide via levels the downregulationand causing DNA damage of TNF-alpha 372and VEGFR-2[16] and inhibits [14]. Stigmasterol the development represses of cholangiocarcinoma the development via of the prostate downregulation cancer by of inducing TNF-alpha p53 and protein 373expression, VEGFR-2 while [14]. inhibiting Stigmasterol p21 represses and p27 proteinthe developmen expressiont of [prostate20] and inducescancer by an inducing increase p53 of proapoptoticprotein 374signals expression, (BAX and p53)while while inhibiting decreasing p21 and antiapoptotic p27 protein signals expression (Bcl-2) [20] in liverand cancer.induces Further,an increase stigmasterol of 375 proapoptotic signals (BAX and p53) while decreasing antiapoptotic signals (Bcl-2) in liver cancer. promotes the mitochondrial apoptosis signaling pathway, including the upregulation of caspase-8 and 376 Further, stigmasterol promotes the mitochondrial apoptosis signaling pathway, including the -9, in hepatoma. Additionally, stigmasterol induces DNA damage and increases apoptosis in HepG2 377 upregulation of caspase-8 and -9, in hepatoma. Additionally, stigmasterol induces DNA damage and 378cells [13increases]. Similarly, apoptosis our in results HepG2 illustrated cells [13]. thatSimilarly, stigmasterol our results induced illustrated the activitythat stigmasterol of apoptotic induced proteins, 379including the activity cleaved of caspase-3,apoptotic proteins, cleaved including caspase-9, cleaved cytochrome caspase-3,c cleaved, BAK, caspase-9, and BAX. cytochrome c, BAK, 380 Mitochondriaand BAX. are important organelles that control cell survival [25]. Mitochondria regulate 381the cleavageMitochondria of caspases, are important production organelles of cytochrome that contro c,l cell and survival activation [25]. Mitochondria of BAX and regulate BAK to the induce 382programmed cleavage cellof caspases, apoptosis production [26]. Moreover, of cytochrome increased c, and mitochondrial activation of calcium BAX and levels BAK can to promoteinduce the 383 activationprogrammed of proapoptotic cell apoptosis factors [26]. viaMoreover, destruction increased of mitochondrial the mitochondria. calcium Thislevels causes can promote an increase the in 384 activation of proapoptotic factors via destruction of the mitochondria. This causes an increase in calcium concentration in the cytosol and induces cell apoptosis [27]. Increased calcium levels in the 385 calcium concentration in the cytosol and induces cell apoptosis [27]. Increased calcium levels in the 386cytosol cytosol stimulate stimulate proapoptotic proapoptotic signals, signals, while while repressing repressing antiapoptotic antiapoptotic signals signals [28]. [28 Cisplatin]. Cisplatin increases increases 387ROS inROS cancer in cancer cells, cells, leading leading to irreversibleto irreversible cell cell death.death. Excessive Excessive ROS ROS production production activates activates antitumor antitumor 388signals signals and programmed and programmed cell cell death death [29 [29].]. In In ourour study,study, stigmasterol stigmasterol induced induced the theapoptosis apoptosis of ovarian of ovarian 389cancer cancer cells bycells causing by causing mitochondrial mitochondrial malfunction, malfunction, ROS ROS production, production, and and calcium calcium overload overload in the in the 390mitochondria mitochondria and cytosol.and cytosol. In addition, In addition, the subG1 the subG1 phage wasphage significantly was significantly increased increased upon stigmasterolupon 391treatment stigmasterol in ovarian treatment cancer in cells.ovarian Ruthenium cancer cells. red Ruthenium prevents red mitochondrial prevents mitochondrial calcium overloadcalcium [30]. The cotreatment of stigmasterol and ruthenium red reduced mitochondrial depolarization and cell death, compared to treatment with stigmasterol alone. These results suggest that stigmasterol-induced cell apoptosis occurs via the accumulation of mitochondrial calcium in ovarian cancer cells. Pharmaceutics 2020, 12, 488 14 of 16

The ER is an important organelle that controls protein translocation, folding, and post-transcriptional modifications in eukaryotic cells. The accumulation of ER stress can induce cancer cell death [31]. Stigmasterol activated ER stress sensor proteins and ER-mitochondria axis proteins in ovarian cancer cells, indicating that stigmasterol induces anticancer effects mediated by the regulation of the ER-mitochondria axis. Additionally, stigmasterol decreased cell proliferation and inhibited cell cycle progression in ES3 and OV90 cells. The PI3K and MAPK signal cascades are pivotal in cancer cell proliferation and cell cycle progression [32]. In ovarian cancer, MAPK/PI3K signals are frequently activated. PI3K pathway inhibition may be the most useful in combination with the MAPK suppressor in ovarian cancer patients [33] Therefore, the inhibition of PI3K/MAPK signal cascades is useful for the treatment of ovarian cancer. The spheroid model provides a good opportunity to confirm the clinical therapeutic effectiveness of various drugs due to their association with the malignant nature of cancer cells, such as tumorigenicity or chemoresistance [34]. Stigmasterol effectively inhibited the aggregation of ovarian cancer cells. Ovarian cancer cells failed to form tumors and were scattered. VEGFA stimulates cell mitogenesis and cell migration in ovarian cancer cells [35]. PLAU induces migration and metastasis in breast cancer [36]. Metalloproteinases (MMPs) are overexpressed in several tumor environments and can promote metastasis and migration for cancer development [37]. In ES2 and OV90 cells, VEGFA, PLAU, MMP2, MMP9, and MMP14 expression levels were reduced by stigmasterol treatments. Taken together, we are the first to show that stigmasterol exerts a complex anticancer effect in the context of ovarian cancer. This effect can improve the anticancer effect of standard ovarian cancer drugs used to treat recurrent ovarian cancer. Therefore, these findings suggest that stigmasterol can be used as a new treatment option for ovarian cancer.

5. Conclusions Collectively, our results suggest that stigmasterol dose-dependently induced cell apoptotic signals in ES2 and OV90 cells. It also caused an increase in mitochondrial depolarization and ROS generation and induced abnormal increases in calcium levels in the cytosol and mitochondria. ER stress sensor protein levels were increased by stigmasterol (Figure9). As a result, cell death occurred in ovarian cancer cell lines treated with stigmasterol. Additionally, stigmasterol inhibited cell growth by controlling cell proliferation and the cell cycle in the tested ovarian cancer cell lines. Stigmasterol also reduced cell migration and inhibited cell growth-related signaling cascades (e.g., PI3K/MAPK) in ovarian cancer cells. This evidence suggests that stigmasterol could be used as a new treatment modality for ovarian cancer. Pharmaceutics 2020, 12, x FOR PEER REVIEW 15 of 17

428

429 FigureFigure 9. 9.Schematic Schematic illustration of ofthe the mechanisms mechanisms downstream downstream of stigmasterol of stigmasterol in human inovarian human ovarian 430 cancercancer cells. cells.

431 Author Contributions: conceived and designed the culture experiments, G.S.; the cell culture 432 methodology and other all experiments, W.L.; collected the experimental samples and conducted all 433 experiments, H.B.; analyzed and interpreted the data and contributed to the development of the 434 manuscript, W.L. and G.S. All authors contributed to its critical review and agreed on the final 435 version. 436 Funding: This research was supported by a grant of the National Research Foundation of Korea 437 (NRF), grant funded by the Ministry of Science and ICT(MSIT) (grant number 2018R1C1B6009048). 438 Acknowledgments: We would also like to thank the School of Life Sciences and Biotechnology for 439 BK21 PLUS, Korea University for supporting the publication fee. 440 Conflicts of Interest: The authors declare no conflicts of interest.

441 References

442 1. Moreau, R.A.; Whitaker, B.D.; Hicks, K.B. Phytosterols, phytostanols, and their conjugates in foods: 443 Structural diversity, quantitative analysis, and health-promoting uses. Prog. Lipid Res. 2002, 41, 457–500, 444 doi:Pii S0163-7827(02)00006-1 445 2. Garcia-Llatas, G.; Rodriguez-Estrada, M.T. Current and new insights on phytosterol oxides in plant sterol- 446 enriched food. Chem. Phys. Lipids 2011, 164, 607–624, doi:10.1016/j.chemphyslip.2011.06.005. 447 3. Woyengo, T.A.; Ramprasath, V.R.; Jones, P.J. Anticancer effects of phytosterols. Eur. J. Clin. Nutr. 2009, 63, 448 813–820, doi:10.1038/ejcn.2009.29. 449 4. Miras-Moreno, B.; Sabater-Jara, A.B.; Pedreno, M.A.; Almagro, L. Bioactivity of Phytosterols and Their 450 Production in Plant in Vitro Cultures. J. Agr. Food Chem. 2016, 64, 7049–7058, doi:10.1021/acs.jafc.6b02345. 451 5. Ferrer, A.; Altabella, T.; Arro, M.; Boronat, A. Emerging roles for conjugated sterols in plants. Prog. Lipid 452 Res. 2017, 67, 27–37, doi:10.1016/j.plipres.2017.06.002. 453 6. Han, J.H.; Yang, Y.X.; Feng, M.Y. Contents of phytosterols in vegetables and fruits commonly consumed in 454 China. Biomed. Environ. Sci. 2008, 21, 449–453, doi:10.1016/S0895-3988(09)60001-5. 455 7. Sundararaman, P.; Djerassi, C. A convenient synthesis of progesterone from stigmasterol. J. Org. Chem. 456 1977, 42, 3633–3634, doi:10.1021/jo00442a044. 457 8. Kametani, T.; Furuyama, H. Synthesis of vitamin D3 and related compounds. Med. Res. Rev. 1987, 7, 147– 458 171, doi:10.1002/med.2610070202.

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Author Contributions: Conceived and designed the culture experiments, G.S.; the cell culture methodology and other all experiments, W.L.; collected the experimental samples and conducted all experiments, H.B.; analyzed and interpreted the data and contributed to the development of the manuscript, W.L. and G.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by a grant of the National Research Foundation of Korea (NRF), grant funded by the Ministry of Science and ICT(MSIT) (grant number 2018R1C1B6009048). Acknowledgments: We would also like to thank the School of Life Sciences and Biotechnology for BK21 PLUS, Korea University for supporting the publication fee. Conﬂicts of Interest: The authors declare no conﬂicts of interest.

References

1. Moreau, R.A.; Whitaker, B.D.; Hicks, K.B. Phytosterols, phytostanols, and their conjugates in foods: Structural diversity, quantitative analysis, and health-promoting uses. Prog. Lipid Res. 2002, 41, 457–500. [CrossRef] 2. Garcia-Llatas, G.; Rodriguez-Estrada, M.T. Current and new insights on phytosterol oxides in plant sterol-enriched food. Chem. Phys. Lipids 2011, 164, 607–624. [CrossRef][PubMed] 3. Woyengo, T.A.; Ramprasath, V.R.; Jones, P.J. Anticancer eﬀects of phytosterols. Eur. J. Clin. Nutr. 2009, 63, 813–820. [CrossRef][PubMed] 4. Miras-Moreno, B.; Sabater-Jara, A.B.; Pedreno, M.A.; Almagro, L. Bioactivity of Phytosterols and Their Production in Plant in Vitro Cultures. J. Agr. Food Chem. 2016, 64, 7049–7058. [CrossRef] 5. Ferrer, A.; Altabella, T.; Arro, M.; Boronat, A. Emerging roles for conjugated sterols in plants. Prog. Lipid Res. 2017, 67, 27–37. [CrossRef] 6. Han, J.H.; Yang, Y.X.; Feng, M.Y. Contents of phytosterols in vegetables and fruits commonly consumed in China. Biomed. Environ. Sci. 2008, 21, 449–453. [CrossRef] 7. Sundararaman, P.; Djerassi, C. A convenient synthesis of progesterone from stigmasterol. J. Org. Chem. 1977, 42, 3633–3634. [CrossRef]

8. Kametani, T.; Furuyama, H. Synthesis of vitamin D3 and related compounds. Med. Res. Rev. 1987, 7, 147–171. [CrossRef] 9. Cabral, C.E.; Klein, M. Phytosterols in the Treatment of Hypercholesterolemia and Prevention of Cardiovascular Diseases. Arq. Bras. Cardiol. 2017, 109, 475–482. [CrossRef] 10. Feng, S.; Dai, Z.; Liu, A.B.; Huang, J.; Narsipur, N.; Guo, G.; Kong, B.; Reuhl, K.; Lu, W.; Luo, Z.; et al. Intake of stigmasterol and beta-sitosterol alters lipid metabolism and alleviates NAFLD in mice fed a high-fat western-style diet. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2018, 1863, 1274–1284. [CrossRef] 11. Valitova, J.N.; Sulkarnayeva, A.G.; Minibayeva, F.V. Plant Sterols: Diversity, Biosynthesis, and Physiological Functions. Biochemistry 2016, 81, 819–834. [CrossRef][PubMed] 12. Tao, C.; Shkumatov, A.A.; Alexander, S.T.; Ason, B.L.; Zhou, M. Stigmasterol accumulation causes cardiac injury and promotes mortality. Commun. Biol. 2019, 2, 20. [CrossRef][PubMed] 13. Kim, Y.S.; Li, X.F.; Kang, K.H.; Ryu, B.; Kim, S.K. Stigmasterol isolated from marine microalgae Navicula incerta induces apoptosis in human hepatoma HepG2 cells. BMB Rep. 2014, 47, 433–438. [CrossRef] [PubMed] 14. Kangsamaksin, T.; Chaithongyot, S.; Wootthichairangsan, C.; Hanchaina, R.; Tangshewinsirikul, C.; Svasti, J. Lupeol and stigmasterol suppress tumor angiogenesis and inhibit cholangiocarcinoma growth in mice via downregulation of tumor necrosis factor-alpha. PLoS ONE 2017, 12, e0189628. [CrossRef] 15. Pandey, P.; Bajpai, P.; Siddiqui, M.H.; Sayyed, U.; Tiwari, R.; Shekh, R.; Mishra, K.; Kapoor, V.K. Elucidation of the Chemopreventive Role of Stigmasterol Against Jab1 in Gall Bladder Carcinoma. Endocr. Metab. Immune. Disord Drug Targets 2019, 19, 826–837. [CrossRef] 16. Ali, H.; Dixit, S.; Ali, D.; Alqahtani, S.M.; Alkahtani, S.; Alarifi, S. Isolation and evaluation of anticancer efficacy of stigmasterol in a mouse model of DMBA-induced skin carcinoma. Drug Des. Devel. Ther. 2015, 9, 2793–2800. [CrossRef] 17. Li, K.; Yuan, D.; Yan, R.; Meng, L.; Zhang, Y.; Zhu, K. Stigmasterol exhibits potent antitumor effects in human gastric cancer cells mediated via inhibition of cell migration, cell cycle arrest, mitochondrial mediated apoptosis and inhibition of JAK/STAT signalling pathway. J. BUON 2018, 23, 1420–1425. Pharmaceutics 2020, 12, 488 16 of 16

18. Newill, H.; Loske, R.; Wagner, J.; Johannes, C.; Lorenz, R.L.; Lehmann, L. Oxidation products of stigmasterol interfere with the action of the female sex hormone 17beta-estradiol in cultured human breast and endometrium cell lines. Mol. Nutr. Food Res. 2007, 51, 888–898. [CrossRef] 19. Ayaz, M.; Sadiq, A.; Wadood, A.; Junaid, M.; Ullah, F.; Zaman Khan, N. Cytotoxicity and molecular docking studies on phytosterols isolated from Polygonum hydropiper L. Steroids 2019, 141, 30–35. [CrossRef] 20. Scholtysek, C.; Krukiewicz, A.A.; Alonso, J.L.; Sharma, K.P.; Sharma, P.C.; Goldmann, W.H. Characterizing components of the Saw Palmetto Berry Extract (SPBE) on prostate cancer cell growth and traction. Biochem. Biophys. Res. Commun. 2009, 379, 795–798. [CrossRef] 21. Liu, X.; Li, Q.; Zhou, J.; Zhang, S. ATP-binding cassette transporter A7 accelerates epithelial-to-mesenchymal transition in ovarian cancer cells by upregulating the transforming growth factor-beta signaling pathway. Oncol. Lett. 2018, 16, 5868–5874. [CrossRef][PubMed] 22. Siegel, R.L.; Miller, K.D.; Jemal, A. Cancer statistics, 2018. CA Cancer J. Clin. 2018, 68, 7–30. [CrossRef] [PubMed] 23. Bae, H.; Lee, J.Y.; Yang, C.; Song, G.; Lim, W. Fucoidan Derived from Fucus vesiculosus Inhibits the Development of Human Ovarian Cancer via the Disturbance of Calcium Homeostasis, Endoplasmic Reticulum Stress, and Angiogenesis. Mar. Drugs 2020, 18, 45. [CrossRef][PubMed] 24. Lian, I.A.; Loset, M.; Mundal, S.B.; Fenstad, M.H.; Johnson, M.P.; Eide, I.P.; Bjorge, L.; Freed, K.A.; Moses, E.K.; Austgulen, R. Increased endoplasmic reticulum stress in decidual tissue from pregnancies complicated by fetal growth restriction with and without pre-eclampsia. Placenta 2011, 32, 823–829. [CrossRef][PubMed] 25. Lopez, J.; Tait, S.W. Mitochondrial apoptosis: Killing cancer using the enemy within. Br. J. Cancer 2015, 112, 957–962. [CrossRef][PubMed] 26. Pinton, P.; Giorgi, C.; Siviero, R.; Zecchini, E.; Rizzuto, R. Calcium and apoptosis: ER-mitochondria Ca2+ transfer in the control of apoptosis. Oncogene 2008, 27, 6407–6418. [CrossRef] 27. Azimi, I.; Roberts-Thomson, S.J.; Monteith, G.R. Calcium inﬂux pathways in breast cancer: Opportunities for pharmacological intervention. Br. J. Pharmacol. 2014, 171, 945–960. [CrossRef] 28. Wang, H.G.; Pathan, N.; Ethell, I.M.; Krajewski, S.; Yamaguchi, Y.; Shibasaki, F.; McKeon, F.; Bobo, T.; Franke, T.F.; Reed, J.C. Ca2+-induced apoptosis through calcineurin dephosphorylation of BAD. Science 1999, 284, 339–343. [CrossRef] 29. Moloney, J.N.; Cotter, T.G. ROS signalling in the biology of cancer. Semin. Cell Dev. Biol. 2018, 80, 50–64. [CrossRef] 30. Yoon, M.J.; Lee, A.R.; Jeong, S.A.; Kim, Y.S.; Kim, J.Y.; Kwon, Y.J.; Choi, K.S. Release of Ca2+ from the endoplasmic reticulum and its subsequent inﬂux into mitochondria trigger celastrol-induced paraptosis in cancer cells. Oncotarget 2014, 5, 6816–6831. [CrossRef] 31. Larman, M.; Lucy, J. The endoplasmic reticulum: Structure and function. Mol. Membr. Biol. 1999, 16, 313–316. [CrossRef][PubMed] 32. Osaki, M.; Oshimura, M.; Ito, H. PI3K-Akt pathway: Its functions and alterations in human cancer. Apoptosis 2004, 9, 667–676. [CrossRef][PubMed] 33. Cheaib, B.; Auguste, A.; Leary, A. The PI3K/Akt/mTOR pathway in ovarian cancer: Therapeutic opportunities and challenges. Chin. J. Cancer 2015, 34, 4–16. [CrossRef][PubMed] 34. Ishiguro, T.; Ohata, H.; Sato, A.; Yamawaki, K.; Enomoto, T.; Okamoto, K. Tumor-derived spheroids: Relevance to cancer stem cells and clinical applications. Cancer Sci. 2017, 108, 283–289. [CrossRef][PubMed] 35. Jang, K.; Kim, M.; Gilbert, C.A.; Simpkins, F.; Ince, T.A.; Slingerland, J.M. VEGFA activates an epigenetic pathway upregulating ovarian cancer-initiating cells. EMBO Mol. Med. 2017, 9, 304–318. [CrossRef] [PubMed] 36. Tang, L.; Han, X. The urokinase plasminogen activator system in breast cancer invasion and metastasis. Biomed. Pharm. 2013, 67, 179–182. [CrossRef] 37. Jacob, A.; Prekeris, R. The regulation of MMP targeting to invadopodia during cancer metastasis. Front. Cell Dev. Biol. 2015, 3, 4. [CrossRef]

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