Alpinumisoflavone Disrupts Endoplasmic Reticulum and Mitochondria Leading to Apoptosis in Human Ovarian Cancer.

Alpinumisoflavone (Cat No. CFN98440 ) was acquired from ChemFaces and dissolved into dimethyl sulfoxide (DMSO). Cisplatin (cis-diamminedichloroplatinum) (Cat No. P4394, Sigma-Aldrich, St. Louis, MO, USA) was purchased from Sigma-Aldrich, LY294002 (Cat No. 99015) was purchased from Cell Signaling Technology (Danvers, MA, USA), and U0126 (Cat No. BML-EI282) and SB203580 (Cat. No. BML-EI286) were purchased from Enzo Life Sciences (Farmingdale, NY, USA). ES2 and OV90 cells (OC cells) were purchased from ATCC (Manassas, VA, USA). ES2 and OV90 cells were grown in McCoy’s medium (Cat No. SH30200, Hyclone, Carlsbad, CA, USA) with a 10% fetal bovine serum (FBS) (Cat No. SH30919.03, Hyclone) and 1% penicillin–streptomycin solution (P/S) (Cat No. SV30010, Hyclone) and cultured at 37 °C in a 5% CO 2 incubator. CHO-K1 cells, purchased from Korea Cell Line Bank (Seoul, Korea), were grown in RPMI-1640 with HEPES medium (Cat No. SH30255.01, Hyclone), 10% FBS (Cat No. SH30071.01, Hyclone) and 1% P/S at 37 °C in a 5% CO 2 incubator. The cells were cultured using adherent methods. The proliferation of ES2 and OV90 cells was evaluated using the bromodeoxyuridine (BrdU) Cell Proliferation ELISA kit (Cat No. 11647229001, Roche, Basel, Switzerland). Both ES2 and OV90 cells were seeded at a density of 6 × 10 3 cells/100 μL in a 96-well cell culture plate, and then were treated with alpinumisoflavone (0, 0.5, 1, and 2 µM) or cisplatin (4 µM) for 48 h at 37 °C in a 5% CO 2 incubator. After 48 h, BrdU was added to the cell medium and allowed to react with the cells for 2 h at 37 °C in a 5% CO 2 incubator. After incubation for 2 h, the cells were fixed using fixation and anti-BrdU-peroxidase-working solutions were incubated at 25 °C for 1.5 h. Subsequently, the cells were washed and the same amount of substrate solution was added for detection using an ELISA reader. In addition, the cell viability of normal ovarian cells, CHO-K1, was confirmed in response to alpinumisoflavone under the same condition using MTT solution (Cat No. 11465007001, Roche, Basel, Switzerland), by following the manufacturer’s instructions. Finally, the cells were analyzed using the BioTek Epoch Plate Reader and BioTek Gen5 software. Proliferating cell nuclear antigen (PCNA) expression was demonstrated in ES2 and OV90 cells with or without alpinumisoflavone via immunofluorescence analysis. For fluorescence imaging, cells were seeded in confocal dishes (SPL, Daejeon, Korea) and treated with alpinumisoflavone for 48 h. After incubation, the cells were washed and fixed with methanol. Subsequently, the cells were incubated with primary PCNA antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 16 h. Then, we incubated the cells with anti-mouse secondary antibodies conjugated with Alexa 488 for visualization. Next, we washed the cells with PBS and stained the nuclei with DAPI. The cell images were captured using an LSM710 confocal microscope (Carl Zeiss, Oberkochen, Germany). The cells were dropped on the cover of a 100 mm culture dish plate. Alpinumisoflavone-treated and nontreated ES2 and OV90 cells were cultured for 5 days by the hanging drop method. After incubation, the morphology of the spheroids was detected with a DM3000 microscope. Spheroid quantification was performed using the ImageJ software version 1.8. The migration of ES2 and OV90 cells was confirmed using culture-insert 2 well in µ-dish 35 mm (Cat No. 80206, ibidi GmbH, Munich, Germany). The distances of the cell-free gap, in both with or without alpinumisoflavone groups, were captured using a DM3000 microscope (Leica, Wetzlar, Germany) and its width was quantified using the ImageJ software. ES2 and OV90 cells were grown to 60% confluence in a 60 mm cell culture dish. The cells were incubated with alpinumisoflavone (0, 0.5, 1 and 2 µM) for 48 h. Subsequently, the cells were collected and washed twice using 0.1% BSA in PBS, and then fixed with 70% ethanol at 4 °C for 24 h. Next, the cells were collected by centrifugation, washed with 0.1% BSA in PBS, and resuspended using a 1× binding buffer. Furthermore, 100 µL of the cell suspension was added to a brown tube and stained with RNase A (Cat No. R6513, Sigma-Aldrich) and propidium iodide (PI) for 30 min. After incubation, the stained cells were mixed with 1× binding buffer and analyzed using an FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). Relative fluorescence was detected using a PE channel. To detect change in mitochondrial membrane potential, JC-1 dye (Cat No. CS0390, Invitrogen, Carlsbad, CA, USA) was employed to stain the alpinumisoflavone- or cisplatin-treated ES2 and OV90 cells. After staining with the JC-1 dye (5 μg/mL) for 20 min, the cells were washed according to the manufacturer’s instructions. Finally, the relative changes of JC-1 aggregates to JC-1 monomers were analyzed using the FL1 and FL2 channels of the flow cytometer to detect emission wavelengths of 530 nm (green, JC-1 monomer) and 590 nm (red, JC-1 aggregate). ES2 and OV90 cells treated with alpinumisoflavone were validated to calculate apoptotic cells using FITC-annexin V (5 μL for each sample) and 50 mg/mL of PI of the Annexin V Apoptosis Detection Kit (Cat No. 556547, BD Biosciences). After incubation with alpinumisoflavone for 48 h, the cells were collected and stained with both dyes for 15 min. The stained cells were analyzed using the FL1 and FL2 channels of the flow cytometer. ES2 and OV90 cells were treated with alpinumisoflavone (0, 0.5, 1 and 2 µM) for 24 h. Proteins were extracted using a whole cell lysate buffer. Denatured proteins were loaded in each well of 10% acrylamide gel in equal amounts (calculated via the Bradford assay) and subjected to SDS-PAGE. After separating the proteins via electrophoresis, the separated proteins in the gels were transferred onto nitrocellulose membranes. Thereafter, primary and secondary antibodies (Cat No. 5450-00110, SeraCare, MA, USA) were incubated with the membranes to detect the target proteins. The immunoblots were detected via chemiluminescence and visualized using the ChemiDoc EQ system (Bio-Rad, Hercules, CA, USA). The antibodies used in immunoblotting assays are listed in Table 1 . For the mitochondrial stress analysis, ES2 and OV90 cells were seeded in Seahorse XFe24 cell culture microplates at a concentration of 3 × 10 4 cells/100 μL. After the cell confluency reached 80% in each well, the cells were treated with alpinumisoflavone (2 µM) for the next 24 h; then, they were additionally treated with 1.5 µM of oligomycin (an inhibitor of ATP synthase), 0.5 µM of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (also known as FCCP, an uncoupler), and a mixture of 0.5 µM rotenone (a mitochondrial complex I inhibitor) and 0.5 µM antimycin A (a mitochondrial complex III inhibitor) during the measurement, according to the user guide of the Seahorse XF Cell Mito Stress Test Kit (Cat No. 103015-100, Agilent Technologies, Santa Clara, CA, USA) and previous studies [ 13 , 14 ]. The treated cells were evaluated for oxygen consumption rate (OCR) using a Seahorse XFe24 Analyzer (Agilent Technologies). To verify that ES2 and OV90 cells exhibited significant differential effects in response to treatments, all triplicate data results were subjected to analysis of variance by least-squares analysis of variance (ANOVA), according to the general linear model (PROC-GLM) of the SAS program (SAS Institute, Cary, NC, USA). Differences with a probability value of * p < 0.05 were considered statistically significant. The obtained data are presented as the mean ± SEM, unless otherwise stated.

Ovarian cancer (OC) is the second most common gynecological malignancy due to its high mortality rate and frequent relapses [ 1 ]. The American Cancer Society estimates that in 2021, 21,410 women will be diagnosed with OC, and among them, 13,700 are expected to die from the disease [ 2 ]. OC can be classified into three types, depending on the patient’s age. In general, it can be categorized into epithelial cell tumors in women over 50 years of age, stromal cell tumors in women of all ages, and germ cell tumors in girls under 1 year of age or 15–19 years of age [ 3 ]. According to a recent study, among OC patients that are actually diagnosed, more than 70% of them progress to stage III or IV, which is the most severe and advanced phase of the disease, and the 5-year survival rate is known to be approximately 50% [ 4 ]. Factors that increase the incidence of OC are old age, genetic alterations, family history and endometriosis. Among these factors, mutations in tumor suppression genes breast cancer type 1 (BRCA1) and BRCA2 significantly increase the risk of OC, and these cancers can be prevented by chemoprevention and bilateral ovarian resection [ 5 ]. For treating OC patients, it is important to improve the quality of systemic treatment based on surgery and chemotherapy, to increase the survival rate in first line therapy [ 6 ]. In addition, cisplatin and carboplatin are platinum-based drugs that have been used for decades and are established as the standard treatment for ovarian cancer [ 7 ]. However, most advanced OC patients suffer from chemoresistance, relapse and the side effects of therapeutic drugs. Therefore, there is a need to identify a supporting agent that can enhance cisplatin activity. Isoflavonoids are considered as effective sensitizers for the treatment of various cancers and chronic diseases, with less side effects than conventional therapeutic agents [ 8 ]. In addition, isoflavonoids are known as phytoestrogens, as they exhibit estrogenic or antiestrogenic activities in biological responses. For example, genistein, an isoflavone, exhibits its anticancer activities by inhibiting the growth and progression of estrogen-related OC via the regulation of multiple signal transduction pathways, such as PI3K, MAPK, NF-κB and intrinsic/extrinsic apoptosis pathways [ 9 ]. In addition, the prenylation of isoflavonoids improves various pharmacological activities. Alpinumisoflavone is a prenylated isoflavonoid, and it is commonly found in the Cudrania tricuspidate fruit and Genista pichisermolliana . It is regarded as a traditional Chinese medicinal ingredient with various beneficial pharmacological features, including antiosteoporotic, antioxidative, anti-inflammatory, antibacterial and anticancer activities [ 10 , 11 ]. A previous study reported that alpinumisoflavone improves radiation sensitivity in esophageal squamous cell carcinoma (ESCC) [ 10 ]. Moreover, it suppresses the proliferation of lung cancer cells by the activation of intrinsic pathways and the inactivation of NF-κB and ERK1/2 MAPK pathways [ 12 ]. However, no study has been conducted on the effects of alpinumisoflavone on OC. Therefore, in this study, we investigated whether alpinumisoflavone regulates cell growth and programmed cell death in human OC. We demonstrated the effects of alpinumisoflavone on ES2 and OV90 cells, focusing on (1) anti-proliferation via cell cycle arrest; (2) cell death and alteration of MMP (∆Ψm); (3) impairment of mitochondrial respiration; (4) regulation of signal transduction; and (5) synergy with cisplatin. Our results suggest that alpinumisoflavone may enhance therapeutic efficiency against the growth of OC via mitochondrial dysfunction.

Epithelial OC cells (ES2 and OV90) were exposed to alpinumisoflavones (0, 0.5, 1 and 2 µM) for 48 h to estimate cell proliferation. In response to the dose-dependent treatment of alpinumisoflavone, the cell proliferations of ES2 and OV90 gradually decreased ( Figure 1 A,B). Specifically, the cell proliferations decreased to 39% ( p < 0.001) and 69% ( p < 0.001) in ES2 and OV90 cells with 2 µM alpinumisoflavone, respectively. In addition, we exposed CHO-K1 cells to alpinumisoflavone under the same conditions as for ES2 and OV90, to check the toxicity of alpinumisoflavone in normal ovarian cells ( Figure 1 C). The obtained results indicated that alpinumisoflavone did not alter the cell viability of CHO-K1 cells. Based on the reduced proliferation of OC cells by alpinumisoflavone, we compared the immunofluorescence intensity of PCNA in ES2 and OV90 cells, with or without alpinumisoflavone (2 µM). The treatment of alpinumisoflavone significantly decreased the immunoreactive PCNA expression in both ES2 and OV90 cells ( Figure 1 D,E). We further confirmed the suppression of PCNA expression in response to alpinumisoflavone treatment in both cell lines via western blot analysis ( Figure 1 F). Next, we determined whether alpinumisoflavone induces cell cycle arrest in OC cells. The proportion of ES2 and OV90 cells treated with 2 µM alpinumisoflavone in the sub-G1 phase increased by 2.89% and 3.0%, respectively, but the change was only significant in OV90 cells ( Figure 2 A,B). In addition, the proportion of cells in the S-phase of the ES2 population significantly increased ( Figure 2 A). To verify the anti-proliferation effect, we analyzed the spheroid formation of ES2 and OV90 cells in an alpinumisoflavone-included medium. The obtained results indicate that the 3D spheroid density of ES2 and OV90 cells decreased by approximately 80% ( p < 0.001), compared to vehicle-treated spheroid cells ( Figure 2 C). In addition, ES2 and OV90 cells were cultured using culture-insert 2 well in µ-dish to measure cancer cell migration. The alpinumisoflavone treatment inhibited cell migration by increasing the gap distance between cells by 1.2- and 1.7-fold in ES2 and OV90 cell cultures, respectively, compared to vehicle-treated cells ( Figure 2 D). Collectively, alpinumisoflavone suppressed cell growth in ES2 and OV90 cells. We investigated the population of late apoptosis cells using an annexin V staining kit, as well as the change in MMP using JC-1 dye in ES2 and OV90 cells with alpinumisoflavone. First, annexin V staining results indicated that alpinumisoflavone (2 μM) increased the amount of late apoptotic OV90 and ES2 cells by 187% ( p < 0.05) and 165% ( p < 0.05), respectively, compared to the vehicle-treated group ( Figure 3 A,B). Furthermore, MMP was disrupted by 77% ( p < 0.01) and 87% ( p < 0.001) in ES2 and OV90 cells, respectively, in response to alpinumisoflavone treatment ( Figure 3 C,D). Together, the results revealed that alpinumisoflavone triggered cell death with the depolarization of MMP in ES2 and OV90 cells. To detect OCR, we conducted Seahorse XF Cell Mito Stress tests using the Seahorse XFe analyzer. We verified mitochondrial respiration in OC cells using oligomycin (1.5 μM), FCCP (0.5 μM) and rotenone/antimycin A (0.5 μM). Basal and maximal respiration were significantly reduced by approximately 20% in both cell lines following treatment with 2 μM alpinumisoflavone. In addition, alpinumisoflavone significantly reduced ATP generation by approximately 25% in ES2 and OV90 cells compared to control cells ( Figure 4 ). Therefore, alpinumisoflavone inhibited mitochondrial respiration, followed by mitochondrial malfunction. To measure cell proliferation, human OC (ES2 and OV90) cells were treated with alpinumisoflavone, or a combination of alpinumisoflavone with cisplatin, for 48 h. The results indicated that ES2 and OV90 cells treated with alpinumisoflavone (2 μM) and cisplatin (4 μM) exhibited a more significant decrease in proliferation compared to cells treated with alpinumisoflavone alone ( Figure 5 A,B). To verify the synergistic effect of alpinumisoflavone with cisplatin on mitochondrial-regulated cell death, we determined the death of apoptotic cells and the depolarization of MMP in the OC cells ( Figure 5 C,D). The proportion of apoptotic ES2 cells was significantly increased after cisplatin treatment with alpinumisoflavone ( Figure 5 C). In addition, OV90 cells treated with alpinumisoflavone significantly increased upon the additional treatment with cisplatin, compared to those treated with alpinumisoflavone alone. Furthermore, the permeability of MMP in OV90 cells was significantly disrupted by a combination of alpinumisoflavone and cisplatin; however, there was no significant effect on ES2 cells ( Figure 5 D). These results indicate that the combined treatment exhibited complementary effects on the inhibition of cellular proliferation, thereby leading to the death of OC cells. To verify the change in protein levels triggered by alpinumisoflavone, we conducted a western blot analysis. Downstream of the PI3K signaling pathway, the phosphorylation of P70S6K and S6 proteins was gradually reduced in both cell lines in response to alpinumisoflavone ( Figure 6 A,B). Furthermore, the expression of phosphor-P38 protein in alpinumisoflavone-treated cell lines increased, compared to vehicle-treated cells ( Figure 6 C). In addition, the phosphor-ERK1/2 and P90RSK proteins slightly decreased with alpinumisoflavone in both ES2 and OV90 cells ( Figure 6 D,E). Subsequently, we investigated the relative levels of unfolded protein in ES2 and OV90 cells treated with different doses of alpinumisoflavone. The phosphor-eIF2α and GRP78 proteins were promoted by alpinumisoflavone in both cells ( Figure 7 ). In addition, the expression levels of VDAC and IP3R1 proteins, which are involved in the ER–mitochondria axis for regulating calcium ion levels, were upregulated in response to alpinumisoflavone in both ES2 and OV90 cells. In general, alpinumisoflavone altered the levels of signaling molecules regulating proliferation, ER stress and ER-mitochondrial contact in the OC cells. To determine the synergistic effect of alpinumisoflavone with pharmacological inhibitors, we performed annexin V/PI staining and western blot analyses. The amount of late apoptosis cells increased following the pretreatment with LY294002 (PI3K inhibitor), U0126 (ERK1/2 inhibitor) and SB203580 (P38 inhibitor) prior to the treatment of alpinumisoflavone, compared to the sole treatment of each inhibitor in OV90 cells, whereas there were no significant differences between a single treatment of target substance and the combined treatment ( Figure 8 ). Furthermore, in both ES2 and OV90 cells, the phosphorylation of P70S6K was better suppressed by all these inhibitors than with alpinumisoflavone alone ( Figure 9 ). The phosphorylation of S6 was blocked by the inhibitors, except SB203580, in both cell lines. The phosphorylation of ERK1/2 was blocked by a combination of alpinumisoflavone with U0126 in ES2 and OV90 cells. In general, alpinumisoflavone regulated the OC cell proliferation via PI3K and MAPK signaling.

In this study, alpinumisoflavone suppressed cell proliferation, PCNA expression, spheroid formation and cell migration in human OC cells ( Figure 10 ). Furthermore, alpinumisoflavone promoted the loss of MMP and increased the number of late apoptotic cells. In addition, cisplatin, a chemotherapeutic agent administered against cancers, exhibited similar effects in combination with alpinumisoflavone, thus enhancing the efficiency of alpinumisoflavone in treating OC cells. Moreover, alpinumisoflavone triggered mitochondrial dysfunction and anti-proliferative effects in OC cells through the regulation of MAPK, PI3K and ER stress signaling pathways. Collectively, we elucidated the anticancer effects and cellular mechanisms of alpinumisoflavone in human OC cells. Platinum-based anticancer drugs are marketed worldwide, and with approximately one-half of patients being treated with cisplatin, cisplatin is one of the best metal-based chemotherapy drugs [ 15 ]. Although cisplatin has been proven to be effective against a variety of cancers, including sarcoma, cancers of the bone, muscle, etc., the drug’s resistance and significant side effects have inspired a novel treatment strategy for the management of various cancers with the combination of cisplatin and other drugs or supplementation [ 16 ]. In previous studies, natural compounds, such as fucoidan and eupatilin, were adopted to improve chemotherapeutic effects with additional chemotherapy by inhibiting proliferation and angiogenesis [ 17 , 18 ]. In this study, cisplatin increased the mitochondria-regulated cell death in ES2 and OV90 cells when the cells were treated with alpinumisoflavone, compared to the case where cisplatin was used alone. Mitochondrial dysfunction, which is caused by mtDNA mutations or mitochondrial enzyme deficiencies, disrupts cellular bioenergetics and supports cancer cell metabolic reprogramming; in addition, it triggers tumor-promoting alterations mediated by reactive oxygen species, Ca 2+ , or small molecule metabolites released from mitochondria [ 19 ]. Furthermore, mitochondria serve as the cell's powerhouse, producing over 80% of ATP and performing several cellular functions. Mitochondria maintain cell motility and intracellular homeostasis and are closely related to apoptosis [ 20 ]. For example, α,β-thujone disrupt mitochondrial function via metabolic alterations by reducing maximal respiration, ATP production and MMP, leading to cell death in ovarian cancer [ 21 ]. In particular, inhibiting mitochondrial respiration in ovarian cancer could enhance the sensitivity to anticancer drugs even in chemo-resistant cells, because of its high dependence on the OXPHOS system [ 22 ]. Although alpinumisoflavone has a role in inhibiting the proliferation of cancerous cells, the effects of alpinumisoflavone remain unclear. In this study, we identified that alpinumisoflavone induced the disruption of mitochondrial respiration and the depolarization of MMP, which results in the cell death of ES2 and OV90 cells. The PI3K and MAPK signaling system is a significant therapeutic target for OC, and the inhibition of this mechanism can impede cancer growth [ 23 ]. In a previous study, it was reported that the management of the PI3K pathway is a critical strategy for treating OC patients using specific inhibitors [ 24 ]. In addition, the inhibition of PI3K and ERK1/2 pathways exhibited synergistic effects on inhibiting tumor growth in OC [ 25 ]. Similar to previous studies, alpinumisoflavone inhibited the PI3K and ERK1/2 MAPK pathways in OC cells, leading to apoptosis, while pharmacological inhibitors, including LY294002 or U0126, in combination with alpinumisoflavone, improved the antiproliferative effects of alpinumisoflavone on the OC cells. ER is an organelle involved in multiple processes, including protein homeostasis, stress response and Ca 2+ homeostasis [ 26 ]. GRP78 aids in the stabilization of ER proteins and triggers the unfolded protein response. In addition, surface-associated GRP78 is involved in cytoprotection and mediated cytoskeletal remodeling, while secreted GRP78 is involved in immunomodulation [ 27 ]. High levels of GRP78 can increase the activities of matrix metalloproteinases in pancreatic cancer metastasis and invasion by activating signaling pathways, such as JNK and FAK [ 28 ]. In addition, ER stress leading to excessive GRP78 and eIF2α expression was determined to be closely related to the activation of the P38 protein in tumor cells [ 29 , 30 ]. VDACs located in the outer mitochondrial membrane are responsible for the release of cytochrome C from the mitochondrial membrane space into the cytoplasm, which is an early step in the apoptosis process [ 31 ]. IP3R1, located in the endoplasmic reticulum, is known to play important roles, such as cell proliferation, metabolism and apoptosis, by pumping calcium from intracellular calcium stores. In addition, IP3R1 overexpression induces an increase in cytoplasmic Ca 2+ levels and apoptosis [ 32 ]. Likewise, the IP3R-mediated increase in calcium ion flux between the ER and mitochondria sensitized cisplatin sensitivity and activated ER stress in OC [ 33 ]. In OC cells, alpinumisoflavone activated ER stress sensor proteins and ER–mitochondria axis proteins with the activation of P38 protein, thus indicating that alpinumisoflavone has anticancer effects mediated by the ER–mitochondria axis.

In summary, we first demonstrated the anticancer effects of alpinumisoflavone on OC cells, and further explained the working mechanisms of alpinumisoflavone in OC cells. Therefore, we suggest alpinumisoflavone as a potential therapeutic agent against OC cells. Considering its synergistic effect with cisplatin, it can be used as a supplementary agent to treat chemoresistance. However, since our studies were limited to cell lines, further in vivo and clinical verification and validation will be needed for future applications.