Human ovarian adenocarcinoma OVCAR8 cells were purchased from American Type Culture Collection (Manassas, VA, USA). OVCAR8 cells are highly resistant to cisplatin (26), and respond poorly to high-dose platinum-based chemotherapy. Additionally, OVCAR8 cells are resistant to cadmium (26). Mangiferin was purchased from Shanghai PureOne Bioechnology (Shanghai, China; www.pureonebio.com), with a purity of >95%. RPMI-1640 medium and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). MTT (cat. no. M5655), paraformaldehyde (PFA; cat. no. 16005), cisplatin (cat. no. 1134357), dimethyl sulfoxide (DMSO; cat. no. D2650), Annexin V-fluorescein isothiocyanate (FITC) Apoptosis detection kit (cat. no. APOAF), HEPES (cat. no. H3375), Triton X-100 (cat. no. H9284), 2 mmol/l sodium orthovanadate (cat. no. S6508), sodium fluoride (cat. no. S7920), 1 mmol/l edetic acid (cat. no. E9884), PMSF (cat. no. 78830), aprotinin (cat. no. A11530) and leupeptin were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Hoechst 33342 (cat. no. C1025) and a Bradford protein assay (cat. no. P0006) were purchased from Beyotime Institute of Biotechnology (Suzhou, China). NuPAGE^® Bis-Tris gels (cat. no. NP0327BOX) were purchased from Thermo Fisher Scientific, Inc. Polyvinylidene difluoride (PVDF) membranes (cat. no. ISEQ00010) and electrochemiluminescence (ECL) reagents (cat. no. 345818) were purchased from EMD Millipore (Billerica, MA, USA). Mouse monoclonal β-actin antibody (cat. no. sc-47778; 1:5,000 dilution) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Matrigel (cat. no. 356234) was purchased from BD Biosciences (Franklin Lakes, NJ, USA). Rabbit polyclonal cleaved caspase-3 antibody (cat. no. 9661; 1:500 dilution) and rabbit polyclonal YAP antibody (cat. no. 4912; 1:1,000 dilution) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Mouse monoclonal TEA domain transcription factor 4 (TEAD4) antibody (cat. no. ab58310; 1:1,000 dilution) and rabbit monoclonal cleaved poly(ADP-ribose) polymerase 1 (PARP1) antibody (cat. no. ab32138; 1:1,000 dilution) were purchased from Abcam (Cambridge, MA, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse immunoglobulin G (IgG) polyclonal antibody (cat. no. 115-035-003; 1:5,000 dilution) and HRP-conjugated goat anti-rabbit polyclonal IgG (cat. no. 111-035-003; 1:5,000 dilution) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).

OVCAR8 cells were cultured in RPMI-1640 which contained 20% FBS, 10 µg/ml bovine insulin, 100 µg/ml streptomycin (Thermo Fisher Scientific, Inc.), 100 U/ml penicillin (Thermo Fisher Scientific, Inc.) and 0.03% L-glutamine (Sigma-Aldrich; Merck KGaA).

OVCAR8 cells at a logarithmic growth phase were seeded in a 96-well plate (3×10⁴ cells per well) and incubated at 37°C for 24 h. For the cell proliferation experiments examining the inhibitory effect of mangiferin via the YAP pathway, OVCAR8 cells were treated with mangiferin (25 µg/ml) at 37°C for 24 h following transfection by Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) with either an empty vector (pcDNA3; Addgene, Inc., Cambridge, MA, USA) or YAP overexpression plasmids for 12, 24, 36 and 48 h. For the experiments examining cisplatin sensitivity, OVCAR8 cells in three groups were respectively treated at 37°C for 6 h with cisplatin (1 µg/ml) or cisplatin (1 µg/ml) and mangiferin (25 µg/ml) combined, following transfection by Lipofectamine 2000 with either an empty vector (pcDNA3) or YAP overexpression plasmids at 37°C for 48 h. Subsequently, cells in each well were treated with 0.05 mg MTT (10 µl of 5 mg/ml) and incubated at 37°C for 4 h. Subsequent to incubation, the supernatant was removed. For the suspended cells, following centrifugation at 200 × g for 5 min at 4°C, the medium was discarded and 150 µl DMSO was added to each well. The plate was thoroughly agitated by hand shaking for 10 min. Finally, the absorbance was measured at a wavelength of 570 nm using a spectrophotometer (Model 3550 Microplate Reader; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cell viability was determined via the following equation: Cell viability (%)=[optical density (OD) 570 nm (drug)/OD 570 nm (control)] ×100%. Experiments were repeated three times, and data are representative of replicate experiments.

Cells were plated in triplicate wells in 6-well plates for 14 days for a flat colony formation assay. For the soft agar assay, 2×10³ cells were plated in complete medium (RPMI-1640 medium plus 10% FBS) in addition to 0.6% agar in triplicate wells in 6-well plates. Subsequent to transfection with either an empty vector (pcDNA3) or YAP overexpression plasmids, mangiferin (25 µg/ml) was added to the medium. Medium was replaced every 48 h and visible colonies were counted using light microscopy after 21 days.

OVCAR8 cells were seeded into a 6-well culture plate at a density of 4×10⁵ cells/well and cultured for 24 h. Cells were treated with mangiferin (25 µg/ml) subsequent to the transfection with either the empty vector (pcDNA3) or YAP overexpression plasmids. The cell morphology was observed under phase contrast microscopy (×400 magnification). Hoechst 33342 staining was applied to further detect viable cells. In brief, cells were fixed with 4% PFA for 30 min at room temperature, and then cells were washed twice with PBS. Hoechst 33342 (5 µg/ml) was added and incubated for 15 min at 37°C, and then the cells were washed and analyzed immediately with a fluorescence microscope (Olympus Corporation).

OVCAR8 cells were treated with mangiferin (25 µg/ml) for 24 h following transfection with either an empty vector (pcDNA3) or YAP overexpression plasmids. Then, the detached and adherent cells were collected at 300 × g at 4°C for 5 min. The cells were labelled for 15 min at room temperature using an Annexin V-FITC and PI apoptosis detection kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. In detail, the cells were washed twice with PBS and centrifuged at 300 × g at 4°C for 5 min. The cells were resuspended with binding buffer and then the Annexin V and PI staining solution was added. At least 1×10⁵ cells were analyzed on a FACS Aria II cytometer (BD Biosciences). Data analyses were performed using FlowJo Software version 9.7.1 (FlowJo LLC, Ashland, OR, USA). Experiments were repeated three times, and data are representative of replicate experiments.

The migration of cells in the logarithmic growth was examined through a wound healing assay. Cells were cultured in a 12-well plate at a density of 5×10⁴ cells/well until a confluent monolayer was formed. A 200 µl pipette tip was used to scratch the wells and the initial with of the scratch was ~100 µm. Then, the wells were rinsed with phosphate buffered saline (PBS) and cultured in RPMI-1640 medium with 10% FBS at 37°C for 24 h. The cells were treated with 25 µg/ml mangiferin at 37°C for 24 h subsequent to transfection with either an empty vector (pcDNA3) or YAP overexpression plasmids. Images were obtained through light microscopy (Olympus CKX31; Olympus Corporation, Tokyo, Japan). The cell migration was measured by Image J software (version 1.48; National Institutes of Health, Bethesda, MD, USA) with a contrasting quantification of pixels in the area of the scratch at 24 h. Migration inhibition rate (%)=(1-experimental group pixels/control group pixels) ×100%.

A Matrigel cell invasion assay was performed using Transwell cell culture chambers (8-mm pore size; EMD Millipore). Transwell membranes were firstly coated with 100 µl Matrigel matrix (1 mg/ml; BD Biosciences). OVCAR8 cells (5×10⁴ cells/well) were added to serum-free RPMI-1640 medium and placed in the upper chamber of the Transwell insert and incubated at 37°C for 24 h with 25 µg/ml mangiferin subsequent to transfection with either an empty vector (pcDNA3) or YAP overexpression plasmids. RPMI-1640 containing 20% FBS was added to the lower chamber as a chemoattractant. After 24 h incubation at 37°C, the upper surface of the chambers was scraped using a cotton swab. The cells on the lower surface were fixed with 4% PFA in PBS for 30 min at 37°C. Subsequent to this, the chambers were rinsed with PBS and cells in the lower chamber were manually counted and analyzed under a light microscope at a ×200 magnification.

OVCAR8 cells were transfected with either an empty vector (pcDNA3) or YAP overexpression plasmids using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) for 24 h at 37°C, and then incubated with 25 µg/ml mangiferin for a further 24 h. Adherent and floating cells were collected. The cell pellets were resuspended in a lysis buffer and lysed at 4°C for 15 min. The lysis buffer consisted of 50 mmol/l HEPES (pH 7.4), 1% Triton X-100, 2 mmol/l sodium orthovanadate, 100 mol/l sodium fluoride, 1 mmol/l edetic acid, 1 mmol/l PMSF, 10 mg/l aprotinin and 10 mg/l leupeptin. Centrifugation at 12,000 × g for 15 min at 4°C was performed and followed by the determination of supernatant protein content using a Bradford protein assay (cat. no. P0006; Beyotime Institute of Biotechnology). Equal quantities (10 µg) of the total protein were separated using 4–12% NuPAGE^® Bis-Tris gels (Thermo Fisher Scientific, Inc.) and were transferred onto PVDF membranes (EMD Millipore). The membranes were soaked in blocking buffer (5% bovine serum albumin; Sigma-Aldrich; Merck KGaA). Then the proteins of interest were detected using primary antibodies (incubated overnight at 4°C) and secondary antibodies (incubated for 1 h at room temperature), which was finally visualized using ECL (EMD Millipore). Experiments were repeated three times. Data are representative of replicate experiments and analyzed using ImageJ software (version 1.44; National Institutes of Health, Bethesda, MD, USA).

OVCAR8 cells were transfected with either an empty vector (pcDNA3) or YAP overexpression plasmids by Lipofectamine 2000 for 24 h at 37°C, and then incubated with 25 µg/ml mangiferin for another 24 h. Cells were grown in RPMI-1640 medium supplemented with 20% FBS and puromycin (Thermo Fisher Scientific, Inc.) for selection. A total of 5×10⁴ cells in 24-well plates were transfected at 37°C for 24 h with 0.5 µg synthetic 8×GTIIC TEAD luciferase promoter plasmids (Addgene, Inc.) and 0.1 ug Renilla luciferase control reporter (Promega Corporation, Madison, WI, USA), using Lipofectamine 2000 (Thermo Fisher Scientific, Inc.). Single (5′-TGTGGAATGTGT-3′) and tandem (5′-TGTGGAATGTGTGGAATGTGT-3′) TEAD4 binding sites were cloned upstream of the CMV promoter of the pGL4-hRluc vector. A luciferase assay was performed 24 h after transfection using a Dual-Luciferase Reporter Assay system (Promega Corporation) according to the manufacturer's protocol. Luminescent signaling was detected using a GloMax-96 Microplate Luminometer (Promega Corporation) according to the manufacturer's protocol.

The polymerase chain reaction (PCR) products for YAP with a restriction enzyme cutting site (EcoRI and XbaI) and pcDNA3 (Addgene, Inc.) with the same sticky ends were obtained and ligated by T4 DNA ligase (Tiangen Biotech, Co., Ltd., Beijing, China). In detail, the RNA of OVCAR8 cells were extracted using a Qiagen RNeasy kit (Qiagen, Inc., Valencia, CA, USA), according to the manufacturers protocol and reverse transcribed to cDNA using a reverse transcription system (Promega Corporation), which was used as the template. For the reverse transcription protocol, RNA was incubated at 70°C for 10 min, centrifuged at 1,000 x g at 4°C for 20 sec and then placed on ice. Then with the random primers provided in the kit, dNTP mixture, AMV reverse transcriptase, MgCl₂, buffer and nuclease free water were added to the microcentrifuge tube. The reaction mixture was incubated at room temperature for 10 min, then incubated at 42°C for 15 min. The sample was heated at 95°C for 5 min, then incubated at 5°C for 5 min. The YAP gene was amplified using PCR from cDNA using the following primers: Forward (containing an EcoRI restriction site as underlined), 5′-GAATTCGAGGCAGAAGCCATGG-3′ and reverse (containing a XbaI restriction site as underlined), 5′-TAGAGCTCTATAACCATGTAAGAAAGCT-3′. The thermocycling conditions were as follows: 95°C for 180 sec to open the template, then 95°C for 30 sec, 55°C for 30 sec and 72°C for 60 sec for 35 cycles. Further extension was performed at 72°C for 10 min, and then maintained at 4°C. Then the PCR products were electrophoresed using 1% agarose (Sigma-Aldrich; Merck KGaA), stained with ethidium bromide (10 mg/ml; Tiangen Biotech, Co., Ltd.), visualized under ultraviolet light, and then purified using a gel purification kit (Tiangen Biotech, Co., Ltd.), according to the manufacturers protocols. All restriction enzymes and the T4 DNA ligase were purchased from Tiangen Biotech, Co., Ltd. The insertion of YAP in pcDNA3 was performed and confirmed by sequencing.

The PCR product of YAP (as described above) was cloned into pTY linkers. Third-generation vectors were used in this experiment. A total of 2 µg YAP lentiviral vectors were transiently transfected into 1×10⁵ OVCAR8 cells using Lipofectamine 2000 at 37°C for 48 h. Briefly, OVCAR8 cells, cultured in DMEM medium plus 10% FBS, were co-transfected with 2 µg vector plasmids, including a helper construct, envelope plasmid, tat plasmid and pTY linker containing YAP. Then the viral supernatant was harvested at 48 h, filtered through a 0.45-µm filter, subjected to ultracentrifugation (113,000 × g at 4°C for 2 h) for a 100-fold concentration and stored at −80°C. Then, the lentiviral supernatant was thawed at 37°C and diluted in 0.9% saline (Sichuan Kelun Pharmaceutical, Co., Ltd., Chengdu, Sichuan, China) and polybrene (8 µg/ml final concentration; Sigma-Aldrich; Merck KGaA) to produce a dose of 1.6×10⁷ transducing units in a 50 µl injection volume. The virus was injected intravenously into the BALB/c nude mice with a 30-gauge needle on a 1-cc syringe. A total of 3 consecutive injections were administered at 3-day intervals (n=6).

A total of 5×10⁶ OVCAR8 cells were injected into BALB/c nude female mice (n=60 in total and n=20 per group; 5–6 weeks old; 16–18 g body weight; purchased from the Affiliated Laboratory Animal Center of Sichuan Academy of Medical Science and Sichuan Provincial People's Hospital, Chengdu, China). All animals were maintained at 26°C at 40–60% humidity in a 12 h light/dark cycle with ad libitum access to water and food, which was in accordance with the individually ventilated cages requirements at the Sichuan Academy of Medical Science and Sichuan Provincial People's Hospital. Prior to the initiation of daily treatment, the tumors were allowed to grow to a size of 100–550 mm³. No mouse bearing multiple tumors was observed in the present study. During the present study, the maximum body loss in an animal due to cachexia was 11.3%, and the longest diameter exhibited by a single subcutaneous tumor was 1.2 cm, which is smaller compared with the Institutional Animal Care and Use Committee Guidelines (2.0 cm) of the American Association for Laboratory Animal Science (https://www.aalas.org/), therefore it was confirmed that the tumor burden did not exceed the recommended dimensions. Subsequent to tumor formation by OVCAR8 cells, mice were randomly divided into three groups: i) A cisplatin group, where cisplatin (10 mg/kg) was intraperitoneally administered; 2) A cisplatin and mangiferin plus empty virus transfection group, where cisplatin (10 mg/kg) and mangiferin (50 mg/kg) were intraperitoneally administered; 3) A cisplatin and mangiferin plus YAP overexpression lentivirus transfection group, where cisplatin (10 mg/kg) and mangiferin (50 mg/kg) were intraperitoneally administered. The treatment lasted for a further two weeks. All experiments were ethically approved by the ethics committee of Sichuan Academy of Medical Science and Sichuan Provincial People's Hospital.

The body weight of the mice was measured daily prior to and following the mangiferin treatment. Then, 5 mice in each group were sacrificed, and 5 mice in each group were raised for a survival assay. For the sacrificed mice, the subcutaneous tumors were removed and weighed, while the volume of the tumors was determined in 3 dimensions with venire calipers according to the following formula: Tumor volume=length × width × depth ×0.5. After 20 days of treatment, mice were sacrificed by cervical dislocation, and subcutaneous tumor masses were determined.

All data were expressed as the mean ± standard error of the mean from at least three independent experiments. Data analysis was performed using GraphPad Prism 5.0 software (Graphpad Software, Inc., La Jolla, CA, USA), and P<0.05 was considered to indicate a statistically significant difference. One-way analysis of variance followed by Bonferroni's post-hoc test, two-way analysis of variance followed by Bonferroni's post-hoc test and a Student's t-test (paired) were performed to determine the statistical significance. All experiments were performed at least in triplicate and repeated at least three times, and the data are representative of replicate experiments.

One previous study revealed that mangiferin may inhibit the proliferation and induce the apoptosis of ovarian cancer cells through the regulation of Notch3, which is a downstream effector of YAP (24). Thus, it was hypothesized that YAP may also be involved in the antitumor efficacy of mangiferin. To verify this hypothesis and elucidate the mechanism of mangiferin inhibiting cell proliferation, YAP protein expression was assessed. As presented in Fig. 1A, a comparatively decreased expression of YAP was observed in mangiferin-treated cells. To further validate that YAP was involved in mangiferin-inhibited cell proliferation, a time course study for the mangiferin-treated group and YAP-overexpressed mangiferin-treated group (abbreviated as the YAP-overexpressed group) was performed. As predicted, compared with the mangiferin-treated group, the inhibitory rate of the YAP-overexpressed group was significantly decreased (P<0.01; Fig. 1B). In addition, based on cell fluorescent staining and cell morphology images, OVCAR8 cells treated with mangiferin demonstrated shrinkage of the cytoplasm and a condensed nucleus. However, a greater number of viable cells were observed in YAP overexpressed cells, which indicated that mangiferin may mediate cell apoptosis via the inhibition of YAP (Fig. 1C). Additionally, the quantified cell colony numbers revealed a significantly higher number of colonies in the YAP-overexpressed group compared with the mangiferin-treated group (P<0.01) further substantiated that YAP may facilitate cell growth by counteracting the effects of mangiferin (Fig. 1D).

As demonstrated in Fig. 2A, increased caspase-3 cleavage and PARP cleavage were produced in mangiferin-treated cells, compared with in the mangiferin-treated YAP-overexpressed cells. To further demonstrate that mangiferin may induce apoptosis through the inhibition of YAP, cells were stained with Annexin V and PI, and the apoptotic cell percentages were analyzed using flow cytometry. As presented in Fig. 2B, there were more early apoptotic cells (Annexin V⁺/PI⁻) and late apoptotic cells (Annexin V⁺/PI⁺) in the mangiferin-treated group compared with the YAP-overexpressed cells (Fig. 2B). Quantified apoptotic percentages further revealed that mangiferin-induced apoptosis was significantly inhibited by the overexpression of YAP (P<0.05; Fig. 2C).

To further investigate the migration capability of tumor cells and to address the association between mangiferin-inhibited cell migration and YAP, a wound healing assay was performed and the images of the cell migration were captured through microscopy (Fig. 3A and B). After 24 h of treatment, cell fusion was observed in the mangiferin-treated YAP-overexpressed group, with a significant increase in the fold increase of migrated cells compared with the mangiferin-treated group (P<0.01). Conversely, a scratch was clearly observed in the mangiferin-treated group at 24 h. These data indicated that mangiferin may effectively suppress tumor cell migration, and that this suppression may be reversed by YAP overexpression. Additionally, a cell invasion assay was performed. As revealed in Fig. 3C, the data suggested that YAP was able to significantly reverse the mangiferin-suppressed invasion capability (P<0.05), further validating the involvement of YAP in mangiferin-inhibited migration and invasion events in OVCAR8 cells.

To further investigate the molecular mechanism of mangiferin, a luciferase assay was performed. As presented in Fig. 4A, mangiferin reduced TEAD4-dependent luciferase activity, whereas YAP overexpression resulted in the significant activation of TEAD4-dependent luciferase activity (P<0.01). Furthermore, western blot analysis of TEAD4 was performed. It was revealed that TEAD4 protein levels were decreased upon mangiferin treatment and elevated in YAP-overexpressed cells (Fig. 4B).

To address the effect of YAP on the enhanced chemotherapy sensitivity induced by mangiferin, the proliferation of YAP-overexpressing OVCAR8 cells in the presence of mangiferin and cisplatin was assessed. As presented in Fig. 5A, cisplatin combined with mangiferin was able to inhibit cell proliferation, which may be abrogated by YAP overexpression. To further validate this hypothesis, cell morphology was observed under phase contrast microscopy. As presented in Fig. 5B, compared with the cisplatin-treated group, fewer viable cells were observed in the mangiferin and cisplatin combined-treated cells, whereas a greater number of viable cells were observed in the YAP-overexpressed OVCAR8 cells. Altogether, these results suggested that mangiferin enhanced the sensitivity of OVCAR8 cells to cisplatin via the inhibition of YAP.

To evaluate the effect of YAP on the anti-neoplastic properties of mangiferin and mangiferin-increased tumor sensitivity to cisplatin in an in vivo tumor xenograft model system, OVCAR8 cells were xenografted into nude mice. As presented in Fig. 6A, the xenograft tumor size decreased significantly in the empty vector-transfected mangiferin and cisplatin combined-treated group following a 20-day treatment, compared with that of the cisplatin monotherapy group (P<0.05). However, in the YAP-overexpressed mangiferin and cisplatin combined-treated group, the xenograft tumor size was increased. Similarly, the empty vector and mangiferin and cisplatin-treated mice also demonstrated significantly decreased xenograft tumor weights compared with the cisplatin monotherapy group (P<0.05; Fig. 6B), suggesting that mangiferin may increase the sensitivity of ovarian tumor types to cisplatin. In accordance with the decreased tumor size, the body weight of the mice in the mangiferin and cisplatin-treated group also demonstrated a significant increase compared with the cisplatin monotherapy group (P<0.05; Fig. 6C). Furthermore, the survival days for the YAP-overexpressed mangiferin and cisplatin-treated mice were 15, 17, 26, 53 and 69 days. The survival days for mice treated with mangiferin and cisplatin were 36, 39, 75, 100 and 100. But for the control mice treated with cisplatin alone, the survival days were 15, 33, 53, 79 and 100 days. Therefore, judging from the survival data, mangiferin in addition to cisplatin treatment may increase the life span of ovarian cell xenograft mice compared with any other groups (Fig. 6D). Altogether, the in vivo results suggested that mangiferin increased cisplatin sensitivity via inhibition of the YAP pathway.