Adiponectin Reverses the Proliferative Effects of Estradiol and IGF-1 in Human Epithelial Ovarian Cancer Cells by Downregulating the Expression of Their Receptors

OVCAR-3, SKOV-3, and Caov-3 were purchased from the American Type Culture Collection (Manassas, VA, USA). COV434 cells were obtained from the European Collection of Authenticated Cell Cultures (ECACC, Sigma-Aldrich, St. Louis, MO, USA). The human ovarian serous carcinoma cell lines OVCAR-3, SKOV-3, and Caov-3 were cultured in RPMI 1640, McCoy’s, and Dulbecco’s modified Eagle’s medium (DMEM), respectively. Media were supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific Inc., Carlsbad, CA, USA). The human metastatic granulosa cell line COV434 was cultured in DMEM supplemented with 2 mM l-glutamine (Biowest, Nuaillé, France) and 10% FBS. All cell cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂.

Human adiponectin, 17β-estradiol, progesterone, and insulin-like growth factor 1 were purchased from Sigma-Aldrich. BPA (AccuStandard, New Haven, CT, USA) and E2 were dissolved in absolute ethanol. TBBPA and TCBPA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and dissolved in DMSO. The final concentration of ethanol and DMSO in the medium was 0.1%. Viability of cells was not affected at this concentration.

COV434, OVCAR-3, SKOV-3, and Caov-3 cells were seeded in 96-well plates and cultured until 70% confluent in the indicated media for 24 h. The culture medium was then removed, and the cells were rinsed with PBS and stored at − 20 °C. To investigate the effects of E2, P4, IGF-1, BPA, TBBPA, and TCBPA on the expression of AdipoR1 and AdipoR2, the culture medium was replaced with fresh medium 24 h before the cells were treated with compounds. The cells were then exposed to vehicle (medium, 0.1% DMSO, or 0.1% ethanol), E2 (10 nM), P4 (100 μM), IGF-1 (100 ng/ml), or BPA, TBBPA, or TCBPA (1, 10, and 100 nM) for 24 h. To investigate the effects of adiponectin on the expression of ERα, ERβ, PR, and IGF1R, the culture medium was replaced with fresh medium 24 h before the cells were treated with adiponectin at 25 μg/ml for 24 h.

Total RNA was isolated from control and treated cells, followed by cDNA synthesis using the TaqMan Gene Expression Cells-to-CT Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. The lysis solution contained DNase I to digest contaminating genomic DNA. The resulting cDNA was analyzed by real-time PCR using a StepOnePlus Real-Time PCR System (Applied Biosystems) and TaqMan Gene Expression Assays together with a TaqMan Gene Expression Master Mix and ROX Reference Dye (Applied Biosystems). The thermal cycling conditions were as follows: 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 60 s. Duplicate control cDNA-free samples were prepared for each gene. The TaqMan Gene Expression Assays used for real-time PCR were as follows: adiponectin [ADIPOQ, assay nos. Hs00605917_m1 and Hs00977214_m1], AdipoR1 [ADIPOR1, assay no. Hs01114951_m1], AdipoR2 [ADIPOR2, assay no. Hs00226105_m1], ERα [ESR1, assay no. Hs00174860_m1], ERβ [ESR2, assay no. Hs001100353_m1], PR [PGR, assay no. Hs01556702_m1], and IGF1R [IGF1R, assay no. Hs00609566_m1]. Expression levels were normalized to that of GADPH (assay no. 4310884E), and the relative expression was quantified using the 2^−ΔΔCt method [28].

COV434, OVCAR-3, and SKOV-3 cells were seeded in 24-well plates and cultured until 70% confluent in the indicated media for 24 h. The cells were then removed from plates, transferred to ice-cold lysis buffer, and stored at − 20 °C. The protein concentrations of the lysates were determined by the Bradford method (Bio-Rad Protein Assay, Bio-Rad Laboratories, Hercules, CA, USA). An equal amount of protein (60 μg) from each sample was assayed. Proteins were separated by 10% SDS-PAGE, and then transferred to polyvinylidene fluoride (PVDF) microporous membranes (Millipore, Billerica, MA, USA) using the Bio-Rad Mini-Protean 3 System. The membranes were blocked for 1 h in 0.02 M Tris-buffered saline containing 5% bovine serum albumin and 0.1% Tween 20, and then incubated overnight at 4 °C with antibodies specific for human AdipoR1 (cat. no. sc-46749, Santa Cruz Biotechnology), human AdipoR2 (cat. no. sc-46751, Santa Cruz Biotechnology), ERα (cat. no. sc-542, Santa Cruz Biotechnology), PR (cat. no. sc-539, Santa Cruz Biotechnology), IGF1R (cat. no. ab39675, Abcam, Cambridge, UK), and PARP (cat. no. #9542, Cell Signaling Technology). The membranes were then washed three times in Tris-buffered saline containing 0.1% Tween 20 (TBST) and incubated for 1 h at room temperature with a species-compatible horseradish peroxidase-conjugated secondary antibody (cat. no. sc-2020, Santa Cruz Biotechnology). β-Actin (cat. no. A5316, Sigma-Aldrich) was used as the loading control. Immunopositive bands were visualized using WesternBright Quantum HRP Substrate (cat. no. K-12043 D20, Advansta Inc., Menlo Park, CA, USA). Quantification of protein bands was performed by densitometry using VisionWorks LS Acquisition and Analysis software (UVP, LLC Upland, CA).

Cell proliferation was measured using AlamarBlue Cell Viability Reagent (Invitrogen, Paisley, UK), according to the manufacturer’s instructions. OVCAR-3 and SKOV-3 cells were exposed to adiponectin (0.025, 0.25, 2.5, or 25 μg/ml) for 48 h. In co-treatment experiments, OVCAR-3 cells were treated with adiponectin (25 μg/ml) and E2 (10 nM), P4 (100 μM), or IGF-1 (100 ng/ml) for an additional 48 h. The AlamarBlue stock solution was aseptically added to the wells after 48 h of culture in an amount equaling 10% of the volume of culture medium. The reduction of resazurin to resorufin was determined after 4 h of incubation by measuring the fluorescence at an excitation wavelength of 560 nm and an emission wavelength of 590 nm using an FLx800 fluorescence microplate reader (BioTek Instruments, Winooski, VT, USA). Data were analyzed using KC JUNIOR Software (BioTek Instruments).

Twenty-four hours before each experiment, the culture medium was replaced with serum-free medium. Treatments consisted of vehicle or adiponectin (0.025, 0.25, 2.5, or 25 μg/ml) for 24 h. The medium was then removed, and the plates were stored at − 70 °C. Cells were lysed in caspase assay buffer (50 mM HEPES, pH 7.4, 100 mM NaCl, 0.1% CHAPS, 1 mM EDTA, 10% glycerol, and 10 mM DTT). The protein concentrations of the lysates were determined by the Bradford method (Bio-Rad Protein Assay). An equal amount of the cytosolic extract (100 μg of protein) from each sample was analyzed. The assay was carried out by adding 100 μM Ac-DEVDAMC (Sigma-Aldrich) and incubating the plates at 37 °C. The amount of fluorescent product was monitored continuously for 120 min with a spectrofluorometer (FLx800 BioTek Instruments) at an excitation wavelength of 355 nm and an emission wavelength of 460 nm. Data were analyzed using KC JUNIOR Software, and normalized to fluorescence levels in vehicle-treated cells.

Before investigating the effects of adiponectin on human epithelial ovarian cancer cells, we examined the expression of adiponectin and its receptors in epithelial ovarian cancer cell lines (OVCAR-3, SKOV-3, and Caov-3) and compared the mRNA levels with those in the granulosa tumor cell line (COV434). By real-time PCR analyses, all cancer cell lines failed to express adiponectin (data not shown). Both ADIPOR1 and ADIPOR2 receptors were expressed by all cell lines. The relative quantity (RQ) of each transcript (ADIPOR1 and ADIPOR2) expressed by the granulosa tumor cell line (COV434) was arbitrarily set at 1. The basal ADIPOR1 mRNA level was lower in the epithelial ovarian cancer cells (3.5-, 2-, and 1.3-fold in OVCAR-3, SKOV-3, and Caov-3, respectively) than in the granulosa tumor cell line (Fig. 1a; p < 0.05). Similarly, the basal ADIPOR2 transcript level was lower in OVCAR-3, SKOV-3, and Caov-3 cells (6-, 4.3-, and 2.2-fold in OVCAR-3, SKOV-3, and Caov-3, respectively) than in COV434 cells (Fig. 1b; p < 0.05). The basal AdipoR1 and AdipoR2 protein levels were examined by Western blot analysis in COV434, OVCAR-3, and SKOV-3 cells, and the results mirrored those from real-time PCR analyses (Fig. 1c).

A previous study reported that BPA regulates adiponectin production and secretion in 3T3-L1 adipocytes. For this reason, we assessed the effects of BPA, TBBPA, and TCBPA on the expression of AdipoR1 and AdipoR2 in SKOV-3 and OVCAR-3 cells by real-time PCR analysis. BPA, TBBPA, and TCBPA at all tested concentrations (1, 10, and 100 nM) had no effects on ADIPOR1 and ADIPOR2 expression in SKOV-3 (Fig. 2a) and OVCAR-3 cells (Fig. 2b).

We examined the ability of adiponectin to affect OVCAR-3 and SKOV-3 cell proliferation. After 48 h of treatment, we observed a dose-dependent inhibition in the proliferation of both OVCAR-3 and SKOV-3 cells. Compared with that of control cells, there was a statistically significant decrease in proliferation after exposing OVCAR-3 cells to adiponectin at concentrations of 2.5 and 25 μg/ml (85 ± 1.5% and 76 ± 3% relative to the control, respectively) (Fig. 3a; p < 0.01, p < 0.001). Adiponectin at concentrations of 0.025 and 0.25 μg/ml had no effect on the proliferation of OVCAR-3 cells. Similarly, adiponectin decreased SKOV-3 cell proliferation dose-dependently (87 ± 1.7%, 79 ± 2.9% and 75 ± 2.5% at 0.25, 2.5, and 25 μg/ml relative to the control, respectively) (Fig. 3b; p < 0.05, p < 0.001).

Epithelial ovarian cancer cells differ from granulosa tumor cells in terms of their morphology and clinical behavior. We found that ADIPOR1 and ADIPOR2 expression was lowest in OVCAR-3 cells compared with that in the other cells. Moreover, our finding suggested that adiponectin inhibit OVCAR-3 and SKOV-3 cell proliferation in a similar manner. For this reason, OVCAR-3 cells were chosen as an in vitro model of serous epithelial ovarian cancer for further analysis.

In a parallel experiment that employed a caspase-3 activity assay, we evaluated the effect of adiponectin on caspase-3 activity and cleaved PARP protein expression in OVCAR-3 cells. Adiponectin at all tested concentrations had no effect on OVCAR-3 caspase-3 activity as well as cleaved PARP protein expression (Fig. 4).

Previous studies reported E2, P4, and IGF-1 to affect ovarian cancer development, which prompted us to examine the effects of adiponectin and E2, P4, or IGF-1 on the proliferation of cancer cells. Consistent with previous results, we found that E2 and IGF-1 stimulated OVCAR-3 cell proliferation (115 ± 3% and 138 ± 5% relative to that of the control, respectively) (Fig. 5; p < 0.05). However, adiponectin reversed E2- and IGF-1-induced OVCAR-3 cell proliferation; the combined treatment of adiponectin and E2 or adiponectin and IGF-1 decreased cell proliferation to the basal adiponectin level after 48 h, (75 ± 7% and 86 ± 9%, respectively, vs 80 ± 5%), (Fig. 5; p < 0.05). Although P4 had no effect on OVCAR-3 cell proliferation, the combined treatment of adiponectin and P4 decreased cell proliferation to below the basal adiponectin level (67 ± 5% vs 80 ± 5%), (Fig. 5; p < 0.05).

To investigate the mechanism by which adiponectin reduced E2- and IGF-1-induced cell proliferation, we measured the mRNA levels of AdipoR1 and AdipoR2 in OVCAR-3 cells after E2, IGF-1, and P4 treatment. As shown in Fig. 6, E2 and IGF-1 had no effect on the basal AdipoR1 and AdipoR2 levels. However, the levels of AdipoR1 and AdipoR2 were higher in OVCAR-3 cells cultured in the presence of P4 than in control cells (35 ± 6% for AdipoR1 and 31 ± 8% for AdipoR2 compared with the respective basal levels) (Fig. 6).

Next, we examined whether adiponectin (25 μg/ml) could affect the mRNA expression of ESR1, ESR2, IGF1R, and PGR in OVCAR-3 cells. We observed no difference in ESR2 expression between adiponectin-treated and control cells (data not shown). By contrast, the levels of IGF1R, ESR1, and PGR were significantly lower in adiponectin-treated cells than in control cells (0.83 ± 0.04, 0.74 ± 0.06, and 0.43 ± 0.1 vs 1 RQ, respectively). The ERα, PR, and IGF1R protein levels were examined by Western blot analysis, and the results mirrored those from real-time PCR analyses (Fig. 7).