Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco, Thermo Fisher Scientific (Waltham, MA, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and celastrol (Fig. 1) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Annexin V/propidium iodide (PI) staining kit was purchased from BD Biosciences (San Jose, CA, USA). The bicinchoninic acid (BCA) protein assay kit was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).

OVCAR3 human ovarian carcinoma cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultivated in DMEM with 10% FBS, 100 U/ml penicillin and 100 µg/ml streptomycin (Sangon Biotech Co., Ltd., Shanghai, China) at 37°C in a 5% CO₂ incubator.

OVCAR3 cells (4,000 cells/well) were seeded and cultured in 96-well microplates overnight. Subsequently, OVCAR3 cells were treated with varying concentrations of celastrol (0, 0.25, 0.5, 1, 2, 4 and 6 µM) for 0, 1, 2 and 3 days. A total of 10 µl MTT (5 mg/ml) solution was added to each well and incubated at a temperature of 37°C in a humidified atmosphere of 5% CO₂. Following this, 100 µl of the resolving solution [10% sodium dodecyl sulfate (SDS) and 0.1 mM HCl] was added to each well and incubated for 10 min at room temperature whilst the plate was agitated. The absorbance of the plate was measured at 570 nm using the Multiskan EX Primary EIA V.2.1–1 spectrophotometer.

Following treatment with celastrol (1, 2 and 4 µM) for 2 days, OVCAR3 cells were harvested and washed with phosphate-buffered saline. OVCAR3 cells were re-suspended with 1X binding buffer prior to staining with Annexin V for 30 min at room temperature in the dark. OVCAR3 cells were then double-stained with PI for 30 min at room temperature in the dark. The apoptotic OVCAR3 cells were quantitatively counted by a flow cytometer.

OVCAR3 cells (1×10⁶ cells/well) were seeded and cultured in 6-well microplates overnight at 37°C in a 5% CO₂ incubator. Subsequently, the OVCAR3 cells were treated with varying concentrations of celastrol (1, 2 and 4 µM) for 2 days. Following treatment with celastrol, the OVCAR3 cells were prepared in cell lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) for 30–60 min and centrifuged at 12,000 × g for 10 min at 4°C. The protein concentration was measured using a BCA protein assay kit. An equal quantity of total protein extract was mixed with the reaction buffer (acetyl-Leu-Glu-His-Asp-p-nitroanilide for caspase-9 and acetyl-Asp-Glu-Val-Asp-p-nitroanilide for caspase-3) (Beyotime Institute of Biotechnology) for 4–6 h. Caspase-9 and −3 activity was measured at an absorbance of 405 nm.

OVCAR3 cells (1×10⁶ cells/well) were seeded and cultured in 6-well microplates overnight at 37°C. Subsequently, OVCAR3 cells were treated with varying concentrations of celastrol (1, 2 and 4 µM) for 2 days. Following treatment with celastrol, OVCAR3 cells were prepared in cell lysis buffer for 30–60 min and centrifuged at 12,000 × g for 10 min at 4°C. Total RNA was extracted from the cell lysate using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA (1–2 µg) was reversed transcribed into to cDNA using PrimeScript RT Master Mix (Takara Bio, Inc., Otsu, Japan). qPCR was conducted using the ABI 7500 system (Takara Bio, Inc.). The cycling conditions were as follows: One cycle at 94°C for 5 min, followed by 30 cycles at 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec. The relative expression of miRNA-21 was measured using a Bulge-Loop miRNA qRT-PCR kit (Invitrogen; Thermo Fisher Scientific, Inc.). The primers used were as follow: miRNA-21, forward 5′-GCCCGCTAGCTTATCAGACTGATG-3′ and reverse 5′-GCCCGCTAGCTTATCAGACTGATG-3′; and U6 forward 5′-GTTGACATCCGTAAAGACC-3′ and reverse 5′-GGAGCCAGGGCAGTAA-3′.

OVCAR3 cells (1×10⁶ cells/well) were seeded and cultured in 6-well microplates overnight at 37°C in a 5% CO₂ incubator. Subsequently, OVCAR3 cells were treated with varying concentrations of celastrol (1, 2 and 4 µM) for 2 days. Following treatment with celastrol, the OVCAR3 cells were prepared in radioimmunoprecipitation assay lysis buffer with protease and phosphatase inhibitors (Beyotime Institute of Biotechnology) for 30–60 min and centrifuged at 12,000 × g for 10 min at 4°C. The protein concentration was measured using a BCA protein assay kit. A total of 30 µg of total protein lysate was loaded and electrophoresed onto 10% SDS-polyacrylamide gels (Sangon Biotech Co., Ltd.) and the separated proteins were transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% fat-free milk in Tris-buffered saline with 0.1% Tween-20 (TBST) for 2 h at room temperature. The membranes were then incubated with anti-PI3K (sc-67306; 1:500; Santa Cruz Biotechnology, Inc.), anti-phosphorylated-Akt (p-Akt; sc-7985-R and sc-1618; 1:1,000; Santa Cruz Biotechnology, Inc.), anti-NF-κB (sc-109; 1:1,000; Santa Cruz Biotechnology, Inc.) and anti-β-actin (sc-130657; 1:500; Santa Cruz Biotechnology, Inc.) overnight at 4°C. Following washing with TBST three times for 2 h at room temperature, secondary fluorescent antibodies were incubated with the membranes at room temperature for 2 h. Proteins were visualized using an LI-COR Odyssey scanner (LI-COR, Inc., Lincoln, NE, USA).

Anti-miRNA-21 plasmids were chemically synthesized by BeastBio Co., Ltd. (Shanghai, China). OVCAR3 cells (1×10⁶ cells/well) were seeded and cultured in 6-well microplates overnight. A total of 100 pmol/l anti-miRNA-21 plasmid was transfected into the OVCAR3 cells with Lipofectamine 2000 serum medium (Invitrogen; Thermo Fisher Scientific) for 24 h. Subsequently, the transfected OVCAR3 cells were treated with celastrol for 48 h.

Statistical analysis was conducted with SPSS software, version 18.0 (SPSS, Inc., Chicago, IL, USA). Values are presented as the mean ± standard error. Experiments were conducted a minimum of three times. Differences between the groups were assessed by two-way analysis of variance and no post hoc tests were used. P<0.01 was considered to indicate a statistically significant difference.

The effects of celastrol on the cellular proliferation of ovarian carcinoma cells was investigated using an MTT assay. OVCAR3 cells were treated with a range of celastrol concentrations (0, 0.25, 0.5, 1, 2, 4 and 6 µM) for 0, 1, 2 and 3 days. As presented in Fig. 2, the proliferation of OVCAR3 cells was inhibited following treatment with celastrol in a dose- and time-dependent manner. Treatment with celastrol (1, 2, 4 and 6 µM) for 3 days or celastrol (2, 4 and 6 µM) for 2 days resulted in significant reductions in cellular proliferation (P<0.01; Fig. 2). These results suggest that celastrol exerted clear anticancer effects on human ovarian carcinoma cells.

The effects of celastrol on apoptosis in ovarian carcinoma cells was investigated using Annexin V/PI staining. As presented in Fig. 3, celastrol induced apoptosis in ovarian carcinoma cells in a dose-dependent manner, with a significant increase in the levels of apoptotic cells following celastrol treatment for 2 days at 2 and 4 µM (P<0.01; Fig. 3).

To further investigate the effects of celastrol treatment on apoptosis in ovarian carcinoma cells, the activity levels of caspase-9 and −3 was investigated. As presented in Fig. 4, celastrol increased the activity levels of caspase-9 and −3 in ovarian carcinoma cells in a dose-dependent manner. Treatment with celastrol (2 and 4 µM) for 2 days resulted in a significant increase in the activity levels of caspase-9 and −3 (P<0.01; Fig. 4).

To explore the mechanisms involved in the effect of celastrol on ovarian carcinoma cells, miRNA-21 expression was measured using qPCR. The relative expression levels of miRNA-21 in OVCAR3 cells was reduced following treatment with celastrol (2 and 4 µM) for 2 day (P<0.01; Fig. 5). These results indicated that the anticancer effect of celastrol may be involved with reducing miRNA-21 levels.

To investigate the mechanisms involved in the effect of celastrol treatment on ovarian carcinoma cells, the expression levels of PI3K/Akt were measured in ovarian carcinoma cells using western blot analysis. Following treatment with celastrol (2 and 4 µM) for 2 days, the protein expression levels of PI3K and p-Akt were reduced in OVCAR3 cells (P<0.01; Fig. 6). These results indicate that the anticancer effects of celastrol may be associated with the suppression of the PI3K/Akt signaling pathway.

To further investigate the mechanisms involved in the effect of celastrol treatment on ovarian carcinoma cells, NF-κB protein expression was measured by western blot analysis. Following treatment with celastrol (2 and 4 µM) for 2 days, the expression levels of NF-κB in OVCAR3 cells were significantly reduced (P<0.01; Fig. 7). These results indicated that the anticancer effect of celastrol may be associated with the suppression of the NF-κB signaling pathway.

To further analyze the mechanisms involved in the effect of celastrol treatment on ovarian carcinoma cells, anti-miRNA-21 plasmids were transfected into OVCAR3 cells. As presented in Fig. 8A, the anti-miRNA-21 plasmids significantly reduced the relative expression levels of miRNA-21 in OVCAR3 cells. In addition, the anti-miRNA-21 plasmids were able to reduce the cellular proliferation of OVCAR3 cells (Fig. 8B). Notably, the anti-miRNA-21 plasmids suppressed the PI3K/Akt-NF-κB signaling pathway in ovarian carcinoma cells (Fig. 8C-F).