Oridonin and cisplatin were obtained from the Shifeng Biocorporation (Shanghai, China). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-lium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). The A2780/DDP cisplatin-resistant human ovarian cancer subline (Huiying Corporation, Shanghai, China) and SKOV3/DDP (Yunnan Tumor Hospital, Kunming, China) were used. The non-resistant cells lines, A2780 and SKOV3 were obtained from Wuhan Boster Biological Technology, Ltd. (Wuhan, China). The cells were cultured in Dulbecco's modified Eagle's medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) enriched with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences) at 37°C and 5% CO₂. Cisplatin was obtained from Qilu Pharmaceutical Co., Ltd. (Jinan, China) and 0.5 µg/ml cisplatin was added into the medium to maintain chemoresistance in the resistant cells.

The inhibitory effect of oridonin alone, cisplatin alone and oridonin + cisplatin on A2780/DDP and SKOV3/DDP human ovarian cancer cells was measured using the MTT assay. Cells were transferred to cispl-atin-free medium 3 days prior to the experiments. The cells (1.0×10⁴ cells/well) were plated into 96-well plates and allowed to attach overnight. The cells were treated with different concentrations of oridonin (10, 40, 80 and 160 µmol/l) or cisplatin (1, 2, 5, 10, 20, 40, 80, 200, 500 or 1,000 µM) for 24, 48 and 72 h, respectively. Control cells were administered an equal quantity of dimethyl sulfoxide (DMSO). MTT (20 µl; 5 mg/ml) was added to each well and incubated for 4 h at 37°C in the dark. The supernatant was removed and the formazan crystals were dissolved in 100 µl DMSO and mixed thoroughly prior to determining absorbance at a wavelength of 490 nm using an AquaMate-Plus ultraviolet spectrophotometer (Thermo Fisher Scientific, Inc.). All in vitro experiments were conducted in triplicate.

Flow cytometry was used to detect the apoptosis rate. A2780/DDP cells (2×10⁴ cells/well) were plated into 6-well plates. They were cultured for 6 to 8 h and treated with cisplatin alone, oridonin alone, or cisplatin in combination of oridonin for 48 h. The oridonin concentrations used were: 0, 10, 20, 40, 80 and 160 µmol/l and 50 µM cisplatin. The cells were analyzed following treatment with RNase (Sigma-Aldrich) and stained with Annexin V and propidium iodide (PI; Sigma-Aldrich) for flow cytometry (BD FACSCalibur™; BD Biosciences, Franklin Lakes, NJ, USA).

Western blotting was performed as described previously (23–25). Briefly, a total protein extract for each tissue sample or cell line was dissolved in lysis buffer and equal quantities of protein (60 µg) were analyzed by immu-noblotting. Rabbit anti-human polyclonal antibodies against Bcl-2 (cat. no. sc-492), rabbit anti-human polyclonal antibodies against Bax (cat. no. 526), rabbit anti-human polyclonal antibodies against MMP-2 (cat. no. sc-10736) and goat anti-human polyclonal IgG antibodies against MMP-9 (cat. no. sc-6840) were purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA) and used at a dilution of 1:1,000. The horseradish peroxidase-conjugated goat anti-rabbit secondary antibody was obtained from Abgent, Inc. (San Diego, CA, USA; cat. no. ASS1006).

All data were processed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Statistical analysis was performed using analysis of variance and Student's t-test for continuous data. The data are presented as the mean ± standard error of the mean and P<0.05 was considered to indicate a statistically significant difference.

In order to detect the anti-tumor effects of cisplatin on ovarian cancer cells, the A2780 and SKOV3 human ovarian cancer cell lines and the cisplatin-resistant sublines, A2780/DDP and SKVO3/DDP, were used as cell models. The ovarian cancer cells were treated with increasing concentrations of cisplatin and the inhibitory rate was determined by an MTT assay. The structure of oridonin and cisplatin is presented in Fig. 1. Cisplatin had an increasing antitumor effect in A2780 and A2780/DDP human ovarian cancer cell lines in a dose- and time-dependent manner. As presented in Fig. 2, the IC₅₀ values of cisplatin were 88.89, 13.20 and 9.55 µM for 24, 48 and 72 h in the A2780 sensitive cell line, respectively. However, the IC₅₀ values were 350.50, 50.96 and 25.39 µM in the A2780/DDP cisplatin-resistant cell line following treatment for 24, 48 and 72 h, respectively. IC₅₀ values in SKOV3 and SKOV3/DDP were 105.10, 51.73, 16.13 and 446.70, 135.00, 66.70 µM, respectively, for 24, 48 and 72 h.

The inhibitory effects of oridonin in ovarian cancer cells were also detected by an MTT assay. The A2780/DDP cisplatin resistant ovarian cancer cells were treated with increasing concentrations (10, 20, 40, 80 and 160 µM) of oridonin for 24, 48 and 72 h, respectively. As presented in Fig. 3A, the inhibitory effects increased in a dose-dependent manner.

In order to determine whether oridonin exerts synergistic antitumor effects with cisplatin in ovarian cancer cells, 20 µM was selected as the appropriate concentration for oridonin as the inhibition rate was effective at ~30% but relatively low. The concentrations of cisplatin used were 5, 10, 25, 50 and 100 µM. Compared with the group treated with oridonin alone, the inhibitory effects were significantly increased at 10, 25 (P<0.05) and 50 µM (P<0.01), which demonstrated that oridonin and cisplatin demonstrate synergistic antitumor effects in ovarian cancer cells.

Results from the present study demonstrated that oridonin synergistically increased the antitumor effects of cisplatin in A2780/DDP and SKOV3/DDP cells. The cells were treated with increasing concentrations of cisplatin in combination with 20 µM oridonin for 48 h. The samples had two replicates and the experiments were conducted twice. The untreated cells were used as negative controls. As presented in Fig. 4, the IC₅₀ values were calculated as 26.12 and 73.00 µM for 48 h. The ratio of IC₅₀ values was downregulated by ~1.95 and 1.84-fold in A2780/DDP and SKOV3/DDP cells treated with cisplatin + 20 µM of oridonin, respectively, which demonstrated that oridonin significantly (P<0.01) decreased the resistance to cisplatin in A2780/DDP and SKOV3/DDP cells.

The present study demonstrated that the cell death rates were significantly increased in the cisplatin and oridonin group. In order to identify whether cell apoptosis in A2780/DDP cells was induced by treatment with oridonin alone, cisplatin alone and oridonin in combination with cisplatin for 48 h, fluorescence-activated cell sorting (FACS) analysis was performed to detect the cell apoptosis rate in A2780/DDP cells. As presented in Fig. 5A, the cells were treated with 50 µM cisplatin alone, 50 µM oridonin alone, or 50 µM cisplatin + 50 µM oridonin for 48 h, and the late apop-tosis rate was 32.48% in the oridonin + cisplatin group, which was markedly higher than that of the oridonin or cisplatin alone groups. As presented in Fig. 5B, the early and late cell apoptosis rates were 42.67, 40.73 and 71.24% in the oridonin, cisplatin and oridonin + cisplatin groups, respectively. All the results demonstrated that oridonin and cisplatin act synergistically with cisplatin in inducing cell apoptosis in A2780/DDP cells.

In order to further elucidate the underlying mechanism of cell apoptosis in cisplatin-resistant ovarian cancer cells, the A2780/DDP cells were treated with oridonin at 10, 20, 40, 80 and 160 µM for 48 h. As presented in Fig. 6A, the result demonstrated that oridonin induced cell apoptosis of A2780/DDP cells, which increased in a dose-dependent manner.

The cell cycle distribution of A2780/DDP cells was also analyzed by the PI-staining method. As presented in Fig. 6B, G₀/G₁ phase arrest of the cells was induced and the number of cells in G₀/G₁ phase increased in a dose-dependent manner, while the number of cells in G₂/M phase decreased and the number in S phase was not markedly changed.

The expression levels of Bcl-2 family proteins were detected by western blot analysis. As presented in Fig. 6C, treatment with oridonin resulted in downregulation of Bcl-2 protein expression levels and the upregulation of Bax protein expression levels. This was consistent with cells that were treated with cisplatin alone. Notably, in A2780/DDP cells treated with 20 µM oridonin and 10 µM cisplatin for 48 h, the ratio of Bax/Bcl-2 was markedly higher than that of cells treated with cisplatin or oridonin alone. All data indicated that oridonin and cisplatin synergistically downregulated the protein expression levels of Bcl-2 and upregulated the protein expression levels of Bax.

MMPs, including MMP-2 and MMP-9, are involved in the invasion and metastasis in a number of types of human malignancy, as degradation of collagen IV in the basement membrane and extracellular matrix facilitates tumor progression. The expression levels of MMP-2 and MMP-9 in A2780/DDP cells treated with an increasing concentration of oridonin were detected. A2780/DDP cells were treated with increasing concentrations (10, 20, 40 and 80 µM) of oridonin for 48 h. As presented in Fig. 7, the protein expression levels of MMP-2 and MMP-9 decreased with increasing concentration of oridonin suggesting that oridonin may suppress the invasion and metastasis of human ovarian cancer cells.