Cell culture and transfection

SKOV3 cells were grown in McCoy’s 5A (Modified) Medium (Gibco), and A2780 cells were grown in RPMI-1640 Medium (Hyclone), under 5% CO₂ at 37°C. The two media above were supplemented with 10% fetal bovine serum (Hyclone), 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco). Cells were plated at 2 × 10⁵ cells per well in 6-well plates for siRNA transfection. Transfection was performed using Lipofectamine 3000 (Invitrogen), following the manufacturer’s instructions. Cells were transfected with siRNAs at a final concentration of 100 nM. siRNAs were ordered from Genepharma (Shanghai, China). FOXP3 siRNA: 5′-GGCGGACCAUCUUCUGGAUdTdT-3′.

Preparation of oridonin solution

Oridonin was bought from Abcam. Oridonin powder was dissolved in DMSO (Sigma) at 50 mM and stored at –80°C. Storage oridonin solution was diluted to 10 mM before use.

Western blot

Cells were harvested, washed with phosphate buffered saline (PBS) and lysed with lysis buffer (Sigma). The protein concentration of cell lysate was determined using the Bicinchoninic acid (BCA) protein assay (Invitrogen). Forty micrograms of proteins were resolved by electrophoresis on 8% or 10% Tris-glycine polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked in 2.5% skimmed milk for 1 hour and incubated overnight with the primary antibody to MMP-2, FAK, p-mTOR (Ser2448), mTOR (Cell Signaling, 1 : 1000 dilution), MMP-9, FOXP3 (abcam, 1 : 1000 dilution) or GAPDH (Bioworld, 1 : 2000 dilution) at 4°C. After washing three times, the membranes were incubated with the second antibody (ZSGB-Bio, 1 : 4000 dilution) for 2 h at room temperature. Blots of proteins were detected using a chemiluminescence detection system (CWBIO).

Cell proliferation and cytotoxicity assay

Cells were plated at 3 × 10³ cells per well in a 96-well plate 24 h before treatment. After treatment, cell viability was assessed using a CCK-8 Kit (Dojindo) following the manufacturer’s instructions. In brief, CCK-8 reagent was diluted in serum free medium in advance (1 : 10). Medium of samples was removed from the 96-well plate. Cells were washed with PBS, then CCK 8 reagent (100 μl/well) was added into the 96-well plate. Samples were incubated at 37°C, and the intensity of absorbance was assessed using a Multimode Reader after 2 h.

Transwell assay

Cells were suspended in serum free medium containing 0.1% BSA, and placed in transwell inserts (Corning Life Sciences) at a concentration of 1 × 10⁵ cells per insert. The final volume of medium is 200 μl for each transwell insert. Put transwell inserts in a 24-well plate containing complete medium. After 12 h’ incubation, erase cells in the inner side of the membrane. Migrated cells were fixed with 4% paraformaldehyde and stained with crystal violet (0.1%). Then remove the membrane and count cells. Images were taken of five areas of the membrane. An average cell count of the five images was then used in statistical analysis.

Cell apoptosis assay

Apoptotic cells were assessed using an ApoDETECT annexin V-FITC apoptosis detection kit (Sigma-Aldrich) by flow cytometry following the kit manual. Cells treated with different concentrations of oridonin were digested, washed twice with cold PBS, and resuspended in binding buffer. Add 5 μl of FITC-labeled annexin V in 190 μl of cell suspension, mix gently, then add 5 μl propidium iodide (PI) solution. Incubate cells in dark for 10 min at room temperature. Cells were analyzed on a flow cytometer (BD Biosciences).

Cell cycle assay

SKOV3 and A2780 cells were plated at the density of 5 × 10⁵ cells in 6 cm dishes and treated with different concentrations of oridonin. After 24 h, cells were collected and fixed in ice-cold 70% ethanol (v/v) overnight at 4°C. Wash cells with PBS and collect cells, resuspend cells in PBS containing 50 μg/ml PI, 100 μg/ml RNase A, 0.2% Triton X-100 and stain for 30 min in the dark, at 4°C. Then cells were analyzed by a flow cytometer (BD Biosciences). The results were calculated by the FlowJo software.

Statistical analysis

All data are shown as mean±SEM. Statistical analysis was performed with Student’s t-test to evaluate single-factor differences between two sets of data, or with ANOVA followed by the Bonferroni post-hoc test for multiple comparisons.

Oridonin inhibited cell proliferation and induced cell cycle arrest in human ovarian cancer cells

SKOV3 and A2780 cells were treated independently with oridonin at different concentrations for 24–72 h. After treatment, we used CCK8 assay to assess the cell viability of different cell lines. The proliferation of SKOV3 cells was almost abolished by 15 μM oridonin (Figure 1 A). The suppression of cell viability of A2780 was also strengthened along with the concentration of oridonin treatment (Figure 1 B). For the human normal liver cells, HL-7702 cells, only weak suppression of proliferation was detected compared with SKOV3 and A2780 cells (Figure 1 C). These results showed that cancer cells could be more sensitive to the treatment of oridonin compared with normal cells, indicating that oridonin is a potential anti-cancer drug, which is hypotoxic to normal tissue.

Figure 1

Oridonin inhibited cell proliferation and induced cell cycle arrest in human ovarian cancer cells. A–C – Cell viability of SKOV3 (A), A2780 (B), and HL-7702 (C) assessed using CCK8 assay after oridonin treatment (0–50 μM). Red: 24 h; Blue: 48 h; Green: 72 h. Data were normalized with control group (0 μM). The assays were performed in triplicate. D, E – Cell cycle distribution of SKOV3 (D) and A2780 (E) cells was assessed by flow cytometry after 24 h oridonin treatment (0–20 μM)

To further study whether the proliferation inhibition is related to cell cycle arrest, we assessed the cell progression of human ovarian cancer cells treated with different concentrations of oridonin by flow cytometry. In SKOV3 cells, treatment with oridonin (5–20 μM) induced an increased S phage cell population, from 35.85% to 43.17% (20 μM), while the cell population of G1 phage had no obvious increase (Figure 1 D, Table I). In A2780 cells, we observed that the G1 phage cell population increased from 70.75% to 87.10% (20 μM), while the cell population of S phage and G2 phage decreased (Figure 1 E, Table I). Combining the results above, the cell progression of human ovarian cancer cells was affected by oridonin, and the cell cycle was arrested in G1/S phage.

Oridonin induced human ovarian cancer cells apoptosis

To determine whether oridonin induces cell apoptosis in human ovarian cancer cells other than the inhibition of cell proliferation, we examined the apoptosis of human ovarian cancer cell lines after 12 h of oridonin treatment (5–20 μM) by flow cytometry. As Figure 2 shows, in SKOV3 (Figures 2 A, C) and A2780 cells (Figures 2 B, D), both early and late apoptosis rates were increased by oridonin treatment in two cell lines, in a dose-dependent manner (Figures 2 A, B). When treating human ovarian cancer cells with 10 μM or 20 μM oridonin, we observed significant apoptosis in SKOV3 and A2780 cells (Figures 2 C, D). Thus, oridonin induced cell apoptosis in human ovarian cancer cells.

Figure 2

Oridonin induced apoptotic cell death in human ovarian cancer cells. A, B – Representative images of flow cytometry analysis in SKOV3 (A) and A2780 (B) cells. Human ovarian cancer cells were treated with oridonin (0–20 μM) for 12 h, then stained with annexin V-FITC/PI, and analyzed by flow cytometry. The early and late apoptosis was quantified and indicated in Q3 and Q2 gates, respectively. C, D – Plot of the percentage of apoptotic cells of SKOV3 (C) and A2780 (D)

Results are presented as the mean ± SEM of three independent experiments. **P < 0.01.

Oridonin inhibited metastasis of human ovarian cancer cells through the mTOR pathway

Metastasis plays an important role in the development of cancer, and it is also a key standard in the assessment of new anti-cancer drugs. To evaluate the effect of oridonin on the metastasis of human ovarian cancer cell lines, we performed a transwell assay to assess the metastasis rates of oridonin-treated SKOV3 cells (1.25–5 μM). Twelve hours after oridonin treatment, erasing the cells in the inner side of the membrane, migrated cells were fixed and stained (Figure 3 A). Cell counting results showed that the treatment with oridonin suppressed the metastasis of SKOV3 cells significantly in the concentration of 2.5 μM and 5 μM (Figure 3 B). We also assessed the expression level of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) and focal adhesion kinase (FAK) by western blot. The expression of these metastasis-related factors decreased after treatment with oridonin in both SKOV3 and A2780 cells (Figures 3 C, D). These data indicated that oridonin not only inhibited the survival and proliferation, but also suppressed the metastasis of human ovarian cancer cells.

Figure 3

Oridonin suppressed the metastasis of human ovarian cancer cells. A – Representative images of migrated SKOV3 cells in transwell assay. SKOV3 cells were treated with oridonin (0–5 μM). After 12 h, the cells in the inner side of the membrane were erased, and migrated cells were fixed and stained. B – Plot of the metastasis rate of SKOV3 cells in transwell assay.**P < 0.01. C, D – Western blot analysis of MMP-2, MMP-9, FAK, FOXP3, p-mTOR and mTOR genes in SKOV3 (C) and A2780 (D) cells treated with oridonin (0–5 μM). GAPDH was used as an internal control

Additionally, we found that the mTOR pathway was suppressed by oridonin treatment. With a consistent level of total mTOR expression, the level of p-mTOR decreased along with the concentration of oridonin treatment (Figures 3 C, D). In order to illuminate whether this pathway participated in the anti-metastatic process induced by oridonin, we treated human ovarian cancer cells with an activator of the mTOR pathway, 3BDO, and found that the level of p-mTOR was increased in both SKOV3 (Figure 4 A) and A2780 (Figure 4 B) cells. Using a transwell assay, we assessed the metastasis rate of SKOV3 cells with treatment with oridonin and/or 3BDO. The metastasis rate rose after treatment with only 3BDO for 12 h (Figure 4 C). For the group co-treated with oridonin and 3BDO, the metastasis rate was reduced compared with the 3BDO only group. Also the pretreatment with 3BDO suppressed the anti-metastatic effect of oridonin in SKOV3 cells (Figure 4 C). Therefore, oridonin inhibited metastasis of human ovarian cancer cells via suppressing the mTOR pathway.

Figure 4

Activation of mTOR pathway attenuated the anti-metastatic effect of oridonin. A, B – Western blot analysis of p-mTOR, mTOR and FOXP3 genes in SKOV3 (A) and A2780 (B) cells treated with 3BDO (30 μM). GAPDH was used as an internal control. C – Representative images of migrated SKOV3 cells in transwell assay. SKOV3 cells were pretreated with or without 3BDO (30 μM) for 12 h, then oridonin was added (0 or 5 μM). After 12 h, the cells in the inner side of the membrane were erased, and migrated cells were fixed and stained

On the other hand, it is unexpected that we also found that FOXP3 was up-regulated when SKOV3 and A2780 cells were treated with oridonin, and the FOXP3 expression level increased along with the raised concentration of oridonin (Figures 3 C, D). FOXP3 is a member of the forkhead/winged-helix family of transcriptional regulators, and has been demonstrated to play important roles in the development and function of regulatory T cells [25–28]. In addition, studies have recently shown that FOXP3 is also expressed in some tumor cells, and influenced the development of different cancers [29–34]. To clarify the correlation between the up-regulation of FOXP3 and the suppression of metastasis, we performed experiments to investigate whether the low level of FOXP3 influences the metastasis of human ovarian cancer cells. We designed siRNA targeting FOXP3, transfected siRNA for 24 h in SKOV3 and A2780 cells, added 2.5 μM oridonin, treated for 12 h, and assessed the expression levels of MMP-2, MMP-9, FOXP3, mTOR and p-mTOR by western blot. We found under the condition of a lower level of FOXP3 that the anti-metastatic effect of oridonin was suppressed. The suppression of MMP-2 and MMP-9 expression was rescued partially in both SKOV3 and A2780 cells (Figures 5 A, B). At the same time, we performed a transwell experiment to assess the metastasis rate of SKOV3 cells co-treated with siRNA targeting FOXP3 and oridonin. Cells were plated 24 h after the siRNA transfection. Twelve hours after oridonin treatment, we found that cells transfected with FOXP3 siRNA had a higher metastasis rate compared with the cells transfected with NC siRNA (Figures 5 C, D). These results indicated that FOXP3 plays an important role in the anti-metastatic process of oridonin.

Figure 5

Low level of FOXP3 weakened the anti-metastasis effect of oridonin. A, B – Western blot analysis of MMP-2, MMP-9, FOXP3, p-mTOR and mTOR genes in SKOV3 (A) and A2780 (B) cells co-treated with oridonin and FOXP siRNA. SKOV3 cells were transfected with or without FOXP siRNA (50 nM) for 24 h, then oridonin was added (0 or 2.5 μM). After 12 h, cells were harvested for western blot. GAPDH was used as an internal control. C – Representative images of migrated SKOV3 cells co-treated with oridonin and FOXP siRNA in transwell assay. D – Plot of the metastasis rate of SKOV3 cells in transwell assay. **P < 0.01

Meanwhile, we found that siRNA targeting FOXP3 did not disrupt the level of p-mTOR or mTOR expression (Figures 5 A, B), while, with the treatment with 3BDO, we observed that FOXP3 was down-regulated along with the activation of the mTOR pathway (Figures 4 A, B). Thus, FOXP3 was negatively regulated by the mTOR pathway. Combine with the former results, oridonin inhibited the mTOR pathway, and then up-regulated the downstream gene FOXP3, and induced suppression of metastasis.