Oridonin (>98%) was purchased from National Institutes for Food and Drug Control (Beijing, China). Doxorubicin was obtained from Pharmacia Italia S.p.A., Gaggiano, Italy. Hydroxycamptothecin (HCPT) was provided by Shanghai Longxiang Biological Medicine Development Co. Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO), trypsin, Tris, glycine, acrylamide, methylene diacrylamide and Tween-20 were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-1640 cell culture medium was purchased from Gibco (Grand Island, NY, USA). Fetal calf serum (FCS) was purchased from Sijiqing Hangzhou Bio Engineering Co., Ltd. (Hangzhou, China). Hoechst33342, Annexin V-FITC apoptosis detection kit, DAB Horseradish Peroxidase Color Development kit were obtained from the Beyotime Institute of Biotechnology (Jiangsu, China). Antibodies for cytochrome c, Bcl-2, Bax, caspase-3, cleaved-caspase-3, β-actin, and the secondary antibodies were purchased from ZSGB-BIO (Beijing, China). All other chemicals and solvents used were the highest purity grade.

The human gastric cancinoma SGC-7901 cell line was obtained from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in RPMI-1640 supplement with 10% (v/v) fetal bovine serum (FBS) and antibiotics (100 IU/ml of penicillin and 100 μg/ml of streptomycin) at 37°C in a humidified atmosphere containing 5% CO₂.

The cultured cells at the exponential growth phase were harvested from the culture flasks by trypsin and then re-suspended in fresh RPMI-1640 medium. The cell suspensions were dispensed into a 96-well microplate at 100 μl/well and placed in an incubator with 5% CO₂ at 37°C. After 24 h, 100 μl various concentrations of oridonin were added and incubated for 72 h. Then the medium was discarded and 100 μl of MTT stock solution (1 mg/ml) was added. After incubation for 4 h, DMSO (150 μl) was added to each well to solubilize the water-insoluble purple formazan crystals. The amount of MTT-formazan is directly proportional to the number of living cells and was determined by measuring the optical density (OD) at 570 nm using microplate reader (model 680; Bio-Rad Laboratories, Hercules, CA, USA). The percentage of cytotoxic activity compared to the untreated cells was determined, and the IC₅₀ was calculated by the Logit method.

Morphology of apoptotic cell was observed by Hoechst 33342 staining assay. The cells were washed in phosphate-buffered saline (PBS) and fixed in formaldehyde solution (4%, w/v) for 30 min. Then the fixed cells were stained with 10 mg/ml Hoechst 33342 for 10 min, and nuclear morphology was observed under a fluorescence microscopy (Leica, Wetzlar, Germany) equipped with a digital camera.

Qualitative experiment of apoptosis was observed by confocal laser scanning microscopy after staining cells with the Annexin V-FITC apoptosis detection kit (MultiSciences Biotech Co., Ltd., Hangzhou, China). SGC-7901 cells (1.5×10⁵ cells/well) were placed on 6-well plates and incubated with oridonin for 24 h. The cells were stained by Annexin V-FITC (green fluorescence) in the dark for phosphatidylserine (PS) examination. Then cells were stained with PI (red fluorescence) in the dark for nucleus examination. Stained cells were visualized by confocal laser scanning microscopy (Leica, SP2, Wetzlar, Germany) equipped with 488 nm Argon lasers (31).

Early apoptosis rate were measured using the Annexin V-FITC apoptosis detection kit (MultiSciences Biotech Co. Ltd., China) as described in the supplier instructions. After exposure to oridinin (0, 20, 40 and 80 μM) for 24 h, cells were harvested by centrifugation, washed twice with PBS, and resuspended in Binding Buffer, 5 μl of Annexin V-FITC and 5 μl of propidium iodide (PI, 50 mg/ml) was added and incubated at room temperature in the dark. The data acquisition and analysis were performed using MultiCycle software flow cytometry (Beckman Coulter, XL, USA).

SGC-7901 cells were treated with different concentration of oridonin. For isolation of total protein fractions, cells were collected, washed twice with cold PBS, and lysed with cell lysis buffer (50 mM Tris-Cl, pH 8.0, 120 mM NaCl, 50 mM NaF, 200 μM sodium vanadate, 0.5% NP-40, 10 mM phenylmethylsulfonyl fluoride (PMSF), 2 μg/ml aprotinin 0.2 μl, 10 μg/ml leupeptin 10 μl). The lysates were centrifuged at 12000 × g for 10 min at 4°C, the supernatant was saved at −20°C. Protein concentrations of cell lysates were detected by Bradford assay (32). Total protein samples were separated by SDS-PAGE. The separated proteins were transferred to NC membranes. After being blocked with blocking solution (5% skim milk in TBS, 10 mM Tris-HCl, 150 mM NaCl, pH 7.5 plus 0.1% Tween-20) at room temperature for 2 h. Each membrane was incubated with primary antibodies overnight at 4°C. Afterwards, the membranes were probed with the appropriate horseradish-peroxidase conjugated secondary antibody for 2 h at room temperature. Detection was performed by the DAB Horseradish Peroxidase Color Development kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Bands were recorded and relative density units of the bands were analyzed by Gel Imaging System (Tanon, GIS-2019, Beijing, China). Densitometrical data of multiple experiments are shown.

The data are presented as the mean ± SD. Statistical significance was calculated using Student's t-test. P-values of ≤5% were considered to indicate statistically significant differences.

In order to evaluate the effect of oridonin on proliferation of the SGC-7901 cells, the cells were treated with different concentrations of oridonin for 72 h, the cell viability was quantitated by MTT assay. The results showed that oridonin inhibited the proliferation of SGC-7901 cells, and the IC₅₀ was 22.74 μM. The results are shown in Table I.

The results above can significantly demonstrate that oridonin possessed notable antitumor activity on human gastric cancer SGC-7901 cells. To determine whether the antitumor activity of oridonin was due to induction of apoptosis, SGC-7901 cells were stained with Hoechst 33342 to examine the nuclear morphological changes. The results showed that cells of the control group had normal nuclear morphology and the dye of Hoechst 33342 was evenly distributed under fluorescent microscope, which indicated that the chromatin was equivalently distributed in the nucleus. However, after treatment with different concentrations of oridonin for 24 h, the characteristic features of apoptosis (including marked nuclear fragmentation, nuclear blebbing, condensation of chromatin, and emitting brighter fluorescence) was clearly detected in the SGC-7901 cells under the inverted fluorescence microscope (Fig. 2Ac–e). These results indicated that oridonin induced cell apoptosis in human gastric carcinoma.

In order to confirm whether oridonin induced cell apoptosis, we applied the assay of Annexin V-FITC stain for detection of phosphatidylserine (PS), the biochemical marker of apoptosis. PS is normally located in the inner plasma membrane, however, in the early apoptosis the PS is transferred to its outer surface. Annexin V-FITC combined with PS of the outer surface of the membrance and emit green fluorescent. After treatment with different concentrations of oridonin for 24 h, SGC-7901 cells were stained by Annexin V-FITC (green fluorescence) and PI (red fluorescence), and observed and photographed by laser scanning confocal microscopy. The results showed that a large number of cells treated with oridonin were positively stained by Annexin V-FITC (Fig. 2Bc–e). Which showed that oridonin was able to induce SGC-7901 cell apoptosis.

To quantify the apoptotic rate of oridonin on SGC-7901 cells, the Annexin V-FITC/PI staining and flow cytometry was adopted. The data obtained showed that oridonin induced early apoptosis of SGC-7901 cells in a dose-dependent manner (Fig. 3 and Table II). When the cells were treated with 0, 20, 40, 80 μmol/l oridonin for 24 h, the average proportion of Annexin V-staining positive cells and PI-staining negative cells (early apoptotic cells) significantly increased from 1.53%±0.67% in control to 3.33%±0.29, 84.80%±0.82 and 96.43%±0.51%, respectively (Table I). Fig. 3B shows the graphic representation of the increase in the early apoptotic cells with increase in the dose of oridonin.

Whether or not oridonin induced apoptosis in SGC-7901 cell through the effects of apoptosis-associated protein, western blots were adopted to examined the protein expression of mitochondrial pathway of SGC-7901 cell treated with 2, 4, 8 μM oridonin. The analysis results showed that all concentrations of oridonin (2, 4, 8 μM) resulted in a significant increase of cytochrome c (Fig. 4A) and remarkable cleavage of caspases-3 were detected compared with the control group (Fig. 4B). Mitochondrial dysfunction is regulated by Bcl-2 family proteins, thus the Bcl-2 family proteins were examined in the study. Oridonin caused a significant reduction in Bcl-2 expression whereas the expression of Bax was significantly increased, which led to decrease of Bcl-2/Bax in a concentration-dependent manner (Fig. 4C). Thus, oridonin could induce apoptosis in SGC-7901 cells with mitochondrial pathway involved.