Oridonin was obtained from the Beijing Institute of Biological Products (Beijing, China). The purity of oridonin was measured by high-performance liquid chromatography (HPLC) and determined to be 99.4%. Oridonin was dissolved in dimethyl sulfoxide (DMSO) to create a stock solution at a concentration of 10 mmol/l and stored at −20°C. The DMSO concentration was kept below 0.1% in all the cell cultures and did not exert any detectable effect on cell growth or cell death. Gemcitabine (Gemzar) from Eli Lilly was stored at 4°C and dissolved in sterile phosphate-buffered saline (PBS) at a concentration of 0.2 mmol/l on the day of use. Fetal bovine serum (FBS), trypsin-EDTA, Roswell Park Memorial Institute-1640 (RPMI-1640) and the Cell Counting kit-8 (CCK-8) were obtained from Gibco, the Annexin V-FITC/PI apoptosis detection kit was from Biological Development Co, Ltd. Nanjing KGI, the RNA extraction kit was from Life Technologies Co., the cDNA first-strand synthesis kit was from Fermentas and the 2X Taq PCR MasterMix was from Tiangen. Ribonuclease A (RNase A), propidium iodide (PI) and DMSO were obtained from Sigma. Antibodies against p53, phosphorylated p53 (P-p53), p38, phosphorylated p38 (P-p38), β-actin and horseradish peroxidase (HRP)-conjugated secondary antibodies (goat-anti-rabbit and goat-anti-mouse) were purchased from Sigma.

The PaCa cell line, BxPC-3, was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). All cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. Cells were maintained at 37°C in a humidified atmosphere of 5% CO₂. The medium was changed every 2–3 days, and the cells were subcultured when confluency reached 70–80% by 0.25% trypsin at 37°C.

Cell survival was determined using CCK-8. Briefly, the logarithmic phase PaCa cells were plated in 96-well culture plates (7×10³ cells per well). After 24 h of incubation, the cells were treated with the vehicle alone (0.1% DMSO) and various concentrations (20, 40, 60, 80 and 100 μM) of oridonin, followed by 24, 48 and 72 h of cell culture. Each group had 6 wells. A total of 10 μl CCK-8 was added to each well 1 h before the end of incubation. The absorbance at 450 nm was read using the Bio-Tek ELx800 Absorbance Microplate Reader. The experiment was repeated 3 times. The degree of cellular inhibition by each drug was calculated by the following formula: relative % inhibition = 1- (dosing absorbance - blank absorbance)/(control absorbance - blank absorbance) ×100%.

Apoptosis induction was assessed using the Annexin V-FITC kit according to the manufacturer’s instructions. BxPC-3 PaCa cells were exposed to the control (DMSO-treated), the desired concentration of oridonin (40 μM) and gemcitabine (20 μM) alone or in combination with oridonin for 48 h, and the floating and adherent cells were collected by centrifugation at 1,000 x g for 5 min. Pooled cells were washed with the binding buffer supplied by the manufacturer. Approximately 5×10⁴ cells were suspended in 200 μl of PBS, and mixed with 5 ml of Annexin V-FITC and 10 ml of PI. After 15 min of incubation in the dark, the fluorescence intensities of oridonin of >10,000 viable cells from each cell sample were analyzed by using a Coulter Epics XL flow cytometer with excitation and emission settings of 488 and 525 nm, respectively. The data were analyzed using CellQuest software.

The effect of oridonin on cell cycle distribution was determined by flow cytometry analysis of the DNA content in the cell nuclei following staining with PI. Asynchronized (70–80%) confluent cells were treated with 40 μM oridonin for 48 h. The control cells were treated with 0.1% DMSO only. After incubation at 37°C for a specified time, the floating and adherent cells were collected by using 0.05% trypsin, washed twice with cold PBS and then fixed with ice-cold 70% ethanol overnight at 4°C. The fixed cells were subsequently centrifuged at 300 x g for 10 min and the pellets were washed with PBS. The cells were then treated with 80 mg/ml RNase A for 30 min at 37°C. The cells were chilled over ice for 10 min and stained with PI (50 mg/ml final concentration) for 1 h in the dark. The stained cells were analyzed using a Coulter Epics XL flow cytometer. Approximately 20,000 cells were evaluated for each experiment. In all the determinations, cell debris and clumps were excluded from the analysis. The cell cycle data were reanalyzed using ModFit software.

PaCa cells were treated with the assigned concentration oridonin (40 μM) and gemcitabine (20 μM) alone or in combination for 48 h. The control cells were treated with 0.1% DMSO only. Subsequently, total cellular RNAs were isolated from the cells with TRIzol reagent, and the RNA content was measured using a UV spectrophotometer under 260 nm. cDNA was synthesized with 1 μg of total RNA and oligo(dT) primer according to the manufacturer’s instructions. PCR amplification conditions were as follows: p38, 94°C 60 sec, 61.8°C 60 sec, 72°C 60 sec, 35 cycles; p53, 94°C 45 sec, 51.9°C 1 min, 72°C 90 sec, 35 cycles; GAPDH, 94°C 45 sec, 58°C 1 min, 72°C 1 min, 35 cycles. GAPDH was used as the internal control. The primer pairs used for the amplification are listed in Table I. The PCR product (5 μl) was analyzed by 1% agarose gel electrophoresis and the results were photographed.

Briefly, PaCa cells were incubated with the allocated concentration of oridonin (40 μM) and gemcitabine (20 μM) alone or in combination for 48 h; the cells in medium alone were used as the controls. Subsequently, the cells were harvested, washed twice with ice-cold PBS and then the cell pellets were resuspended in lysis buffer consisting of 50 mM HEPES (pH 7.4), 1% Triton-X 100, 2 mM sodium orthovanadate, 100 mM sodium fluoride, 1 mM edetic acid, 1 mM PMSF, 10 mg/l aprotinin (Sigma), and 10 mg/l leupeptin (Sigma) and lysed at 4°C for 60 min. After centrifugation at 13,000 × g for 15 min, the protein content of the supernatant was determined by the bicinchoninic acid (BCA) assay kit (Sigma) according to the manufacturer’s instructions. The protein lysates (20 μg/lane) were separated by electrophoresis on 12% SDS polyacrylamide gel and blotted onto a nitrocellulose membrane. Each membrane was blocked with 5% skim milk and then incubated with the indicated primary antibodies against p53, P-p53, p38, P-p38 and β-actin overnight at 4°C. Subsequently, the membrane was incubated with the secondary antibodies, goat anti-rabbit and goat anti-mouse IgG conjugated with HRP, for 1 h at room temperature and the formed immunocomplex was visualized by enhanced chemiluminescence reagent and exposed to X-ray films. Quantitative data are expressed as the means ± SD of the relative levels of the objective protein and control β-actin of each group of cells from 3 independent experiments.

Female nude mice [4–6 weeks old, BALB/cA-nu (nu/nu), weight 18–20 g] were purchased from the Shanghai Cancer Institute for Tumor Implantation and maintained in a specific-pathogen-free environment at the Wenzhou Medical College Experimental Animal Center. The animals were kept in a controlled environment with a temperature of 25±1°C and relative humidity of 40–60%. Ethical approval for this study was given by the Ethics Committee at Wenzhou Medical College.

BxPC-3 cells in the log-phase were suspended with serum-free culture medium (5×10⁶ cells in 200 μl), and tumor xenografts were established by a subcutaneous inoculation of BxPC-3 PaCa cells into the right abdominal flanks of the nude mice. Tumors were allowed to develop for 4–5 weeks until they reached a size of 100–150 mm³, and then treatment was initiated. All the mice were randomly divided into 4 groups and treated intraperitoneally (IP) with saline, gemcitabine (80 mg/kg) alone, oridonin (40 mg/kg) alone, or gemcitabine (80 mg/kg) and oridonin (40 mg/kg) in combination every 3 days for up to 30 days post-implantation in a volume of 0.2 ml (n=10 per group). Tumor volume was measured using calipers and estimated according to the formula: tumor volume (mm³) = L × W²/2, where L is the length and W is the width. Two weeks after the last treatment, the mice were sacrificed and the tumors were excised. Xenograft tumors of the treated and control mice, were harvested and fixed in 4% formalin, embedded in paraffin, and cut into suitable sections for western blot and RT-PCR analysis.

Protein was routinely extracted from tumor tissues using radioimmunoprecipitation assay (RIPA) buffer. Total protein concentration was measured using the BCA assay kit (Sigma) with bovine serum albumin as the standard, according to the manufacturer’s instructions. Tumor tissue extracts containing 80 μg total protein were subjected to 12% SDS/PAGE, and the resolved proteins were transferred electrophoretically to polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% fat-free milk, the membranes were probed with individual primary antibodies overnight at 4°C and the bound antibodies were detected with HRP-conjugated goat anti-rabbit IgG for 1 h. The formed immunocomplex was visualized by enhanced chemiluminescence reagent (ECL; Pierce).

Total RNA was extracted from the tumor tissues using the TRIzol reagent. The amount and purity of the extracted RNA was quantified using spectrophotometry. The value of A₂₆₀/A₂₈₀ was measured to evaluate the quality of the RNA. cDNA was synthesized with 5 μg total RNA and oligo(dT) primer according to the manufacturer’s instructions. The PCR amplification reaction was amplified using the GeneAmp PCR System 9600 (Perkin-Elmer Corp. Norwalk, CT, USA). The amplification conditions and the primer pairs used for the amplification were the same as those used for the in vitro analysis. The PCR products (5 μl) were analyzed by electrophoresis on 1% agarose gel with ultraviolet (UV) illumination and the results were photographed.

All results were confirmed by at least 3 separate experiments. The data are expressed as the means ± SD. Statistical comparisons were performed by one-way ANOVA and the Student’s t-test for differences between two sample means using the SPSS v17.0 software. A P-value <0.05 was considered to indicate statistically significant differences.

We first investigated the effect of oridonin on the proliferation of the PaCa cell line, BxPC-3. Oridonin inhibited the growth of PaCa cells (BxPC-3) in a dose- and time-dependent manner (Fig. 1). These tumor cells showed sensitivity to the oridonin treatment. The half maximal inhibitory concentration (IC₅₀) value of oridonin was determined as 38.86 μM for the BxPC-3 cells following treatment for 48 h. Oridonin was used at the dose of 40 μM.

To determine whether oridonin enhances the induction of apoptosis by gemcitabine, we investigated the occurrence of apoptosis in the cell lines. Flow cytometry analysis was performed using Annexin V-FITC/PI-stained PaCa cells. The cells were cultured with oridonin and gemcitabine, alone or in combination for 48 h. The results showed that, at a dose at which oridonin and gemcitabine alone were minimally effective, the combination of both was highly effective (Fig. 2).

We also investigated the mechanisms by which oridonin potentiates the effects of gemcitabine in these cells. The DNA content of the PaCa cells treated for 48 h with 40 μM oridonin or the vehicle (0.1% DMSO) was analyzed using a flow cytometer. Fig. 3 shows that there were marked and consistent changes in the cell cycle at 48 h. The number of cells in the G₁ phase increased significantly with a concomitant decrease in the number of treated cells in the S and G₂ phase when compared with the control. These results suggest that oridonin treatment suppresses the growth of PaCa cells, and synergizes the apoptotic effects of gemcitabine.

The tumor suppressor gene product, p53, has been reported to mediate apoptosis in a number of experimental systems. The MAPK family members, including the extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, play important roles in the regulation of apoptosis (30). Therefore, in order to confirm whether MAPK-p38 and its downstream p53 protein are involved in the anti-cancer effects of oridonin, western blot analysis was carried out. After treatment of the PaCa cells with oridonin and gemcitabine alone or combination for 48 h, the results showed that both oridonin (P<0.05) and gemcitabine (P<0.05) alone significantly increased the level of P-p38 and P-p53 (Fig. 4), as compared with that of the control cells, and that the combination of both agents was even more effective (P<0.01). The unphosphorylated forms of p38 and p53 were not altered by oridonin and gemcitabine alone or combination treatment (data not shown). These results suggest that oridonin can improve the p38 activation of gemcitabine in PaCa cells, which contributes to the further activation of p53. This could further increase PaCa cell apoptosis.

To further clarify whether the possible mechanism is relevant with the p38 and p53 gene, RT-PCR was carried out to detect the p38 and p53 mRNA expressions. In the PaCa cell line, BxPC-3, when compared with the control, gemcitabine alone significantly upregulated the expression of p38 and p53 mRNA (P<0.05). Treatment with oridonin alone or in combination with gemcitabine significantly upregulated the expression of p38 and p53 mRNA (P<0.05). The upregulation induced by the combined treatment of oridonin and gemcitabine was even more evident than the other groups (P<0.05) (Fig. 5). These results indicate that p38 and p53 participate in the apoptosis induced by oridonin and gemcitabine in PaCa cells.

Our in vitro data prompted us to examine whether the effects of oridonin and gemcitabine are equally demonstrable in vivo. We examined the effects of oridonin and gemcitabine, alone or in combination, on the growth of subcutaneously implanted pancreatic tumors in nude mice. The experimental protocol is depicted in Fig. 6. We found that the oral administration of oridonin alone at 40 mg/kg significantly inhibited tumor growth (P<0.05 when compared to the control) (Fig. 6). Gemcitabine alone was as effective as oridonin (P<0.05 when compared to the control, P>0.05 when compared to the oridonin alone group), and the combination of both agents was even more effective in reducing the tumor burden. The tumor volume in the combination group was significantly lower than in the oridonin alone (P<0.05) or gemcitabine alone group (P<0.05) (Fig. 6).

To determine whether the impact on the tumor growth inhibition of oridonin was related to the p38 and p53 activation, we evaluated the effects of oridonin and gemcitabine on P-p38 and P-p53 levels, by western blot analysis in the pancreatic tumor tissues. The results showed that both oridonin (P<0.05) and gemcitabine (P<0.05) alone significantly upregulated the expression of P-p38 and P-p53 in the PaCa tissue as compared to the control group and that the combination of both was even more effective (P<0.05 when compared to gemcitabine alone or the control) (Fig. 7).

In order to further confirm our conclusion in pancreatic tumor tissues. we investigated the expression of p38 and p53 mRNA levels in pancreatic tumor tissues. The results showed that oridonin either alone or in combination with gemcitabine (P<0.05 vs. the mice treated with gemcitabine or the control) was effective in boosting the expression of p38 and p53 mRNA in PaCa tissue (Fig. 8).