Antibodies against caspase-3, -9, Bcl-2, Bax, survivin, cytochrome C, cathepsin D, p53 and NF-κB were purchased from Santa Cruz Biotechnology, Inc. Anti-mouse or anti-rabbit antibodies were purchased from Beijing Solarbio Science & Technology Co., Ltd.

Panc-28, a human pancreatic cancer cell line, was cultured in DMEM/F12 supplemented with 10% FBS plus antibiotics of penicillin and streptomycin (Sigma) at 37°C in 5% CO₂ in air. Cells were grown in 96-, 24- and 6-well plates.

OA (Sigma) and 5-FU (Sigma) were dissolved in dimethylsulfoxide (DMSO). Cells were pretreated with OA for 12 h. Then, the supernatant fluid containing OA was removed and the fresh medium containing 5-FU was added, and were incubated at 37°C for another 24 h and harvested for the following examinations.

Inhibition of cell proliferation by OA was measured by MTT assay (20). Briefly, cells were grown in a 96-well plate (5×10³/200 μl/well) overnight, and were treated with OA (20, 30 and 40 μg/ml) for 12 h, and then the supernatant fluid was removed. The fresh medium with 5-FU (10 μg/ml) was added. After incubation for another 24 h, 40 μl of MTT solution (5 mg/ml) was added to each well. After incubation for additional 4 h, the formazan precipitate was dissolved in 150 μl DMSO, and then the absorbance at 490 nm was measured using ELx808™ Absorbance Microplate Reader (BioTek, USA). The nature of the interactions between studied drugs was estimated by using published methods (21,22), based on the principles described by Chou and Talalay (23,24). Briefly, the expected value of combination effect between treatment with OA and treatment with 5-FU is calculated by the following formula: [(observed treatment with OA value)/(control value)] × [(observed treatment with 5-FU value)/(control value)] × (control value), and the coefficient of drug interaction (CDI) is calculated as (observed value)/(expected value). CDI of <1 indicates a synergistic effect and CDI of >1 indicates a less than additive or an antagonistic effect.

Flow cytometry analysis was performed as described by Luo et al (25). Briefly, apoptosis was assessed by the binding of annexin V-FITC to phosphotidylserine, which was externalized to the outer leaflet of the plasma membrane. Cells treated with OA (30 μg/ml) and 5-FU (10 μg/ml) were resuspended in the binding buffer (Nanjing KeyGen Biotech Co., Ltd., China) and reacted with 5 μl of annexin V-FITC reagent and 5 μl of PI for 30 min at room temperature in the dark. Stained cells were analyzed by flow cytometry.

DNA fragmentation was analyzed by agarose gel electrophoresis (26). Cells untreated or treated with 5-FU, OA or their combination were collected by centrifugation and washed with PBS. Total DNA was purified with a universal genomic DNA extraction kit (Takara Biotechnology Co., Ltd, China) according to the manufacturer’s instructions. The DNA was resolved in 2% agarose gel and visualized by ultraviolet illumination (the Bio-Rad Chemi Doc XRS imaging system, USA) after staining with ethidium bromide.

The uptake of the cationic fluorescent dye rhodamine 123 was used for the estimation of mitochondrial membrane potential (27). Cells untreated or treated with reagents as described above were harvested and washed twice with PBS. The cell pellets were then resuspended in 2 ml fresh medium containing rhodamine 123 (2.0 μM) and incubated at 37°C for 10 min with gentle shaking. Cells were collected by centrifugation and washed twice with PBS, then analyzed by fluorescence microscopy and fluorescence spectrophotometer (excitation 507 nm, emission 529 nm).

LMP was measured by the lysosomotropic base and metachromatic fluorochrome acridine orange as described previously (28). Briefly, cells untreated or treated with reagents as described above were stained with acridine orange (0.005 μg/ml, Sigma) for 6 min at 37°C without CO₂. Immediately after staining, cells were analyzed by fluorescence microscopy.

Western blot analysis was performed as previously described (29) to determine the effect of OA and 5-FU on anti-/pro-apoptotic proteins, cells untreated or treated with OA, 5-FU or their combination were harvested by centrifugation at 3000 g for 10 min, washed twice with ice-cold PBS, and lysed with RIPA buffer containing fresh protease inhibitor mixture (50 μg/ml aprotinin, 0.5 mM phenylmethanesulfonyl fluoride (PMSF), 1 mM sodium orthovanadate, 10 mM sodium fluoride and 10 mM β-glycerolphosphate). Proteins were quantified using the BCA protein assay (Biocolor BioScience & Technology, China). Protein samples were resolved on 10–15% SDS-polyacrylamide gels transferred to nitrocellulose membranes and probed with protein specific antibodies followed by treatment with HRP-conjugated secondary antibody. The relative quantity of protein were analyzed by Gel-Pro Analyzer software.

All of the experiments were performed at least three independent experiments, and the data are presented as the mean ± SD. The statistical significance of the mean difference between the control and treated groups was determined by a paired t-test. P<0.05 was considered statistically significant.

In order to determine the effect of OA and 5-FU on Panc-28 cells, the respective cell viability of OA and 5-FU were measured by MTT assay. As shown in Fig. 1A, the proliferation was decreased to 98, 95 and 64% in Panc-28 cells treated with 20, 30 and 40 μg/ml OA, respectively, while the viability of Panc-28 cells treated with 10 μg/ml 5-FU was ~84%. However, in the present of OA (20, 30 and 40 μg/ml), the cell viability was decreased to 57, 41, and 15%, respectively. The CDIs were 0.69, 0.58 and 0.37 respectively (Fig. 1B). This result indicates that the combined treatment increases the inhibition effect on Panc-28 cells, and OA synergistically potentiates the effect of 5-FU on the growth inhibition of human pancreatic Panc-28 cells.

As have been confirmed earlier, OA or 5-FU induced cell death in apoptotic pathway (19,30,31). We next determine if the combination of the two compounds increases the apoptotic effect. As shown in Fig. 2, the average percentage of apoptotic cells increased significantly when treating the cells with either OA or 5-FU; the apoptotic rate was 4.78 and 7.14% in cells treated with OA (30 μg/ml) or 5-FU (10 μg/ml) respectively. However, combinations of the two reagents produced significant increase in apoptotic cells; the apoptotic rate induced by combination treatment increased to 35.33% (Fig. 2A and B). In addition, fragments of degraded DNA were visible in Panc-28 cells after treatment with the combination of OA (30 μg/ml) and 5-FU (10 μg/ml), while OA or 5-FU alone did not induce DNA fragmentation (Fig. 2C). The result reveals that the combined treatment with OA and 5-FU could enhance cell death via apoptotic pathway in Panc-28 cells.

To determine the underlining mechanism of apoptosis induced by the combination of OA and 5-FU, we measured the MMP change and the level of the cytochrome C in cytosol. As shown in Fig. 3A and B, treatment of the cells with OA at 30 μg/ml or 5-FU at 10 μg/ml did not affect the fluorescence intensity significantly, and the fluorescence intensity of the cells treated with the combination of OA and 5-FU resulted in slight decrease of the fluorescence intensity as determined by fluorescence microscopy (Fig. 3A) and fluorescence spectrophotometer (Fig. 3B). We then checked the expression of cytochrome C and the results showed that the release of cytochrome C from mitochondria to cytosol was not changed in Panc-28 cells either treated with OA, 5-FU or treated with the combination of OA and 5-FU (Fig. 3C). The results suggested that the enhancement of apoptosis induced by the combination of OA and 5-FU might not be mediated by the mitochondrial pathway.

To determine if LMP is affected by the combination of OA and 5-FU, we analyzed the alterations in Panc-28 cells using acridine orange staining. The results showed that the fluorescence intensity of the cells treated with OA or 5-FU alone did not have a significant change, while the cells treated with the combination of OA and 5-FU displayed less intense red fluorescence and more intense green fluorescence (Fig. 4A). We next detected the level of cathepin D in Panc-28 cells, and the result showed that the level of cathepsin D in cytosol was elevated significantly in cells treated with the combination of OA and 5-FU compared to OA or 5-FU alone (Fig. 4B). The results indicated that the combined use of OA and 5-FU could enhance the LMP following the increase of cathepin D leakage and that LMP pathway may play an important role in the apoptosis induced by the combination of OA and 5-FU.

Since caspases are the important mediators of apoptosis, we next checked the expression of caspases. As shown in Fig. 5, treatment the cells with the combination of OA and 5-FU resulted in a significant increase in caspase-3 activity compared to that treated with OA or 5-FU alone, while the activity of caspase-9 did not have a significant change (Fig. 5). The results further confirmed that the enhancement of apoptosis induced by the combination of OA and 5-FU was not dependent on the caspase-9 mediated mitochondrial pathway.

The expression of Bax, and Bcl-2 was determined by western blot analysis. As shown in Fig. 6, the expression of Bax was increased, while the expression of Bcl-2 was decreased significantly in cells treated by the combination of OA and 5-FU compared to that treated with OA or 5-FU alone (Fig. 6). Additionally, the combined treatment with OA and 5-FU inhibited the level of survivin in cytosol significantly (Fig. 6). Thus, the data confirmed that combination of OA and 5-FU could regulate the level of some apoptosis-related proteins.

The expression of p53 and NF-κB was also determined by western blot analysis. Treatment of human pancreatic Panc-28 cells with OA alone did not affect the p53 expression (Fig. 7) and the p53 expression was increased in cells treated with 5-FU either in the presence or absence of OA (Fig. 7), suggesting that OA did not affect 5-FU induced p53 expression. However, in the presence of OA, the expression of NF-κB induced by 5-FU was significantly increased (Fig. 7). These results suggest that NF-κB plays an important role in the apoptosis induced by the combination of OA and 5-FU in Panc-28 cells.