Role of reactive oxygen species in brucein D-mediated p38-mitogen-activated protein kinase and nuclear factor-kappaB signalling pathways in human pancreatic adenocarcinoma cells

Abstract

Background: In human pancreatic adenocarcinoma, nuclear factor-kappa-B (NF-kappaB) transcription factor is constitutively activated that contributes to the resistance of the tumour cells to induced apoptosis. In our earlier studies, we have shown that brucein D (BD) mediated apoptosis through activation of the p38-mitogen-activated protein kinase (MAPK) signalling pathway in pancreatic cancer cells. This study investigated the function of reactive oxygen species (ROS) in BD-mediated p38-MAPK and NF-kappaB signalling pathways in PANC-1 cells.

Methods: Glutathione and dihydroethidium assays were used to measure the antioxidant and superoxide levels, respectively. The protein expression of p22(phox), p67(phox) and p38-MAPK were examined by western blot. The NF-kappaB activity was evaluated by electrophoretic mobility shift assay.

Results: Treatment with BD depleted the intracellular glutathione levels in PANC-1 cells. Brucein D triggered the activation of NADPH oxidase isoforms, p22(phox) and p67(phox) while enhancing the generation of superoxide. Increases in both intracellular ROS and NADPH oxidase activity were inhibited by an antioxidant, N-acetylcysteine (NAC). Brucein D-mediated activation of p38-MAPK was also inhibited by NAC. However, inhibition of NF-kappaB activity in BD-treated cells was independent of ROS. In vivo studies showed that BD treatment effectively reduced the rate of xenograft human pancreatic tumour in nude mice with no significant toxicity.

Conclusion: These data suggest that BD is an apoptogenic agent for pancreatic cancer cells through activation of the redox-sensitive p38-MAPK pathway and inhibition of NF-kappaB anti-apoptotic activity in pancreatic cancer cells.

Figure 1

Chemical structure of BD, a C-20 quassinoid.

Figure 2

Effects of BD on redox balance in PANC-1 cells. (A) BD attenuates antioxidant levels in PANC-1 cells. Cells were treated with BD for 2 h and GSH levels were determined. (B, D) Representative diagram showing dihydroethidium (DHE) staining of PANC-1 cells after treatment with vehicle (a–c), 250 μM NAC (d–f), 30 μM BD (g–i) and 250 μM NAC + 30 μM BD (j–l) for 1 or 2 h. Cells were fixed in 4% PFA and then incubated with DHE to detect the superoxide production (red). Nuclei were counterstained with DAPI (blue). Merged images are shown for each treatment (c, f, i and l). (C, E) Effects of BD on superoxide production in PANC-1 cells. Cells were pre-incubated with 250 μM NAC for 1 h and then treated with 30 μM BD for 1 or 2 h. The intensity of DHE staining was measured. ^*P<0.05 vs vehicle-control group; ^#P<0.05 and ^###P<0.001 vs 30 μM BD-treatment group. The results were obtained from at least three independent experiments. (The colour reproduction of this figure is available on the html full text version of the manuscript.)

Figure 3

Brucein D-treated results in an increase in the expression of NADPH oxidase. Effects of BD on the protein expression of NADPH oxidase p22^phox (A) and p67^phox (B) in PANC-1 cells in the absence and presence of 250 μM NAC. Representative images and statistical analysis of band intensity are shown in upper and lower panels, respectively. The results were obtained from at least three independent experiments. ^**P<0.01 vs vehicle-control group; ^###P<0.001 vs 30 μM BD-treatment group. The results were obtained from at least three independent experiments.

Figure 4

Effects of pre-treatment with NAC of the phosphorylation of p38-MAPK after treatment with BD. Cells were pre-treated with 250 μM NAC for 1 h, then incubated with 30 μM BD for 15 min to 2 h. (A) Western bolt analysis of samples using total p38 (p38-MAPK) and phosphorylation-specific p38 (p-p38-MAPK) antibodies. The results were obtained from three independent experiments. (B) Quantitative analysis of western blot band intensities. ^*P<0.05 vs vehicle control, ^**P<0.01 vs control and ^##P<0.01 vs 30 μM BD treatment.

Figure 5

Effects of BD on the activation of NF-κB. PANC-1 cells were treated with 3 and 30 μM BD for 2–4 h. (A, B) Western blots of cytoplasmic protein extracts (20 μg per lane) using antibodies specific for IκB-α and phospho-IκB-α. (C) Western blots of nuclear protein extracts (20 μg per lane) using a phospho-NF-κB p65 antibody. (D) RT–PCR analysis for the mRNA expression of NF-κB-regulated genes, bcl-2 and XIAP. ^*P<0.05 and ^**P<0.01 vs vehicle control. The results were obtained from three independent experiments.

Figure 6

Brucein D-induced inhibition of NF-κB activation is independent of ROS in PANC-1 cells. Cells were pre-treated with 250 μM NAC or vehicle for 1 h, then incubated with 30 μM BD for 15 min to 2 h. Western blot analysis was performed on cytoplasmic lysate using anti-phospho-IκBα antibodies (A) and nuclear lysate using anti-phospho-NF-κB p65 antibodies (B). ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs vehicle control. The results were obtained from three independent experiments. Quantitative analysis of band intensities is shown in lower panels, respectively. (C) Representative image showing the results from EMSA of nuclear extracts of PANC-1 cells after BD treatment for 2 h. Lane 1: distilled water (negative control); Lane 2: HeLa cell extract (positive control); Lane 3: vehicle control; Lane 4: 250 μM NAC treatment; Lane 5: 30 μM BD treatment; Lane 6: 250 μM NAC + 30 μM BD treatment. Similar results were obtained in three independent experiments.

Figure 7

In vivo analysis of the anti-tumour effects of BD. CAPAN-2 cells were xenografted by subcutaneous inoculation into nude mice. (A) Mice were treated with BD daily at the indicated concentrations through i.v. administration for 10 consecutive days. The tumour volume (TV) of each mouse was determined using the formula: TV=4/3πr³; n=4, where ⁺P<0.05 and ⁺⁺P<0.01 comparing 0.375 mg kg^–1 per day vs vehicle control; ^**P<0.01 and ^***P<0.001 comparing 0.75 mg kg^–1 per day vs vehicle control; ^##P<0.01 and ^###P<0.001 comparing 1.5 mg kg^–1 per day vs vehicle control. (B) Effects of BD on tumour xenograft growth in nude mice after 4 days of treatment at the indicated concentrations. The percentage of tumour growth was calculated after 4 days BD treatment; n=4, ^**P<0.01 and ^***P<0.001 vs vehicle control. (C) The body weight of each mouse was measured for 10 days.