Bufalin enhances the antitumor effect of gemcitabine in pancreatic cancer

Abstract

Bufalin, an active component of the Chinese medicine chan'su, has been reported to have an inhibitory effect on the growth of various types of cancer cells. In the present study, we investigated whether gemcitabine combined with bufalin enhanced the antitumor efficacy in pancreatic cancer. Three pancreatic cancer cell lines (Bxpc-3, Mia PaCa-2 and Panc-1) were treated with gemcitabine and/or bufalin in vitro. The combination treatment demonstrated greater inhibition of cellular growth and apoptosis. The activity of apoptosis signal-regulating kinase 1 (ASK1)/JNK was upregulated in gemcitabine-induced apoptosis when combined with bufalin. We also observed that tumor growth was significantly inhibited by the combination therapy in a tumor-bearing mouse model, and upregulation of ASK1 activity was validated by immunohistochemical staining. These results suggest that bufalin may be a potential chemotherapeutic agent for pancreatic cancer, which could enhance the antitumor efficacy of gemcitabine when used in combination, possibly through the activation of ASK1/JNK.

Figure 1.

Bufalin inhibits the proliferation of three pancreatic cancer cell lines. (A) Bxpc-3, Mia PaCa-2 and Panc-1 cells were treated with bufalin for 24, 48 and 72 h. The viability was assessed by MTT. Bufalin was shown to induce a dose- and time-dependent loss in all three cell lines. (B) Cells treated with bufalin (0.01 μM) and/or gemcitabine [Bxpc-3 (0.5 μg/ml), Panc-1 and Mia PaCa-2 (5 μg/ml), respectively] for 48 h. Compared with treatment of bufalin or gemcitabine alone, the combination group was statistically different (^*P<0.05). Columns, mean of three experiments; bars, standard error (SE); ^*P<0.05 compared with respective group.

Figure 2.

Potentiation of gemcitabine-induced apoptosis by bufalin in three pancreatic cancer cell lines. (A) Bxpc-3, Mia PaCa-2 and Panc-1 cells were treated with bufalin and/or gemcitabine for 48 h, and then analyzed by flow cytometry. A significant difference was observed with the combination treatment compared with bufalin or gemcitabine treatment alone.(B) Protein of cleaved caspase-3 and bcl-2 in the three pancreatic cancer cell lines was extracted and analyzed by western blot analysis. Cleaved caspase-3 was activated and bcl-2 was downregulated. β-actin served as the internal control. Data are the results of three independent experiments.

Figure 3.

The role of ASK1 in the apoptotic pathway induced by bufalin in Bxpc-3, Mia PaCa-2 and Panc-1 cells. (A) Dose response of bufalin upregulated the expression of ASK1 in Mia PaCa-2 cells. Total proteins were prepared from cells after incubation with different concentrations of bufalin for 48 h as described in Materials and methods. (B) Time course of upregulation of ASK1 induced by bufalin in Bxpc-3, Mia PaCa-2 and Panc-1 cells. The cells were incubated with 0.01 μM bufalin and the total protein was extracted for evaluation of ASK1 induction.

Figure 4.

Bufalin inhibited the ASK1/JNK signaling pathway in Mia PaCa-2 cells. (A) Mia PaCa-2 cells were treated with gemcitabine (5 mg/ml), bufalin (0.01 μM) and the combination for 48 h. The expression of ASK1, p-JNK and JNK were investigated. (B) Mia PaCa-2 cells were transfected with various siRNAs as indicated, and the expression of ASK1 and p-JNK was determined after treatment with bufalin and gemcitabine for 48 h. A reduced level of p-JNK was detected by western blot analysis. β-actin protein served as the loading control.

Figure 5.

The combination treatment of gemcitabine with bufalin inhibited tumor growth in xenograft model. (A) The tumor volume in pancreatic cancer xenograft model after treatment. There was a statistically significant difference between the combination treatment group and the control group (P<0.05). (B) The expression of Ki-67 was decreased and ASK1 was increased in the combination group.