Pristimerin causes G1 arrest, induces apoptosis, and enhances the chemosensitivity to gemcitabine in pancreatic cancer cells

Abstract

Despite rapid advances in chemotherapy and surgical resection strategies, pancreatic cancer remains the fourth leading cause of cancer related deaths in the United States with a 5-year survival rate of less than 5%. Therefore, novel therapeutic agents for the prevention and treatment of pancreatic cancer are urgently needed. The aim of this study was to investigate the effect of pristimerin, a quinonemethide triterpenoid compound isolated from Celastraceae and Hippocrateaceae, on inhibition of cell proliferation and induction of apoptosis in three pancreatic cancer cells, BxPC-3, PANC-1 and AsPC-1, in both monotherapy and in combination with gemcitabine. Treatment with pristimerin decreased the cell proliferation of all three pancreatic cancer cells in a dose- and time-dependent manner. Treatment of pancreatic cancer cells with pristimerin also resulted in G1-phase arrest which was strongly associated with a marked decrease in the level of cyclins (D1 and E) and cyclin-dependent kinases (cdk2, cdk4 and cdk6 ) with concomitant induction of WAF1/p21 and KIP1/p27. Pristimerin treatment also resulted in apoptotic cell death, cleavage of caspase-3, modulation in the expressions of Bcl-2 family proteins, inhibition of the translocation and DNA-binding activity of NF-κB. In addition, pristimerin potentiated the growth inhibition and apoptosis inducing effects of gemcitabine in all three pancreatic cancer cells, at least in part, by inhibiting constitutive as well as gemcitabine-induced activation of NF-κB in both its DNA-binding activity and transcriptional activity. Taken together, these data provide the first evidence that pristimerin has strong potential for development as a novel agent against pancreatic cancer.

Figure 1. Chemical structure of pristimerin.

Figure 2. Effect of pristimerin on cell growth.

BxPC-3, PANC-1 and AsPC-1 cells were grown in the absence or presence of increasing concentration of pristimerin for 24 h, 48 h and 72 h and then the viability of cells was measured by CCK-8 assay as described in Materials and Methods. Data shown are representative of at least three independent experiments. *P<0.05, compared with control. **P<0.01, compared with control.

Figure 3. Effect of pristimerin on cell cycle distribution and expression of cell cycle-related proteins.

(A) Effect of pristimerin on cell cycle distribution in pancreatic cancer cells. BxPC-3, PANC-1 and AsPC-1 cells were cultured in complete medium and treated with either pristimerin (200, 400 or 600 nM) or DMSO (control) for 48 h. After treatment, cells were collected by trypsinization, washed with ice-cold PBS, and digested with RNase. Cellular DNA was stained with propidium iodide and flow cytometric analysis was performed for the detection of the percentage of cells in the different phases of the cell cycle as described in the Materials and Methods. *P<0.05, compared with control. **P<0.01, compared with control. (B) Effect of pristimerin on the protein level of cyclin D1, cyclin E, cdk 2, cdk 4, cdk 6, WAF1/p21 and KIP1/p27 in pancreatic cancer cells. As detailed in Materials and Methods, pancreatic cancer cells (BxPC-3, PANC-1 and AsPC-1) were treated with either pristimerin (200, 400 or 600 nM) or DMSO (control) for 48 h and then harvested. Total cell lysates were prepared and subjected to SDS-PAGE followed by Western blot analysis for G1 cell cycle regulatory proteins (cyclin D1, cyclin E, cdk2, cdk4, cdk6, WAF1/p21 and KIP1/p27). β-actin was detected as protein loading control. The immunoblots shown here are representative of at least three independent experiments with similar results.

Figure 4. Effect of pristimerin on apoptosis induction in pancreatic cancer cells.

(A) Effect of pristimerin on apoptosis induction assessed by Annexin V/PI method using flow cytometry. BxPC-3, PANC-1 and AsPC-1 cells were cultured in complete medium and treated with either pristimerin (200, 400 or 600 nM) or DMSO (control) for 48 h. Apoptosis rate was determined by flow cytometry on annexin V-FITC. (B) Representative dot-plots from cytometrically illustrating apoptotic status in BxPC-3 (upper panel), PANC-1 (middle panel) and AsPC-1 (lower panel) cells. (C) Effect of pristimerin on apoptosis induction assessed by fluorescence microscopy. Cells were also viewed under a fluorescence microscopy. Representative photographs were taken from Annexin V/PI-stained pancreatic cancer cells under certain treatment. (D) Effect of pristimerin on apoptosis induction assessed by caspase-3 activity assay. Cell lysates were assayed for caspase-3 activity as described in “Materials and methods”. (E) Effect of pristimerin on cleavage of caspase-3. As detailed in Materials and Methods, pancreatic cancer cells (BxPC-3, PANC-1 and AsPC-1) were treated with either pristimerin (200, 400 or 600 nM) or DMSO (control) for 48 h and then harvested. Total cell lysates were prepared and subjected to SDS-PAGE followed by Western blot analysis. β-actin was detected as protein loading control. The immunoblots shown here are representative of at least three independent experiments with similar results. *P<0.05, compared with control. **P<0.01, compared with control.

Figure 5. Effect of pristimerin on the protein level of Bcl-xl, Bcl-2 and Bax.

As detailed in Materials and Methods, pancreatic cancer cells (BxPC-3, PANC-1 and AsPC-1) were treated with either pristimerin (200, 400 or 600 nM) or DMSO (control) for 48 h and then harvested. Total cell lysates were prepared and subjected to SDS-PAGE followed by Western blot analysis. β-actin was detected as protein loading control. The immunoblots shown here are representative of at least three independent experiments with similar results.

Figure 6. Effect of pristimerin on NF-κB activation in pancreatic cancer cells.

(A) Effect of pristimerin on protein levels of NF-κB/p65 in nuclear lysates and total cell lysates of pancreatic cancer cells. As detailed in Materials and Methods, pancreatic cancer cells (BxPC-3, PANC-1 and AsPC-1) were treated with either pristimerin (200, 400 or 600 nM) or DMSO (control) for 48 h and then harvested. Nuclear lysates and total cell lysates were prepared and subjected to SDS-PAGE followed by Western blot analysis for the protein level of NF-κB/p65, p-IκB-α (S32/36) and IκB-α. β-actin was detected as protein loading control. The immunoblots shown here are representative of at least three independent experiments with similar results. (B) Effect of pristimerin on NF-κB/p65 DNA-binding activity in pancreatic cancer cells. As detailed in Materials and Methods, pancreatic cancer cells (BxPC-3, PANC-1 and AsPC-1) were treated with either pristimerin (200, 400 or 600 nM) or DMSO (control) for 48 h and then harvested. Nuclear lysates were prepared and the NF-κB DNA-binding activity was determined using the Trans-Am NF-κB ELISA Kit. *P<0.05, compared with control. **P<0.01, compared with control.

Figure 7. Potentiation of the effect of gemcitabine by pristimerin in pancreatic cancer cells.

(A) Potentiation of gemcitabine-induced cell growth inhibition by pristimerin. Cells were grown in the absence or presence of pristimerin (200 nM), gemcitabine (500 nM) or their combination for 48 h. The viability of cells was measured by CCK-8 assay as described in Materials and Methods. Combination index (CI) versus fraction affected (Fa) plots obtained from median-effect analysis of Chou-Talalay. CI values: >1, antagonism; 1, additivity; <1, synergism. (B) Potentiation of gemcitabine-induced apoptosis by pristimerin. Cells were grown in the absence or presence of pristimerin (200 nM), gemcitabine (500 nM) or their combination for 48 h. Apoptosis rate was determined by flow cytometry on annexin V-FITC. *P<0.05, compared with control. **P<0.01, compared with control. ^#P<0.05, compared with single gemcitabine group. ^##P<0.01, compared with single gemcitabine group.

Figure 8. Abrogation of constitutive and gemcitabine-induced NF-κB activation by pristimerin in pancreatic cancer cells.

(A) Effect of pristimerin and gemcitabine on protein levels of NF-κB/p65 in nuclear lysates and total cell lysates of pancreatic cancer cells. As detailed in Materials and Methods, pancreatic cancer cells (BxPC-3, PANC-1 and AsPC-1) were grown in the absence or presence of pristimerin (200 nM), gemcitabine (500 nM) or their combination for 48 h and then harvested. Nuclear lysates and total cell lysates were prepared and subjected to SDS-PAGE followed by Western blot analysis for the protein level of NF-κB/p65. β-actin was detected as protein loading control. The immunoblots shown here are representative of at least three independent experiments with similar results. (B) Effect of pristimerin and gemcitabine on NF-κB/p65 DNA-binding activity in pancreatic cancer cells. As detailed in Materials and Methods, pancreatic cancer cells (BxPC-3, PANC-1 and AsPC-1) were grown in the absence or presence of pristimerin (200 nM), gemcitabine (500 nM) or their combination for 48 h and then harvested. Nuclear lysates were prepared and the NF-κB DNA-binding activity was determined using the Trans-Am NF-κB ELISA Kit. *P<0.05, compared with control. **P<0.01, compared with control. ^#P<0.05, compared with single gemcitabine group. ^##P<0.01, compared with single gemcitabine group.

Figure 9. Proposed mechanisms of pristimerin-mediated G1 arrest, apoptosis and chemosensitivity of human pancreatic cancer cells.