Inhibition of hTERT/telomerase contributes to the antitumor activity of pristimerin in pancreatic ductal adenocarcinoma cells

Abstract

Pristimerin (PM) is a promising anticancer agent that has exhibited strong antiproliferative and apoptosis-inducing activity in various types of cancer cells. In the present study, we investigated the role of telomerase in mediating the antitumor activity of PM in pancreatic ductal adenocarcinoma (PDA) cells. PM inhibited cell proliferation, arrested cells in the G1 cell cycle phase and induced apoptosis in MiaPaCa-2 and Panc-1 PDA cells. These antitumor activities of PM correlated well with the inhibition of human telomerase reverse transcriptase (hTERT), the gene that codes for the catalytic subunit of telomerase complex. Gene knockin and knockdown approaches demonstrated that hTERT regulates the response of PDA cells to PM. PM inhibited hTERT expression by suppressing the transcription factors Sp1, c-Myc and NF-κB which control hTERT gene expression. PM also inhibited protein kinase Akt, which phosphorylates and facilitates hTERT nuclear importation and its telomerase activity. These findings identified hTERT (telomerase) as a potential therapeutic target of PM for the treatment of PDA.

Figure 1

PM inhibits cell proliferation and cell cycle progression in PDA cells. (A) Effect on cell proliferation. MiaPaCa-2 and Panc-1 cells (1×10⁴) were seeded in each well of 96-well plates. After incubation for 24 h, the cells were treated with PM at concentrations ranging from 0 to 5 μM for 72 h in triplicate. Cell viability was measured by the MTS assay using the CellTiter AQueous Assay System. ^*P<0.05 compared to the control cells. (B) Effect on cell-cycle progression. MiaPaCa-2 and Panc-1 cells treated with PM (0–5 μM) for 24 h were fixed and stained with PI. Cellular DNA content was analyzed by a flow cytometry and cell distribution in various cell cycle phases was analyzed. PM, pristimerin; PDA, pancreatic ductal adenocarcinoma; PI, propidium iodide.

Figure 2

PM induces apoptosis in PDA cells. (A) MiaPaCa-2 and Panc-1 cells were treated with PM at 0–5 μM for 24 h. The cells were then reacted with 5 μl of Annexin V-FITC and 5 μl PI for 30 min and the percentage of Annexin V-FITC binding cells was determined by flow cytometry. (B) MiaPaCa-2 and Panc-1 cells were treated with PM as above and the cell lysates were analyzed for the cleavage of PARP-1 by western blotting. Similar results were obtained in two independent experiments. PM, pristimerin; PDA, pancreatic ductal adenocarcinoma.

Figure 3

PM inhibits hTERT mRNA and hTERT protein in PDA cells. (A) Effect on hTERT gene expression. MiaPaCa-2 and Panc-1 cells were treated with PM (0–5 μM) for 48 h and total cellular RNA was prepared using TRIzol reagent. Cellular RNA (1 μg) was reverse transcribed using oligo(dT) primer and high fidelity reverse transcriptase. cDNA (1 μl) was amplified using hTERT or GAPDH primers. Amplified products were separated on a 2% DNA agarose gel. Gels were stained with ethidium bromide and amplified DNA fragments were identified by base pair sizes. (B) Effect on hTERT protein. MiaPaCa-2 and Panc-1 cells were treated with PM and cell lysates were analyzed for hTERT and p-hTERT protein by western blotting. (C) Effect on telomerase activity. MiaPaCa-2 and Panc-1 cells treated with PM (0–5 μM) for 48 h were extracted in CHAPS buffer. The telomerase activity in the cell extracts was measured by the PCR-based TRAP assay as described in Materials and methods. Negative (−), no cell extract. Each experiment was repeated at least twice. PM, pristimerin; PDA, pancreatic ductal adenocarcinoma.

Figure 4

hTERT mediates response to PM in PDA cells. MiaPaCa-2 and Panc-1 cells were transfected with (A) hTERT expression vector (pCI-neo-hTERT) or (B) siRNA-hTERT for 48 h using Lipofectamine Plus reagent. hTERT levels were determined by immunoblotting (insets) and sensitivity of control and transfected cells to PM was assessed in the MTS assay. Each experiment was repeated twice. PM, pristimerin; PDA, pancreatic ductal adenocarcinoma.

Figure 5

PM inhibits hTERT regulatory proteins in PDA cells. (A) MiaPaCa-2 and Panc-1 cells were treated with PM (0–5 μM) for 48 h and the cell lysates were analyzed for (A) transcription factors c-Myc, Sp1 and NF-κB or (B) inhibitors of hTERT transcription (CTCF, E2F1 and Mad-1) or (C) signaling proteins p-Akt and p-mTOR by western blotting. This experiment was repeated twice. PM, pristimerin; PDA, pancreatic ductal adenocarcinoma.