PM was purchased from Sigma Chemicals (St. Louis, MO, USA). Antibodies against PARP-1, Akt, p-Akt (Ser⁴⁷³), Sp1, c-Myc, NF-κB and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Anti-hTERT and p-TERT antibodies were obtained from Abcam, Inc. (Cambridge, MA, USA). The CellTiter 96^® AQueous One Solution Proliferation Assay System was purchased from Promega (Madison, WI, USA). The Annexin V-FITC Apoptosis Detection Kit II was obtained from BD Biosciences Pharmingen (San Diego, CA, USA) and the TRAPeze Telomerase Detection kit was purchased from Millipore (Temecula, CA, USA).

A stock solution (100 mM) of PM was prepared in dimethyl-sulfoxide (DMSO) and test concentrations were prepared by diluting the stock solution in tissue culture medium.

MiaPaCa-2 and Panc-1 PDA cell lines were obtained from the American Type Culture Collection (ATCC; Rockville, MD, USA). The two cell lines were grown in Dulbecco’s modified Eagle’s medium (DMEM) tissue culture medium (Gibco-BRL, Rockville, MD, USA) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 25 mM HEPES buffer. The cells were incubated at 37°C in a humidified atmosphere consisting of 5% CO₂, 95% air and maintained by splitting the cultures twice a week.

Tumor cells (1×10⁴) in 100 μl of tissue culture medium were seeded in each well of 96-well plates. After a 24-h incubation to allow cells to adhere, the cells were treated with PM at concentrations ranging from 0 to 5 μM. The cultures were incubated for an additional 72 h and cell viability was then determined by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazo lium (MTS) assay using the CellTiter 96 AQueous One Solution Proliferation Assay System. This assay measures the bioreduction of the tetrazolium compound MTS in the presence of the electron-coupling reagent phenazine methosulfate by intracellular dehydrogenases. MTS and phenazine methosulfate were added to the culture wells, and the cultures were incubated for 2 h at 37°C. The absorbance, which is directly proportional to the number of viable cells in the cultures, was measured at 490 nm using a microplate reader.

The distribution of cells in various cell cycle phases was analyzed by measuring cell DNA content. Untreated (control) (2×10⁶) or PM-treated cells were fixed in 70% ethanol overnight at 4°C. The cells were washed twice and resuspended in 0.8 ml of phosphate-buffered saline (PBS). In total, 100 μl of DNAse free RNAse (500 μg/ml) and 100 μl of propidium iodide (PI) (500 μg/ml) were added to each tube and the tubes were incubated at room temperature in the dark for 30 min. Cell DNA content was determined by flow cytometry using an Accuri C6 flow cytometer (Accuri Cytometers, Inc., Ann Arbor, MI, USA).

Induction of apoptosis was assessed by the binding of Annexin V to phosphatidylserine, which was externalized to the outer leaflet of the plasma membrane early during induction of apoptosis. Briefly, MiaPaCa-2 and Panc-1 cells treated with PM (0–5 μM) for 24 h were resus-pended in the binding buffer provided in the Annexin V-FITC Apoptosis Detection Kit II. The cells were mixed with 5 μl of the Annexin V-FITC reagent, 5 μl of PI and incubated for 30 min at room temperature in the dark. The stained cells were analyzed by flow cytometry.

Cell lysates were prepared by CHAPS detergent lysis [1% Triton X-100 (v/v), 10 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 10% glycerol, 2 mM sodium vanadate, 5 μg/ml leupeptin, 1 μg/ml aprotinin, 1 μg/ml pepstatin A and 10 μg/ml 4-2-aminoethyl-benzenesulfonyl fluoride]. The lysates were clarified by centrifugation at 14,000 × g for 10 min at 4°C, and protein concentrations were determined by the Bradford assay. Samples (50 μg) were boiled in an equal volume of sample buffer [20% glycerol, 4% SDS, 0.2% bromophenol blue, 125 mM Tris-HCl (pH 7.5) and 640 mM 2-mercaptoethanol] and separated on 10% SDS-polyacrylamide gels. Proteins resolved on the gels were transferred to nitrocellulose membranes. The membranes were blocked with 5% milk in 10 mM Tris-HCl (pH 8.0), 150 mM NaCl with 0.05% Tween-20 (PBS) and incubated with protein-specific antibodies followed by an HRP-conjugated secondary antibody. Immune complexes were visualized with an enhanced chemiluminescence detection system from Amersham Corp. (Arlington Heights, IL, USA) and protein bands were imaged.

For hTERT overexpression, semi-confluent cell cultures were transfected with 10 μg of empty or hTERT expression plasmid (pCI-neo-hTERT) DNA using Lipofectamine Plus reagent. After transfection for 48 h, the cells were analyzed for the expression of hTERT by immuno-blotting.

For the silencing of hTERT, the cells were transfected with double-stranded siRNA-hTERT or a non-targeting siRNA sequence using a SignalSilence siRNA kit (Cell Signaling Technology, Beverly, MA, USA). Briefly, 2×10⁶ tumor cells were plated in a 60-mm Petri dish for 24 h and treated with 3 ml of transfection medium containing 20 μg Lipofectamine and 100 nM siRNA for 48 h. The hTERT expression was analyzed by immunoblotting.

Data are presented as means ± SD. The differences between the control and treatment groups were analyzed using the Student’s t-test. P<0.05 was considered to indicate a statistically significant result.

The effect of PM on proliferation of PDA cells (MiaPaCa-2 and Panc-1 cells) was examined using the MTS assay. Briefly, the cells were treated with PM at concentrations of 0–5 μM for 72 h and the viability of the cultures was determined. As shown in Fig. 1A, a significant (MiaPaCa-2, 71%) to measurable (Panc-1, 19%) reduction in viability was observed at 0.625 μM PM. In the MiaPaCa-2 cells the viability reached a plateau (85–87%) at 1.25-5 μM PM. On the other hand, reduction of viability in the Panc-1 cells occurred in a dose-dependent manner (51, 76 and 85% at 1.25, 2.5 and 5 μM PM). Thus, PM significantly reduced the proliferation of the two PDA cell lines at PM concentrations of 1.25–5 μM.

The effect of PM on cell cycle progression was then analyzed. MiaPaCa-2 and Panc-1 cells were treated with PM (0–5 μM) for 24 h, stained with PI and cell DNA content of the cells was measured by flow cytometry. As shown in Fig. 1B, treatment with PM resulted in cell cycle arrest in G1-phase with a cell distribution of 67.3, 76.2, 79, 80.3 and 80.1% in MiaPaCa-2 cells and 69.7, 64, 70.5, 82 and 87.5% in Panc-1 cells at 0, 0.625, 1.25, 2.5 and 5 μM PM, respectively.

Whether the cell cycle arrest leads to the induction of apoptosis was investigated by measuring the binding of Annexin V-FITC and cleavage of PARP-1 in tumor cells treated with PM. Thus, MiaPaCa-2 and Panc-1 cells were treated with PM (0–5 μM) for 24 h and the binding of Annexin V-FITC was determined by flow cytometry. As shown in Fig. 2, only a small percentage of untreated MiaPaCa-2 and Panc-1 cells bound to Annexin V-FITC (9–4%, respectively). By contrast, the percentage of Annexin V-FITC binding cells in the two cell lines increased in a dose-dependent manner following treatment with PM at 0.625–5 μM (MiaPaCa-2, 57–91%; Panc-1, 7–81%).

The induction of apoptosis was confirmed by the cleavage of PARP-1. Thus, the tumor cells were treated with PM as described above and cleavage of PARP-1 was determined by western blotting. As shown in Fig. 2B, treatment with PM induced the cleavage of native PARP-1 (110-kDa fragment) as identified by the emergence of an 89-kDa cleaved PARP-1 fragment in the two cell lines treated with PM. The increase in Annexin V-FITC binding and the cleavage of PARP-1 demonstrated induction of apoptosis in PDA cells by PM.

Reactivation of telomerase promotes the proliferation of tumor cells. Therefore, we determined the effect PM exerts on hTERT expression and its telomerase activity. Briefly, hTERT mRNA was analyzed by RT-PCR and hTERT protein by western blotting following treatment of the tumor cells with PM. Treatment with PM at concentrations of 1.25-5 μM for 48 h completely inhibited hTERT mRNA in the two cell lines (Fig. 3A). PM at these concentrations, however, had no effect on the expression of the housekeeping gene GAPDH.

Treatment with PM (1.25–5 μM) also resulted in partial to complete reduction in the native and phosphorylated-hTERT (Ser⁸²⁶) protein in the two cell lines.

To determine whether PM affects hTERT telomerase activity, MiaPaCa-2 and Panc-1 cells were treated with PM (0–5 μM) for 48 h and the cells were extracted in CHAP lysis buffer. The telomerase activity in the extracts was measured using the PCR-based TRAP assay. As shown in Fig. 3C, the telomerase activity in the MiaPaCa-2 cells was completely abolished even at the lowest concentration of 0.625 μM PM, as determined from the complete loss of DNA laddering. Telomerase activity was also markedly reduced in Panc-1 cells at 1.25 and 2.5 μM PM and completely abolished at 5 μM.

To identify the involvement of telomerase in mediating the antiproliferative and apoptosis-inducing activity of PM, we genetically altered the expression of hTERT in MiaPaCa-2 and Panc-1 cells and measured their response to PM. To increase hTERT expression, the tumor cells were transfected with pCI-neo-hTERT expression vector for 48 h and the response to PM was measured in the MTS assay. As expected, the transfected cells showed higher levels of hTERT (Fig. 4A) and demonstrated a significantly reduced sensitivity to PM, particularly at lower concentrations of PM (0.625 and 1.25 μM) compared to the control cells (MiaPaCa-2, 5 vs. 61%, and 54 vs. 83%; Panc-1, 3 vs. 22%, and 33 vs. 70%). Similarly, to investigate the effect of a reduced expression of hTERT on the response to PM, tumor cells were transfected with hTERT siRNA for 48 h. As shown in Fig. 4B, transfection with siRNA-hTERT significantly reduced the levels of hTERT in the two cell lines (insets). A decrease in hTERT levels significantly (p<0.05) increased the sensitivity of the two cell lines to PM at concentrations that were otherwise inactive or only slightly active (MiaPaCa-2, 19.6, 23 and 72% vs. 2, 1.4 and 53%; Panc-1, 38, 42 and 52% vs. 0, 4 and 18% at 0.157, 0.312 and 0.625 μM PM). Transfection with empty plasmid or non-targeting siRNA had no effect on response of cells to PM (data not shown). These data demonstrated that hTERT regulates the response to PM in pancreatic cancer cells.

Transcription factors such as c-Myc, Sp1, NF-κB and STAT-3 control the transcription of the hTERT gene (26,27) and post-translationally, the phosphorylation of hTERT on Ser²²⁷ and Ser⁸²⁶ residues by Akt is necessary for the activation of telomerase activity and the nuclear translocation of hTERT (28,29). Therefore, we assessed the effect of PM on the levels of these hTERT regulatory proteins. Treatment with PM (0.625–5 μM) for 48 h partially to completely inhibited Sp1, c-Myc and NF-κB (Fig. 5A). Of note, the inhibitory factors that negatively regulate the transcription of the hTERT gene (CTCF, E2F1 and Mad-1) were also reduced or completely inhibited following treatment with PM (Fig. 5B). Furthermore, p-Akt and p-mTOR that post-translationally regulate hTERT were also inhibited by PM (Fig. 5C). These data demonstrated that PM downregulates hTERT expression and activity through the inhibition of transcription factors (Sp1, c-Myc and NF-κB) and signaling molecules p-Akt and p-mTOR, respectively.