Inhibition of telomerase activity by oleanane triterpenoid CDDO-Me in pancreatic cancer cells is ROS-dependent

Abstract

Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) is a synthetic derivative of oleanolic acid, a triterpene, with apoptosis-inducing activity in a wide range of cancer cells. Induction of apoptosis by CDDO-Me is associated with the generation of reactive oxygen species (ROS) and inhibition of telomerase activity. In the present study, we investigated the role of ROS in inhibition of telomerase by CDDO-me. Treatment of MiaPaCa-2 and Panc-1 pancreatic cancer cell lines with CDDO-Me induced the production of hydrogen peroxide and superoxide anions and inhibited the telomerase activity. Pretreatment of cells with N-acetylcycsteine, a general purpose antioxidant or overexpression of glutathione peroxidase (GPx) or superoxide dismutase-1 (SOD-1) blocked the telomerase inhibitory activity of CDDO-Me. Furthermore, blocking ROS generation also prevented the inhibition of hTERT gene expression, hTERT protein production and expression of a number of hTERT-regulatory proteins by CDDO-Me (e.g., c-Myc, Sp1, NF-κB and p-Akt). Data also showed that Akt plays an important role in the activation of telomerase activity. Together, these data suggest that inhibition of telomerase activity by CDDO-Me is mediated through a ROS-dependent mechanism; however, more work is needed to fully understand the role of ROS in down-regulation of hTERT gene and hTERT-regulatory proteins by CDDO-Me.

Figure 1

CDDO-Me induces ROS production in pancreatic cancer cells and NAC blocks it. (A) MiaPaCa-2 cells and Panc-1 cells pretreated or not with NAC (3 mM) for 2 h were treated with CDDO-Me (1.25 μM) for 2 h. Cells were then reacted with 5 μM H₂DCFDA for 30 min at 37 °C and DCF fluorescence was measured by flow cytometry; (B) For DHE staining, after treatment with CDDO-Me as described above, MiaPaCa-2 cells and Panc-1 cells were reacted with DHE for 30 min. Cells were counter stained with Hoechst dye and observed under fluorescent microscope (200× magnification). NAC completely blocked the DHE fluorescence induced by CDDO-Me. * p < 0.05.

Figure 2

NAC blocks inhibition of pancreatic cancer cell proliferation by CDDO-Me. (A) 1 × 10⁴ MiaPaCa-2 or Panc-1 cells pretreated or not with NAC (3 mM) for 2 h were treated with CDDO-Me at concentrations ranging from 0 to 5 µM for 72 h in triplicate in 96-well microtiter plates. Cell viability was measured by MTS assay using CellTiter AQueous assay system from Promega. Data are presented as percent reduction in viability obtained in three independent experiments; (B) CDDO-Me induces apoptosis in pancreatic cancer cells and NAC blocks it. For annexin V-FITC binding, MiaPaCa-2 and Panc-1 cells were treated or not with NAC (3 mM) for 2 h prior to treating with CDDO-Me at concentrations of 0 to 5 µM for 24 h. Cells were then reacted with 5 μL of annexin V-FITC + PI for 30 min at room temperature. The percentage of annexin V-FITC positive tumor cells was determined by flow cytometry; (C) PARP-1 cleavage in MiaPaCa-2 cells and Panc-1 cells treated or not with NAC (3 mM) for 2 h prior to treatment with CDDO-Me for 24 h was analyzed by Western blotting. Each experiment was repeated two times.

Figure 3

CDDO-Me inhibits telomerase activity and antioxidants block it. (A) Effect of NAC. MiaPaCa-2 and Panc-1 cells pretreated or not with NAC (3 mM) for 2 h were treated with CDDO-Me (0–10 μM) for 48 h and telomerase activity of cell extracts was measured by TRAP assay as described in Materials and Methods. DNA laddering patterns under different treatment conditions are shown. Experiments were repeated two times. NC, negative control (no cell extract); (B,C) Effect of overexpression of antioxidant enzymes GPx and SOD-1. MiaPaCa-2 and Panc-1 cells were transfected with GPx or SOD-1 expression plasmids using LipofectAMINE Plus reagent for 48 h. Overexpression of enzymes was confirmed by immunoblotting and cells were then treated with CDDO-Me at 0.625–5 µM for 48 h and telomerase activity was measured by TRAP assay.

Figure 4

NAC blocks the inhibition of hTERT expression and hTERT related transcription factors by CDDO-Me. (A) Effect on hTERT gene expression. Mia PaCa-2 and Panc-1 cells pretreated or not with NAC (3 mM) were treated with CDDO-Me (0–5 μM) for 48 h and total cellular RNA was prepared using TRI-zole reagent. 1 μg of cellular RNA was reverse transcribed using oligo-dt primer and high fidelity reverse transcriptase. 1 μL of cDNA was amplified using hTERT or GAPDH primers. Amplified products were separated on 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. Mia PaCa-2 and Panc-1 cells were treated with CDDO-Me (0–5 μM) for 48 h as above and cell lysates were analyzed for hTERT and p-hTERT protein by western blotting; (C) Effect on hTERT transcription factors. MiaPaCa-2 and Panc-1 cells were treated with CDDO-Me (0–5 μM) for 48 h as in A and cell lysates were analyzed for c-Myc, Sp1, and NF-κB by Western blotting. Each experiment was repeated at least two times.

Figure 5

NAC blocks inhibition of p-Ak by CDDO-Me and inhibition of p-Akt inhibits telomerase activity of hTERT. (A) MiaPaCa-2 and Panc-1 cells were pretreated with NAC (3 mM) for 2 h followed by treatment with CDDO-Me (0–5 μM) for 48 h. Cell lysates were analyzed for p-Akt and total Akt by western blotting; (B) Inhibition of p-Akt inhibits telomerase activity. Panc-1 cells were transfected with siRNA-Akt or treated with Akt inhibitor SH6 before treating with CDDO-Me (0–5 μM) for 48 h. Cell lysates were analyzed for Akt, p-Akt and p-TERT by western blotting and telomerase activity was measured by TRAP assay.