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. 2012;10(1):51-64.

Inhibition of cell proliferation and induction of apoptosis by CDDO-Me in pancreatic cancer cells is ROS-dependent

Dorrah Deeb  1 , Xiaohua Gao, Yong Bo Liu, Subhash C Gautam
Affiliations

Inhibition of cell proliferation and induction of apoptosis by CDDO-Me in pancreatic cancer cells is ROS-dependent

Dorrah Deeb et al. J Exp Ther Oncol. 2012.

Abstract

Oleanolic acid-derived synthetic triterpenoids are broad spectrum antiproliferative and antitumorigenic agents. In this study, we investigated the role of reactive oxygen species (ROS) in induction of apoptosis and inhibition of prosurvival Akt, NF-kappaB and mTOR signaling pro-teins by methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) in pancreatic cancer cells. Micromolar concentrations of CDDO-Me inhibited proliferation and induced apoptosis in MiaPaCa-2 and Panc-1 pancreatic cancer cells. Treatment with CDDO-Me caused the generation of hydrogen peroxide and superoxide anion and pretreatment of cells with NADPH oxidase inhibitor diphylene iodonium (DPI) or respiratory chain complex 1 inhibitor rotenone prevented ROS generation. Pretreatment with N-acetylcysteine (NAC) or overexpression of glutathione peroxidase (GPx) or superoxide dismutase-1 (SOD-1) blocked the antiproliferative effects of CDDO-Me. Likewise, NAC prevented the induction of apoptosis (annexin V-FITC binding and cleavage of PARP-1 and procaspases-3,-8 and -9) and reversed the loss of mitochondrial membrane potential and release of cytochrome c from mitochondria by CDDO-Me. CDDO-Me down-regulated p-Akt, p-mTOR and NF-kappaB (p65) but increased the activation of Erk1/2 and NAC blocked the modulation of these cell signaling proteins by CDDO-Me. Thus, the results of this study indicate that the antiproliferative and apoptosis inducing effects of CDDO-Me are mediated through a ROS-dependent mechanism and the role of ROS in modulation of signaling proteins by CDDO-Me warrants further investigation.

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Figures

Figure 1
Figure 1. CDDO-Me inhibits the proliferation of pancreatic cancer cells and NAC blocks it
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 plate. 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. Morphological changes in cell cultures of MiaPaCa-2 cells and Panc-1 cells treated or not with CDDO-Me for 48 h with or without pretreatment with NAC as visualized by light microscopy. Similar results were obtained in two independent experiments. *p<0.05 compared to control cells (no CDDO-Me).
Figure 2
Figure 2. CDDO-Me induces ROS production in pancreatic cancer cells and NAC blocks it
A. Subconfluent MiaPaCa-2 cells and Panc-1 cells were treated with CDDO-Me (2.5 μM) for 2 h with or without pretreatment with NAC (3 mM) for 2 h. Cells were reacted with 5 μM H2DCFDA for 30 min at 37°C and DCF fluorescence was measured by flow cytometry. Flow cytographs and bar graphs show significant increase in mean fluorescence intensity in both cell lines treated with CDDO-Me (p<0.05), which was completely blocked by NAC. B. DHE fluorescence. MiaPaCa-2 cells and Panc-1 cells were pretreated or not with NAC before treating with CDDO-Me (2.5 μM) and then reacted with fluorescent probe DHE for 30 min. Cells were counter stained with Hoechst dye and staining of cells was observed by fluorescent microscope. Fluorescent micrographs show bright DHE fluorescence of cells treated with CDDO-Me, which was completely blocked by NAC (200X magnification). Bar graphs show percent of MiaPaCa-2 and Panc-1 cells with DHE fluorescence following various treatments. *p<0.05.
Figure 3
Figure 3. Effect of respiratory chain inhibitors rotenone (Rot) and DPI on ROS generation by CDDO-Me
MiaPaCa-2 and Panc-1 cells were pretreated with rotenone (100 nM) (A) or DPI (5 μM) for 30 min before treating with CDDO-Me (2.5 μM) for 2 h. ROS generation was measured from an increase in DCF fluorescence resulting from oxidation of H2DCFDA by flow cytometry. Rotenone and DPI significantly prevented the oxidation of H2DCFDA in both cell lines (p<0.05).
Figure 4
Figure 4. Antioxidant enzymes and SOD/catalase mimetic protect pancreatic cancer cells from-CDDO-Me-induced growth inhibition
A. Effect of overexpression of glutathione peroxidase (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 (insets). Cells were then treated with CDDO-Me at 0.625–2.5 μM for 72 h and growth inhibition measured by MTS assay. *p<0.05 compared to control cells. B. Effect of SOD/catalse mimetic MnTBAP. MiaPaCa-2 and Panc-1 cells were pretreated with MnTBAP (100 μM) for 2 h before treating with CDDO-Me at concentrations ranging from 0 to 2.5 μM for 72 h. Cell viability was measured by MTS assay as described above. *p<0.05 compared to control cells.
Figure 5
Figure 5. CDDO-Me induces apoptosis in pancreatic cancer cells and NAC blocks it
A. Binding of annexin V-FITC. MiaPaCa-2 cells and Panc-1cells 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 20 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. B. Cleavage of PARP-1 in MiaPaCa-2 cells and Panc-1 cells by CDDO-Me with or without pretreatment with NAC. C. Processing of procaspases-3,-8 and -9 by CDDO-Me in MiaPaCa-2 cells and Panc-1 cells with or without pretreatment with NAC. Each experiment was repeated twice. *p<0.05 compared to CDDO-Me only treated cells.
Figure 6
Figure 6. NAC prevents CDDO-Me-induced mitochondrial depolarization
A. MiaPaCa-2 and Panc-1 cells were treated with NAC (3mM) for 2 h prior to treating with CDDO-Me (0–5.0 μM) for 20 h. 1×106 cells were resuspended in 1ml of culture medium and loaded with mitochondrial potential sensor JC-1 (10 μg/ml) for 10 minutes at 22°C. Cells were analyzed by flow cytometry for fluorescence emission. Data are shown as flow cytographs of cells fluorescing red (FL2 channel) or green (FL1 channel). B. Histograms showing percentage of cells with loss of mitochondrial potential. Similar results were obtained in two separate experiments. *p<0.05 compared to CDDO-Me only treated cells.
Figure 7
Figure 7. NAC blocks the modulation of survival signaling proteins by CDDO-Me
A. MiaPaCa-2 and Panc-1 cells were pretreated or not with NAC (3 mM) for 2 h before treating with CDDO-Me (0–5.0 μM) for 20 h. Levels of p-Akt, p-mTOR, NF-κB (p65), p-Erk1/2 and β-actin (loading control) were analyzed by Western blotting. B. Immunoblots and bar graphs show the effect of Erk1/2 inhibitor PD98059 on activation of Erk1/2 and inhibition of growth of pancreatic cancer cells by CDDO-Me.
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