Synthetic triterpenoids inhibit growth, induce apoptosis and suppress pro-survival Akt, mTOR and NF-{kappa}B signaling proteins in colorectal cancer cells

Abstract

Lack of apoptotic cell death has been implicated in malignant transformation and resistance to anticancer therapies. The promotion of apoptosis in cancer cells could potentially lead to the regression and improved prognosis of refractory colorectal cancer. Synthetic triterpenoids have shown strong antitumorigenic activity towards diverse cancer cell types, but have not been investigated for colorectal cancer. In the present study, we tested the apoptosis-inducing activity of oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) and its C-28 methyl ester (CDDO-Me) and C-28 imidazole (CDDO-Im) derivatives in colorectal cancer cells lines. Cell growth/viability assay (MTS) demonstrated that colorectal cancer cells are highly sensitive to CDDO-Me at concentrations of 1.25 to 10 microM. The primary mode of tumor cell destruction was apoptosis as demonstrated by the cleavage of PARP-1, activation of procaspases -3, -8, and -9 and mitochondrial depolarization. Induction of apoptosis by CDDO-Me was associated with the inhibition of pro-survival Akt, NF-kappaB and mTOR signaling proteins and NF-kappaB-regulated anti-apoptotic Bcl-2, Bcl-xL, Bad and survivin. These studies provide rationale for clinical evaluation of CDDO-Me for the treatment of advanced chemotherapy refractory colorectal cancer.

Figure 1

Effect of CDDO, CDDO-Im and CDDO-Me on the viability of colorectal cancer cells. A: 1×10⁴ colon cancer cells (HT-29, HCT-15, HCT 8, HCT 116, Colo 201 or Colo 205) were seeded in each well of a microtiter plate in 0.1 ml of culture medium. Cells were allowed to adhere for 24 h before treating with CDDO or CDDO-Im or CDDO-Me at concentrations of 0 to 10 μM in triplicates for 72 h. Cell viability was measured by MTS assay using CellTiter AQueous assay system from Promega. Similar results were obtained in 4 independent experiments. B: Morphological changes in cell cultures (HT-29 and HCT-15 cells) treated or not with CDDO-Me for 72 h as visualized by light microscopy. Similar results were obtained in two independent experiments.

Figure 2

Treatment with CDDO-Me induces apoptosis in colorectal cancer cells. A: Binding of annexin V-FITC. HT-29 and HCT-15 cells were treated with CDDO-Me at concentrations of 1.25 to 5 μM for 20 h. Cells were then reacted with 5 μl of annexin V-FITC reagent for 30 min at room temperature. The percentage of annexin V-FITC positive tumor cells was determined by flow cytometry. Results are presented as percentage of annexin V-FITC-binding cells. B: Cleavage of PARP-1. Cells were treated with CDDO-Me as described above and PARP-1 was analyzed by immunoblotting. Each experiment was repeated at least twice.

Figure 3

Treatment with CDDO-Me cleaves procaspases-3, -8 and -9. HT-29 and HCT-15 cells were treated with CDDO-Me at concentrations of 0 to 5 μM. After incubation for 20 h, cellular lysates prepared from untreated (control) and treated cells were fractionated on 10% SDS-PAGE gel (50 μg/lane). Proteins were transferred from the gel to nitrocellulose membrane and first reacted with antibody to caspase-3, caspase-8 and caspase-9 or β-actin (loading control) followed by HRP-conjugated second antibody. Signals were visualized with enhanced chemiluminescence. Numbers above signal bands denote percentage suppression compared to control (0 CDDO-Me).

Figure 4

CDDO-Me induces mitochondrial depolarization in colon cancer cells. HT-29 and HCT-15 cells were treated with CDDO-Me at 0 to 5 μM for 20 h, then 1×10⁶ cells were resuspended in 1 ml 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). Histograms showing the percentage of cells with loss of mitochondrial potential difference. Similar results were obtained in two separate experiments.

Figure 5

CDDO-Me inhibits prosurvival p-Akt, NF-κB and p-mTOR and antiapoptotic Bcl-2, Bcl-xL, Bad and survivin. HT-29 and HCT-15 cells were treated with CDDO-Me at 0 to 5 μM for 20 h. After treatment, cell lysates were prepared and analyzed by Western blotting using anti-p-Akt antibody, anti-NF-κB (p65) antibody, anti-p-mTOR antibodies (A) or anti-Bcl-2, Bcl-xL, Bad or survivin (B) or anti-β-actin antibody (loading control). Numbers above the signal bands denote percent suppression compared to control (0 CDDO-Me).