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. 2015 Dec;47(6):2100-6.
doi: 10.3892/ijo.2015.3212. Epub 2015 Oct 19.

CDDO-Me inhibits tumor growth and prevents recurrence of pancreatic ductal adenocarcinoma

Xiaohua Gao  1 , Dorrah Deeb  1 , Yongbo Liu  1 , Patricia Liu  1 , Yiguan Zhang  2 , Jiajiu Shaw  2 , Subhash C Gautam  1
Affiliations

CDDO-Me inhibits tumor growth and prevents recurrence of pancreatic ductal adenocarcinoma

Xiaohua Gao et al. Int J Oncol. 2015 Dec.

Abstract

Methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me) has shown potent antitumorigenic activity against a wide range of cancer cell lines in vitro and inhibited the growth of liver, lung and prostate cancer in vivo. In the present study, we examined the antitumor activity of CDDO-Me for pancreatic ductal adenocarcinoma (PDAC) cells with and without activating K-ras mutations. Treatment of K-ras mutant MiaPaCa-2 and K-ras normal BxPC-3 cells with CDDO-Me elicited strong antiproliferative and proapoptopic responses in both cell lines in culture. The inhibition of cell proliferation and induction of apoptosis was accompanied by the inhibition of antiapoptotic/prosurvival p-Akt, NF-кB and p-mTOR signaling proteins. For testing efficacy of CDDO-Me in vivo heterotopic and orthotopic xenografts were generated by implanting BxPC-3 and MiaPaCa-2 cells subcutaneously and in the pancreatic tail, respectively. Treatment with CDDO-Me significantly inhibited the growth of BxPC-3 xenografts and reduced the levels of p-Akt and p-mTOR in tumor tissue. In mice with orthotopic MiaPaCa-2 xenografts, treatment with CDDO-Me prolonged the survival of mice when administered following the surgical resection of tumors. The latter was attributed to the eradication of residual PDAC remaining after resection of tumors. These preclinical data demonstrate the potential of CDDO-Me for treating primary PDAC tumors and for preventing relapse/recurrence through the destruction of residual disease.

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Figures

Figure 1
Figure 1
CDDO-Me inhibits the proliferation of PDAC cells. MiaPaCa-2 or BxPC-3 cells (1×104) in 100 μl of culture medium were seeded in each well of a 96-well microtiter plate and allowed to adhere for 24 h. Cells were treated with CDDO-Me at concentrations ranging from 0 to 5 μM for 72 h in triplicate. 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. *p≤0.05 compared to control cells (no CDDO-Me).
Figure 2
Figure 2
CDDO-Me induces apoptosis in PDAC cells. (A) Binding of Annexin V-FITC. MiaPaCa-2 cells and BxPC-3 cells were treated with CDDO-Me at concentrations of 0–5 μM for 24 h. Cells were then reacted with 5 μl of Annexin V-FITC and 5 μl PI for 30 min at room temperature in the dark. The percentage of Annexin V-FITC-positive tumor cells was determined by flow cytometry. (B) Cleavage of PARP-1. MiaPaCa-2 cells and BxPC-3 cells were treated with CDDO-Me (0–5 μM) for 24 h. Cell lysates were analyzed for cleavage of PARP-1 by western blotting. Experiment was repeated twice.
Figure 3
Figure 3
CDDO-Me downregulates prosurvival signaling proteins in PDAC cells. MiaPaCa-2 and BxPC-3 cells were treated with CDDO-Me (0–5.0 μM) for 24 h. Levels of p-Akt, NF-κB (p65), p-mTOR, and β-actin (loading control) were analyzed by western blotting. Experiment was repeated two times.
Figure 4
Figure 4
CDDO-Me inhibits growth of PDAC xenografts and expression of prosurvival signaling proteins in tumor tissue. (A) BxPC-3 cells (1×106) in 50 μl of PBS were injected in the flank of seven-week-old female Scid/Ncr mice. Starting on day 7 after implantation of tumor cells mice were treated with CDDO-Me at a dose of 15 μmol/kg by oral gavage as described in Materials and methods. Control mice were treated with vehicle alone without CDDO-Me. Tumor dimensions (length and width) were measured with a caliper at weekly intervals. Data are plotted as tumor volumes vs days past tumor cell implantation ± SD. (B) At termination of the experiment (day 40), tumor tissue from control and CDDO-Me treated mice was harvested and levels of p-Akt and p-mTOR were analyzed by western blotting.
Figure 5
Figure 5
Bioluminescent imaging of orthotopic PDAC xenografts and efficacy of CDDO-Me in preventing recurrence. Female Scid/Ncr mice were injected with 1×106 MiaPaCa-2 cells stably expressing luciferase gene (MiaPaCa-2-Luc cells) in the pancreatic tail. The growth of xenografts was monitored by non-invasive bioluminescent imaging (A and B) and a growth curve was generated by plotting BLI signals from tumors (photon emission/sec/mm2) vs time (days) after implantation of MiaPaCa-2-Luc cells (C). Size and histology of a tumor harvested on day 35 is also shown (D and E). (F) To determine efficacy of CDDO-Me for preventing relapse, all visible tumor growth was resected on day 28 after implantation of MiaPaCa-2-Luc cells and mice were treated with CDDO-Me at a dose of 15 μmol/kg orally, 5 days a week until day 100. Control mice were treated with vehicle alone without CDDO-Me. In a separate group, mice were also treated with gemcitabine i.p., twice a week at a dose of 2 mg/kg to compare the activity of CDDO-Me to that of a standard agent for PDAC. Kaplan-Meier plots (F) represent survival data of vehicle only, gemcitabine or CDDO-Me treated animals.
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