Methyl 2-cyano-3,12-dioxooleana-1,9-dien-28-oate decreases specificity protein transcription factors and inhibits pancreatic tumor growth: role of microRNA-27a

Abstract

The anticancer agent 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and its methyl ester (CDDO-Me) typically induce a broad spectrum of growth-inhibitory, proapoptotic, and antiangiogenic responses. Treatment of Panc1, Panc28, and L3.6pL pancreatic cancer cells with low micromolar concentrations of CDDO or CDDO-Me resulted in growth inhibition, induction of apoptosis, and down-regulation of cyclin D1, survivin, vascular endothelial growth factor (VEGF), and its receptor (VEGFR2). RNA interference studies indicate that these repressed genes are regulated by specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and Western blot analysis of lysates from pancreatic cancer cells treated with CDDO and CDDO-Me shows for the first time that both compounds decreased the expression of Sp1, Sp3, and Sp4. Moreover, CDDO-Me (7.5 mg/kg/day) also inhibited pancreatic human L3.6pL tumor growth and down-regulated Sp1, Sp3, and Sp4 in tumors using an orthotopic pancreatic cancer model. CDDO-Me also induced reactive oxygen species (ROS) and decreased mitochondrial membrane potential (MMP) in Panc1 and L3.6pL cells, and cotreatment with antioxidants (glutathione and dithiothreitol) blocked the formation of ROS, reversed the loss of MMP, and inhibited down-regulation of Sp1, Sp3, and Sp4. Repression of Sp and Sp-dependent genes by CDDO-Me was due to the down-regulation of microRNA-27a and induction of zinc finger and BTB domain containing 10 (ZBTB10), an Sp repressor, and these responses were also reversed by antioxidants. Thus, the anticancer activity of CDDO-Me is due, in part, to activation of ROS, which in turn targets the microRNA-27a:ZBTB10-Sp transcription factor axis. This results in decreased expression of Sp-regulated genes, growth inhibition, induction of apoptosis, and antiangiogenic responses.

Figures

Fig. 1.

CDDO and CDDO-Me inhibit cell growth and induce apoptosis in pancreatic cancer cell lines. Inhibition of Panc1, Panc28, and L3.6pL cell growth by CDDO (A) and CDDO-Me (B). Cells were treated with DMSO (solvent control), CDDO (0.5, 1.0, or 2.5 μM), or CDDO-Me (0.01, 0.1, 0.5, or 1.0 μM), and effects on cell growth were determined over a period of 6 days as described under Materials and Methods. Induction of PARP cleavage by CDDO (C) and CDDO-Me (D). Panc1, Panc28, and L3.6pL cells were treated with DMSO, CDDO (1.0, 2.5, or 5.0 μM), or CDDO-Me (0.5, 1.0, or 1.25 μM) for 24 h, and whole-cell lysates were analyzed by Western blot analysis as described under Materials and Methods. β-Actin served as a loading control.

Fig. 2.

CDDO and CDDO-Me decrease the expression of VEGF, VEGFR2, cyclin D1 (CD1), and survivin proteins in Panc1 (A), Panc28 (B) and L3.6pL (C) pancreatic cancer cell lines. Cells were treated with DMSO, CDDO (1.0, 2.5, or 5.0 μM), or CDDO-Me (0.5, 1.0, or 1.25 μM) for 24 h, and whole-cell lysates were analyzed by Western blot analysis as described under Materials and Methods. β-Actin served as a loading control. The gels were typical of results of at least two replicate determinations per treatment group.

Fig. 3.

CDDO-Me down-regulates Sp proteins in a proteosome-independent manner. CDDO-Me decreases Sp protein expression in Panc1 (A), Panc28 (B), and L3.6pL (C). Cells were with DMSO, CDDO (1.0, 2.5, or 5.0 μM), or CDDO-Me (0.5, 1.0, or 1.25 μM) for 24 h, and whole-cell lysates were analyzed for Sp1, Sp3, and Sp4 by Western blot analysis as described under Materials and Methods. D, proteosome-independent down-regulation of Sp proteins by CDDO-Me. Cells were treated with DMSO and CDDO-Me (1.0 μM) in the presence or absence of proteasome inhibitor MG132 (10 μM), and the effects on Sp protein degradation were determined after treatment for 24 h by Western blot as described under Materials and Methods. β-Actin served as a loading control.

Fig. 4.

Role of oxidative stress and MMP in mediating the effects of CDDO-Me on Sp proteins in pancreatic cancer cells. Effect of CDDO-Me on ROS (A) and MMP (B and C). Panc1 and L3.6pL cells were treated with DMSO and CDDO-Me (0.5, 1.0, or 1.25 μM) for 24 h in the presence or absence of antioxidant GSH. ROS was measured using BioTek Synergy 4 plate reader using CM-H2DCFDA (10 μM) dye as described under Materials and Methods, and normalized fluorescence intensity against control is plotted as a bar diagram. MMP was determined using JC-1 dye, and quantitation of the ratio of red to green fluorescence was measured using ImageJ Software as described under Materials and Methods. D, reversal of CDDO-Me-mediated down-regulation of Sp proteins by thiol antioxidants. Cells were treated with DMSO or CDDO-Me (1.0 μM) in the presence or absence of DTT or GSH for 24 h, and whole-cell lysates were analyzed by Western blots as described under Materials and Methods. β-Actin served as a loading control. Results in A and C are expressed as means ± S.E. for three replicate determinations for each treatment group, and significant (P < 0.05) CDDO-Me-mediated decreases (*) or increases (**) after cotreatment with antioxidants compared with the solvent (DMSO) control are indicated.

Fig. 5.

Effect of CDDO-Me on the expression miR-27a and ZBTB10 mRNA and role of ZBTB10 overexpression on Sp proteins in pancreatic cancer cells. CDDO-Me decreases miR-27a (A) and induces ZBTB10 mRNA (B). Panc1 and L3.6pL cells were treated with the indicated doses of CDDO-Me for 24 h, and miR-27a and ZBTB10 levels were analyzed by real-time PCR as described under Materials and Methods. C, effect of ZBTB10 overexpression on Sp proteins and Sp-dependent genes. Panc1 and L3.6pL cells were transfected with empty vector (pCMV6-XL4) or 4 μg/well ZBTB10 expression plasmid pCMV6-XL4 vector, and whole-cell lysates were analyzed by Western blots as described under Materials and Methods. D, effect of GSH on CDDO-Me-mediated miR-27a and ZBTB10 mRNA expression. Panc1 and L3.6pL cells were treated with the indicated doses of CDDO-Me in the presence or absence of GSH for 24 h, and miR-27a and ZBTB10 mRNA levels were analyzed by real-time PCR as described under Materials and Methods. Results in A, B, and D are expressed as means ± S.E. for three replicate determinations for each treatment group, and significant (P < 0.05) inhibition (*) or induction (**) of responses are indicated.

Fig. 6.

Growth inhibition and induction of apoptosis by as-miR-27a, ZBTB10, and Sp knockdown. Panc1 and L3.6pL cells were transfected with as-miR-27a (A), ZBTB10 expression plasmid (B), and small inhibitory RNAs against Sp transcription factors (C and D), and their effects on cell proliferation and induction of PARP cleavage were determined as described under Materials and Methods. Western blots are typical of at least two replicate determinations. Cell numbers are given as means ± S.E. of three replicate determinations. Significant (P < 0.05) growth inhibition is indicated (*). RNA interference decreased expression of individual Sp proteins approximately 60 to 80% as described previously (Chadalapaka et al., 2008a, 2010).

Fig. 7.

CDDO-Me inhibits pancreatic tumor growth and down-regulates Sp proteins and Sp dependent genes. Tumor weights (A) and volume (B). Male athymic nude mice bearing orthotopic pancreatic (L3.6pL) tumors were treated with corn oil or CDDO-Me (7.5 mg/kg) for 4 weeks, and tumor weights and tumor volumes (in cubic millimeters) were determined as described under Materials and Methods. Significant (P < 0.05) inhibition (*) is indicated in results as means ± S.E. for five animals per treatment. Western blot analysis of tumor lysates for Sp proteins (C) and Sp-dependent proteins (D). Lysates from three mice in the treated and control groups were analyzed by Western blots as described under Materials and Methods. β-Actin served as loading control.