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.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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This study was supported by the National Institutes of Health National Cancer Institute [Grants R01-CA108718, P20-CA10193, and R01-CA136571]; and Texas A&M AgriLife Research.
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.110.064451.
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ABBREVIATIONS:
- CDDO
- 2-cyano-3,12-dioxoleana-1,9-dien-28-oic acid
- CDDO-IM
- imidazole ester of 2-cyano-3,12-dioxoleana-1,9-dien-28-oic acid
- CDDO-Me
- methyl ester of 2-cyano-3,12-dioxoleana-1,9-dien-28-oic acid
- CDODA
- 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oic acid
- CDODA-Me
- methyl ester of 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oic acid
- GSH
- glutathione
- MMP
- mitochondrial membrane potential
- PPARγ
- peroxisome proliferator-activated receptor γ
- ROS
- reactive oxygen species
- Sp
- specificity protein
- VEGF
- vascular endothelial growth factor
- DMEM
- Dulbecco’s modified Eagle’s medium
- FBS
- fetal bovine serum
- PARP
- poly(ADP-ribose) polymerase
- TBP
- TATA binding protein
- ZBTB10
- zinc finger and BTB domain containing 10
- PCR
- polymerase chain reaction
- DMSO
- dimethyl sulfoxide
- TBST
- Tris-buffered saline/Tween 20
- MG132
- N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal
- DTT
- dithiothreitol
- as-miR-27a
- antisense microRNA-27a
- CM-H2DCFDA
- 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester.
- Received March 2, 2010.
- Accepted May 13, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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