PPARγ is an orphan nuclear receptor that is overexpressed in multiple tumor types and cancer cell lines, and this receptor is a potential target for cancer chemotherapy [37
]. Different structural classes of PPARγ agonists, including the thiazolidinediones (TZDs), 15-deoxy-Δ12,14
-prostaglandin J2 (PGJ2), CDDO-Me and PPARγ-active 1,1-bis(3′-indolyl)-1-(p
-substituted phenyl)methanes (C-DIMs), activate overlapping and compound-specific growth inhibitory and proapoptotic responses in pancreatic cancer cells [17
]. Many of these compounds induce p21 and/or p27 expression, downregulate cyclin D1 protein, and cause G0
to S phase arrest and these effects are both cell context- and structure-dependent. For example, troglitazone, a TZD, induces p27 and not p21 protein expression in several pancreatic cancer cell lines, whereas in another study TZD induces p21 and differentiation markers in a number of pancreatic cancer cells [14
]. PPARγ-active C-DIMs also induce p21 but not p27 in Panc28 cancer cell lines and this is accompanied by a significantly higher percentage of cells in G0
(28%) and a decreased percentage in S phase (21%) after treatment for 24 hr [17
]. The induction of p21 in Panc28 cells by PPARγ-active C-DIMs is inhibited by PPARγ agonists; however, most other studies in pancreatic cancer cells have not investigated the role of this receptor in mediating these responses.
In this study, CDODA-Me inhibited Panc1 and Panc28 cell proliferation (), and comparative studies with DODA-Me demonstrated the importance of the 2-cyano group which markedly enhanced antiproliferative activity. The 2-cyano group was necessary for the PPARγ agonist activities of the glycyrrhetinic acid derivatives [11
] and structurally related oleane and lupane derivatives [7
]. CDODA-Me induced PPARγ-dependent transactivation and activity (), whereas DODA-Me which does not contain a 2-cyano substituent exhibited decreased antiproliferative activity () and did not exhibit PPARγ agonist activity (data not shown). In addition, CDODA-Me induced differentiation of 3T3-L pre-adipocytes and this is a highly prototypical PPARγ-dependent response (). These results on activation of PPARγ by DODA-Me and CDODA-Me in Panc1 and Panc28 cells are similar to those observed in prostate and colon cancer cells [11
Like other PPARγ agonists, CDODA-Me induced p21 and p27 and decreased cyclin D1 expression in Panc1 and Panc28 cells () and, in Panc1 cells (), this was accompanied by a G0
to S phase arrest. However, studies with the PPARγ antagonist GW9662 indicated that these responses were receptor-independent, and this contrasted to the receptor-dependent induction of p21 in Panc28 cells by PPARγ-active C-DIMs and the 2-cyano derivative of betulinic acid which, like CDODA-Me, contains a 1-en-3-one function in the A-ring [17
]. Thus, among the three PPARγ agonists, there was a structure-dependent induction of p21 and similar results have been observed for induction of KLF4, suggesting that CDODA-Me, PPARγ-active C-DIMs, and the cyano derivative of betulinic acid are selective PPARγ modulators.
CDODA-Me also induces apoptosis (DNA laddering) in Panc1 and Panc28 cells () and previous studies indicate that CDODA-Me, other PPARγ agonists, and anticancer drugs induce the proapoptotic proteins NAG-1 and ATF3 in colon, prostate and other cancer cells [25
]; however, induction of these proteins has not previously been investigated in pancreatic cancer cells. Results in demonstrate that CDODA-Me induced NAG-1 and ATF3 proteins in Panc28 and Panc1 cells and cotreatment with the PPARγ antagonist GW9662 did not affect the induction responses or activation of caspase-dependent PARP cleavage (). Previously, we also observed receptor-independent induction of the proapoptotic proteins NAG-1 and ATF3 by CDODA-Me in LNCaP prostate cancer cells [12
], and these responses were kinase-dependent and the prior induction of Egr-1 was associated with induction of NAG-1. In prostate cancer cells, induction of ATF3 by CDODA-Me was JNK-dependent [12
]; however, in pancreatic cancer cells, JNK and other kinase inhibitors had no effect on ATF3 induction (data not shown) which was not further investigated.
Egr-1 was induced in Panc1 and Panc28 cells within 1 - 2 hr after treatment with CDODA-Me, whereas NAG-1 was induced at later time points in Panc28 (6 - 24 hr) and Panc 1 (24 hr) cells (). This temporal pattern of NAG-1 and Egr-1 induction is similar to that observed in other studies where Egr-1 activates NAG-1 through interactions with the GC-rich proximal region of the NAG-1 promoter [26
]. CDODA-Me induces phosphorylation of several kinases (PI3K, p38/p42MAPK and JNK) in both Panc28 and Panc1 cells () as previously observed in prostate cancer cells [12
]; however, induction of Egr-1 was p42MAPK-dependent (). In Panc1 cells, the p42MAPK inhibitor PD98059 also inhibited induction of NAG-1 which is consistent with a role for Egr-1 in mediating the induction of NAG-1 by CDODA-Me. However, the PI3-K and p38 MAPK inhibitors LY294002 and SB203580, respectively, also decreased induction of NAG-1, demonstrating the contributions of Egr-1-independent pathways in Panc1 cells. Kinase inhibitor studies in Panc28 showed that induction of NAG-1 by CDODA-Me was primarily Egr-1-independent and was inhibited by LY294002 and SB20358 which had no effect on induction of Egr-1 in this cell line. This is one of the first examples of drug-dependent activation of both Egr-1 and NAG-1 in which induction of the latter gene is Egr-1-independent in one cell line (Panc28) and partially Egr-1-independent in another (Panc1 cells).
In summary, results of this study demonstrate for the first time that CDODA-Me inhibits growth and induces apoptosis in pancreatic cancer cells. Although CDODA-Me activates PPARγ in Panc28 and Panc1 cells, induction of growth inhibitory and proapoptotic proteins and activation of multiple kinase activities is receptor-independent. This is the first report of the induction of the proapoptotic protein NAG-1 in pancreatic cancer cells; however, it was evident from studies with kinase inhibitors that the mechanisms of NAG-1 induction and the role of Egr-1 is cell context-dependent in Panc28 and Panc1 cells and differs from results of previous studies on NAG-1 induction [26
]. Current studies are investigating the interplay between kinase activation, induction of proapoptotic proteins, and apoptosis by CDODA-Me in pancreatic cancer cells and the contributions of other pathways in mediating the proapoptotic effects of CDODA-Me in pancreatic cancer cells and tumors.