CDDO-Me is a multifunctional compound with potent anti-inflammatory and anticarcinogenic
]. CDDO-Me inhibits proliferation and induces apoptisis in diverse cancer cell types in cell culture [25
] and inhibit the growth of tumor implants and prevent development of cancers in mouse models [29
]. The anticarcinogenic
mechanisms of CDDO-Me involves inhibiting a number of prosurvival signaling pathways, such as MAPK (Erk1/2), NF-κB, and Akt/mTOR signaling [32
]. hTERT expression and telomerase activity is elevated in vast majority of cancers including pancreatic cancers [11
]. Reexpression of telomerase provides unlimited proliferative advantage to cancer cells and telomerase inhibition inhibits cell proliferation, inducing cellular senescence or apoptosis. Little is known about the interplay between the anticancer mechanisms of CDDO-Me and telomerase. In a previous study we have shown that inhibition of cell proliferation and induction of apoptosis in pancreatic cancer cells by CDDO-Me is associated with the inhibition of hTERT gene that codes for the catalytic subunit of telomerase and its telomerase activity; however, the molecular mechanism of hTERT inhibition by CDDO-Me was not elucidated. The results of the present study confirm our previous findings that induction of apoptosis even at very low concentrations of CDDO-Me used in this study is associated with the inhibition of hTERT expression and its telomerase activity. CDDO-Me could repress hTERT by inhibiting hTERT gene transcription and/or hTERT protein production. Our data showed inhibition of both hTERT gene expression and protein production. Further, it also inhibited the phosphorylation of hTERT protein. The attenuation of hTERT mRNA, basal hTERT and phospho-hTERT suggested that CDDO-Me might also inhibit telomerase activity. Indeed, our data demonstrated that treatment with CDDO-Me reduced the telomerase activity in both pancreatic cancer cell lines (Figure S2). Although reduction in cellular telomerase activity can be attributed to the inhibition of hTERT gene expression and/or inhibition of phosphorylation of hTERT, these data do not demonstrate whether CDDO-Me is able to directly inhibit the telomerase activity of hTERT. Overall, attenuation of hTERT gene expression, hTERT protein production and phosphorylation and telomerase activity indicated that inhibition of telomerase is part of the mechanism by which CDDO-Me inhibits proliferation and induce apoptosis in pancreatic cancer cells. These findings are in agreement with other reports showing that inhibition of hTERT telomerase activity is necessary for the antiproliferative and apoptosis-inducing activity of natural compounds including genistein, sulforaphane and green tea polyphenols [38
]. However, more work is required to determine whether CDDO-Me directly binds and degrades RNA template of telomerase and if it also causes shortening of telomeres.
A number of factors and molecules that regulate hTERT transcription have been identified. The hTERT core promoter contains binding sites for several transcription factors such as Sp1, c-Myc and NF-κB and STAT-3 [20
]. Inhibition of these transcription factors would likely impact transcription of hTERT gene. Indeed, we found that CDDO-Me inhibited Sp1, c-Myc and NF-κB in Panc-1 and MiaPaCa-2 cells, indicating that diminished hTERT expression and protein production by CDDO-Me may be attributed to the inhibition of these transcription factors. Contrary to our expectations however, various repressors of gene transcription, such as CTCF, E2F-1 and MAD1 that negatively regulate hTERT expression were also reduced in cells treated with CDDO-Me. Since CDDO-Me inhibited transcription factors that both up-regulate and down-regulate hTERT gene expression, how is hTERT gene expression then inhibited? One possibility is that CDDO-Me exerts more inhibitory function on transcription factors that up-regulate hTERT expression (Sp1, c-Myc and NF-κB and STAT-3) than those that down-regulate its expression (e.g., CTCF, E2F-1 and MAD1). This conclusion however requires further elucidation.
As stated before, epigenetic mechanisms play critical roles in regulating hTERT expression. Contrary to the prevalent view that hypermethylation of gene promoters typically inhibits their transcription; hypermethylation of hTERT promoter is associated with increased hTERT expression [41
]. Epigenetically, genes expression can be regulated through processes such as DNA methylation, chromatin remodeling and modulation of the activity of enzymes and factors associated with these processes. Studies have shown that DNA methylation plays an important role in hTERT transcription and DNA methylation is primarily the function of DNMTs [43
]. DNMT1, a maintenance methyltransferase
, maintains hypermethylation of hTERT promoter, whereas DNMT3a and DNMT3b are responsible for de novo
activity. Treatment with CDDO-Me inhibited DNMT1 and DNMT3a in Panc-1 and MiaPaCa-2 cells. As expected, the inhibition of DNMT1 resulted in demethylation
of hTERT promoter. The number of methylated CpGs in hTERT promoter was significantly reduced following treatment with CDDO-Me. These data correlated with the inhibition of hTERT expression and suggest that promoter demethylation plays an important role in inhibition of hTERT expression by CDDO-Me. Demethylation of hTERT promoter allows binding of repressors, such as CTCF or E2F-1 and silencing of hTERT expression [39
]. CDDO-Me not only caused demethylation of hTERT promoter but also suppressed CTCF, E2F-1 and MAD-1. Thus, the exact mechanism by which demethylation of hTERT promoter leads to the inhibition of hTERT expression by CDDO-Me remains elusive.
Besides DNA methylation, histone acetylation and methylation also play critical roles in hTERT expression [44
]. Histone modifications result in loosening of the chromatin, allowing binding of the activators and/or repressors of gene
transcription at the gene promoters. We found decrease in cellular levels of transcriptionally active chromatin
markers acetylated histones H3 and H4. CDDO-Me also affected the methylation of histone, since di-methyl-H3 lysine 4 and trimethyl-H3K9 were also reduced in cells treated with CDDO-Me. The alterations in chromatin markers were also found at the hTERT promoter. ChIP analysis showed decrease in ac-H3, ac-H4, dimethyl-H3 and tri-methy-H3K9 at hTERT promoter in cells treated with CDDO-Me. Together, these data demonstrate that inhibition of epigenetic processes such as DNA methylation and chromatin modifications plays a crucial role in inhibition of hTERT expression by CDDO-Me in pancreatic cancer cells. These findings corroborate the results of other studies in which other anticancer agents also inhibited hTERT expression in tumor cells by interfering with the epigenetic regulatory processes [23