BACKGROUND: Wnt signaling plays an essential role in tumor cell growth, 
including the development of malignant mesothelioma (MM). Epigenetic silencing 
of negative Wnt regulators leading to constitutive Wnt signaling has been 
observed in various cancers and warrants further attention. We have reported 
that a succinate ether derivative of α-tocotrienol (T3E) has potent cytotoxic 
effects in MM cells. Thus, in this study, we investigated whether the anti-MM 
effect of T3E could be mediated via the epigenetic alteration of the Wnt 
antagonist gene, Dickkopf-1 (DKK1).
METHODS: WST-1 and cell analyzers were employed to analyze the effects of T3E on 
cell viability and apoptosis of human MM cell lines (H2452, H28). Real-time PCR 
and Western blot were performed to evaluate the expression at mRNA and protein 
levels. Methylation status and epigenetic modifications of DKK1's promoter 
regions after T3E treatment in MM cells were studied using methylation-specific 
PCR and Chromatin immunoprecipitation. Small interfering RNA-mediated knockdown 
-(siRNA), and specific inhibitors, were used to validate DKK1 as a target of 
T3E.
RESULTS: T3E markedly impaired MM cell viability, increased the expression of 
phosphorylated-JNK and DKK1 and suppressed cyclin D, a downstream target gene of 
Wnt signaling. Knockdown of DKK1 expression by siRNA or a specific JNK inhibitor 
confirmed the contribution of DKK1 and JNK to T3E-induced cytotoxicity in MM 
cells. On the other hand, cytoskeleton-associated protein 4 (CKAP4) expression, 
which promotes cell proliferation as a Wnt-independent DKK1 receptor was 
inhibited by T3E. Silencing CKAP4 by -siRNA did not appear to directly affect MM 
cell viability, thereby indicating that expression of both DKK1 and CKAP4 is 
required. Furthermore, T3E-mediated inhibition of both DNA methyltransferases 
(DNMT1, 3A, and 3B) and histone deacetylases (HDAC1, 2, 3, and 8) in MM cells 
leads to increased DKK1 expression, thereby promoting tumor growth inhibition. 
MM cells treated with Zebularine (a DNMT inhibitor) and sodium butyrate (an HDAC 
inhibitor) exhibited cytotoxic effects, which may explain the inhibitory action 
of T3E on MM cells. In addition, an enhanced expression of DKK1 in MM cells 
following T3E treatment is positively correlated with the methylation status of 
its promoter; T3E decreased DNA methylation and increased histone acetylation. 
Moreover, T3E specifically increased histone H3 lysine 4 (H3K4) methylation 
activity, whereas no effects were observed on histone H3K9 and H3K27.
CONCLUSIONS: Targeting the epigenetic induction of DKK1 may lead to effective 
treatment of MM, and T3E has great potential to induce anti-MM activity.