Graduate School of Medical and Pharmaceutical Sciences, Chiba University, Chiba, Japan.
Research Institute of Life Innovation, Toyo University, Gunma, Japan.
Pharmacology. 2018;102(1-2):26-36. doi: 10.1159/000489128. Epub 2018 May 15.
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).
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.
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.
Targeting the epigenetic induction of DKK1 may lead to effective treatment of MM, and T3E has great potential to induce anti-MM activity.
Wnt 信号通路在肿瘤细胞生长中起着至关重要的作用，包括恶性间皮瘤（MM）的发展。在各种癌症中观察到负 Wnt 调节剂的表观遗传沉默导致组成型 Wnt 信号通路，这值得进一步关注。我们已经报道 α-生育三烯酚（T3E）的琥珀酸醚衍生物在 MM 细胞中具有很强的细胞毒性作用。因此，在这项研究中，我们研究了 T3E 是否可以通过 Wnt 拮抗剂基因 Dickkopf-1（DKK1）的表观遗传改变来介导抗 MM 作用。
采用 WST-1 和细胞分析仪分析 T3E 对人 MM 细胞系（H2452、H28）细胞活力和凋亡的影响。实时 PCR 和 Western blot 用于评估 mRNA 和蛋白质水平的表达。用甲基化特异性 PCR 和染色质免疫沉淀研究 T3E 处理 MM 细胞后 DKK1 启动子区域的甲基化状态和表观遗传修饰。使用小干扰 RNA 介导的敲低（siRNA）和特异性抑制剂来验证 DKK1 是 T3E 的靶标。
T3E 显著损害 MM 细胞活力，增加磷酸化-JNK 和 DKK1 的表达，并抑制 Wnt 信号下游靶基因 cyclin D。用 siRNA 或特定的 JNK 抑制剂敲低 DKK1 表达证实了 DKK1 和 JNK 在 T3E 诱导的 MM 细胞细胞毒性中的作用。另一方面，T3E 抑制细胞骨架相关蛋白 4（CKAP4）的表达，作为 Wnt 非依赖性 DKK1 受体促进细胞增殖。用 siRNA 沉默 CKAP4 似乎不会直接影响 MM 细胞活力，这表明需要同时表达 DKK1 和 CKAP4。此外，T3E 介导的 MM 细胞中 DNA 甲基转移酶（DNMT1、3A 和 3B）和组蛋白去乙酰化酶（HDAC1、2、3 和 8）的抑制导致 DKK1 表达增加，从而促进肿瘤生长抑制。用 Zebularine（一种 DNA 甲基转移酶抑制剂）和丁酸钠（一种组蛋白去乙酰化酶抑制剂）处理的 MM 细胞表现出细胞毒性作用，这可能解释了 T3E 对 MM 细胞的抑制作用。此外，T3E 处理后 MM 细胞中 DKK1 的表达增加与启动子的甲基化状态呈正相关；T3E 降低 DNA 甲基化并增加组蛋白乙酰化。此外，T3E 特异性增加组蛋白 H3 赖氨酸 4（H3K4）的甲基化活性，而对 H3K9 和 H3K27 没有影响。
靶向 DKK1 的表观遗传诱导可能导致 MM 的有效治疗，而 T3E 具有诱导抗 MM 活性的巨大潜力。
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