SAHA及PEITC在死亡受器引發口腔癌細胞凋亡訊息機轉之研究

Molecular mechanisms for SAHA and PEITC sensitization of death receptor mediated apoptosis in oral cancer

葉正昌(Cheng-Chang Yeh)

指導教授 ： 郭彥彬

國立臺灣大學/牙醫專業學院/臨床牙醫學研究所/博士(2011年)

https://doi.org/10.6342/NTU.2011.10809

摘要

Suberoylanilide hydroxamic acid (SAHA)，屬於 HDAC2 的專屬抑制劑，近年來已經被證實在多種腫瘤細胞中，可誘發腫瘤細胞的自殺性凋亡，因此 SAHA 被認為是很有潛力的抗腫瘤藥物。 SAHA 結合放射性治療、cisplatin 和腫瘤壞死因子相關的誘導凋亡配體 (TRAIL)，在多種腫瘤的治療結果中，有很好的效果。此這篇研究，我們證明了 SAHA可以誘發口腔癌細胞進行自殺性凋亡，在西方墨點的實驗結果中，我們發現了 SAHA 增加了 Fas、Fas ligand、DR4 和 DR5 的蛋白表現量，同時也活化了 caspase-8 和 caspase-9 的活性。而 SAHA 所誘發的細胞凋亡可以被 caspase-8 的抑制劑 Z-IETD-FMK 完全抑制，而 caspase-9的抑制劑 Z-LEHD-FMK 則可減少一部分 SAHA 所誘發的細胞凋亡。人類 DR5/Fc 重組抗體 可以有效地抑制 SAHA 所誘發的細胞凋亡，但Fas/Fc 或DR4/Fc 卻無法抑制 SAHA 所誘發的細胞凋亡，這些結果顯示出 SAHA 所誘發的細胞凋亡，主要是透過活化 DR5/TRAIL 的死亡路徑。 此外，針對 SAHA 的次毒性濃度測試顯示，SAHA 可以引起對 TRAIL 有耐受度的兩株人類口腔癌細胞－SAS 和 Ca9-22 對外加的 TRAIL ，重新引起細胞自殺性凋亡，且這個凋亡反應與 p53 基因沒有相關聯。因此，結合 SAHA 和 TRAIL 同時在口腔癌的治療上，在未來可能是一個相當具潛力的新治療方式。
除了SAHA 外，近年來發現，在水田芥植物中所發現的自然化合物，PEITC也屬於 HDAC2 的抑制劑，可以誘發細胞周期的停止和細胞凋亡。 先前的研究顯示出 PEITC 具有顯著預防及治療口腔鱗狀細胞癌的功效。 本研究我們將探討 PEITC 誘發口腔鱗狀細胞癌細胞凋亡的效能與機轉，為了釐清 PEITC 所誘發的細胞凋亡機轉，我們的研究包含了: 死亡接受器、caspases、粒線體的胞色素的釋放和 MAPK 相關的訊號傳遞路徑。 根據西方墨點的實驗結果，當細胞在 PEITC 處理之後會使得 DR4 和 DR5 的蛋白質表現量上升，此外 PEITC 也活化了 caspase-8 和 caspase -9 當加入 caspase-8 和 caspase -9 的專一抑制劑，同時也抑制了 PEITC 所誘發的細胞凋亡。當加入JNK、ERK 和 p38 個別的專一抑制劑時，只有 JNK 的抑制劑－SP600125 可以減少 PEITC 所引發的細胞凋亡，這個結果顯示出 JNK 的活性在細胞凋亡過程中扮演著重要的角色甚至和 DR4 與 DR5 的蛋白質表現量上升並使細胞對 TRAIL 更加敏感有關。
PEITC 會誘發 ROS 的產生並使 JNK 活化，使得細胞進入凋亡的程序，當加入 ROS 的清除試劑 NAC 便抑制了 PEITC 所誘發的細胞凋亡。這個結果顯示 PEITC 所誘發 ROS 的產生，是造成細胞凋亡的主因。此外 PEITC 也會在 SAS 和Ca9-22 細胞中誘發 autophagy 透過活化 AMPK 的訊息路徑， 且 PEITC 所誘發的 autophagy 扮演著促使細胞死亡的角色。
在這篇研究中我們發現 PEITC 可引發ROS 的產生且活化 JNK 的活性，並造成 DR4 和 DR5 的 mRNA 和蛋白質表現量的上升，最後造成細胞凋亡。而且 PEITC 也在 SAS 和 Ca9-22 細胞中引起促進細胞死亡的 autophagy。

關鍵字

細胞凋亡 ； 死亡接受器 ； 口腔癌

並列摘要

Suberoylanilide hydroxamic acid has been shown to selectively induce tumor apoptosis in cell cultures and animal models in several types of cancers and is about as a promising new class of chemotherapeutic agents. In addition, SAHA showed synergistic anticancer activity with radiation, cisplatin, and tumor necrosis factor–related apoptosis–inducing ligand (TRAIL) in some cancers. Here, we report SAHA also induced apoptosis in human oral cancer cells. Western blotting showed SAHA increased Fas, Fas ligand, DR4, and DR5 protein expression and activated caspase-8 and caspase-9. The apoptosis was almost completely inhibited by caspase- 8 inhibitor Z-IETD-FMK and attenuated by caspase-9 inhibitor Z-LEHD-FMK. Human recombinant DR5/Fc chimera protein but not Fas/Fc or DR4/Fc significantly inhibited apoptosis induced by SAHA. These results suggest that SAHA induces apoptosis mainly through activation of DR5/TRAIL death pathway. Furthermore, subtoxic concentrations of SAHA sensitize to TRAIL resistant human oral cancer cells, SAS and Ca9-22, to exogenous recombinant TRAIL-induced apoptosis in a p53-independent manner. Combined treatment of SAHA and TRAIL may be used as a new promising therapy for oral cancer.
Recently, it has been found that phenethyl isothiocyanate (PEITC), a naturally occurring isothiocyanate especially abundant in watercress, could induce cell cycle arrest and apoptotic cell death. Previous studies showed that PEITC have significant chemopreventive and possibly chemotherapeutic effects in OSCC cells. Here we investigated the pro-apoptotic effect of PEITC and the mechanisms of apoptosis induced by PEITC in human OSCC cell lines, SAS and Ca9-22. To understand apoptotic mechanisms induced by PEITC, we studied the role of death receptors, caspases, mitochondria-cytochrome c release, and mitogen-activated protein kinase (MAPK) signaling pathways involved in PEITC-induced apoptosis.
The western blot data shows that DR-5 and DR-4 were up-regulated after PEITC treatment in SAS and Ca9-22 cells. Both the caspase-8 and -9 activities were stimulated by PEITC and the apoptosis was almost completely inhibited either by caspase-8 inhibitor Z-IETD-FMK or caspase-9 inhibitor Z-LEHD-FMK.
PEITC could induce apoptosis in SAS and ca9-22 cells in a time dependent manner via the mitochondria caspase cascade. Three MAPKs, JNK (c-Jun N-terminal kinase), ERK (extracellular signal-regulated protein kinase) and p38 kinase, were evaluated after PEITC treatment in SAS and Ca9-22 cells. JNK activation was obviously induced by PEITC. The activation of JNK is critical for the initiation of the apoptotic processes and sensitive to TRAIL-induced cell death by up-regulating DR5 and DR4 via generation of reactive oxygen species (ROS), leading to rapid induction of TRAIL-mediated signaling and cell death. Importantly, the SP600125 compound, an anthrapyrazolone inhibitor of JNK, but not the ERK and p38 inhibitors, attenuated both DR4 and DR5 up-regulated and suppressed apoptosis caused by PEITC. Here we had shown that apoptosis induction by PEITC in human oral cancer cells is associated with ROS-dependent activation of JNK. PEITC -mediated ROS production, JNK activation, and/or apoptosis in human oral cancer cells were significantly attenuated by pretreatment with NAC. The mechanism of PEITC plays an important role in the killing of cancerous cells and offers a potential therapeutic strategy for its anticancer action in vivo.
This study demonstrated that PEITC not only induces JNK-dependent DR4 and DR5 upregulation dependent of ROS generation but also induces autophagy via the AMPK pathway and the PEITC-induced autophagy plays the role of promoting cell death in SAS and Ca9-22 cells.

並列關鍵字

SAHA ； PEITC ； TRAIL ； DR5 ； ROS ； Apoptosis ； Autophagy

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