組蛋白去乙醯化抑制劑Suberoylanilide Hydroxamic Acid (SAHA)誘導人類口腔癌細胞凋亡機轉之研究

Abstract

口腔癌在台灣死亡率和發生率急劇攀升，是台灣地區死亡年增率最高的癌症。儘管近年在診斷及治療技術上已長足進步，但口腔癌病人之五年存活率並未有顯著改變。因此，迫切需要尋求新的的治療與預防方法來改善整體存活率及生活品質。目前已發現在多種癌症中均可發現組蛋白去乙醯化酶(HDACs)有異常上升表現。最近研究發現，多種HDAC抑制劑能選擇性抑制腫瘤細胞生長、促進細胞分化或凋亡。有效地抑制和殺傷負荷腫瘤動物的腫瘤細胞，並且不伴有明顯的毒性副作用。HDAC抑制劑成為抗癌藥物研發的新潮流之ㄧ。不過，有關HDAC 抑制劑抗腫瘤作用的理論基礎至今仍不甚明白。相同的HDACI對不同種類的腫瘤細胞的作用機轉也不相同。本實驗室前導研究發現在70% 口腔癌病人的組織中有HDAC2過度表現，正常口腔組織則無表現。 因此HDACI可做為未來治療口腔癌的潛力新藥物。本研究以人類口腔癌細胞株SAS及Ca9-22來探討已被證實有很好的口服耐受性的HDACI，SAHA (suberoyl anilide bishydroxamine )對口腔癌細胞的影響，並進一步了解其可能的機制。結果顯示，以0.5-5 μM SAHA處理SAS及Ca9-22 細胞，可以明顯抑制其細胞生長，且濃度愈高或作用時間愈長，抑制效應就愈明顯 (IC50分別為1.5μM&3μM)。藉由流式細胞儀分析細胞之細胞週期結果顯示，以3 μM SAHA處理細胞株12小時後，會明顯增加sub G0/G1細胞數目百分比。而在西方墨點法分析中，SAHA會增加SAS及Ca9-22細胞 PARP cleavage的情形，亦證實SAHA可引起人類口腔癌細胞的細胞凋亡。我們在Ca9-22和SAS細胞中加入Caspase-8和-9的抑制劑（Z-LEHD-FMK, Z-IETD-FMK），發現皆可降低SAHA誘導的細胞凋亡現象發生。顯示SAHA在口腔癌細胞中可以活化內在和外在兩條不同的細胞死亡路徑。西方墨點法實驗中，SAHA會增加SAS中DR5, FADD等蛋白量的表現，活化caspase-8後使得t-BID及細胞質的Cytochrome c表現量上升進而活化caspase-9。而在Ca9-22中Bcl-2的下降及Bax的上升共同作用使得細胞質的Cytochrome c表現量上升，進而活化caspase-9。 我們進一步利用各種抑制劑來探討SAHA誘導SAS及Ca9-22細胞凋亡的機轉。 發現在SAS細胞中加入自由基（ROS）的抑制劑，N-acetylcysteine(NAC)後可降低SAHA所誘發的細胞凋亡反應，顯示自由基（ROS）在SAHA引起的人類口腔癌細胞細胞凋亡中扮演了重要的角色。西方墨點法分析發現NAC可降低SAS中DR5, p-FADD等蛋白量的表現，可能因此降低SAHA所誘發的細胞凋亡。另外，利用20ng的TRAIL與1

Oral cancer is the fourth leading cause of cancer-related deaths in male population in Taiwan. Despite recent advances in radiotherapy and chemotherapy, the survival of patients with oral cancer has not improved significantly. Continued investigation of new chemotherapeutic agents is thus needed. Our recent studies have shown that histone deacetylase 2 (HDAC2) is overexpressed in 70% of oral cancer specimens. Furthermore, recent studies have shown that inhibitors of HDACs (HDACIs) possess antitumor activity and are well tolerated, supporting the idea that their use might develop as a specific strategy for cancer treatment. In this study, we investigated the effects and mechanisms of suberoylanilide hydroxamic acid (SAHA, one of the most potent HDAC inhibitors) on OSCC cell lines SAS and Ca9-22. Here, we demonstrated that SAHA induces apoptosis in human oral cancer cell lines SAS and Ca9-22 as evidenced by nuclear condensation, TUNEL labeling and cleavage of PARP. Apoptosis induced by SAHA was both time- and dose-dependent; however, the mechanisms are different in these two cells. In SAS cells, SAHA treatment induced DR5, FAS/FASL, FADD, caspase-8, -9 activation and Bid cleavage. In addition, SAHA treatment induced reactive oxygen species (ROS) production as detected by H2DCFDA fluorescence. Pretreatment of cells with N-acetyl cysteine (NAC) reduced the up-regulation of DR5, FAS, FADD and completely inhibited SAHA-induced apoptosis. These results indicated that ROS was an important mechanism for SAHA-induced apoptosis in SAS cells. SAHA-induced apoptosis was also completely inhibited in the presence of caspase 8 or caspase 9 inhibitors (Z-LEHD-FMK, Z-IETD-FMK). Taking together, SAHA induced apoptosis via subsequent induction of ROS, DR5, FADD, FAS/FASL, caspase-8 activation, Bid cleavage and then activation of mitochondrial pathway. In Ca9-22 cells, SAHA induced Bax protein expression, caspase 9 activation. In addition, we found SAHA down-regulated the expression of Bcl-2. Treatment caspase 9 inhibitor (Z-IETD-FMK) decreased SAHA induced apoptosis and the result was not more effective when both of caspase 8 and capase 9 inhibitors were treated. These data showed that SAHA-induced apoptosis by activating intrinsic- apoptosis pathway. We further evaluated the potential combinative effect of TRAIL and SAHA in OSCC cell lines. Compared with either TRAIL (20ng/ml) or SAHA (1