https://scholars.lib.ntu.edu.tw/handle/123456789/162558
標題: | PEITC做為未來口腔癌化學預防與治療藥物的發展潛力 Study on the Potential Usage of phenethyl isothiocynate for the Treatment of Oral Cancer |
作者: | 楊靜欽 Yang, Ching-Chin |
關鍵字: | 異硫氰酸苯乙酯;細胞凋亡;口腔癌;細胞週期;PEITC;SAS cell;apoptosis;MAPK kinase;G2/M cell cycle arrest | 公開日期: | 2005 | 摘要: | 根據衛生署民國九十三年的癌症登記報告指出,口腔癌在臺灣男性十大癌症中死亡率與發生率皆位居第四位。儘管近年在診斷及治療之科技上有不少進步,但口腔癌病人之五年存活率並未有明顯改變 。因此,需要一個更好的治療方法來改善整體存活率及生活品質。 流行病學研究指出,多吃蔬菜與水果,可以降低胃癌、食道癌、肺癌、口腔癌等腫瘤的發生率。 進一步研究發現,十字花科蔬菜中含有各種的有機異硫氰酸鹽(-N=C=S, ITC)類,具有抑制化學致癌作用。 食用乙基異硫氰酸酯(Phenylethyl Isothiocyanate, PEITC)的小鼠和大鼠可減少皮膚、直腸、口腔致癌物處理而引起癌瘤形成的機率。 但其可能的作用機制至今尚未明瞭。本研究在以人類口腔癌細胞株SAS探討PEITC對口腔癌細胞的影響,並進一步了解其可能的機制。 我們發現, 以7.5-15 μM PEITC處理SAS 細胞,可以明顯抑制其細胞生長(p<0.05),且具有劑量依賴性 (dose-dependent) 的毒殺效應 (IC50= 7.5 μM)。 藉由流式細胞儀分析細胞之細胞週期結果顯示,以7.5 uM PEITC處理SAS 細胞,可增加G2/M細胞與sub G0細胞 (apoptotic cells)數目的百分比。而DAPI染色、cytochrome C 的釋放,及PARP的活化等,更證實PEITC引起人類口腔癌細胞死亡的方式為細胞凋亡。 在SAS細胞株中,PEITC的處理會促使p53蛋白在細胞核中表現以及p53的下游基因如Bax也會隨時間而增加,減少造成抑制細胞死亡的BcL2蛋白的表現,但並不會影響 BcL-xL 蛋白質之表現。 Fas 以及TRAIL receptor 2 (亦稱DR5) 的表現也隨著PEITC處理的時間有增加的趨勢增加。 PEITC 亦可以同時活化caspase 8 和 caspase 9 ,在 SAS 細胞的培養液中加入 caspase 8和 caspase 9的抑制劑 (Z-LEHD-FMK; Z-IETD-FMK), 皆能減低PEITC引起的細胞凋亡,且有加成效果。 因此PEITC可藉由粒線體及死亡接受器兩條路徑造成口腔癌細胞的凋亡。另外,西方墨點分析與G2/M 相關蛋白質結果發現, PEITC會增加細胞中細胞週期調控之p21、p27 蛋白質之表現,但並不會影響 Cdk1、cyclinB1 蛋白質之表現。 進一步實驗證明PEITC會激發JNK(c-Jun NH2-termianl kinase)及ERK (Extra-cellular regulation kinase)的活性, JNK 的抑制劑SP600125 及ERK 的抑制劑 PD98059可抑制PEITC所引起的細胞凋亡。因此初步認為MAPK kinase的成員對於PEITC造成人類口腔癌細胞的凋亡也扮演了重要角色。對於PEITC是否可當作口腔癌抗癌藥物則需要再以更精確的動物實驗及臨床試驗作更深入的評估其抗癌的功效。 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. Recent studies have shown that feeding phenylethyl isothiocyanate (PEITC) inhibited carcinogen-induced mouse oral carcinogenesis. However, the mechanisms by which it inhibits oral carcinogenesis are not well understood. The present study examined the effects of PEITC on proliferation, cell cycle and apoptosis of oral cancer cell lines SAS using trypan blue assay, flow cytometry analysis, DAPI assay and Western blot analysis. PEITC significantly inhibited the proliferation of SAS oral cancer cell lines in a dose-dependent manner with a 50% inhibition concentration (IC50) of PEITC about 7.5 μM. DNA flow cytometric analysis showed that PEITC treatment induced a G2/M arrest. PEITC treatment also caused significant apoptosis of SAS cells as evidenced by DAPI staining, increase of cytochorme C release and cleavage of PARP. These results indicate that the inhibitory effects of PEITC on oral carcinogenesis may be related to the G2/M phase arrest and induction of apoptosis. The PEITC induced cell cycle arrest was associated with the increase of p21, p27 protein, but not the protein levels of Cdk1 and cyclin B1. We also found PEITC enhanced the expression of p53 and Bax in SAS cells. In addition, PEITC decreased the expression of Bcl-2 in SAS. PEITC treatment also induced the expression of death receptors, Fas and DR5. Furthermore, PEITC could induce the activation of caspase 8 and caspase 9 in SAS cells. The PEITC-induced apoptosis was significantly attenuated in the presence of specific inhibitors of caspase-8 and caspase-9. These results suggest that apoptosis occurred via both the mitochondria mediated and death receptor mediated pathway. The MAPK family of serine/threonine kinases, including extracellular signal-regulated kinase1/2 (ERK1/2), c-jun N-terminal kinase1/2/3 (JNK1/2/3), and p38 MAPK play an important role in cell proliferation and apoptosis in response to different stimuli. We found JNK and ERK were activated shortly after PEITC treatment in SAS cells. In addition, JNK inhibitor SP600125 and the ERK inhibitor PD98059 suppressed apoptosis induced by PEITC. These results indicate that MAPKs involve in apoptosis induction by PEITC in human oral cancer cells. |
URI: | http://ntur.lib.ntu.edu.tw//handle/246246/51308 | 其他識別: | en-US |
顯示於: | 口腔生物科學研究所 |
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