探討吲哚化合物對子宮內膜癌細胞株之腫瘤抑制機轉

The tumor-suppressed mechanism of indole compounds in endometrial cancer cells

吳冀威(Chi-Wei Wu)

指導教授 ： 曾淑玲

中山醫學大學/醫學院/醫學研究所/碩士(2015年)

https://doi.org/10.6834/CSMU.2015.00196

摘要

在美國，子宮內膜癌是相當常見的婦科癌症。而台灣也因飲食逐漸西化，子宮內膜癌發生率也有逐漸增加的趨勢。根據104年台灣衛福部統計資料顯示，103年惡性腫瘤死亡率佔十大死因之首位，子宮頸及部位未明示子宮癌佔女性十大癌症死亡率第七位，因此研發子宮內膜癌的新療法顯得必須且重要。
天然蔬果蘊含的種類多元之特殊化合物稱為植化素，研究顯示植化素對於多種癌症具有預防效果。其中一類植化素為吲哚化合物，文獻研究顯示吲哚化合物對於乳癌細胞MCF-7、MDA-MB-231與前列腺癌細胞PC3及LNCaP皆具有抑制癌細胞生長結果，但於子宮內膜癌細胞株如RL95-2、HEC-1A卻無詳細研究。根據本實驗室先前實驗結果，吲哚化合物處理RL95-2、HEC-1A後觀察到有明顯之細胞生長抑制，兩株細胞株對吲哚化合物之敏感性也不甚相同，本研究延續先前結果，欲探討吲哚化合物如何調控RL95-2、HEC-1A之生長與各別抑癌機轉為何。
吲哚化合物DIM由I3C代謝轉換而來，本實驗室發現DIM抑制子宮內膜癌效果優於I3C，因此選擇DIM 50μM各別處理RL95-2、HEC-1A 24、48、72小時後，觀察到細胞存活比例隨時間增加而減少，RL95-2細胞死亡現象較為明顯。而利用Acridine Orange染色發現DIM未能誘發此兩株細胞的自噬作用，進一步使用hoechst 33342染色法觀察到DIM明顯誘發RL95-2染色質濃縮。利用西方墨點法（Western Blot）觀察到RL95-2處理DIM 50μM 48小時後PARP與caspase 3有活化現象，HEC-1A則無。進一步觀察到DIM透過外源性細胞凋亡路徑活化caspase 8而造成細胞凋亡，內源性凋亡蛋白Bad、Bax、Bcl-2與caspase 9，皆未有明顯變化。而在細胞週期蛋白表現則觀察到DIM能造成HEC-1A之S期細胞週期停滯，其cyclin D1、cyclin B1蛋白表現隨著DIM處理時間增長而減少，P21蛋白表現則於DIM處理後明顯增加。
綜合上述實驗結果，證實吲哚化合物能抑制子宮內膜癌細胞株RL95-2與HEC-1A之生長作用，兩者抗癌機轉亦不相同，藉著誘發外源性細胞凋亡路徑促使RL95-2凋亡，透過調控細胞週期蛋白表現進而抑制HEC-1A的生長。

關鍵字

子宮內膜癌 ； 吲哚化合物 ； 細胞凋亡 ； 凋亡蛋白酶8 ； 細胞週期

並列摘要

Endometrial cancer is the mostcommonly diagnosed gynecologic malignancy in the United State. In Taiwan, westernized diet is becoming the major food culturewhich increases incidence of endometrial cancer steadily. According to statistic data from Ministry of Health and Welfare in 2014, malignant tumor is the first among the top ten leading causes of death. The mortality of cervix cancer anduterine corpus cancer is the seventh among the top ten mortality of cancer in women. All the information above shows that finding out effective anticancer strategy is necessary.
Naturally occurring compounds, known as phytochemicals, is rich in variety of vegetables and fruits. There is one kind of phytochemicals is called Indole compounds, some researches indicated that Indole compounds effectively not only inhibit growth ability in breast cancer cells, MCF-7 and MDA-MB-231, but also in prostate cancer cells, PC3 and LNCaP. However, there is seldom study about how Indole compounds affect the ability of growth in endometrial cancer cells. In our lab, we investigated indole compounds could inhibit cell proliferations of RL95-2 and HEC-1A cell lines, and RL95-2 was more sensitive to indole compounds than HEC-1A was. In this study,we try to figure out the tumorsuppressed mechanism ofindole compounds in RL95-2 and HEC-1A cell lines.
3,3'-Diindolylmethane(DIM),the derivativeproduct fromIndole-3-Carbinol(I3C),had better anticancer effects than I3C, DIM 50 μM was be chosen to investigate its anticancer mechanism for our research. RL95-2 and HEC-1A treated with DIM 50μM for 24, 48 and 72 hours, the rusults showedcell viability of both two cell lines were significant decreased after treated with DIM in time-dependent manner, and RL95-2 was more sensitive to DIM. By Acridine Orange staining assay, DIM could not induce autophagy neither in RL95-2 nor HEC-1A. By hoechst staining, DIM induced DNA condensation in RL-95-2 after 24h and 48h DIM treatments. We also used Western blot assay to detectthe expressions of apoptosis related proteins. In protein level, DIM activatedextrinsic apoptotic pathway in RL95-2. In level of cell cycle related protein expressions, DIM induced S phase cell cycle arrest in HEC-1A and decreased cyclin D1 and cyclin B1 expressionsin a time-dependent manner. Expression of P21 was increased in HEC-1A after treated with DIM.
In summary,we demonstrate that indole compounds inhibited cell proliferationsin RL95-2 and HEC-1A through different mechanisms. It induced apoptosis in RL95-2, in HEC-1A, it might inhibit the cell proliferation by regulating cell cycle.In the future, we will continue to clarify the anticancer mechanism of DIM in these two EC cell lines and finding out effective anticancer strategy in endometrial cancer therapy.

並列關鍵字

endometrial cancer ； RL95-2 ； HEC-1A ； DIM ； apoptosis ； caspase 8 ； cell cycle

參考文獻

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