腺嘌呤藉由細胞的凋亡反應和自噬作用抑制人類大腸癌細胞的生長

Adenine Inhibits the Growth of Human Colon Cancer Cells via Apoptosis and Autophagy

賴馨吾(Hsin-Wu Lai)

指導教授 ： 魏正宗 ； 林俊哲

中山醫學大學/醫學院/醫學研究所/博士(2020年)

https://doi.org/10.6834/CSMU202000016

摘要

研究目的：對於人類晚期大腸癌的治療，目前的治療藥物及治療方式仍有更加精進的發想。近年，自然界存在的腺嘌呤廣泛在許多癌症上被研究，諸多已證實其在抑制癌症上有正面的角色。腺嘌呤參與了多種細胞生物作用，其在藥理學方面之應用也陸續被研究。尤其，它在治療癌症的應用中引起了極大的探討。在本研究中，我們努力研究腺嘌呤在人類大腸癌細胞中的抗癌效應及作用機制。期望研究所得的成果，將來能在對抗人類大腸癌的治療上，有所貢獻。
研究方法：本研究使用人類大腸癌細胞株HT-29和Caco-2。運用MTT試驗(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay)來測定細胞存活率。使用西方墨點法(Western blotting)來測定蛋白質磷酸化和表達的程度。使用定量即時聚合酶鏈鎖反應(Realtime polymerase chain reaction)來評估具影響性基因的信使核糖核酸 (mRNA)表現的變化。
研究結果：腺嘌呤以劑量依赖性的方式顯著地抑制大腸癌細胞株HT-29和Caco-2的細胞存活率。相較於Caco-2 細胞，腺嘌呤誘導之細胞凋亡在HT-29細胞較顯著。數據顯示，在兩種人類大腸癌細胞系中，腺嘌呤可激活腺苷酸活化蛋白激酶(AMPK)訊息傳導，且可經由抑制雷帕霉素靶蛋白(mammalian target of rapamycin，簡稱mTOR)導致細胞自噬性的死亡。
結論：我們的研究結果顯示腺嘌呤可抑制人類大腸癌細胞的生長。腺嘌呤的抗癌活性歸因於凋亡訊息的傳導，以及AMPK途徑的活化而抑制mTOR導致細胞自噬。本研究亦證實，腺嘌呤具有停滯大腸癌細胞週期的能力，進而導致癌細胞死亡。因此，腺嘌呤是具有抗癌效力的代表性天然化合物，未來在治療人類大腸癌的角色上，將扮演著一定程度的助益。

關鍵字

大腸癌 ； 腺嘌呤 ； 細胞凋亡 ； 細胞自噬 ； 雷帕霉素靶蛋白 ； 單磷酸腺苷活化蛋白質激酶

並列摘要

Objective：Because of current treatment methods of terminal stage or metastatic human colon cancer is unsatisfactory. The objective of this study is to examine the anti-cancer effects of adenine and the underlying mechanism in human colon cancer cells. Many studies had showed that adenine involved in a variety of cell biological processes and the pharmacological uses. Its therapeutic use for managing cancer is meaningful issues.
Methods and Materials：In our study, cell viability was measured using MTT assay. Flow cytometry was used to analysis the role of cell cycle arrest of adenine. Levels of phosphorylation and protein expression were determined using Western blotting. qPCR was performed to assess the changes in mRNA expression of genes of interest.
Results：Adenine treatment resulted in a significantly inhibited viability in human colon cancer cells, HT-29 and Caco-2 cells, in a dose-dependent fashion. Adenine induced significant apoptosis in HT-29 cells, whereas Caco-2 cells exhibited less apoptotic responses. The data showed that adenine activated AMP-activated protein kinase (AMPK) signaling contributing to autophagic cell death through mTOR in both colon cancer cell lines.
Conclusion：Our study and findings suggest that adenine inhibits the growth of human colon cancer cells. The anti-cancer activity is attributed to the activation of apoptotic signaling and the activation of AMPK/mTOR pathway. Flow cytometry also confirms that adenine would lead to cell cycle arrest of human colon cancer cells. Adenine represents a natural compound with anti-cancer potency. Therefore, adenine will be an important role in the strategy of treatment of humen colon cancer. It will be an useful compound with less side effect in the colon cancer therapy. In the future, it is inspiring that medication treatment combined with adenine to treat human colon cnacer will represent more effectable outcome and better life quality.

並列關鍵字

Colon cancer ； Adenine ； Apoptosis ； Autophagy ； mTOR ； AMPK

參考文獻

1. Ferlay, J. et al. Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. International journal of cancer 2015;136: 359-386, doi:10.1002/ijc.29210.

2. Arnold M., Sierra M. S., Laversanne M., Soerjomataram I., Jemal A., Bray F. Global patterns and trends in colorectal cancer incidence and mortality. Gut. 2017;66(4):683–691. doi: 10.1136/gutjnl-2015-310912.

3. Jose María Huguet, Patrícia Suárez, Luis Ferrer-Barceló, et al. Screening for colorectal cancer in patients with inflammatory bowel disease. World J Gastrointest Endosc. 2018;10(11):322–325. doi: 10.4253/wjge.v10.i11.322.

4. Yumei Zhang, Zhiyu Chen, Jin Li, et al. The current status of treatment for colorectal cancer in China. Medicine. 2017;96(40): e8242. doi: 10.1097/MD. 0000000000008242.

5. Ragnhammar P, Hafström L, Nygren P, et al. Glimelius B; A systematic overview of chemotherapy effects in colorectal cancer. Acta Oncol. 2001;40(2-3):282-308. dol:10.1080/02841860151116367.