細胞自體吞噬作用影響癌症放射線敏感性與治療結果之研究

The study of the impact of autophagy on the radiosensitivity and treatment outcome of cancer

曾顯群(Hsien-Chun Tseng)

指導教授 ： 周芬碧教授

中山醫學大學/醫學院/生化暨生物科技研究所/博士(2011年)

https://doi.org/10.6834/CSMU.2011.00011

摘要

手術切除是唯一能夠治癒原位肝癌的方法，但只有少數患者能夠接受手術治療。對於無法手術切除的腫瘤，其他治療方法包括：經動脈化學栓塞（transarterial chemoembolization, TACE）、酒精注射療法、高頻電熱滅除（radiofrequency ablation, RFA）、生物治療、化學治療及三度空間順形放射線治療（3-D conformal radiotherapy, 3D-CRT），甚至合併上述療法等多重療法。本研究目的在於探討肝癌細胞對於放射線治療抗性的機轉。方法是使用兩株人類肝癌細胞：HepG2與Hep3B，分別給予不同劑量的放射線照射之後，使用MTT assay分析，分別在不同時間點來比較兩株細胞的存活力（cell viability），做為評估放射線敏感性的參考指標，結果證實Hep3B細胞具有較高的射線敏感性，且DAPI染色法顯示Hep3B細胞呈現較多的凋亡細胞，而且當放射線劑量增加時，凋亡的Hep3B細胞數目愈多。接著以acridine orange stain將細胞染色，在螢光顯微鏡下發現HepG2細胞質內出現許多紅色空泡狀物質，稱之為AVO（acidic vesicular organelles），且隨著放射線劑量增加，AVO數目也增多。Western blotting分析自噬小體（autophagosomes）的標記：LC3-II，結果證實HepG2細胞質內的AVO為自噬小體，電子顯微鏡進一步確認自噬小體構造的存在。本研究證實放射線會誘發肝癌細胞的自體吞噬反應，而且是肝癌細胞的自我防衛機制，免於放射線所誘發的凋亡反應。3-MA,一種autophagy抑制劑，與放射線合併處理HepG2細胞，MTT cell viability assay證實3-MA促進細胞凋亡的進行，不僅在in vitro模式，3-MA合併放射線能夠增加抑制細胞的生長，在HepG2 xenografts的老鼠模式，3-MA合併放射線也增加抑制腫瘤的生長。
近來研究指出癌症幹細胞（CSCs）是造成治療失敗及復發的主要原因，本研究收集了34位直腸癌患者的癌組織，包括切片診斷及經過同步化放療後手術切除的癌組織，以immunohistochemical staining分析癌細胞表達CD133（CSC 標記）及p62（autophagy標記）的程度，由兩位病理科醫師判讀分級。結果顯示術後的癌細胞表達CD133標記的細胞愈多，患者的DFS（disease-free survival）較差，但是不論術前或術後的組織p62表達的強弱，對患者的無病存活率或一般存活率都沒有顯著影響；TRG4（tumor regression grade 4, 意指沒有存活的癌細胞）與DFS呈正相關。
本研究的結果證實了放射線照射肝癌細胞會誘導其autophagy活化，造成放射線治療的抗性，合併3-MA抑制autophagy的保護機制增加放射線毒殺細胞的效果，未來可以提供肝癌放射線治療的新策略。CSC的存在影響直腸癌的預後，如何發展對抗CSC的新治療方法是未來研究的重點，p62的表達與患者的存活無關，未來必須另尋其它調控autophagy相關蛋白是否可以作為療效或是存活的預測標記。

關鍵字

自體吞噬 ； 肝癌 ； 直腸癌 ； 細胞凋亡 ； 放射線敏感性 ； 化學放射治療 ； 癌症幹細胞

並列摘要

The only curative modality for treatment of primary hepatocellular carcinoma is complete surgical removal. However, few patients can receive complete surgical resection because of frequent late stage at diagnosis. For the treatment of unresectable hepatocellular carcinoma at diagnosis, there are several treatment modalities such as: transarterial chemoembolization (TACE)、intratumoral alcohol injection、radiofrequency ablation (RFA)、biological therapy、chemotherapy and 3-D conformal radiotherapy. In this study, 3-methyladenine (3-MA), an autophagy inhibitor, was investigated for its potential to enhance radio-sensitivity under radio-resistant conditions both in vitro and in vivo. Hep3B and HepG2 cells were used to examine the radioresistance of liver cancer cells. The results show that Hep3B cells respond to irradiation with increased apoptotic cell death and that HepG2 is radio-resistant due to the IR-induced autophagy, as verified by DNA fragmentation, electron microscopy, acidic vesicular organelle formation, and Western blot analysis. Application of IR with 3-MA to inhibit autophagy simultaneously suppressed the expression of LC3 and enhanced cell death. The tumor xenograft model in nude mice verified the synergistic cytotoxic effect of 3-MA and IR, which resulted in significant repression of tumor growth. The results demonstrate that IR-induced autophagy provides a self-protective mechanism against radiotherapy in HepG2 cells. In addition, 3-MA enhances the cytotoxicity of IR in cell models and suppresses tumor growth in animal models. Based on these results, application of 3-MA, or other autophagy inhibitors, could be used as an adjuvant for radiotherapy when radio-resistance develops as a result of autophagy response.
Recent studies indicate the existence of CSCs（cancer stem cells）as the main cause of treatment failure and cancer relapse. In our study a total of 34 rectal cancer patients with neoadjuvant chemoradiotherapy and surgery were enrolled. Collection of the cancer tissues including biopsy specimens and surgical specimens was sent for immunohistochemical staining of CD133 ( a cancer stem cell marker) and p62 ( an autophagy marker ). Their reading and grading was executed by two pathologists. Our result showed that high expression of CD133 was correlated to poor patient’s disease-free survival. The expression of p62 marker has no effect on survival. Our data showed TRG4 was significantly related to DFS, but not to OS.
Our study demonstrated that IR induced autophagy in HepG2 cells, which contributed to its resistance to radiation. Combination of 3-MA and IR enhanced the cytotoxicity of HepG2 cells. This may be adopted as a new strategy in considering the radiotherapy of liver cancer. A more effective treatment should be developed to eradicate cancer stem cells. It is our future goal to search a new effective biomarker to predict the treatment response of cancer since p62 expression fails to be relevant to survival. It is also important to determine if there is any potential to target autophagy signaling in the elimination of cancer stem cells.

並列關鍵字

autophagy ； liver cancer ； rectal cancer ； apoptosis ； radiosensitivity ； chemoradiotherapy ； cancer stem cells

參考文獻

12. Cheng JC, Chuang VP, Cheng SH, Huang AT, Lin YM, Cheng

2.Hofseth LJ, Hussain SP, Wang XW, Harris CC.. Hepatocellular cancer: molecular biology and genetics. In: Kelsen DP, Daly JM, Kern SE, Levin B, Tepper JE, editors. Gastrointestinal oncology: principles and practice. Philadelphia: Lippincott Williams & Wilkins. P 539–558（2002）.

3.Staib F, Hussain SP,. Hofseth LJ, Wang XW, and. Harris CC. TP53 and liver carcinogenesis. Human mutation 21: 201-216 (2003).

4.C.M. Lo, H. Ngan, W.K. Tso, C.L. Liu, C.M. Lam, R.T. Poon, S.T. Fan, J. Wong. Randomized controlled trial of transarterial lipiodol chemoembolization for unresectable hepatocellular carcinoma, Hepatology 35: 1164–1171 (2002).

5.R. Murakami, S. Yoshimatsu, Y. Yamashita, K. Sagara, A. Arakawa, M. Takahashi. Transcatheter hepatic subsegmental arterial chemoembolization therapy using iodized oil for small hepatocellular carcinomas. Correlation between lipiodol accumulation pattern and local recurrence, Acta. Radiol. 35:576–580 (1994).