評估reversine對甲狀腺癌細胞株的抑癌效果及其機制之探討

The anti-tumor effect evaluation and mechanistic investigation of reversine on thyroid cancer cell lines

花士哲(Shih-Che Hua)

指導教授 ： 張天鈞

國立臺灣大學/醫學院/臨床醫學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.01585

摘要

研究背景：
在內分泌腫瘤中，甲狀腺癌是最常見的癌症，雖然以手術或合併原子碘治療甲狀腺分化癌預後非常好，但對於已轉移且對原子碘治療無效的分化癌或是甲狀腺未分化癌，目前臨床治療效果非常不理想，急待新藥物之開發。染色體非整倍體變異，為腫瘤細胞的重要特徵，包括甲狀腺癌在內，與惡性度高和預後差有關。Aurora kinase (Aur)為調控細胞週期正常進行的重要激酶之ㄧ。癌細胞會表現過量的Aur，反之抑制Aur則會抑癌生長，Aur抑制劑會導致細胞週期阻滯和染色體多套體化及癌細胞凋亡，目前已有數個Aur抑制劑在進行臨床試驗中。甲狀腺癌也會表現過量的Aur，並會隨著未分化程度增加而表現增加。近來發現reversine為purine類似物的合成小分子，也能有效抑制與腫瘤生長有關的激酶，包括Aur，而去抑制急性骨髓癌和多發性骨髓瘤，但尚未有應用在甲狀腺癌的研究。
研究目的與假說：
針對目前臨床上對於甲狀腺癌的治療困境，研究新的有效治療藥物有其重要性和急迫性。本研究假說是reversine能抑制甲狀腺癌細胞株的腫瘤生長，並干擾細胞分裂週期之進行，和誘發細胞凋亡。
研究方法：
先利用活體外實驗，以MTT分析法檢驗reversine對於人類甲狀腺未分化癌細胞株(ARO)、人類濾泡癌細胞株 (WRO) 及人類分化不良甲狀腺癌細胞株 (SW579)，是否能抑制腫瘤細胞之生長；同時進行動物實驗，利用腫瘤異種移植的裸鼠去探討reversine抑制甲狀腺癌的活體效果；若發現有抑癌效果，則進一步利用流式細胞儀探討reversine導致細胞週期之變化和細胞死亡的抑癌機轉。若發現有細胞凋亡之現象，則利用西方墨點法去偵測是否有caspase 3、caspase 8、或caspase 9的活化，而加入泛caspase抑制劑是否能阻斷reversine所造成的細胞死亡，進一步探討凋亡訊息傳遞是屬粒腺體依賴型或非依賴型路徑。
研究結果：
活體外 reversine能有效抑制甲狀腺癌細胞株生長，但此三細胞株的易感性不同，分化良好的WRO細胞較有抗性；動物實驗研究，只能成功建立ARO腫瘤異種移殖的裸鼠，reversine能有效抑制ARO腫瘤生長，然而reversine的劑量增加並不能增強抑癌效果；細胞週期分析方面，reversine處理後會導致ARO、WRO、SW579等三甲狀腺癌細胞株的G2/M期阻滯；在細胞死亡分析方面，三細胞株死於凋亡比率會隨暴露於reversine的時間和濃度之增加而增加，但三細胞株的易感性亦不同，SW579細胞最為顯著，ARO和WRO細胞則不顯著。進一步探討reversine所造成SW579細胞的凋亡，發現caspase 8和caspase 3會被活化，而caspase 9不會被活化，顯示屬非粒腺體依賴型路徑，而反之泛caspase抑制劑能阻斷reversine的細胞死亡效果。
研究結論：
本研究首次證實reversine對甲狀腺癌細胞株腫瘤有抑癌效果並探討其抑癌機轉，reversine抑制ARO和WRO細胞主要是造成細胞週期進行阻滯，而抑制SW579細胞主要是造成細胞週期進行阻滯和細胞凋亡。Reversine所導致的凋亡訊息傳遞機轉是屬粒腺體非依賴型路徑，而泛caspase抑制劑能抑制凋亡。Reversine為日後進入甲狀腺抗癌藥物臨床試驗的新潛力用藥。

關鍵字

reversine ； 甲狀腺癌 ； aurora kinase ； 細胞週期 ； 凋亡 ； 腫瘤異種移植裸鼠

並列摘要

Background:
Thyroid cancer is the most common cancer among endocrine malignancies. Although current management with surgical resection followed by radioactive iodine therapy has been proved effective in treating differentiated thyroid cancer, but there have been no available effective treatment modalities for metastatic radio-resistant differentiated thyroid cancer and anaplastic thyroid cancer. Aneupolidy represents a hallmark of solid cancers including thyroid cancer and is associated with poor prognosis. Aurora kinases (Aur) are important kinases in the regulation of normal cell cycle, which are overexpressed in various type of cancer cells and aurora kinases inhibition will suppress tumor growth. Currently, several Aur inhibitors are undergoing clinical trials. Thyroid cancer has also been proved to have Aur overexpression which is related to undifferentiated phenotypes. Recently, a small synthetic molecule named reversine, a purine analogue, has proved anti-tumor effective in acute myeloid leukemia cells and multiple myeloma cells. However, up to date, there has been no study aimed at thyroid cancer treatment by reversine.
Objective and hypothesis:
Because of the current clinical treatment dilemma in incurable differentiated thyroid cancer, poorly differentiated thyroid cancer or anaplastic thyroid cancer, a novel and effective treatment modality is urgently needed. We propose reversine could suppress thyroid cancer tumor growth by inducing cell cycle arrest and cell apoptosis.
Methods:
First, we in vitro examined the anti-tumor effects of reversine on human anaplastic thyroid cancer cell line (ARO), human follicular thyroid cancer cell line (WRO), and human poorly-differentiated thyroid cancer cell line (SW579) by MTT assay; simultaneously, we in vivo examined the anti-tumor effects of reversine on ARO by xenograft nude mice model; if anti-tumor effects proved, we further investigated reversine’s effects on cell cycle and cell death by flow cytometry analysis. If apoptosis is proved by flow cytometry, to clarify the apoptotic signal transduction pathway, further activated caspase 3, caspase 8, and caspase 9 would be detected by western blot. Pan-caspase inhibitor would be tested its ability to reverse cell death caused by reversine.
Results:
Reversine suppressed these 3 thyroid cancer lines (ARO, WRO, SW579) growth in vitro, although with different susceptibility, e.g. well-differentiated WRO cells more resistant to reversine. Only ARO xenograft nude mice model was successfully established. Reversine was also validated to suppress ARO tumor growth in vivo, but the anti-tumor was not in a dose-dependent manner. In the aspect of cell cycle analysis, these 3 thyroid cancer cells treated by reversine were shown G2/M phase arrest. In the aspect of cell death analysis, apoptotic ratio increased in a time and dose-dependent manner in these 3 thyroid cancer cell lines, but also with different susceptibility. SW579 cells treated by reversine were susceptible to apoptosis, but neither ARO nor WRO cells. SW579 cells treated by reversine activated caspase 8 and capase 3, not caspase 9. And cell death after treatment of reversine was attenuated by pan-caspase inhibitor.
Conclusion:
This study is the first to prove anti-tumor effects of reversine on thyroid cancer cell lines in vitro and in vivo. Besides, we investigated reversine’s anti-tumor mechanisms. Reversine inhibits ARO and WRO cells mainly by cell cycle progression arrest. However, reversine inhibits SW579 cells mainly by cell cycle arrest and inducing cell apoptosis. The apoptotic mechanisms were via mitochondria-independent extrinsic pathway. Reversine is a novel potential anti-thyroid cancer drug and may enter clinical trials in the future.

並列關鍵字

reversine ； thyroid cancer ； aurora kinase ； cycle cycle ； apoptosis ； xenograft nude mice

參考文獻

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