抗癌藥物造成人類甲狀腺濾泡癌細胞死亡之機制:PPARγ促效劑與蟲草素的作用探討

Studies on the mechanisms of PPARγ agonists- and cordycepin-induced cell death in a Chinese thyroid carcinoma cell line

陳瀅(Ying Chen)

指導教授 ： 王淑美

國立臺灣大學/醫學院/解剖學暨生物細胞學研究所/博士(2009年)

https://doi.org/10.6342/NTU.2009.02817

摘要

由於生活環境與飲食的改變，導致癌症的發生率逐年上升，但是用於治療癌症的化學治療與放射線治療，對於病人的生活品質有很大的影響，所以目前許多研究著重在積極找尋藥物以治療癌症。我們以由甲狀腺癌病人組織所分化出的甲狀腺濾泡癌細胞株CGTH W-2進行藥物試驗，嘗試找尋除了全甲狀腺切除外，對抗癌細胞生長的藥物。
　　在本論文的第一部份，我們以二種peroxisome proliferator-activated receptor γ (PPARγ) 促效劑ciglitazone與15-deoxy-Δ12,14-prostaglandin J2 (15dPGJ2)，探討其作為抑癌藥物的可能性與抑癌機制為何。首先二種PPARγ促效劑皆會經由誘導細胞凋亡及部份細胞壞死而降低CGTH W-2細胞株的存活率。細胞凋亡是透過cytochrome c 活化caspase 3 ， 進而促使poly (ADP-ribose) polymerase (PARP) 斷裂的訊息路徑。另外，我們發現PPARγ促效劑處理後，會降低甲狀腺癌細胞黏附斑蛋白質的表現，包括vinculin 、integrin β1 、focal adhesion kinase (FAK)、paxillin等，造成細胞的黏附能力下降。同時，磷酸化FAK與paxilln的蛋白質表現與染色程度都有下降的現象。此外，PPARg促效劑活化PTEN與PTP-PEST等去磷酸酶，使黏附斑蛋白質去磷酸化，降低黏附斑的穩定，造成細胞自培養基脫離。另外，抗以integrin β1的抗體處理甲狀腺癌細胞會使細胞黏附能力降低，也會引發細胞凋亡。我們更發現， PPARγ促效劑會導致PTEN活化並和Akt表現下降有關係，並且可能也影響到甲狀腺癌細胞的存活。綜合以上結果，二種PPARγ促效劑造成甲狀腺癌細胞凋亡是透過cytochrome ccaspase 3與PTEN-Akt的訊息路徑，而造成細胞壞死則是經由PARP活化的訊息路徑。
　　第二部份， 則是利用冬蟲夏草中的有效成份之一的蟲草素 (3’-deoxyadenosine)，研究其抗癌的效果。目前已知蟲草素抑制癌細胞生長的作用會透過腺苷第三亞型接受器 (A3 adenosine receptor, A3R)，但是其下游的作用分子與其他腺苷接受器是否參與仍然不清楚。我們繼續利用濾泡性甲狀腺癌細胞CGTH W-2，探討腺苷接受器於蟲草素抗癌的作用中扮演角色為何。蟲草素處理會降低甲狀腺癌細胞的存活率，並造成細胞凋亡，但是不引起細胞壞死。甲狀腺癌細胞經由反轉錄聚合酶連鎖反應後，發現其表現四種腺苷接受器，包括 A1R、A2AR、A2BR與A3R。我們利用藥理學方法證實，蟲草素造成細胞凋亡主要是透過A1R與A3R，而部份的作用是透過A2AR與A2BR。此外，我們也利用A1R與A3R的小干擾RNA (small interfering RNA, siRNA)，再次證實A1R與A3R參與蟲草素抑癌作用的重要性。而蟲草素透過A1R與A3R活化下游的caspase 7，造成PARP的斷裂。A1R與A3R的抑制劑或A1R與A3R的小分子干擾RNA也可避免caspase 7活化與PARP斷裂。另一方面，蟲草素可透過A1R與A3R使甲狀腺癌細胞內鈣離子濃度上升，A1R與A3R的抑制劑和鈣離子螯合劑BAPTA/AM，皆可抑制由鈣離子濃度上升而活化的calpain。此外，蟲草素造成的細胞凋亡作用，是透過活化calpain，進而啟動caspase 7-PARP訊息路徑。總結來說，我們發現蟲草素造成甲狀腺癌細胞凋亡，是透過A1R-/A3R-calcium-calpain-caspase 7-PARP的訊息路徑，並且希望這樣的結果能提供治療甲狀腺癌一個新的方向。我們的研究提供除了全甲狀腺切除外，治療甲狀腺癌的另一個方向，也對上述藥物的抑癌機制，提供新的看法。

關鍵字

PPARγ促效劑 ； 蟲草素 ； 細胞壞死 ； 細胞凋亡 ； 腺苷接受器 ； 訊息傳遞

並列摘要

Thyroid carcinoma is the third popular cancer during young women in Taiwan. The major treatment of thyroid carcinoma is total thyroidectomy with radioiodine. However, searching for the anti-cancer drugs to replace the traditional treatments is also important. In the first part of the thesis, we focused on the effects of two peroxisome proliferator-activated receptor γ (PPARγ) agonists, ciglitazone and 15-deoxy-Δ 12, 14-prostaglandin J2 (15dPGJ2), on the survival of thyroid carcinoma CGTH W-2 cells. Both ciglitazone and 15dPGJ2 decreased cell viability in a time- and dose-dependent manner. Cell death was mainly due to apoptosis, with a minor contribution from necrosis. Increased levels of active caspase 3, cleaved poly (ADP-ribose) polymerase (PARP), and cytosolic cytochrome c were noted. Moreover, inhibition of cleaved PARP activity prevented necrosis induced by both PPARγ agonists and 15dPGJ2-induced apoptosis. Inhibition of both PARP activity and caspase activity reduced apoptosis and necrosis caused by PPARγ agonists. In addition, ciglitazone and 15dPGJ2 induced detachment of CGTH W-2 cells from the culture substratum. Immunostaining of focal adhesion proteins, including integrin β1, phosphorylated FAK and paxillin were decreased concomitantly with decreased protein levels and after PPARγ agonist treatment. Moreover, PPARγ agonists induced expression of protein
tyrosine phosphatase-PEST (PTP-PEST) and of phosphatase and tensin homologue deleted on chromosome ten (PTEN). The upregulation of these phosphatases might contribute to the dephosphorylation of FAK and paxillin, since inhibition on the expression of PTP-PEST and PTEN prevented PPARγ agonist-induced dephosphorylation of FAK and paxillin. The downregulation of integrin may also contribute to the disassembly of focal adhesions and, consequently, cell detachment. It was noted that 90% of the apoptotic cells exhibited disassembled focal adhesions, suggesting the presence of crosstalk between apoptosis and integrin-focal adhesion signaling. In addition, upregulation of PTEN was correlated with the dephosphorylation of Akt, which may contribute to cell apoptosis. In conclusion, PPARγ agonists induced apoptosis of thyroid carcinoma cells via the cytochrome c-caspase 3 and PTEN-Akt pathways and induced necrosis via the PARP pathway.
Cordycepin (3’-deoxyadenosine), a bioactive gradient of Cordyceps sinensis, has been shown to exhibit anti-cancer activity. Although activation of A3 adenosine receptor (A3R) has been implicated in the inhibition of tumor cell growth, the downstream events contributing to the cytotoxic effects of cordycepin remain to be elaborated. This study used CGTH W-2, a follicular thyroid carcinoma cell line, to investigate the roles of adenosine receptors in the cordycepin signaling pathway. Cordycepin-induced cell death was due to apoptosis, but not necrosis. Reverse transcription-polymerase chain reaction analyses showed that CGTH W-2 cells expressed A1R, A2AR, A2BR, and A3R. Since the antagonists of A1R and A3R blocked cordycepin-induced apoptosis more effectively than A2AR or A2BR, we focused on the roles of A1R and A3R in this event. Small interfering RNA (siRNA) for A1R and A3R abolished cordycepin-induced apoptosis, confirming the involvement of these two ARs.
Both antagonists and siRNAs of A1R and A3R abolished the cordycepin-induced cleavage of caspase 7 and PARP. Cordycepin also increased intracellular calcium levels, which was prevented by A1R and A3R antagonists. Moreover, cordycepin induced calcium-dependent calpain activation, since depletion of intracellular calcium blocked the cordycepin-induced increase in calpain activity. Calpeptin and BAPTA/AM inhibited the cordycepin-induced cleavage of caspase 7 and PARP, implying the upstream roles of calcium and calpain. In conclusion, the cordycepininduced apoptosis of CGTH W-2 cells is mainly mediated by the A1R-/A3R-calcium-calpain-caspase 7-PARP pathway, and these results provide a new strategy for thyroid cancer therapy.

並列關鍵字

adenosine receptors ； cordycepin ； PPARγ agonists ； necrosis ； apoprosis ； signal transduction

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