吳茱茰鹼於惡性神經膠質細胞瘤U87-MG細胞誘發細胞凋亡與存活性細胞自嗜之分子訊息探討

Molecular Signals of Evodiamine-Induced Apoptosis and Survival Autophagy in Human U87-MG Glioma Cells

劉安正(Ann-Jeng Liu)

指導教授 ： 邱文達

共同指導教授 ： 施純明

臺北醫學大學/醫學院/臨床醫學研究所/博士(2014年)

摘要

惡性膠質細胞瘤(malignant gliomas)為最常見之成人原發性惡性腦瘤，然而因為腦瘤好發於腦部重要位置，無法完全以手術切除，因此給予化療藥物或放射療法做為手術之外的治療方式。由於臨床使用之化癌藥物往往昂貴並有副作用，且因血腦障壁的存在而降低抗癌藥對腦癌之作用，因此科學家致力於從中草藥中發展具有抗癌作用之藥物。吳茱萸鹼(evodiamine, Evo)是由中草藥吳茱萸萃取出來具有生物活性之生物鹼，分子量僅303.3 Daltons，因此具有通過血腦障壁之可能性。文獻證實Evo可導致癌細胞進行細胞凋亡(apoptosis)而具有抗癌作用，並可誘導細胞進行細胞自噬(autophagy)，然其詳細機轉仍不明。因此本論文研究Evo誘導人類惡性神經膠質細胞瘤U87-MG細胞死亡的型式，並探討其中可能參與之訊息傳遞因子。本研究首先利用MTT assay證實Evo可抑制U87-MG細胞生長。利用流式細胞儀搭配annexin V/PI及acridine orange染劑分別偵測細胞apoptosis、necrosis與autophagy比例，並利用immunoblotting方式偵測autophagy之marker蛋白microtubule-associated protein light chain 3 (LC3)-II，證實Evo可誘導腦癌細胞同時進行autophagy及apoptosis。Autophagy雖被歸類為第二型計畫性細胞死亡形式，然而在生理上autophagy可藉分解細胞質內蛋白質或胞器如粒線體或內質網，來維持胞內平衡(homeostasis)及細胞存活，是細胞面對不佳環境的適應生存機制。本實驗證實autophagy inhibitor (3-methyladenine)與Evo併用後，apoptosis比例進一步增加，且存活率降低，顯示Evo所誘導之autophagy扮演保護細胞角色。鈣離子為二級訊息傳遞因子之一，文獻指出，細胞遭受外來刺激時鈣離子濃度會上升而導致apoptosis或autophagy，然而鈣離子在Evo誘導細胞死亡中扮演之角色尚不明確，因此本研究即進一步探討。利用Fluo-3 AM染劑配合流式細胞儀偵測細胞內鈣離子濃度（[Ca2+]i），結果發現Evo作用1小時後，U87-MG細胞內鈣離子濃度即明顯上升。以粒線體膜電位專一性染劑JC-1偵測粒線體膜電位，結果顯示Evo作用會造成粒線體膜電位下降。給予內質網IP3 receptor (IP3R)的抑制劑2-aminoethoxydiphenyl borate (2-APB) 可有效抑制Evo造成的[Ca2+]i上升、粒線體膜去極化及apoptosis等現象，顯示Evo藉由增加鈣離子濃度以造成粒線體傷害，最終導致細胞死亡。此外，2-APB亦可抑制Evo所誘導之autophagy，顯示鈣離子濃度增加亦參與autophagy之訊息傳遞路徑。給予細胞內鈣離子螯合劑BAPTA-AM之實驗結果證實Evo藉鈣離子而促進c-Jun N-terminal kinases (JNK)蛋白活化，而給予JNK抑制劑(SP600125)後則可降低autophagy，並增加apoptosis，顯示Evo透過細胞內鈣離子/JNK之分子機制誘導細胞進行保護性autophagy。文獻指出在glioma cell lines中，細胞膜陽離子通道蛋白transient receptor potential vanilloid 1 (TRPV1)的表現量較一般細胞為高，且其活化會促使鈣離子流入細胞，進而引發細胞凋亡或autophagy，然而其詳細分子機制仍不清楚。Evo為TRPV1之agonist，但TRPV1於Evo所誘導apoptosis或autophagy之角色仍不明，因此本研究探討TRPV1於Evo誘導細胞死亡中之重要性。實驗結果指出，細胞外鈣離子螯合劑EGTA、TRPV1抑制劑capsazepine (CPZ)或轉染TRPV1 siRNA後可抑制Evo造成的[Ca2+]i上升、JNK活化及autophagy，同時apoptosis比例增加，並降低細胞存活率，顯示Evo可活化TRPV1，促使細胞外鈣離子進入細胞內以誘導U87-MG細胞進行保護性autophagy。綜合上述實驗結果，本論文證實Evo促進細胞內質網IP3R釋放鈣離子，或活化TRPV1使鈣離子進入細胞，促使JNK活化，因而誘導腦癌細胞進行保護性autophagy，同時鈣離子濃度增加導致粒線體傷害而造成細胞凋亡。此研究之進行有助於瞭解Evo對抗腦癌細胞之分子機轉，以利提供臨床上抗癌藥物開發之新方向，增加腦癌患者的存活率。

關鍵字

惡性膠質細胞瘤 ； 吳茱萸鹼 ； 細胞自噬 ； 細胞凋亡 ； 鈣離子 ； TRPV1 ； JNK

並列摘要

Malignant gliomas are the primary malignant brain tumors which are difficult to be treated with surgery completely. Adjuvant chemotherapy or radiation is the alternative treatments in addition to surgery. The clinical medicines for treating gliomas have serious side effects, and the presence of blood-brain barrier diminishes the therapeutic effect. These drugs merely extend the survival time for a number of months. Therefore, development of new agents from Chinese herbs against gliomas is worthy of studying. Evodiamine (Evo), an alkaloid with alkaloids biological activity, is extracted from Chinese herbal medicine Evodiae fructus. Evo may have an ability to pass through BBB since the molecular weight of Evo is only 303.3 Daltons. It was reported that Evo has anti-cancer effects through inducing apoptosis. Evo can induce autophagy in tumor cells. However, the detail mechanism is still unclear. Accordingly, in this study, we aimed to investigate the underlying molecular mechanism of Evo-induced cytotoxicity in glioma cells. Firstly, the MTT assay showed that Evo decreased the U87-MG cell viability. Evo induced glioma cells to undergo dose- and time-dependent apoptosis and autophagy in U87-MG cells as examined using flow cytometry with annexin V/PI staining, PI staining, acridine orange staining, respectively, and immunoblotting. Although autophagy is classed as a type II cell death, it is also a crucial process for cells to maintain homeostasis and survival through degradation of cellular proteins and organelles, including mitochondria and endoplasmic reticula (ER). Therefore, the autophagy inhibitor (3-methyladenine) was employed to investigate the role of Evo-induced autophagy. Pharmacological inhibition of autophagy resulted in increased apoptosis and reduced cell viability. The results indicated that Evo induced pro-survival autophagy. Variation of intracellular calcium concentration may involve in the regulation of apoptosis and autophagy. However, the detail molecular mechanism is not well-understood. Using flow cytometry with Fluo-3 AM and JC-1 staining, we showed that the intracellular calcium ([Ca2+]i) increased from 1 h, and mitochondrial depolarization was induced after treatment with Evo. Blockade of ER calcium channel (IP3 receptor) by 2-aminoethoxydiphenyl borate (2-APB) significantly reduced Evo-induced cytosolic calcium elevation, apoptosis, and mitochondrial depolarization, which suggests that Evo induces a calcium-mediated intrinsic apoptosis pathway. Inhibition of ER calcium channel activation also significantly reduced Evo-induced autophagy. Inactivation of c-Jun N-terminal kinases (JNK) suppressed Evo-mediated autophagy accompanied by increased apoptosis. Furthermore, Evo-mediated JNK activation was abolished by BAPTA-AM, an intracellular calcium scavenger, suggesting that Evo mediates autophagy via a calcium-JNK signaling pathway. It has been reported that transient receptor potential vanilloid type 1 (TRPV1) receptor, a non-selective ligand-gated cation channel, is highly expressed in glioma cells. The activation of TRPV1 may lead to apoptosis, as well as lead to autophagy through calcium-mediated signaling. Evo is a TRPV1 agonist, but the role of TRPV1 in Evo-induced apoptosis and autophagy is unclear. Here, we proved that inhibition of extracellular calcium influx by the scavenger (EGTA) or inhibition of TRPV1 by capsazepine (CPZ) or TRPV1 siRNA reduced EVO-induced increase of [Ca2+]i, JNK activation and autophagy accompanied with increase of apoptosis, suggesting that Evo-induced JNK-mediated autophagy was resulted from influx of extracellular calcium through TRPV1 channel. Taken together, these results suggest that Evo increases the intracellular calcium through ER calcium channel or TRPV1 to induce JNK signaling-mediated autophagy and calcium/mitochondria-mediated apoptosis in U87-MG cells. This study reveals the molecular mechanism of Evo-induced cell death and provides a strategy for development of new chemotherapeutic agents against brain cancer.

並列關鍵字

Malignant glioma ； evodiamine ； autophagy ； apoptosis ； calcium ； TRPV1 ； JNK

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