鈣與內質網壓力參與吳茱萸鹼誘導細胞自噬及凋亡之研究

Participation of Calcium and ER Stress in Evodiamine-Induced Autophagy and Apoptosis

江峯(Feng Jiang)

指導教授 ： 施純明

臺北醫學大學/醫學院/醫學科學研究所/碩士(2011年)

摘要

根據行政院衛生署九十九年的癌症死亡人數統計中肺癌占20.0%，仍有約8194人死於肺癌，占罹癌死因之第一位。肺癌在臨床初期不易診斷且無法完全以外科手術切除，預後不良易復發，治療上常併用化學治療及放射治療為輔助療法，但價格昂貴且具副作用。傳統醫學廣泛將中草藥作為其主要治療方法之一，近年來在流病學及學術研究中有越來越多文獻證實，從中草藥萃取的抗癌生物活性成分，可用以發展作為促進化療效果的化學輔助療法 (chemical adjuvants)，同時改善因化學療法所導致的副作用。中草藥吳茱萸可萃取出具生物活性之吳茱萸鹼 (evodiamine)，在中醫上用來治療頭痛、經痛、腸胃不適及腹脹。計畫性細胞死亡分兩種：第一型細胞凋亡 (apoptosis)及第二型細胞自噬 (autophagy)，文獻證實，evodiamine可誘導數種癌細胞進行細胞凋亡，包括黑色素癌、子宮頸癌。但近年研究evodiamine對肺癌細胞之抗癌機轉仍然未知，細胞死亡過程中可能伴隨鈣離子濃度上升，進而導致細胞凋亡；亦有文獻證明inositol 1,4,5-trisphosphate (IP3)會活化內質網endoplastic reticulum (ER)上的inositol 1,4,5-trisphosphate receptor (IP3R)促使鈣離子釋放，進而使細胞質產生鈣離子波動誘導autophagy，進一步促使細胞進行apoptosis；evodiamine是否引發肺癌細胞導致autophagy亦尚未釐清，且autophagy在細胞中扮演細胞保護或促使細胞死亡的角色仍具爭議性。本實驗室研究顯示，利用MTT assay分析evodiamine對肺癌細胞的抑制效果，結果指出細胞存活率具有濃度依存性下降，利用流式細胞儀搭配acridine orange及annexin V/PI分別偵測酸性胞器 acidic vesicular organelle (AVO)及細胞凋亡比例，發現evodiamine可誘導肺癌細胞進行autophagy及apoptosis，autophagy比例於24小時後可達最高百分比43.6 ± 2.3 %, p<0.001，而後降低，apoptosis比例則逐漸增加，於48小時達68.4 ± 0.3 %, p<0.01。利用Fluo-3 AM染劑偵測鈣離子濃度，結果發現鈣離子隨時間增加於48小時達控制組的6.95倍 (p<0.01)，本研究進一步透過鈣離子螯合劑BAPTA-AM及EGTA可顯著抑制evodiamine所誘導的autophagy及apoptosis，透過western blot同時可觀察到內質網壓力相關蛋白：GRP 78及GADD 153蛋白質量的增加，證實細胞內鈣離子及內質網壓力參與evodiamine誘導autophagy之訊息傳遞路徑。
本論文之執行，有助瞭解evodiamine對肺癌細胞抗癌機轉，並探討evodiamine是否藉增加鈣離子及內質網壓力誘導細胞產生autophagy及apoptosis。本論文期望可提供臨床上開發抗癌藥物的新方向，透過化學療法藥物與evodiamine併用促進肺癌細胞死亡，延長肺癌病人存活率。

關鍵字

肺癌 ； 吳茱萸鹼 ； 細胞自噬 ； 細胞凋亡 ； 鈣離子 ； 內質網

並列摘要

Lung cancer is the leading cause of cancer-related death in the Taiwan, with an estimated 8194 deaths in 2010. Lung cancer is quite difficult to remove completely by surgical excision. Chemotherapy and irradiation therapy are often used as the adjuvant therapies. However, the expense is high and exhibits side effects. In recent years, scientists tried to treat and prevent cancer in many ways, particularly in the development of pharmacological effect of Chinese traditional medicine. Chinese herbs are regarded as a new and promising source of potential anti-cancer remidies and new chemotherapy adjuvants to enhance the efficacy of chemotherapy and to ameliorate its side effect. Evodiamine, extracted from Chinese herbal medicine Evodiae fructus, with alkaloids biological activity. It used to treat headache, menstrual pain, stomachache and bloating. There are two subtypes in programmed cell death, including apoptosis (typeⅠcell death) and autophagy (type II cell death). It has been reported that evodiamine induced apoptotic cell death in various cancer cells, including melanoma and cervix cancer. However, the anti-cancer effects in lung cancer and the role of autophagy are still unclear. Calcium released from inositol 1,4,5-trisphosphate receptor (IP3R) of endoplastic reticulum (ER) results in calcium signals, and induce autophagy which protect cell from apoptosis. However, It is still debated that autophagy plays a protective role or mediates cells undergo cell death. Our lab demonstrated that evodiamine significantly inhibited H-460 cell growth in a dose dependent manner via MTT assay. As revealed by flow cytometry with acridine orange stain and annexin V/propidium iodide (PI) double staining, we demonstrated that autophagy and apoptosis were both induced after treatment with evodiamine. The percentage of autophagy reached to a plateau at 24-h (43.6 ± 2.3 %, p<0.001) and then decreased as time elapsed. A parallel experiment indicated that apoptosis was increased in a time-dependent manner and reached to a peak after 48-h exposure of evodiamine (68.4 ± 0.3 %, p<0.01). Using Fluo-3 AM staining, we demonstrated that intracellular calcium was increased to 6.95 fold compared with control in a time-dependent manner suggested that calcium participated in evodiamine-induced autophagy. Further, we demonstrated that Ca2+-chelation strongly inhibits autophagy and apoptosis in lung cancer cells by co-treatment with BAPTA-AM and EGTA significantly. We finally indicated that ER stress may participate in evodiamine-induced autophagy through Western blot. Our research can expend the knowledge of anti-cancer mechanisms of evodiamine, and the role of evodiamine-mediated autophagy. This study will provide a novel strategy for anti-cancer therapy in clinical via combination of chemical therapy and evodiamine.

並列關鍵字

Lung cancer ； Evodiamine ； Autophagy ； Apoptosis ； Calcium ； Endoplasmic reticulum

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