黑殭菌素B誘導人類肺腺癌細胞凋亡經由Bcl-2家族分子活化粒線體路徑

Destruxin B-induced apoptosis via a Bcl-2 family-dependent mitochondrial pathway in human lung adenocarcinoma A549 cells

陳詠晴(Yung-Ching Chen)

指導教授 ： 吳俊錡

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2011年)

https://doi.org/10.6834/CSMU.2011.00100

摘要

近年來肺癌因其高發生率及高致命性穩居全球惡性腫瘤前十名，在台灣，則多次蟬連十大死因之首。目前臨床上雖有許多治療肺癌的藥物 ( ex: 艾瑞莎 Iressa )，但受限於其價格與所引發之副作用，因此，開發新型藥物成為現階段醫藥研發之目標。環狀胜肽物質 ( Cyclodepsipeptides ) 為廣泛存於自然界的一類物質，其具有多樣的生物活性，如：免疫抑制、抗真菌、抗發炎與抗腫瘤等活性。因此，許多此類的物質被運用於探討生物細胞調節過程的研究上。而黑殭菌素 B 是由昆蟲病原性真菌 ( Entomopathogenic fungus ) ─ Oospora destructor 中所分離萃取出的真菌二次代謝物，其亦為環狀縮合胜肽的一種，並具有毒殺昆蟲的活性。先前的研究中顯示，艾瑞莎合併黑殭菌素 B ( Destruxin B ) 在腫瘤治療上具有加成效果，但對於黑殭菌素B單獨使用的效果則無相關報導。因此，本論文將探討黑殭菌素 B 誘發 A549 人類肺癌細胞毒殺效果及其可能之分子機轉。實驗之初先檢測黑殭菌素 B 對正常、纖維化與癌細胞的毒殺效果與半致死劑量 ( IC50 )。在此我們選用 HUVEC ( 人類臍靜脈內皮細胞 )、MLF ( 小鼠肺纖維母細胞 )、MRC-5 ( 人類肺纖維母細胞 ) 及 A549 ( 人類肺腺癌細胞 ) 四株細胞；由生長曲線可求出黑殭菌素 B 抑細胞生長之 IC50 分別為：HUVEC 7.0μM、MLF 3.3 μM、MRC-5 7.5 μM、A549 4.9 μM。各細胞其 IC50 差異並不大，代表黑殭菌素 B 對細胞的毒殺效果並無特異性。藉由流式細胞儀分析以黑殭菌素 B 處理之 A549 肺癌細胞之細胞週期，發現細胞週期並未有顯著的停滯，而是直接造成細胞凋亡。進一步檢測在細胞凋亡中扮演關鍵角色的 caspase 活性，發現經黑殭菌素 B 處理後， caspase-2、-3 及 -9 的活性明顯上升，但 caspase-8 及 -12 並沒有被活化，因此，推斷黑殭菌素 B 所引發之細胞死亡是經由粒線體的凋亡路徑 ( Mitochondrial pathway )。隨後以西方墨點法 ( western blot ) 偵測粒腺體細胞凋亡相關分子，隨著黑殭菌素 B 劑量的增加，促凋亡分子 PUMA 蛋白質表現量上升；抑凋亡分子 Mcl-1 表現量減少，然細胞凋亡機制中的主要分子 Bax 卻無顯著變化。透過免疫螢光染色 ( immunofluorescence ) 的結果，發現 Bax 由細胞質轉位 ( translocation ) 至粒線體膜 ( Mitochondria membrane ) 上，由此可知，黑殭菌素 B 所誘導的粒線體凋亡作用是經由 Bax 的轉位傳遞訊息，而非 Bax 表現量的增減。此外，在黑殭菌素B的刺激下，p53 的表現量亦會增加，但選用 A549 ( p53 wild-type )、A549-shp53 與 H1299 ( p53 null ) 三株 p53 表現不同的細胞，分別投與不同劑量的黑殭菌素 B 後，發現抑制生長的現象隨黑殭菌素 B 劑量的遞增而更加顯著。由此推論 p53 在細胞內表現的狀況與黑殭菌素 B 所誘導的細胞凋亡相關性不大。綜合以上結果，可得知黑殭菌素 B 所誘導的人類肺腺癌細胞凋亡是經由 Bcl-2 家族分子活化粒線體路徑 。

關鍵字

黑殭菌素B ； 粒線體凋亡途徑 ； Bcl-2家族分子 ； Mcl-1 ； PUMA

並列摘要

Lung cancer is the top ten causes of death worldwide due to its high incidence and mortality rates. In Taiwan, lung cancer had even become the leading cause of death in the last decade. Some target drugs such as Iressa were applied in the treatment of lung cancer patients clinically; the high-priced and severed side-effects of this kind of target drugs, however, limited their medical application. Lots of efforts have been made to pursuit of new compounds which are capable of eliminating lung cancer cells more effectively. Cyclodepsipeptides, a wide variety of cyclic peptides of natural origin, have a wide range of biological activities, such as immunosuppressant, antibiotic, antifungi, antiinflammatory or antitumoral activities. Besides, many of these cyclic depsipeptides represent useful tools for the research of biological processes involved in cellular regulation. Destruxin B, isolated from entomopathogenic fungus, is one of cyclodepsipeptides with insecticidal and phytotoxic activities. Previous report showed that the combination of Iressa with Destruxin B increased the killing effect in lung cancer cells; the effect of destruxin B alone on lung cancer cells, however, has not been reported yet. In this study, the toxic effect and the underlying mechanisms of destruxin B on A549 lung cancer cells was investigated. Our results showed that destruxin B inhibited the growth of HUVEC (Human Umbilical Vein Endothelial Cell), MLF (Mouse Lung Fibroblast), MRC-5 (Human Lung Fibroblast) and A549 (Human Lung Cancer Cells) in both dose- and time-dependent manners, and the IC50 was determined as 7.0 μM、3.3 μM、7.5 μM and 4.9 μM for each cell line at 48 h treatment. Data from flow cytometric analysis showed that destruxin B induced apoptotic cell death evidenced by increasing of subG1 population. Moreover, the activity of caspase - 2, - 3 and -9, but not caspase-8 and -12, were induced by destruxin B-treated A549 cells. Additionally, destruxin B increased the expression levels of PUMA, a pro-apoptotic protein, while decreased Mcl-1 levels, which is recognized as an anti-apoptosis molecule. Interestingly, Bax translocated from cytoplasm to mitochondrial membrane instead of increasing protein levels after administration of destruxin B. Knockdown of Bax by shRNA effectively attenuated destruxin B-triggered apoptosis in A549 cells. These results pointed that the destruxin B increased the expression levels of PUMA and decreased Mcl-1expression, which might subsequently induce mitochondrial translocation of Bax, and then activates the caspase cascade, finally leds to apoptotic cell death in A549 cells. In addition, destruxin B also increase the expression levels of p53, a tumor suppressor. Destruxin B also inhibited the growth however, A549-shp53 and H1299 ( p53 null ) cells as well, indicated that p53 might not involve in destruxin B-induced apoptosis in lung cancer cells. Taken together, our findings suggest that destruxin B-induced apoptosis in A549 lung cancer cells is via Bcl-2 family-dependent mitochondrial pathway.

並列關鍵字

Destruxin B ； Mitochondria-mediated apoptosis pathway ； Bcl-2 family moleculars ； Mcl-1 ； PUMA

參考文獻

Hsieh RK (2003). Molecular Targeted Therapy for Solid Tumors. Formosan. J. Med. 7: 222-6.

陳拯裕 (2005). 黑殭菌素B對於人類大腸癌及肝癌細胞株之抗癌機制與探討 朝陽科技大學/生物技術研究所.

蔡勇勝, 高穗生. 黑殭菌素在蟲生真菌致病過 程中所扮演的角色. 行政院農業委員會農業藥物毒物試驗所技術專刊第 9104 號.

Adachi K, Nanba H, Kuroda H (1987). Potentiation of host-mediated antitumor activity in mice by beta-glucan obtained from Grifola frondosa (maitake). Chem Pharm Bull (Tokyo) 35: 262-70.

Alberg AJ, Samet JM (2003). Epidemiology of lung cancer. Chest 123: 21S-49S.