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標題: | Moscatilin 之衍生物 MT-6 在人類卵巢癌細胞引起細胞凋亡之機轉探討 The apoptotic mechanisms of MT-6, a derivative of moscatilin, in human ovarian cancer cells |
作者: | Yi-Chiu Kuo 郭憶萩 |
指導教授: | 鄧哲明(Che-Ming Teng) |
關鍵字: | 細胞凋亡,卵巢癌, apoptosis,ovarian cancer, |
出版年 : | 2010 |
學位: | 碩士 |
摘要: | 卵巢癌病人當被診斷出時大多已是後期,導致卵巢癌病人的存活率極差,目前卵巢癌的死亡率在女性排名為第八位。為了研究環草石斛成分 moscatilin 之衍生物 MT-6 是否在卵巢癌細胞具有抑制作用,及探討可能之作用機轉,我們做了以下實驗。利用 SRB 和 MTT 試驗發現 MT-6 在48 小時有濃度依賴性的抑制癌細胞的增生與生長,在 SKOV3 和 OVCAR3 細胞株的 GI50 分別為 0.041 μM 和 0.062 μM,在 SKOV3 和OVCAR3 細胞株的 IC50 分別為 0.073 μM 和 0.192 μM。接著我們利用 PI 染色合併流式細胞儀分析,發現 MT-6 有濃度及時間依賴性地使細胞聚集到 G2/M 期,伴隨 sub-G1 細胞比例的增加,此現象可能是 MT-6 影響微管的動態所造成。在許多微管抑制藥物皆會造成細胞停滯在 M 期而非 G2 期,所以我們檢查 M 期的指標蛋白MPM2 及 Histone-3 ser10 磷酸化的現象。在卵巢癌細胞加入 MT-6 後,的確可以看到 MPM2 及 Histone-3 ser10 磷酸化增加的現象。另一方面因為 MT-6 使 caspase-3、-7、-8、-9 及 -10 活化,所以代表其不僅活化細胞凋亡的內生性途徑也活化了外生性途徑。因此我們往細胞凋亡途徑的上游尋找,像是死亡受體與 Bcl-2 家族蛋白,結果發現 MT-6 能增加卵巢癌細胞 DR5 的表達,引起 Bid cleavage,另外促使 Bcl-2 磷酸化,及降低 Mcl-1 及 Bcl-xL 的表達。同時我們也確認 Akt 及 MAPK 在 MT-6 造成的細胞凋亡所扮演的角色,發現 MT-6 能同時在 SKOV3 及 OVCAR3 細胞造成 JNK 活化,因此在 MT-6 所造成的細胞凋亡中,JNK的角色相對於 Akt、Erk 及 p38 可能較為重要。最後我們發現 JNK 抑制劑 SP600125 能避免 MT-6 所造成的細胞凋亡及 M 期滯留,而這抑制現象也會出現在 taxol 及 vincristine 處理過的細胞,此與我們的 MT-6 作用相呼應。綜合以上結果,MT-6 改變微管蛋白的動態,使細胞週期停留在 M 期,造成 JNK 活化,並經由活化 caspase 來造成細胞凋亡,所以 MT-6 具有潛力發展成新的抗癌藥物。 Ovarian cancer patients are always diagnosed at an advanced stage resulting in poor survival rates. So far ovarian cancer is the eighth cause of cancer deaths among women. To study the effects of MT-6, a derivative of moscatilin, on human ovarian cancer cells, several experiments were performed. The data showed that MT-6 inhibited cell proliferation in a concentration-dependent manner using sulphorodamine B (SRB) and 3-(4,5-dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (MTT) assay. After 48 hours of treatment, the GI50 values of SKOV3 and OVCAR3 are 0.041 μM and 0.062 μM, respectively, by SRB assay; the IC50 values are 0.073 μM and 0.192 μM, respectively, by MTT assay. Cell cycle analysis of SKOV3 cells was determined by flow cytometric analysis with propidium iodide (PI) staining. We found that MT-6 induced a concentration- and time-dependent arrest of the cell-cycle at G2/M phase followed by an increase of cell proportion at sub-G1 phase. The effect was caused by alternation of tubulin dynamics by in vitro tubulin assay and confocal microscopic examination. To further determine whether MT-6 induced G2 or mitotic arrest of the cell cycle, the two mitotic markers, MPM2 and Histone-3 ser10 were examined in this study. The data demonstrated that MT-6 induced a dramatic increase of phosphorylation of MPM2 and Histone-3 at ser10. Moreover, both intrinsic and extrinsic apoptosis pathways were activated by the detection of catalytic cleavage of caspase-3, -7, -8, -9, -10, poly (ADP)-ribose polymerase-1 (PARP, the substrate of caspase-3 and -7). We further examined the upstream regulators of apoptotic pathways, such as death receptors and Bcl-2 family proteins. As a result, MT-6 increased DR5 expression and caused bid cleavage. Besides, MT-6 induced Bcl-2 phosphorylation and downregulation of anti-apoptotic protein Bcl-xL and Mcl-1 expression. The role of Akt and MAPK apoptotsis was also examined. The results showd that MT-6 activated JNK, whereas inhibited or had no effect on Akt, Erk and p38 in SKOV3 and OVCAR3 cells, depending on cell types. Of note, the data showed that SP600125, a JNK inhibitor, inhibited MT-6-induced caspase activation and mitotic arrest. Similarly, SP600125 also inhibited taxol- or vincristine-induced mitotic arrest and apoptosis. In summary, the results suggest that MT-6 is able to change tubulin dynamics, leading to mitotic arrest of the cell cycle and JNK activation which ultimately causes apoptosis through the intrinsic and extrinsic apoptotic pathways. The study provides evidence that MT-6 is a potential anticancer agent for further development. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/22515 |
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顯示於系所單位: | 藥理學科所 |
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