中藥及天然物成分在人類轉移性去勢療法抗性前列腺癌之抗癌作用機轉研究

Abstract

人類轉移性去勢療法抗性前列腺癌(metastatic castration-resistant prostate cancer，MCRPC)為一種具侵略性且無法以賀爾蒙療法治療的惡性癌症，故開發能被應用於治療MCRPC的藥物極為重要。本論文共分為兩章，在第一章節中，我們由原先並非用於癌症治療的特定中藥方劑中取得十二個精萃產物E001~E012。利用sulforhodamine B (SRB) assay進行一系列藥物篩選實驗後，發現E002能顯著加乘臨床上廣泛使用於抗癌化學療法的藥物vincristine對於PC-3細胞的生長抑制作用。為了進一步了解其中的抗癌機制，以propidium iodide (PI) 染劑與流式細胞儀觀察細胞分裂情形，發現合併使用E002與vincristine會造成處於sub-G1期的細胞增加，進而造成細胞凋亡。利用西方墨點法的分析，亦可同時觀察到Bcl-2蛋白質家族表現量下降與磷酸化，cleaved-caspase-3及cleaved-PARP增加等與細胞凋亡相關的訊號。合併使用E002與vincristine也能有效增加粒線體膜電位的喪失，顯示了粒線體膜電位流失於其所造成的抗癌機轉中扮演著重要角色。而利用in vivo tubulin assay，我們了解到E002能加強vincristine直接與微管蛋白作用。西方墨點法的結果中合併E002與vincristine能顯著增加MPM-2蛋白質表現量，此結果證實了該組合對於磷酸化微管蛋白的影響。而在第二章節中，短柄香苦草(Hyptis brevipes)中的天然物成分(-)-brevipolide H具有複雜的結構與抗癌活性。根據SRB assay的數據，(-)-brevipolide H能有效抑制PC-3的生長 (GI50=2.72±0.15μM)，且其抑制癌細胞生長的效果隨濃度增加。而colony formation的結果顯示(-)-brevipolide H長時間作用(一週)抑制PC-3細胞生長的效果更為明顯(GI50=0.37±0.06 μM)。利用carboxyfluoresceinsuccinimidyl ester (CFSE) 染劑與流式細胞儀觀察，可以觀察到(-)-brevipolide H能有效抑制PC-3細胞的增生情形。以PI染劑與流式細胞儀觀察觀察細胞分裂情形，證實(-)-brevipolide H會造成細胞週期停滯於G0/G1期與並引發細胞凋亡。西方墨點法與免疫螢光染色的分析結果亦顯示了細胞週期調控相關蛋白如c-Myc、cyclin D1及cyclin E表現量下降等與細胞週期停滯於G0/G1期的相關訊號。此外，(-)-brevipolide H透過抑制磷酸化作用位點來抑制Akt/mTOR/p70S6K/4E-BP1的訊息傳遞路徑。以轉染技術過量表現myristylated-Akt後我們發現myristylated-Akt可以逆轉原先(-)-brevipolide H對於Akt/mTOR/p70S6K/4E-BP1訊息傳遞路徑的抑制，代表Akt於(-)-brevipolide H的抗癌作用機轉中扮演重要角色。進一步研究後發現，(-)-brevipolide H短時間內透過引發細胞內氧化壓力(Oxidative stress，ROS)與細胞內鈣離子的過載來調控Akt/mTOR/p70S6K/4E-BP1訊息傳遞路徑中的訊號。(-)-Brevipolide H亦藉由降低Bcl-2 家族蛋白的表現量與造成粒線體膜電位的喪失來引發細胞凋亡。綜合第一及第二章節的研究結果，顯示了合併E002與vincristine能經由提升抗微管蛋白活性、粒線體膜電位的喪失與caspase蛋白的活化來抑制PC-3細胞的生長，最終導致mitotic arrest及細胞凋亡的發生。而(-)-brevipolide經由造成細胞內ROS與鈣離子增加，對於Akt/mTOR/p70S6K/4E-BP1訊息的抑制及降低細胞週期調控蛋白來抑制PC-3細胞的增生並造成G0/G1 arrest與細胞凋亡。

Metastatic castration-resistant prostate cancer (MCRPC) is a type of progressive prostate cancer unresponsive to hormone therapy. Therefore, maximizing treatment options for MCRPC is of importance. This thesis is divided into two chapters. In the first chapter, E002 is a partial-purified extract derived from one traditional Chinese medicine formula used for the treatment of cancer unrelated diseases. The data demonstrated that the combinatorial treatment of E002 and vincristine, one of the chemotherapeutic drugs clinically used to treat several cancers, induced a concentration-dependent and synergistic inhibition of cell proliferation in MCRPC PC-3 by the sulforhodamine B (SRB) assay. To investigate the mechanism, the flow cytofluorometric analysis of cell cycle with propidium iodide (PI) staining demonstrated that combination of E002 and vincristine caused a synergistic increase of sub-G1 population that was confirmed accordingly by phosphorylation of Bcl-2 family proteins (Bcl-2 and Bcl-xL), activation of caspase-3, and the cleavage of caspase-9 and PARP-1. Furthermore, the synergistic loss of mitochondrial membrane potential (ΔΨm) was induced by combinatorial treatment of E002 and vincristine, indicating the role of mitochondrial stress in the sensitization mechanism. Moreover, combinatorial treatment of E002 and vincristine was found to directly interact with tubulins through performing the in vivo tubulin assay. The sensitization effect was validated by a synergistic increase of phosphorylated mitotic proteins identified by MPM-2 antibody. In the second chapter of the thesis, a total synthesis has been performed successfully to obtain (-)-brevipolide H, which is a natural component from Hyptis brevipes, with complex structure and cytotoxic activity. The data demonstrated that (-)-brevipolide H induced a concentration-dependent inhibition of cell proliferation with a GI50 of 2.72±0.15 μM in MCRPC PC-3 by the SRB assay. A long-term exposure to (-)-brevipolide H resulted in a stronger anti-proliferative effect against anchorage-dependent colony formation with a GI50 of 0.37±0.06 μM. Flow cytometric analysis of cell division by carboxyfluoresceinsuccinimidyl ester (CFSE) staining also confirmed the anti-proliferative effect of (-)-brevipolide H. Furthermore, flow cytofluorometric analysis of cell cycle with PI staining showed arrest of the cell cycle at G0/G1 phase to (-)-brevipolide H action and an induction of subsequent cell apoptosis. The G0/G1 arrest was validated to be attributed to the down-regulated protein expressions of c-Myc, cyclin D1 and cyclin E by using Western blotting and confocal immunofluorescence microscopic examination. Moreover, (-)-brevipolide H inhibited the signaling pathway of Akt/mTOR/p70S6K/4E-BP1 through the inhibition of phosphorylation at several stimulatory sites. Overexpression of myristylated-Akt, a constitutively activated Akt, partly rescued the Akt/mTOR signaling cascades to (-)-brevipolide H action. Of note, (-)-brevipolide H induced an increase of intracellular Ca2+ levels and ROS production. It also induced the down-regulation of anti-apoptotic Bcl-2 family proteins (Bcl-2 and Bcl-xL) and the loss of mitochondrial membrane potential indicating the contribution of mitochondrial damage stress to cell apoptosis. In conclusion, the data suggest that combinatorial treatment of E002 and vincristine induces synergistic anticancer activity in PC-3 cells through the sensitization of anti-tubulin activity, mitochondrial stress and caspases activation. In addition, (-)-brevipolide H induces anti-proliferative and apoptotic effects in MCRPC through the intracellular Ca2+ overload and oxidative stress that cooperate with the down-regulation of cell cycle regulators and inhibition of Akt/mTOR/p70S6K pathway, leading to G0/G1 arrest, mitochondrial damage stress and cell apoptosis