2016-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/647919摘要：多形性膠質母細胞瘤(glioblastoma multiforme，GBM)是一種高度惡性的膠質瘤，也是神經系統中非常具侵略性的腫瘤。多形性膠質母細胞瘤經由多種抗藥性機轉，對於放射性及化療產生抗藥性，而增加 P-glycoprotein 的表現及活性，則是對於化療藥物產生抗藥性的主因。強心配醣體是廣為所知，可抑制Na+/K+-ATPase，造成細胞內鈉離子濃度增加，進而增加鈣離子濃度。雖然強心配醣體的安全範圍較窄，限制了其臨床治療，卻也有許多研究指出，他們對於許多種腫瘤具有抗癌活性。許多回顧性的臨床研究皆指出，強心配醣體對於惡性腫瘤確實有好的作用，然而，其抗癌活性卻需要深入研究。我們的合作夥伴利用了活性所引導的成分分離策略，從臺灣梭羅木Reevesia formosana)根部分離出一些新穎的強心配醣體- reevesioside 化合物，我們的研究指出這些化合物對於人類血癌及賀爾蒙抗性前列腺癌，經由不同作用機轉而具有強的抗癌作用，其中Reevesioside F 引發粒線體相關的作用，而造成血癌細胞凋亡；reevesioside A 則是對前列腺癌細胞作用最強，可以抑制 c-myc 的 mRNA 及蛋白表現，進而抑制E2F1 (一種轉殖因子)的作用，目前我們的初步結果則發現，epi-reevesioside F 對於多形性膠質母細胞瘤 T98 及 U87 的作用比已知的強心配醣體 ouabain 更強，其抑制增生的 IC50 約為 40 nM，然而，對於另一株 A172 細胞則不具活性。Epi-reevesiosdie F 會造成 T98 及 U87 的細胞內鈉離子濃度上升，若在高鉀的培養液，則這個作用會被抑制掉，值得注意的一點是，epi-reevesioside F 並不會增加細胞內鈣離子濃度，此外，我們的研究也顯示 epi-reevesioside F 並不是P-glycoprotein 的受質，這些結果顯示，epi-reevesioside F 對抗多形性膠質母細胞瘤的作用，可能是經由抑制 Na+/K+-ATPase 的作用而來，但卻不會影響細胞內鈣離子濃度，然而，其抑制 Na+/K+-ATPase 的作用以後，細胞的訊息傳遞發生了什麼變化，則需要深入研究。本研究計畫的特殊目的如下，第一年為探討細胞的作用標的(例如:Na+/K+-ATPase 次單體或是其他標的)，以及抗多形性膠質母細胞瘤的作用機轉。我們的合作夥伴也會修飾糖基，來半合成及得到更多 reevesioside衍生物，我們也會測試這些衍生物對於多形性膠質母細胞瘤的抗癌活性；第二年則是會根據第一年找出的細胞標的，設計其篩選試驗模式(screening templates)，來檢測後續衍生物的活性，此外，我們也會與化療藥物(如:temozolomide)來做合併療法，研究其加乘作用及機轉；第三年則是會建立多形性膠質母細胞瘤的原位異種移植動物模式，研究單獨給藥或是合併療法的作用，此外，也會研究動物體內的作用機轉。因為強心配醣體對於穿透血腦障壁(blood brain barrier，BBB)的程度不同，因此，本計畫也將了解化合物在腦組織的濃度。期待本計畫的執行成果能達到預期目的，發現有潛力的新穎作用物來做更好的開發。<br> Abstract: Glioblastoma multiforme (GBM) is a highly malignant glioma and an aggressive cancer of central nervous system. GBM is resistant to both radiation and chemotherapy through various resistance pathways. Increased expression and pumping function of P-glycoprotein in glioblastoma are responsible to decreased responses to chemotherapeutic drugs. Cardiac glycosides are well recognized to inhibit Na+/K+-ATPase, leading to an increase of intracellular Na+ levels which, in turn, drives an increase of intracellular Ca2+ concentrations. Although cardiac glycosides show a narrow therapeutic index limiting their therapeutic application, they are well documented to induce anticancer activities in a wide spectrum of cancers. Several retrospective clinical studies have demonstrated that cardiac glycosides would have a positive impact on incidence/clinical outcome of malignancies. However, real anticancer potential of cardiac glycosides needs fully investigated. Recently, our colleagues have performed bioassay-guided fractionation of the root of Reevesia formosana, leading to isolation of new cardiac glycosides. Our studies show that several reevesioside compounds display potent anticancer activities against human leukemia and hormone-refractory prostate cancers (HRPC) through distinct signaling pathways. Reevesioside F induced mitochondria-involved apoptotic events in leukemic cells. Reevesioside A is the most effective among reevesioside compounds against HRPC. It inhibits c-myc expression in both mRNA and protein levels and suppresses E2F1 (a transcription factor) activity. Currently, our preliminary data showed that epi-reevesiosdie F displays a more potent activity than ouabain (a well-known cardiac glycoside) against human glioblastoma T98 and U87 with IC50 values of 40 nM; whereas, it remains ineffective toward glioblastoma A172 cells. Epi-reevesiosdie F induces a significant elevation of intracellular Na+ which is significantly inhibited by supplement of extracellular K+. Notably, epi-reevesioside F does not modify intracellular Ca2+ concentration in both T98 and U87 cells. Furthermore, the data demonstrate that epi-reevesiosdie F is not a P-glycoprotein substrate. The data suggest that the anti-glioblastoma activity induced by epi-reevesiosdie F is attributed to inhibition of Na+/K+-ATPase but not the increase of intracellular Ca2+ levels. The underlying mechanism after Na+/K+-ATPase blockade warrants further study. Specific goals in this project are as follows: the 1st year, to identify primary target (e.g., Na+/K+-ATPase subunit and/or others) and signaling cascades on anti-glioblastoma effects. Our colleagues will semi-synthesize more reevesioside derivatives with modified sugars associated. Test of the derivatives on anti-glioblastoma activity will be performed accordingly. The 2nd year, to create compound examination templates based on discovered molecular targets for more and more derivatives. Combination treatment of reevesioside derivative with clinical therapeutic drugs (e.g., temozolomide) will be examined in anti-glioblastoma effect. Signaling cascades of the combined therapy will be also studied. The 3rd year, to create an in vivo orthotopic xenograft tumor model inoculated with tumorigenic glioblastoma cells. Single as well as combination treatment will be tested in the model. Besides, mechanism study will be also conducted in the in vivo model. Because cardiac glycosides show varied abilities across blood brain barrier (BBB), the concentrations of test derivatives in brain tissue will be examined accordingly. Hopefully, we can achieve the specific goals and discover potential derivatives for advanced development.