Ascleposide對人類荷爾蒙不依賴型轉移性前列腺癌之抗癌機轉探討

Abstract

Ascleposide 由梧桐科的台灣梭羅木（Reevesia formosana）根部萃取而來，為一種含有五元內酯環的強心類固醇（cardenolide）。以 sulforhodamine B（SRB）assay檢測細胞生長，結果顯示ascleposide 顯著地抑制荷爾蒙不反應型前列腺癌細胞株PC-3（GI50 = 27.23 ± 1.05 nM）與 DU-145（GI50 = 65.92 ± 2.41 nM），且與濃度呈正向關係。相比之下 ascleposide 無法對大鼠心肌細胞株 H9c2 產生作用，表示ascleposide 對癌細胞具有選擇性。以carboxyfluorescein succinimidyl ester（CFSE） staining 與流式細胞儀分析細胞分裂情形，證實 ascleposide 可以對細胞週期與細胞生長造成干擾。實驗結果觀察到經 ascleposide 作用後會增加 cleaved caspase-9、cleaved caspase-3 與 cleaved PARP-1 等與細胞凋亡相關的訊號，另外也觀察到抗細 胞凋亡蛋白 Bcl-2 和 Mcl-1L 表現量減少與促細胞凋亡蛋白 Bak 增加，顯示ascleposide 在 PC-3 細胞主要是透過活化內生性途徑引起細胞凋亡。Ascleposide 在PC-3 細胞引起的細胞週期延遲與細胞週期調控的相關蛋白 cyclin D1、cyclin A、CDK4、c-Myc 以及 phosphorylated Rb 的抑制有關。此外 ascleposide 可以抑制 Akt 在 Ser473 的磷酸化與 4E-BP1 在 Thr37/46 和 Thr70 的磷酸化。另外值得注意的是ascleposide 可以透過引起 p38 的磷酸化作用來活化 MAP kinase 訊息傳遞路徑。利用轉殖技術過量表現 myristylated-Akt 和 c-Myc 來探討這些訊息傳遞路徑間是否具 有關聯性，然而結果發現 Akt 和 c-Myc 的過量表現無法對細胞週期調控蛋白與細胞生長造成影響。已知強心苷會增加胞內鈉、鈣與氧化壓力（oxidative stress）。然而我們的實驗結果在 PC-3 細胞中並無觀察到 ROS 含量的改變。鈉含量則在 PC-3和 DU-145 細胞中加藥 24 小時後有顯著的上升。Ascleposide 也可以增加 PC-3 細 胞中的鈣離子，但在 DU-145 細胞中此現象並不明顯，只有 100 nM ascleposide 可以增加 DU-145 中的鈣離子。藉由添加高濃度的胞外 KCl 增加 Na+/K+-ATPase 的活性後可以逆轉 ascleposide 造成的細胞週期調控蛋白表現量下降。另外高濃度鉀離子也可以回復上升的胞內鈉離子，證實 Na+/K+-ATPase 在 ascleposide 對細胞所造成 的影響中具有關鍵的角色。我們觀察到 ascleposide 會造成 PC-3 細胞中的Na+/K+-ATPase α1 subunit 減少，免疫螢光染色影像顯示在藥物作用下會造成Na+/K+-ATPase α1 isoform 的胞吞現象。相對之下，α3 和 β1 的表現量則不受影響。總結來說，ascleposide 透過選擇性地抑制 Na+/K+-ATPase α1 而導致細胞週期調控蛋 白表現量下降，因此最終造成荷爾蒙不反應型前列腺癌細胞株的細胞週期停滯與凋亡。雖然 ascleposide 的作用機轉仍需要更多研究，但其抗癌作用使它具有後續的開發潛力。

Ascleposide, isolated from the root of Reevesia formosana (Sterculiaceae), is a cardenolide containing a five-membered lactone ring. The data demonstrated that ascleposide induced a significant concentration-dependent inhibitory effect on the growth of hormone refractory metastatic prostate cancer (HRMPC) cells PC-3 (GI50 = 27.23 ± 1.05 nM) and DU-145 (GI50 = 65.92 ± 2.41 nM) using sulforhodamine B (SRB) assay. In contrast, rat cardiomyocyte cell line H9c2 was not affected by the compound, indicating the selectivity toward cancer cells of ascleposide. Flow cytometric analysis of cell division by carboxyfluorescein succinimidyl ester (CFSE) staining suggested that ascleposide could result in the perturbation of cell cycle progression and of cell proliferation. The increase of apoptosis-related cellular signals, including cleaved caspase-9, -3 and PARP-1, was observed under ascleposide action. In addition, anti-apoptotic proteins Bcl-2 and Mcl-1L were down-regulated while pro-apoptotic family member Bak was up-regulated in the presence of ascleposide, indicating that ascleposide-induced apoptosis was predominantly through the activation of intrinsic apoptotic pathway. Ascleposide provoked a profound cell cycle delay in PC-3 cells associated with a decrease of protein expression of cyclin D1, cyclin A, CDK4, c-Myc and phosphorylated Rb. Furthermore, ascleposide inhibited Akt/PKB phosphorylation at Ser473 and 4E-BP1 phosphorylation at Thr37/46 and Thr70. Moreover, ascleposide could also activate p38 MAP kinase. However, overexpression of myristylated-Akt and c-Myc did not rescue ascleposide-induced effects. Given that cardiac glycosides are capable of inducing the elevation of cytosolic Na+ , Ca2+ and oxidative stress, these cellular signals have been checked thereafter. The results showed that albeit the levels of ROS were not modified, ascleposide induced increased levels of intracellular Na+ and Ca2+ in PC-3 cells whereas an increase of intracellular Na+ was observed but not Ca2+ in DU-145 cells unless high concentration of 100 nM ascleposide was exposed. An introduction of high extracellular KCl levels to drive Na+/K+-ATPase activity significantly rescued ascleposide-mediated effects on down-regulation of cell cycle regulators as well as an elevated intracellular Na+ , confirming the key role of Na+/K+-ATPase to ascleposide action. The expressions of Na+/K+-ATPase subunits were detected in PC-3 cells. Immunofluorescence imaging showed that ascleposide induced an endocytosis of Na+ /K+-ATPase α1 isoform other than α3 and β1 subunits. In conclusion, the data suggests that ascleposide selectively targets α1 subunit to inhibit Na+/K+-ATPase activity, leading to overloads of intracellular Na+ and Ca2+ and perturbation of cell cycle regulators and induction of cell cycle arrest that ultimately induces apoptosis in HRMPCs. Although considerable challenges remain, the study in ascleposide may enable important strategy for anti-HRMPC development.