強心內酯配醣體衍生物在人類荷爾蒙不依賴型前列腺癌細胞之抗癌作用機轉探討

Abstract

強心內酯配糖體衍生物Reevesioside A，是從台灣梭羅木（Reevesia Formosana）的根部萃取而來，從sulforhodamine B assay偵測細胞的生長，發現Reevesioside A顯著的抑制荷爾蒙不反應型前列腺癌細胞株PC-3及DU-145的生長，並與濃度呈正向關係，其GI50分別為21及14 nM。接著使用Reevesioside A處理 PC-3細胞，做更深入的抗癌機制探討。使用流式細胞儀分析CFSE staining的方法，也證實了reevesioside A所造成的生長抑制作用。對於細胞週期的分析，則是先以高濃度thymidine處理的方法，使細胞週期同步化，結果顯示Reevesioside A會引起細胞停留在G1期，也造成Sub-G1期細胞族群增加。利用西方墨點法觀察細胞週期上游調控者c-Myc及細胞週期調控者cyclin D1、cyclin E、phospho-RbSer807/811蛋白質表現量，分別於加藥處理二小時及六小時後，發現其表現量顯著的減少，與早期G1期停滯結果相符合。利用轉殖技術將Myc過量持續表現，發現可以有效的逆轉Reevesioside A引起的cyclin D1、cyclin E、phospho-RbSer807/811蛋白質表現量減少的現象。此外，Reevesioside A可以顯著的抑制AKTThr308及AKTSer473的磷酸化(活化)作用，但以轉殖技術來持續活化AKT （myristylated-AKT），並無法抑制的作用。而Reevesioside A活化了mTOR途徑，如增加phospho-mTORSer2448、phospho-p70S6KThr389及phospho-GSK-3βSer9蛋白質表現量。隨著Reevesioside A處理時間越久，發現可以引起越多的粒線體膜電位喪失的現象。值得注意的是，傳統強心配糖體類的藥物具有心臟毒性，而Reevesioside A對於大鼠心肌母細胞株H9c2，則不具有毒性。總結來說，Reevesioside A透過抑制Myc、減少cyclin D1 及 cyclin E的蛋白質表現量，最終造成荷爾蒙不反應型前列腺癌細胞停滯於細胞週期的G1期，而抑制其生長及造成細胞作用，故具有後續開發潛力。

Reevesioside A, a cardenolide glycoside isolated from the roots of Reevesia Formosana, induced a concentration-dependent inhibition of cell proliferation in human hormone-refractory prostate cancers PC-3 and DU-145 cells by sulforhodamine B assay. The GI50 values are 21 and 23 nM, respectively. The submaximal concentration of 50 nM reevesioside A was used for the mechanism study in PC-3 cells accordingly. Flow cytometric analysis of CFSE staining confirmed that reevesioside A induced an anti-proliferative activity. The progression of cell cycle was detected using thymidine block to synchronize cells. The data demonstrated that reevesioside A induced a profound G1-phase arrest and an increased subG1 phase of the cell cycle . The detection of cell cycle regulators revealed that reevesioside A induced a dramatic down-regulation of c-Myc, cyclin D1, cyclin E and phospho-RbSer807/811, which was well correlated with the induction of G1 phase arrest. The overexpression of Myc (e.g., constitutive Myc activation) inhibited reevesioside A-induced downregulation of cyclin D1, cyclin E and phospho-RbSer807/811 expression. Moreover, reevesioside A significantly inhibited both phospho-AKTSer473 and phospho-AKTThr308. The overexpression of myristylated- AKT (e.g., constitutive AKT activation) could not inhibit reevesioside A-induced effect. Although reevesioisdie A induced the loss of MMP in PC-3 cells, it also resulted in an activation of mTOR signaling pathway, including the increase of the protein levels of phospho-mTORSer2448, phospho-p70S6KThr389 and phospho-GSK-3βSer9. Of note, reevesioside A showed preferential selectivity against prostate cancer cells other than cardiac H9c2 cells. It is of particular significance because traditional cardiac glycosides always show cardiac toxicity. In contrast, reevesioside A did not cause toxic effect in cardiac cells. In conclusion, the data suggest that reevesioside A displays anticancer activity through the down-regulation of c-Myc, cyclin D1 and cyclin E which cause G1 phase arrest of the cell cycle and, ultimately, apoptosis in human hormone-refractory prostate cancers cells. The data also indicate the potential of reevesioside A for further development.