YC-1經由活化JNK途徑而誘發腎癌細胞 A498凋亡之機轉探討

Abstract

目前，腎癌病人之致死率相當高，但是有效治療的藥物非常少。本篇論文主要利用 YC-1 對腎癌細胞 A498 具有相當高敏感度的特性，進一步了解 YC-1 誘發腫瘤細胞凋亡之機轉，和在動物活體之藥效。實驗結果發現，使用MTT及SRB檢測法，YC-1對於 A498細胞之IC50約在10-7 ~ 10-8 M左右，且具有濃度依賴性的關係。而利用DAPI做螢光染色的結果，證實YC-1可以引起 A498細胞 chromatin condensation 的現象。進一步以流式細胞儀來觀測細胞週期變化，發現 YC-1 引起 A498 細胞在sub-G1期數目的增加具有時間依賴性的關係。在Bcl-2家族蛋白及caspases方面之探討， anti-apoptotic 之 Bcl-xL、Bcl-2 蛋白的表現有減少，而pro-apoptotic 之 Bax 蛋白的表現並無明顯的變化；另外也發現 Bid 受 YC-1 的刺激而被裂解。而caspase-3與caspase-8都有被活化的現象，然而對 caspase-9 卻沒有影響。同樣的caspase-3的受質，PARP，也有觀察到同步被caspase-3活化的結果。而使用caspases抑制劑，z-VAD-fmk則可以有效的逆轉YC-1對 A498細胞所造成的細胞凋亡作用。利用調控細胞凋亡的 protein kinases 之抑制劑進一步探討 YC-1 所支配的訊息傳遞。結果發現只有 SP600125 (a JNK inhibitor) 可顯著地逆轉由 YC-1 所誘發之細胞凋亡，而 SB203580 (a p38 inhibitor) 並無作用。以西方點墨法進一步觀察到 YC-1 在3小時的時間點就能使JNK 磷酸化，並證實SP600125的確能有效的抑制YC-1所造成的細胞凋亡作用，表示JNK為YC-1造成細胞凋亡作用之重要調控途徑。由於YC-1可以促成caspase-8的活化，接著探討死亡受體及其ligands的表現。結果顯示 YC-1 可促使 FasL 蛋白質增加，對於 death receptors (Fas，DR4 和 DR5) 及TRAIL並無顯著的影響。另外透過螢光顯微照相的結果，發現 YC-1 亦可在短時間內造成 Fas clustering的現象。在裸鼠動物實驗中更進一步證實 YC-1 於體內具有抗癌的活性。總而言之，在藥理機轉研究上，YC-1 乃藉由活化 JNK 進而增加 DILs (death-inducing ligands)，另一方面促使 Fas clustering 現象，進一步驅動 caspase-8 和 caspase-3 的活化造成細胞凋亡；並且發現在動物體內具有抗腫瘤生長的作用。因此，YC-1 為一個具有潛力發展成為良好的抗癌候選藥物。

The survival rate of patients affected with renal cancer carcinoma was very low and effective drugs were very few. In this study, we used the character of high sensitivity for YC-1 in A498 cells and tried to understand the mechanism of YC-1-inducing tumor cell apoptosis and the potency in vivo studies. We found that the 50% inhibition of cell survival or cell growth（IC50）of YC-1 in A498 cells was 10-7 ~ 10-8 M as measured by MTT and SRB assay. The YC-1-induced chromatin condensation was identified by DAPI staining technique in A498 cells. By using flow cytometry to detect the progression of cell cycle, we found that the population in subG1 phase dramatically increased in response to YC-1 in a concentration-dependent manner. By using Western blotting analysis to investigate the expressions of Bcl-2 family of proteins and caspase cascades, the data showed that the anti-apoptotic protein, Bcl-xL and Bcl-2, but not other proteins was down-regulated in response to YC-1 treatment. Furthermore, Bid was cleaved in response to YC-1 stimulus. The data also demonstrated that YC-1 significantly induced the cleavage of caspase-3 and caspase-8 but had little influence on caspase-9. The substrate of caspase-3, PARP, in the same way, was cleaved by caspase-3. Additionally, the caspase inhibitor, z-VAD-fmk, could effectively reverse YC-1-induced apoptosis in A498 cells. Then, we used the protein kinase inhibitors, and the regulators in apoptosis pathway to understand which pathway maybe involved. The data showed that SP600125 (a JNK inhibitor) but not SB203580 (a p38 inhibitor) could significantly reverse YC-1-induced apoptosis in A498 cells. By using Western blotting analysis, our data demonstrated the phosphorylation of JNK after YC-1 treatment. Besides, we also confirmed that SP600125 could effectively inhibit apoptosis induced by YC-1. These data demonstrated that JNK is an important factor in YC-1-induced apoptosis in A498 cells. Since caspase-8 was activated, we therefore examine the expressions of death receptors and their ligands. The data revealed that YC-1 induced FasL and up-regulation but had no significant influence on expressions of death receptors（Fas, DR4 and DR5）and TRAIL. Our data also confirmed that YC-1 induced Fas receptor clustering within a short time. Furthermore, YC-1 showed an in vivo anticancer efficacy in xenograft models. It is suggested that YC-1 induces JNK phosphorylation and then up-regulation of DILs（death-inducing ligands）. On the other hand, YC-1 also induces Fas receptor clustering and then promotes the activation of caspase-8 and caspase-3, resulting in apoptosis. Our data also demonstrate the anti-tumor effect of YC-1 in vivo study. These data reveal that YC-1 is an excellent candidate for the development of anticancer drug.