| 摘要: | 隨著全球飲食習慣的改變，大腸直腸癌迅速攀升為全球第二常見致死癌症， 在早期診斷可經由手術治療所治癒。 大腸癌後期使用傳統化學或放射性治療， 但經常伴隨著復發和致死的可能性。 有鑑與此， 新治療方式或藥物的使用， 在大腸癌的治療上是一個很必要的研究方向。
在癌症研究上， 傳統中藥醫學參與在癌症治療中很長時間與成效， 以及扮演一個重要的角色。 其中Osthole 是由中藥蛇床子所萃取， 已在體外和活體實驗研究參與在改善血管功能、抗癌或抗發炎功能、高血壓或高血酯、與骨質疏鬆皆不錯效果。 為更進一步提升Osthole抗癌效果， 由Osthole結構合成18種不同鏈長和取代基位置的Osthole 衍生物，在大腸癌細胞進行毒性測試中篩選出WJ1376-1和WJ1398-1並研究此對抗大腸癌之分子機轉。
在大腸直腸癌細胞SW480和SW620中比較Osthole衍生物生物毒性結果發現Osthole衍生物 WJ1376-1或 WJ1398-1的IC50約 5?n?嵱， 比起Osthole原態具有更高活性。 利用顯微鏡觀察細胞型態和流式細胞儀偵測細胞週期有多核與多倍體出現。 Osthole衍生物經由誘導大腸直腸癌細胞DNA損傷經由ATM-and Rad3-related (ATR)激酶訊息傳導路徑活化檢查點激酶 (checkpoint kinase 2, Chk2) 磷酸化 ， 抑制細胞週期中B1週期素、 cdc2、 cdc25 與 Aurora A激酶活性， 造成細胞G2/ M週期進行停止。 最終造成Bad蛋白大量表現和活化caspase3, 7, 9證實細胞程式性凋亡。在活體實驗中，WJ1376-1和 WJ1398-1不影響活體體重下有效減緩癌細胞腫瘤生長。
此外，發現在低劑量WJ1376-1 和 WJ1398-1 (1 ?嵱) 明顯抑制人類大腸直腸癌細胞移行和侵襲作用。利用轉換生長因子???n(Transforming growth factor beta, TGF-??) 誘導細胞表皮-間質轉換作用模式，WJ1376-1 和WJ1398-1有效降低轉錄因子Snail1，間質細胞蛋白vimentin和MMP9，增加表皮細胞蛋白E-cadherin。WJ1376-1和 WJ1398-1有效抑制Smad2和Akt 在Serine473位置磷酸化，更降低Smad2和Snail1移入細胞核。利用短暫轉染作用，WJ1376-1和WJ1398-1有效抑制由TGF-?珨冗仉mad reporter plasmid 活性。另外也發現WJ1376-1和WJ1398-1降低TGF-?狻珨冗仈GF-???n第一型受體磷酸化。
綜合以上結果得知WJ1376-1和WJ1398-1可使大腸癌細胞誘發DNA損傷造成細胞多倍體形成，G2/M週期進行停止與細胞凋亡，另外也可能具有抑制癌細胞表皮-間質轉換作用經由阻擋TGF-?狻MTGF-?珩臚@型受體結合進而無法傳遞下游Smad2 和Akt訊息路徑，在未來大腸癌研究中具有極大治療潛力。
Because of changes in lifestyle and eating habits in recent years, colorectal cancer is now the world’s second most common and lethal disease. Although the pathology of the cancer is well known, the treatment is still limited. Traditional chemical or radiation therapy for advanced colorectal cancer is often accompanied by recurrence and lethality. Therefore, the development of new treatments or drugs is necessary for colorectal cancer therapy.
Osthole is isolated from the Chinese herbs Cnidium monnieri and Angelica pubescens and was found to have antitumor activity in vitro and in vivo. To improve the antitumor activity, we obtained 18 osthole derivatives and found that the derivatives WJ1376-1 and WJ1398-1 had the greatest antitumor potential in human colon adenocarcinoma cells. In this study, we aimed to understand the anti-cancer effects of WJ1376-1 and WJ1398-1 and the underlying molecular mechanisms.
MTT assay of the human colorectal adenocarcinoma cells SW480 and SW620 revealed an IC50 of about 5 ?嵱 for WJ1376-1 and WJ1398-1. Microscopy and flow cytometry revealed that in contrast to the parent osthole, both WJ1376-1 and WJ1398-1 induced multinucleation and polyploidy. WJ1376-1 and WJ1398-1 significantly activated ataxia telangiectasia and rad3-related (ATR) kinase, which triggered the checkpoint kinase 2 (Chk2) signaling pathway and downregulated Cdc25 phosphatase and Cdc2–cyclin B complex activities. WJ1376-1 and WJ1398-1 also inhibited the phosphorylation of Aurora A kinase, associated with important processes during mitosis. WJ1376-1 and WJ1398-1 ultimately induced apoptosis, as evidenced by the upregulation of Bad and activation of caspases-3, -7, and -9. Furthermore, WJ1376-1 and WJ1398-1 attenuated tumor growth without affecting the body weight of xenograft nude mice.
WJ1376-1 and WJ1398-1 strongly inhibited migration and invasion in human colorectal cancer cells at doses as low as 1 ?嵱. In the epithelial-to-mesenchymal transition model induced by transforming growth factor ?? (TGF-???w, WJ1376-1 and WJ1398-1 potently downregulated the transcription factor Snail1, the mesenchymal protein vimentin, and matrix metalloprotease-9 (MMP-9) but upregulated the epithelial protein E-cadherin. WJ1376-1 and WJ1398-1 also inhibited the TGF-?牷Vinduced phosphorylation of Smad2 and Akt (Ser 473), and the nuclear translocation of Smad2 was substantially lower in WJ1376-1- and WJ1398-1-treated cells than control cells. In transient transfection experiments, WJ1376-1 and WJ1398-1 strongly inhibited TGF-?牷Vstimulated activity of a Smad reporter. Finally, WJ1376-1 and WJ1398-1 blocked TGF-?牷Vinduced phosphorylation of the TGF-???ntype I receptor (TGF-?淯I) and activity of TGF-?? Type II receptor (TGF-?淯II).
In conclusion, the anticancer activities of WJ1376-1 and WJ1398-1 were dissimilar to that of the parental osthole: the derivatives can induce cell polyploidy and G2/M cell cycle arrest in colon adenocarcinoma cells and also inhibit cell migration by suppressing TGF-?淯I phosphorylation, thereby hindering both Smad2 and phosphatidylinositol 3-kinase/Akt signaling pathways, to prevent cancer cell migration and invasion. These results may provide a potential drug for colon cancer therapy. |
