阿里山十大功勞 ( Mahonia oiwakensis ) 對於人類肺癌細胞凋亡作用在活體外( in vitro )及活體內( in vivo ) 的研究

肺癌是在臨床治療上仍是棘手的，發現時多已轉移，失去外科切除的機會。因此多以化學治療為主。所以開發新藥以治療，或是利用天然藥物以預防，是針對肺癌最有效率的。目前已有多種中草藥已被確認具有抗發炎，抗過敏及抗癌的活性，且其中具有生物活性的有效成份，部分已被單離並經分析鑑定。中草藥對癌症所引起的諸多症狀的治療能有所助益，而中草藥的抗癌有效成份之作用機轉，常有效地促使癌細胞凋亡及抑制癌細胞增生。本篇研究的中草藥叫做「阿里山十大功勞」，是屬於小蘗科植物，學名為：Mahonia oiwakensis ，它在中藥上有十大功效，包括根、莖、葉都可以入藥，因此稱為「十大功勞」。Mohonia 其他族系在國外文獻的研究中，有抗菌、抗發炎及治療慢性皮膚病的功效，其中小蘗鹼 ( berberine ) 為主要成份，而此成份近年來被認為有抗癌的潛力。阿里山十大功勞為本土特有品系，希望藉此研究找出其抗癌有效成份，以及促使癌細胞凋亡及抑制癌細胞增生之作用機轉。實驗首先以 HPLC 及 ESI-TOF-MS 分析阿里山十大功勞萃取物的主要成份，作為將來研究的參考。實驗選擇四種肺癌細胞株，包括 A549 ，H1355 ，H1299 ，H226。在 MTT 實驗中發現，上述肺癌細胞株會隨著 MOE 濃度增加而降低，而正常肺部組織細胞對上述濃度的 MOE 沒有致死的反應。所以假設 MOE 誘使細胞凋亡 ( apoptosis ) 而非毒殺 ( cytotoxicity )。MTT 實驗中 A549 肺腺癌細胞株是最敏感的，在 MOE 對四種肺癌細胞株的 IC50 也得到同樣結果。所以選擇 A549 肺癌細胞株作為後續實驗的標的物。以 100 μg/mL MOE 處理的 A549 肺癌細胞株，使用流式細胞儀分析，發現 sub-G1 phase 增加，合併有 G1 phase arrest 的情形。後續實驗從早期到晚期 MOE-induced A549 cells intrinsic apoptotic pathway，包括 DAPI 染色，以顯微鏡觀察發生凋亡步驟的形態學變化，細胞膜 Annexin-V 染色增加，Bax/Bcl-2 比例上升，Bid 的活化形態 tBid 增加，粒腺體膜電位 (△Ψm) 降低致 cytochrome C 釋放至 cytosol 誘發下游一連串 caspase 的反應。Caspase 8、9、3 的活化態上升，PARP 活化態增加，DNA 的 fragmentation 等，都在實驗中得到證實。最後加入 pan-caspase inhibitor z-VAD-fam，證實可以reverse MOE- treated A549 cells apoptosis 的狀況。 所以 MOE-induced A549 apoptosis 可以透過 intrinsic apoptotic pathway 。對於 FAS/CD95 , FAS ligant 及TRIAL、DR4、DR5 receptors 的研究，發現 FAS/CD95 , FAS ligand 有上升的情形，但是並沒有同時發生於 TRIAL、DR4、DR5 receptors 上。推論 MOE-induced A549 apoptosis 也同時可以透過 extrinsic pathway，是經由 FAS/ CD95 ，FAS ligand 途徑，而非透過 TRIAL、DR4、DR5 receptors。活體實驗中將A549細胞異種殖入裸鼠，以MOE餵食的鼠tumor size比較對照組有明顯減少。把收集的tumor cell做後續實驗，發現Ki-67 protein的染色結果計算，其 proliferation Index 呈現下降，TUNEL染色則positive呈現上升。由以上的實驗結論，阿里山十大功勞萃取物可以透過intrinsic 及extrinsic pathway使A549肺腺癌細胞株走向凋亡。所以經由研究阿里山十大功勞的主要成份，可能發現新的肺腺癌治療用藥。

Both the root and stem bark of Mahonia species were popular folk medicines. The plant has several proven biological activities including anti-bacterial, anti-fungal, and anti-inflammatory effects. However, Mahonia has not been studied for its anticancer effects. In the present study, we made extracts from Mahonia oiwakensis ( MOE ), an endemic selected species in Taiwan, and investigated their effects on various human lung cells. Amount the four selected experimental cell lines, including A549, H1299, H1355 and H225, We found that A549 was the most sensitive cell line to MOE. The MTT assay showed decreased viability of all four kinds of malignant cells we chose in dose- dependent manner under treatment of MOE, but no influences on normal lung tissue cell line such as MRC-5. Interestingly, A549 is also the only normal expression of p53 in those four selected cell lines. So A549 was chosen to proceed following experiment. The data showed that MOE-induced apoptotic death in human A549 non-small cell lung carcinoma ( NSCLC ) in a dose- dependent manner. About the cell- cycle study, we treated with MOE also caused an increase in the sub-G1 fraction of cells, G1- phase arrest and decreased with S- phase population simultaneously. The morphology of MOE- treated A549 cells demonstrated the characteristics of the apoptotic procedures, such as sell shrinkage, cell surface blebbing, nuclear condensation and formation of apoptotic bodies. The mitochondrial- mediated pathway was implicated in this MOE-induced apoptosis as evidenced by activation of the caspase cascade 3,8 and 9, cleavage of poly ( ADP-ribose ) polymerase ( PARP ), disruption of mitochondrial membrane potential, and then release of cytochrome C in cytosol. A higher ratio of Bax/ Bcl-2 proteins and cleavage of Bid were also observed in MOE-induced A549 cell apoptosis. Surprisingly, we surveyed if the extrinsic apoptotic pathway play a role in the MOE- induced apoptosis of A549 tumor cell. The Fas/ CD95 and Fas ligand were both increased in MOE- treated A549 cell line. However, the same reaction did not show in the TRAIL, DR4 and DR5 receptors. We speculated that MOE- induced apoptosis of A549 cells though both the intrinsic and extrinsic apoptotic pathway. Furthermore, we prescribed the pan- caspase inhibitor z-VAD-fmk to A549 cells before treating with MOE. The reversal of apoptosis was noted in our experiment. Then, in vivo experiment of A549 tumor-xenografted nude mice, MOE also restricted proliferation (P<0.05) and induced apoptosis in tumor cells, as shown by a decrease in Ki-67-positive staining (P<0.05) and increased transferase-mediated dUTP nick-end labeling ( TUNEL )-positive staining (P<0.05). In conclusion, MOE inhibits the growth of human lung cancer cells in vitro and in vivo though both intrinsic and extrinsic apoptotic pathway, suggesting that it may have therapeutic potential against human lung cancer.