| 摘要: | Solanum Lyratum Thunb (白英) ，為中國傳統中藥之一，在臨床治療上曾用於治療黃疸、腹瀉、水腫，也被當作治療肝癌、肺炎、食道癌的抗癌藥物。雖然白英具有抗腫瘤之效果，但尚未有研究指出白英對於人類骨癌的抗癌相關機轉。因此，本研究主要為探討白英乙醇粗抽物 ( SLE ) 針對人類惡性骨肉瘤U-2 OS細胞，誘導細胞走向凋亡之分子機制。
本實驗中，U-2 OS細胞處理不同濃度之白英乙醇粗抽物 ( SLE )，並培養不同時間，以流式細胞儀來檢測細胞的存活率；另觀察細胞週期之分佈，以及細胞凋亡相關的分子層面之檢測，包含利用Annexin Ⅴ偵測初期的細胞凋亡、活性氧族群 ( ROS )、一氧化氮 (NO) 、粒線體膜電位變化 (MMP, ΔΨm) 、鈣離子釋放及等。並以DAPI 染色法及Comet assay來觀察DNA受損情形，另利用Western Blot觀察蛋白質表現。
實驗結果顯示，當人類惡性骨肉瘤U-2 OS細胞經由白英乙醇粗抽物 ( SLE ) 處理後，細胞存活率會隨著藥物濃度增加而下降，細胞週期停滯於G0/G1期，型態上可觀察到細胞皺縮，由Comet assay和DAPI staining 也可發現有DNA受損之情形，鈣離子及一氧化氮 (NO) 在短時間內釋放，粒線體膜電位下降，Annexin V凋亡細胞增加，caspase-8, -9被活化；而凋亡相關蛋白Bax及caspase-3的活化也證明SLE確實可誘導U-2 OS細胞凋亡。根據實驗結果得知，白英乙醇粗抽物 ( SLE ) 是經由多重路徑，使U-2 OS細胞生長抑制及誘導細胞凋亡。
Solanum Lyratum Thunb, which is one kind of Chinese herb medicine, not only used in clinical therapy including jaundice, diarrhea, edema, but also used as an anticancer drug to treat liver cancers, lung cancers, esophagus cancers and so on. Yet, there has been no report as to whether Solanum lyratum Thumb could induce the apoptosis of bone cancer. In this study, we investigated to examine whether or not Solanum Lyratum ethanol extraction (SLE) induced apoptosis in human osteosarcoma U-2 OS cells.
In this study, we investigated the SLE from ethanol extraction whether or not cause human osteosarcoma cells U-2 OS growth inhibition and induce apoptosis. After treated with SLE in various dosages and time interval, we observed the cell morphology and use flow cytometry to examine cell proliferation. Then also use flow cytometry to detect cell cycle distribution, Annexin V and intracellular change of the mitochondrial membrane potential (Δψm), calcium ion release, free radical generation including reactive oxygen species (ROS) and nitric oxide (NO) as well as caspase-8, -9 activity. DNA damage was determined by DAPI staining and Comet assay, the protein expression measured by Western Blot. Our results indicated that SLE induce cell death, DNA damage in dose-dependent manner, and cell cycle arrest in G0/G1 phase. Calcium ion release and NO generation, MMP decrease, apoptotic cells increase by Annexin V and caspase-8, -9 activate, and apoptosis associate protein expression including Bcl-2–associated X protein (Bax) and caspase-3 activations to confirm the SLE cause U-2 OS cells apoptosis. Overall, we suggest the SLE through multiple mechanisms to inhibit the growth and induce apoptosis of the U-2 OS cells. |
