紫草素藉由細胞凋亡及細胞壞死機制抑制人類肺癌上皮細胞增生

Shikonin Suppresses Human Lung Carcinoma Epithelial Cells Proliferation by Apoptosis and Necrosis

李書芬(Shu-Fen Lee)

指導教授 ： 許立松

中山醫學大學/醫學院/生化暨生物科技研究所/碩士(2009年)

摘要

本研究主要探討紫草素對人類肺癌上皮細胞株—A-549之抗癌效果，以顯微鏡及MTT分析觀察0∼8.0 μM不同濃度紫草素處理A-549細胞24及48小時後的細胞型態及細胞增殖情形，結果發現A-549細胞隨著處理濃度及時間的增加，不規則型態的細胞減少，圓形細胞卻增多；而細胞增殖率也明顯受到影響，呈現劑量及時間相關。A-549細胞株處理0∼4.0 μM不同濃度紫草素24及48小時，經ApopNexin™ Biotin Apoptosis Detection Kit染色後，用流式細胞儀測定，發現24小時處理紫草素的情況下，細胞凋亡的數目隨著紫草素處理濃度的增加而減少，細胞壞死的數目卻隨著紫草素處理濃度的增加而增加，但在48小時處理紫草素的情況卻完全相反。另一方面，經propidium iodide染色後以流式細胞儀測定，發現停滯在sub-G1的細胞數均隨著處理濃度及時間的增加，具有劑量及時間相關性。但以DNA電泳觀察經紫草素處理之細胞之DNA片段化的現象，卻不明顯。用傷口癒合實驗觀察紫草素抑制A-549細胞移行的情形，結果發現隨著處理濃度及時間的增加，傷口癒合有被抑制的情形，但圓形細胞也隨之增加。總括實驗結果顯示紫草素對於A-549細胞的抗癌效果，包括直接使A-549細胞停滯在sub-G1期，引發細胞凋亡及細胞壞死兩個路徑。

關鍵字

紫草素 ； 肺癌 ； 細胞凋亡 ； 細胞壞死

並列摘要

The purpose of this study was discussed the anti-tumor effect of shikonin on the human lung carcinoma epithelial cells, A-549. The cell morphology was observed by microscope and the cell proliferation was analyzed by MTT assay after treated with different concentration shikonin for 24 and 48 hours. Morphological analysis indicated that the amount of irregular cells was decreased but that of round cells was increased. Cell proliferation was reduced by treating shokonin in dose- and time- dependent manner. After treated with a serial concentration of shikonin for 24 and 48 hours, cells were stained via ApopNexin™ Biotin Apoptosis Detection Kit, and the apoptotic and necrotic cells were assayed by flow cytometer analysis. While A-549 cells treated with shikonin for 24 hours, the amounts of apoptotic were decreased and that of necrotic cells were increased in proportioned to the concentration of shikonin. On the contrary, the amounts of apoptotic were raised and that of necrotic cells were diminished in cells treated with increasing concentrations of shikonin for 48 hours. The effect of shikonin on the cell cycle progression was stained by propidium iodide and analyzed by a flow cytometer. The amount of cells at sub-G1 phase was increased following the increase of concentration and shikonin. The amount of cells arrested in S phase was raised at low shikonin concentration, but decreased at higher concentration. Moreover, no DNA fragmentation phenomenon was found in cell treated with different concentration of shikonin for 24 or 48 hours. Our results shown that wound heal was inhibited and the amount of round cells was increased following cells treated with the increase of shikonin concentration and treated time. In Cells treated with shikonin were increased sub-G1 population, induced apoptosis and necrosis.

並列關鍵字

shikonin ； lung cancer ； apoptosis ； necrosis

參考文獻

1. Chen, X. et al. Shikonin, a component of chinese herbal medicine, inhibits chemokine receptor function and suppresses human immunodeficiency virus type 1. Antimicrob Agents Chemother 47, 2810-6 (2003).

2. Chen, X., Yang, L., Oppenheim, J.J. & Howard, M.Z. Cellular pharmacology studies of shikonin derivatives. Phytother Res 16, 199-209 (2002).

3. Shen, C.C. et al. Antimicrobial activities of naphthazarins from Arnebia euchroma. J Nat Prod 65, 1857-62 (2002).

4. Sasaki, K., Abe, H. & Yoshizaki, F. In vitro antifungal activity of naphthoquinone derivatives. Biol Pharm Bull 25, 669-70 (2002).

5. Singh, F., Gao, D., Lebwohl, M.G. & Wei, H. Shikonin modulates cell proliferation by inhibiting epidermal growth factor receptor signaling in human epidermoid carcinoma cells. Cancer Lett 200, 115-21 (2003).