| 摘要: | 大黃酚（chrysophanol, 1,8-Dihydroxy-3-methyl-9,10-anthracenedion）是從蓼科植物大黃萃取得來的蒽醌類（anthraquinone）衍生物的生物鹼。一些相關文獻顯示大黃酚具有抗炎、抗菌、抗氧化、抗老化、降血脂、治療動脈粥狀硬化、抗記憶衰退、與抗癌作用。大黃酚的抗癌分子作用機轉尚未被詳細報導。
本研究即針對人類肝癌細胞株Hep3B及肺癌細胞株A549來探討大黃酚是否具有抑制這兩株細胞的增殖及轉移相關表現的能力。由存活率結果顯示，在處理大黃酚24小時後，大黃酚對Hep3B細胞與A549細胞的增殖就能有效的抑制，其IC50皆大約為50μM。
由細胞形態變化、DAPI 試驗陽性表現及彗星試驗的拖尾現象，顯示大黃酚對於此兩株細胞的DNA 具有破壞的能力，進而導致細胞死亡。
大黃酚使細胞內的活性氧化物（ROS）增加、鈣離子（Ca2+）濃度增加，進而粒線體膜電位改變及ATP 濃度下降；但Apaf-1 (Apoptosis protease activating factor-1) 、caspase 8與caspase 9蛋白質表現量無顯著變化，綜合上述實驗結果，大黃酚誘使Hep3B細胞與A549細胞死亡是以壞死性的細胞死亡方式進行。
在細胞週期的檢測結果顯示，發現大黃酚促使細胞傾向於S期的比例增長，檢測細胞週期調控蛋白的表現；隨著加藥的時間增加，p21、p27及p53蛋白質表現量上升，而cyclin D，CDK2、thymidine synthase等蛋白表現下降，可能由於DNA損傷後被磷酸化，使的在細胞週期過程中從S期到G2期的推移蛋白cyclin A/CDK2 complex受到抑制。
在移動和穿透性試驗的結果顯示，大黃酚能抑制兩株細胞在在轉移相關的表現，且會抑制轉移相關調控蛋白PI3K、ERK、MMPs及VEGF、EPO、NF-κB的表現。
綜合以上結果，大黃酚對於Hep3B細胞及A549細胞的增殖及轉移的相關表現確實有顯著的抑制作用，值得進一步研究發展成為抗癌藥物。
Chrysophanol, an anthraquinone-derivative alkaloid, is extracted from Rheum palmatum L. It has been reported that chrysophanol may exhibit several regulatory functions including anti-inflammation, antiseptic, antioxidation, anti-aging, prevention of atherosclerosis, anti-dementia, and anti-tumor activity. However, the molecular mechanism of anti-tumor effects of chrysophanol has not been well defined. In this study, we used human hepatic carcinoma Hep3B cells and lung carcinoma A549 cells as two cell models to examine the inhibitory effects of chrysophanol on tumor cell growth and migration. Cell viability assay revealed that chrysophanol indeed prevented cell proliferation in these two cell lines. To further investigate the cellular mechanism of chrysophanol, we measured the changes of cellular physiological parameters in chrysophanol-treated cells. We found that the treatment of chrysophanol induced ROS production and increased Ca2+ concentration, results in a significant change of mitochondrial membrane potential and the reduction of ATP concentration in mitochondria. There is no significant change in the expression of Apaf-1 (Apoptosis protease activating factor-1), caspase-8 and caspas-9. Moreover, the chrysophanol-induced cell death in Hep3B cells and A549 cells was accompanied with several characteristics of necrosis, including the necrotic morphology, DNA fragmentation as well as the enhancement of DAPI positive cells. In addition, flow -cytometry analysis showed that the S phase of cell cycle was blocked in chrysophanol-treated cells. Western blotting results also revealed that the expression levels of cell cycle regulatory proteins, such as p21, p27 and p53, were significantly changed in a time-dependent manner. The protein expression of p21 was increased while CDK2 and thymidylate synthase were decreased. We hypothesized that, with the chrysophanol treatment, CDK2 and thymidylate synthase were phosphorylated in response to DNA damage and could not activate cyclin A/CDK2 complex to facilitate the cell cycle progression. In terms of anti-metastasis effects of chrysophanol, our results showed that in chrysophanol-treated cells, the cell migration and invasion were lower than the controls. The expression of PI3K, ERK, and MKK, which are involved in metastasis of cancer cells, was also down-regulated under chrysophanol treatments. Our studies provided the evidence for the inhibitory effects of chrysophanol in cell proliferation and metastasis, and suggested the application of chrysophanol as a potential anticancer reagent in the future. |
