丹蒽醌對人類惡性黑色素瘤細胞株(A375.S2)誘發細胞週期停滯及細胞凋亡之影響

Effects of Danthron on the Induction of Cell Cycle Arrest and Apoptosis in Human Malignant Melanoma Cell Line (A375.S2)

吳育欣(Yu-Hsin Wu)

指導教授 ： 楊仁宏

中山醫學大學/醫學院/醫學研究所/碩士(2008年)

摘要

惡性黑色素瘤是一日漸增加且致命性高的惡性腫瘤，早期診斷及手術切除對於原位癌及初期患者大多可以治癒；但對於較晚期、侵襲性較高的腫瘤患者，除非接受全身性化學治療或免疫療法，其預後是相當不理想的。全身性化學治療或免疫療法的副作用都相當大，如噁心、嘔吐、落髮、骨髓抑制等。
丹蒽醌是中藥材—大黃的萃取物，大黃素、蘆薈大黃素、大黃酸皆為蒽醌類的活性化合物，亦都是大黃的主成分之一。研究者發現大黃素、蘆薈大黃素及大黃酸可經由阻礙細胞週期來抑制細胞生長，並主要經由粒線體路徑誘導多種癌細胞株細胞凋亡，也發現它們有明顯的抑制轉移作用。本研究在探討丹蒽醌對人類惡性黑色素瘤細胞株誘發細胞週期停滯及細胞凋亡之影響。實驗結果指出，丹蒽醌可經由細胞週期停滯在G0/G1 phase及誘導細胞凋亡，對人類惡性黑色素瘤細胞株產生細胞毒殺作用。DAPI染色及Comet assay觀察到DNA受損情形。流式細胞儀及即時聚合酶連鎖反應發現丹蒽醌經由降低粒線體膜電位後釋放cytochrome c、AIF及Endo G到細胞質中；也可增加Bax及抑制Bcl-xL/Bcl-2的表現並活化caspase-9及caspase-3後啟動caspase-dependent或caspase-independent的凋亡路徑。細胞內鈣離子濃度的增加可能導致粒線體膜電位下降及活性氧濃度上升。另外，丹蒽醌也可經由活化caspase-8及Bid/tBid促使cytochrome c釋放。因此，丹蒽醌可經由粒線體路徑、內質網路徑及死亡受體路徑誘發人類惡性黑色素瘤細胞株（A375.S2）的凋亡。

關鍵字

丹蒽醌人類惡性黑色素瘤細胞株 ； 細胞凋亡 ； 細胞週期

並列摘要

Malignant melanoma is an increasing common malignancy and with high mortality. Melanoma in situ and early stage melanoma could be cured if early diagnosis and surgical excision. Unless chemotherapy or immunotherapy, the prognosis is quite poor in patient of late staging and highly invasive malignancy. But the side effects of chemotherapy or immunotherapy are severe, such as nausea, vomiting, hair loss and bone marrow suppression.
Danthron is one of the extracts of Rhubarb that is a traditional Chinese herb. Danthron, Emodin, Aloe-emodin, and Rhein are all active compounds of Rhubarb. Researchers find that Emodin, Aloe-emodin, and Rhein can suppress cell growth via arresting cell cycle. They also can induce apoptosis of a lot of cancer cell lines via mitochondrial pathway and inhibit metastasis. This research is being probed into effects of Danthron on the induction of cell cycle arrest and apoptosis in human malignant melanoma cell line (A375.S2). The result suggests that Danthron can kill A375.S2 cells associated with G0/G1 phase arrest and induction apoptosis. DNA damage was found by DAPI stain and Comet assay. Flow cytometric assay and real time PCR showed that Danthron can decrease mitochondrial membrane potential and let releasing cytochrome c, AIF and Endo G to cytoplasm. Danthron increased expression of Bax and suppressed expression of Bcl-xL/Bcl-2 then active caspase-9 and caspase-3. Finally, it induced apoptosis of A375.S2 cells through caspase-dependent or caspase-independent pathway. Elevation of intracellular calcium concentration may decrease mitochondrial membrane potential and increase reactive oxygen species. Otherwise, Danthron could make cytochrome c to release via activating caspase-8 and Bid/tBid. In conclusion, Danthron can induce apoptosis through mitochondrial, endoplasmic reticulum and death receptor pathway in human malignant melanoma cell line (A375.S2).

並列關鍵字

Danthron ； A375.S2 cells ； apoptosis ； cell cycle

參考文獻

2. Bellamy CO, Malcomson RD, Harrison DJ, Wyllie AH: Cell death in health and disease: the biology and regulation of apoptosis. Semin Cancer Biol 1995; 6(1): 3-16.

3. Michalides RJ: Cell cycle regulators: mechanisms and their role in aetiology, prognosis, and treatment of cancer. J Clin Pathol 1999; 52(8): 555-68.

4. Senderowicz AM: Targeting cell cycle and apoptosis for the treatment of human malignancies. Curr Opin Cell Biol 2004; 16(6): 670-8.

5. Yuspa SH: Cutaneous chemical carcinogenesis. J Am Acad Dermatol 1986; 15(5 Pt 1): 1031-44.

6. Molho-Pessach V, Lotem M: Viral carcinogenesis in skin cancer. Curr Probl Dermatol 2007; 35: 39-51.