探討熊果酸誘發自噬作用對乳癌細胞凋亡的影響

熊果酸 (Ursolic acid；UA)是一種存在於天然植物中的五環三萜類化合物，常見於迷迭香、蘋果、藍莓等植物中，具有抗癌、抗發炎、和抗血管新生等藥理活性。研究指出，熊果酸能引起細胞週期停滯、誘導細胞凋亡和細胞自噬作用發生，但熊果酸誘發兩種作用對癌細胞的影響至今仍不清楚。因此，我們以乳癌細胞為例，嘗試釐清熊果酸誘發自噬和細胞凋亡的互動關聯。首先，在MTT的試驗分析中，我們發現熊果酸有效抑制MDA-MB-231和MCF7乳癌細胞增生，且呈現濃度和時間的依賴關係。以西方墨點法分析，熊果酸抑制乳癌細胞生長是藉由抑制PI3K/AKT/mTOR路徑。我們也發現經熊果酸處理的乳癌細胞會發生自噬作用(autophagy)，並伴隨細胞自噬作用Beclin1、LC3B和p62蛋白表現，共軛焦免疫螢光分析也觀察到LC3B和AVO染色結果顯著增加 (P < 0.001)。利用caspase抑制劑 Z-VAD-FMK偕同與熊果酸處理乳癌細胞，並不會誘發細胞壞死作用(necroptosis)，其標的蛋白RIP1表現量也未顯著改變。相較於單純以熊果酸處理的乳癌細胞，在熊果酸和自噬作用抑制劑chloroquine (CQ)同時添加的乳癌細胞的凋亡現象更加顯著 (P < 0.05)。綜合以上，我們釐清抑制細胞自噬作用能有效增加乳癌細胞對熊果酸抑癌的敏感性。

Ursolic acid (UA) is a natural pentacyclic triterpenoid compound that is present in many plants, such as rosemary, apple, and blueberry. It has been shown that UA exhibits numerous pharmacological activities, including anti-inflammatory, anti-angiogenic, and anti-tumor. It has been reported that UA caused autophagy and apoptosis in different types of cancers. The presented data in this study aims at understanding the effect of UA-induced autophagy in relation to apoptosis of breast cancer cell in vitro. Based on the MTT assay, we observed that UA induced MDA-MB-231 and MCF-7 cell death by a dose-dependent and time-dependent manner. After 48 h incubation, IC50 values of MCF-7 and MDA-MB-231 cells with were 21μM and 32.9 μM, respectively. UA treatment (25μM) for 48 h decreased PI3K/AKT/mTOR pathway. On the contrary, autophagy-related proteins, such as LC3B, p62 and Beclin1 were significantly increased in UA-treated MDA-MB-231 cells. Besides, level of the RIP1 protein did not change in cells co-treated UA and Z-VAD-FMK, indicating UA did not promote necroptosis in UA-treated breast cancer cells. Compared to UA-treated MDA-MB-231 cells, the percentage of cell death was more significant in those ones treated with UA and autophagy inhibitor, Chloroquine (CQ) (P<0.05) and the increased cell number with positive staining of LC3B and AVO are more significant in MDA-MB-231 cells treated with UA and CQ. In conclusion, our data indicate that inhibition the nature of autophagy can effectively enhance UA sensitivity of breast cancer.