蘆薈大黃素經由調控雌激素受體、細胞週期蛋白依賴性激酶和抗藥性蛋白等相關路徑使乳癌細胞重新具有fulvestrant敏感性

Aloe-emodin re-sensitizes breast cancer cells to fulvestrant by regulating the related pathways of estrogen receptors, cyclin-dependent kinases and drug resistance proteins

楊媛婷(Yuan-Ting Yang)

指導教授 ： 柯俊良

中山醫學大學/醫學院/醫學研究所/博士(2022年)

https://doi.org/10.6834/csmu202200211

摘要

研究目的：雌激素受體陽性（estrogen receptor-positive；ER+）乳癌患者約占整個乳癌族群的70%，臨床上常用抗雌激素藥物治療這類患者。然而，約有40-50%的患者在治療期間會出現轉移或復發，被認為是對治療產生抗藥性。當患者使用tamoxifen後無效，便會改用芳香酶抑制劑；當患者對芳香酶抑制劑也產生抗藥性時，則可能改用fulvestrant、CDK4/6抑制劑或mTOR抑制劑治療。但這些抑制劑亦可能促使癌細胞轉往替代性訊息傳遞路徑，再次產生抗藥性，使其有能力繼續增生或對抗細胞凋亡。過去研究發現蘆薈大黃素，一種來自蘆薈和大黃的天然蒽醌類，可有效毒殺各種亞型的乳癌，並能經由阻擋Ras/ERK和PI3K/mTOR路徑而增強tamoxifen對ER+乳癌細胞的毒殺效果。因此，本研究欲進一步探討蘆薈大黃素對ER+抗藥性乳癌的影響及調控機制。
研究方法及資料：本研究選用ER+乳癌細胞株MCF-7/S0.5及其具有fulvestrant抗性的子細胞株MCF-7/182R-6進行實驗。以細胞毒性分析和細胞群落形成分析確認fulvestrant與蘆薈大黃素併用對細胞生長和增殖的影響以及其藥物交互作用，並進一步利用細胞凋亡分析、粒線體膜電位偵測、西方墨點法以及傷口癒合實驗比較藥物作用後的表現變化以分析其分子生物機制。
研究結果：實驗結果發現，蘆薈大黃素對MCF-7/182R-6細胞的生長造成劑量與時間依賴性的抑制。在經fulvestrant處理的MCF-7/182R-6中加入蘆薈大黃素可提升細胞對fulvestrant的敏感性，並協同誘導細胞凋亡，減少抗凋亡蛋白Bcl-xL表現及增加細胞凋亡相關蛋白cleavage caspase 7及PARP表現，伴隨粒線體膜電位的喪失，推測是經由粒線體依賴的細胞凋亡路徑。細胞經蘆薈大黃素合併fulvestrant處理後，CDK4和CDK6的表現亦明顯下調，而且通過阻斷FAK和TACE路徑減弱細胞的移動性；同時降低抗藥性蛋白TARBP2的表現，表明有抑制腫瘤侵襲的可能。此外，傷口癒合分析結果顯示，相較於單獨使用蘆薈大黃素或fulvestrant，兩藥同時使用可明顯抑制細胞移動。
結論與建議：本研究結果顯示蘆薈大黃素可能通過多種機制逆轉乳癌細胞對fulvestrant的抗藥性，包括促進癌細胞凋亡與抑制癌細胞的增殖，顯示蘆薈大黃素可能具有克服fulvestrant抗藥性的潛力。未來仍待進一步的實驗加以確認其在動物與人體的成效。

關鍵字

蘆薈大黃素 ； 乳癌 ； 內分泌抗性 ； fulvestrant ； 細胞凋亡

並列摘要

Objective：Estrogen receptor (ER)-positive breast cancers account for about 70% of all breast cancers and are usually treated clinically with hormone therapy that primarily targets ER itself or its related molecular pathways. Most of ER-positive breast cancer patients can obtain good curative effect in the initial hormone therapy; however, 40% to 50% of patients will develop endocrine resistance, which constitutes a great clinical threat. Previous studies have found that aloe-emodin can enhance the effect of tamoxifen on ER-positive breast cancer cells by blocking the Ras/ERK and PI3K/mTOR pathways. Here, This study intended to further explore the effect and regulatory mechanism of aloe-emodin on endocrine-resistant breast cancer.
Methods and Materials：The ER-positive breast cancer cell line MCF-7/S0.5 and its fulvestrant-resistant subline MCF-7/182R-6 were selected for this study. Cytotoxicity assay and clonogenicity assay were performed to confirm the effects of fulvestrant in combination with aloe-emodin on cell growth and proliferation as well as the drug interaction. After apoptosis analysis, mitochondrial membrane potential detection and wound healing assay, we further compared changes in protein expression after drug treatment by western blotting.
Results：Combined treatment with fulvestrant and aloe-emodin inhibited the growth of MCF-7/182R-6 cells more effectively than fulvestrant alone, showing dose- and time-dependence, accompanied by cell shrinkage and rupture associated with apoptosis that was subsequently shown to be mitochondrial-dependent. Combined treatment with fulvestrant and aloe-emodin not only downregulated CDK4 and CDK6, but also impaired cell mobility by blocking FAK and TACE-related pathways; meanwhile, combined treatment also reduced TARBP2 expression, suggesting the possibility of tumor invasion inhibition.
Conclusion and Suggestion：These results demonstrate the potential of aloe-emodin against endocrine resistance in breast cancer. Further studies and evaluations are needed.

並列關鍵字

aloe-emodin ； breast cancer ； endocrine resistance ； fulvestrant ； apoptosis

參考文獻

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