Induction of cell cycle arrest and apoptosis by betulinic acid-rich fraction from Dillenia suffruticosa root in MCF-7 cells involved p53/p21 and mitochondrial signalling pathway
Induction of cell cycle arrest and apoptosis by betulinic acid-rich fraction from Dillenia suffruticosa root in MCF-7 cells involved p53/p21 and mitochondrial signalling pathway
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DOI：
10.1016/j.jep.2015.03.039
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发表时间：
2015-05-26
影响因子：
5.4
通讯作者：
Abdullah, Rasedee
中科院分区：
文献类型：
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作者：
Foo, Jhi Biau;Yazan, Latifah Saiful;Abdullah, Rasedee
民族药理学相关性：Dillenia suffruticosa（科：Dilleniaceae）或在马来西亚通常被称为“Simpoh air”，传统上用于治疗癌症生长，包括乳腺癌。据报道，在caspase-3缺陷的MCF-7乳腺癌细胞中，木草根二氯甲烷提取物（DCM-DS）诱导G（0）/G（1）期细胞周期停滞和凋亡。本研究旨在研究p53/p21和线粒体途径在DCM-DS处理的MCF-7细胞中的参与，以及鉴定负责DCM-DS的细胞毒性的生物活性化合物。采用连续溶剂法对木粉根进行提取。采用GeXP多重荧光检测系统检测DCM-DS对MCF-7细胞p53、p21、Bax和Bcl-2基因表达的影响。然后使用Western印迹分析测定蛋白质表达。采用柱色谱法分离DCM-DS中的生物活性化合物。化合物的结构用核磁共振波谱法进行了鉴定。采用MTT法检测化合物对MCF-7细胞的细胞毒性。结果：DCM-DS能显著上调p53蛋白的表达。DCM-DS处理后，MCF-7细胞中p21在基因和蛋白水平的表达均显著上调，提示通过p53/p21途径诱导MCF-7细胞发生G（0）/G（1）期细胞周期阻滞。Bcl-2蛋白表达下调，但在mRNA水平无变化，推测Bcl-2蛋白的降解可能是由于翻译后修饰所致。总的来说，DCM-DS处理增加了Bax/Bcl-2的比例，这促使细胞发生凋亡。从DCM-DS中分离得到3个三萜类化合物。结论：DCM-DS通过p53/p21途径诱导MCF-7细胞周期阻滞和凋亡。此外，DCM-DS通过增加Bax/Bcl-2的比值诱导细胞凋亡。桦木酸是主要化合物之一，是DCM-DS细胞毒性的原因。因此，BA可作为D.灌木DCM-DS也可作为富含BA的D. suffruticosa用于乳腺癌的管理。(c)2015爱思唯尔爱尔兰有限公司版权所有。
Ethnopharmacological relevance: Dillenia suffruticosa (Family: Dilleniaceae) or commonly known as "Simpoh air" in Malaysia, is traditionally used for treatment of cancerous growth including breast cancer.Aim of the study: D. suffruticosa root dichloromethane extract (DCM-DS) has been reported to induce G(0)/G(1) phase cell cycle arrest and apoptosis in caspase-3 deficient MCF-7 breast cancer cells. The present study was designed to investigate the involvement of p53/p21 and mitochondrial pathway in DCM-DS-treated MCF-7 cells as well as to identify the bioactive compounds responsible for the cytotoxicity of DCM-DS.Materials and methods: Extraction of D. suffruticosa root was performed by the use of sequential solvent procedure. GeXP-based multiplex system was employed to investigate the expression of p53, p21, Bax and Bcl-2 genes in MCF-7 cells treated with DCM-DS. The protein expression was then determined using Western blot analysis. The bioactive compounds present in DCM-DS were isolated by using column chromatography. The structure of the compounds was elucidated by using nuclear magnetic resonance spectroscopy. The cytotoxicity of the isolated compounds towards MCF-7 cells was evaluated by using MTT assay. The percentage of betulinic acid (BA) in DCM-DS was determined by HPLC analysis.Results: The expression of p53 was significantly up-regulated at protein level. The expression of p21 at both gene and protein levels was significantly up-regulated upon treatment with DCM-DS, suggesting that the induction of G(0)/G(1) phase cell cycle arrest in MCF-7 cells was via p53/p21 pathway. Bcl-2 protein was down-regulated with no change at the mRNA level, postulating that post-translational modification has occurred resulting in the degradation of Bcl-2 protein. Overall, treatment with DCM-DS increased the ratio of Bax/Bcl-2 that drove the cells to undergo apoptosis. A total of 3 triterpene compounds were isolated from DCM-DS. Betulinic acid appears to be the most major and most cytotoxic compound in DCM-DS.Conclusion: DCM-DS induced cell cycle arrest and apoptosis in MCF-7 cells via p53/p21 pathway. In addition, DCM-DS induced apoptosis by increasing the ratio of Bax/Bcl-2. Betulinic acid, which is one of the major compounds, is responsible for the cytotoxicity of the DCM-DS. Therefore, BA can be used as a marker for standardisation of herbal product from D. suffruticosa. DCM-DS can also be employed as BA-rich extract from roots of D. suffruticosa for the management of breast cancer. (c) 2015 Elsevier Ireland Ltd. All rights reserved.