Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
Laboratory of Molecular Biomedicine, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia; Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
J Ethnopharmacol. 2015 May 26;166:270-8. doi: 10.1016/j.jep.2015.03.039. Epub 2015 Mar 19.
Dillenia suffruticosa (Family: Dilleniaceae) or commonly known as "Simpoh air" in Malaysia, is traditionally used for treatment of cancerous growth including breast cancer.
D. suffruticosa root dichloromethane extract (DCM-DS) has been reported to induce G0/G1 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.
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.
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 G0/G1 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.
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.
杜茎山（杜茎山科）或在马来西亚通常称为“Simpoh air”，传统上用于治疗包括乳腺癌在内的癌性生长。
据报道，杜茎山根二氯甲烷提取物（DCM-DS）可诱导 caspase-3 缺陷型 MCF-7 乳腺癌细胞进入 G0/G1 期细胞周期停滞和细胞凋亡。本研究旨在探讨 DCM-DS 处理的 MCF-7 细胞中 p53/p21 和线粒体途径的参与情况，并鉴定负责 DCM-DS 细胞毒性的生物活性化合物。
采用顺序溶剂法提取杜茎山根。使用 GeXP 多重系统研究 DCM-DS 处理的 MCF-7 细胞中 p53、p21、Bax 和 Bcl-2 基因的表达。然后使用 Western blot 分析测定蛋白质表达。使用柱色谱法分离 DCM-DS 中的生物活性化合物。使用核磁共振波谱法阐明化合物的结构。使用 MTT 测定法评估分离化合物对 MCF-7 细胞的细胞毒性。通过 HPLC 分析确定 DCM-DS 中桦木酸（BA）的百分比。
蛋白质水平上 p53 的表达显著上调。DCM-DS 处理后，p21 在基因和蛋白质水平上的表达均显著上调，表明 MCF-7 细胞中 G0/G1 期细胞周期阻滞的诱导是通过 p53/p21 途径。Bcl-2 蛋白表达下调，mRNA 水平无变化，提示发生了翻译后修饰，导致 Bcl-2 蛋白降解。总的来说，用 DCM-DS 处理后，Bax/Bcl-2 的比值增加，导致细胞凋亡。从 DCM-DS 中分离出 3 种三萜类化合物。桦木酸似乎是 DCM-DS 中最主要和最具细胞毒性的化合物。
DCM-DS 通过 p53/p21 途径诱导 MCF-7 细胞周期停滞和凋亡。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，负责 DCM-DS 的细胞毒性。因此，BA 可作为杜茎山药材标准化的标志物。DCM-DS 也可作为杜茎山根中富含 BA 的提取物用于乳腺癌的治疗。
