Liu Chia-Chi, Lin Wei-Wen, Wu Chun-Chi, Hsu Shih-Lan, Wang Chi-Yen, Chung Jing-Gung, Chiang Chi-Shiun
Department of Biochemical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan, R.O.C.
Cardiovascular Center, Taichung Veterans General Hospital, Taichung, Taiwan, R.O.C.
In Vivo. 2018 Sep-Oct;32(5):1119-1127. doi: 10.21873/invivo.11354.
BACKGROUND/AIM: The treatment of human glioma tumor is still an unmet medical need. Natural products are always promising resources for discovery of anticancer drugs. Lauryl gallate (LG) is one of the derivatives of gallic acid, widely present in plants, that has been shown to induce anticancer activities in many human cancer cell lines; however, it has not been studied in human glioma cell lines. Thus, the effects of LG on human glioblastoma U87 cells were investigated in the present in vitro study.
Cell morphology and viability were examined by phase-contrast microscopy. Annexin V/Propidium iodide (PI) double staining were performed and assayed by flow cytometry to confirm that viable cell number reduction was due to the induction of apoptosis. Furthermore, U87 cells were exposed to LG in various concentrations and were analyzed by caspase activity assay. To further confirm that LG induced apoptotic cell death, the expression of apoptosis-associated proteins in LG-treated U87 cells was tested by western blot.
LG induced morphological changes and decreased viability in U87 cells. Annexin V/PI double staining revealed that LG induced apoptotic cell death in U87 cells in a dose-dependent manner. The increased activities of caspase-2, -3, -8 and -9 demonstrated that LG induced U87 cell apoptosis through a caspase-dependent pathway. In terms of molecular level, LG increased pro-apoptotic proteins Bax and Bak and decreased anti-apoptotic protein Bcl-2 in U87 cells. Furthermore, LG also suppressed the expression of p-Akt, Pak1, Hif-1α and Hif-2α, β-catenin and Tcf-1 in U87 cells.
These results suggest that LG induced apoptotic cell death via the caspase-dependent pathway in U87 cells.
背景/目的:人类胶质瘤的治疗仍是未被满足的医疗需求。天然产物一直是发现抗癌药物的有前景的资源。没食子酸月桂酯(LG)是没食子酸的衍生物之一,广泛存在于植物中,已显示在多种人类癌细胞系中具有抗癌活性;然而,尚未在人类胶质瘤细胞系中进行研究。因此,在本体外研究中考察了LG对人胶质母细胞瘤U87细胞的作用。
通过相差显微镜检查细胞形态和活力。进行膜联蛋白V/碘化丙啶(PI)双染色并通过流式细胞术进行分析,以确认活细胞数量减少是由于凋亡诱导所致。此外,将U87细胞暴露于不同浓度的LG,并通过半胱天冬酶活性测定进行分析。为进一步证实LG诱导凋亡性细胞死亡,通过蛋白质免疫印迹法检测LG处理的U87细胞中凋亡相关蛋白的表达。
LG诱导U87细胞形态变化并降低其活力。膜联蛋白V/PI双染色显示LG以剂量依赖性方式诱导U87细胞凋亡性细胞死亡。半胱天冬酶-2、-3、-8和-9活性增加表明LG通过半胱天冬酶依赖性途径诱导U87细胞凋亡。在分子水平上,LG增加了U87细胞中促凋亡蛋白Bax和Bak的表达,并降低了抗凋亡蛋白Bcl-2的表达。此外,LG还抑制了U87细胞中p-Akt、Pak1、Hif-1α和Hif-2α、β-连环蛋白和Tcf-1的表达。
这些结果表明,LG通过半胱天冬酶依赖性途径诱导U87细胞凋亡性细胞死亡。