Department of Medical Genetics, College of Medicine, Hanyang University, 17 Haengdang-Dong, Sungdong-Gu, Seoul 133-791, Republic of Korea.
J Ethnopharmacol. 2010 May 4;129(1):135-9. doi: 10.1016/j.jep.2010.02.010. Epub 2010 Feb 26.
In the course of our screening for novel modulators on cell cycle progression and apoptosis as anticancer drug candidates, we isolated a novel compound HY253 with the molecular structure of 7,8a-divinyl-2,4a,4b,5,6,7,8,8a,9,9a-decahydro-1H-fluorene-2,4a,4b,9a-tetraol from the roots of Aralia continentalis. This study was designed to evaluate the detailed mechanisms of cell cycle arrest and the apoptotic induction of HY253 in human lung cancer A549 cells.
To investigate the effects of HY253 on cell cycle progression in A549 cells, we measured DNA content of A549 cells treated with 35 microM of HY253 using flow cytometric analysis. Furthermore, TUNEL assay was used to examine apoptotic induction in A549 cells treated with 70 microM of HY253 for 24 and 48 h. The effects of HY253 on apoptosis-associated and cell cycle regulatory proteins in A549 cells were examined using Western blot analysis.
The flow cytometric analysis revealed an appreciable G(1) phase arrest in A549 cells treated with 35 microM of HY253. This HY253-induced G(1) phase arrest is associated with decreased expression of cyclin D and up-regulation of p21(CIP1), via p53 phosphorylation at Ser-15, which resulted in increased hypophosphorylated pRb in A549 cells. Furthermore, TUNEL assay and Western blot analysis revealed an appreciable apoptotic induction in A549 cells treated with 70 microM of HY253 for 48 h. This apoptotic induction in HY253-treated A549 cells is also associated with cytochrome c release from mitochondria which in turn resulted in the activation of caspase-9 and -3, and the cleavage of poly(ADP-ribose) polymerase (PARP).
These results demonstrate that HY253, a novel antiproliferative compound isolated from the roots of Aralia continentalis, induces cell cycle arrest at the G(1) phase and apoptosis in A549 cells. Based on these results, we suggest that HY253 may be a potent cancer chemotherapeutic candidate for use in treating human lung cancer cells via up-regulation and activation of p53 gene.
在筛选新型细胞周期进展和凋亡调节剂作为抗癌候选药物的过程中，我们从辽东楤木的根部分离得到了一种具有 7,8a-二乙烯基-2,4a,4b,5,6,7,8,8a,9,9a-十氢-1H-芴-2,4a,4b,9a-四醇结构的新型化合物 HY253。本研究旨在评估 HY253 对人肺癌 A549 细胞周期阻滞和凋亡诱导的详细机制。
为了研究 HY253 对 A549 细胞周期进展的影响，我们使用流式细胞术分析测量了用 35μM HY253 处理的 A549 细胞的 DNA 含量。此外，使用 TUNEL 检测法检测了用 70μM HY253 处理 24 和 48 小时的 A549 细胞的凋亡诱导。使用 Western blot 分析检测了 HY253 对 A549 细胞中凋亡相关和细胞周期调节蛋白的影响。
流式细胞术分析显示，用 35μM HY253 处理的 A549 细胞出现明显的 G1 期阻滞。这种 HY253 诱导的 G1 期阻滞与细胞周期蛋白 D 的表达减少和 p21(CIP1)的上调有关，这是通过 p53 在 Ser-15 位点的磷酸化实现的，导致 A549 细胞中低磷酸化的 pRb 增加。此外，TUNEL 检测法和 Western blot 分析显示，用 70μM HY253 处理 48 小时的 A549 细胞发生明显的凋亡诱导。在 HY253 处理的 A549 细胞中，这种凋亡诱导也与细胞色素 c 从线粒体释放有关，这反过来又导致 caspase-9 和 caspase-3 的激活，以及多聚（ADP-核糖）聚合酶（PARP）的裂解。
这些结果表明，HY253 是一种从辽东楤木根部分离得到的新型增殖抑制化合物，可诱导 A549 细胞在 G1 期停滞并凋亡。基于这些结果，我们认为 HY253 可能是一种有效的癌症化学治疗候选药物，可通过上调和激活 p53 基因用于治疗人类肺癌细胞。
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