Xiao Qian, Zheng Fang, Wu JingJing, Tang Qing, Wang Wei, Hann Swei Sunny
Laboratory of Tumor Biology, Guangzhou, China.
Department of Gastrointestinal Surgery, Guangdong Provincial Hospital of Chinese Medicine, The Second Clinical Medical Collage, Guangzhou University of Chinese Medicine, Guangzhou, China.
Cell Physiol Biochem. 2017;43(6):2353-2366. doi: 10.1159/000484387. Epub 2017 Oct 27.
BACKGROUND/AIMS: Atractylodes macrocephula Koidz is an important ingredient in traditional Chinese herbs. One major bioactive compound, atractylenolide-1 (ATL-1), was reported to have anti-inflammatory and anti-tumor activities. However, the underlying molecular mechanism associated to this has not been well elucidated.
Cell viability and cell cycle distribution were measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometry assays, respectively. Western blot analysis was performed to examine the phosphorylation and protein expression of extracellular signaling-regulated kinase 1/2 (ERK1/2), signal transducer and activator of transcription 3 (Stat3), 3-phosphoinositide dependent protein kinase-1 (PDK1) and transcription factor SP1. QRT-PCR was used to examine the mRNA levels of PDK1 gene. Exogenously expressions of Stat3, PDK1 and SP1 were carried out by transient transfection assays. PDK1 promoter activity was measured by Secrete-Pair Dual Luminescence Assay Kit. A nude mice xenograft model was used to confirm the findings in vitro.
We showed that ATL-1 inhibited human lung cancer cell growth and induced cell cycle arrest. Furthermore, we found that ATL-1 stimulated phosphorylation of ERK1/2, inhibited phosphorylation and protein expressions of Stat3 and SP1; the latter were abrogated in the presence of MEK/ERK inhibitor PD98059. Moreover, ATL-1 reduced the protein, mRNA expression and promoter activity of PDK1. Intriguingly, exogenously expressed Stat3 and SP1 overcame ATL-1-inhibited SP1 and Stat3, and PDK1 protein expressions, respectively. Moreover, overexpression of PDK1 resisted the ATL-1-inhibited lung cancer cell growth. In consistent with the results in vitro, ATL-1 inhibited tumor growth, protein expressions of Stat3, SP1 and PDK1, and induced phosphorylation of ERK1/2 in vivo.
In summary, our results show that ATL-1 inhibits lung cancer cell growth through activation of ERK1/2, followed by suppressing SP1 protein expression. ATL-1 also reduces phosphorylation and protein levels of Stat3. These are mutual regulation between Stat3 and SP1 proteins affected by ATL-1. This ultimately suppresses PDK1 gene expression. This study reveals a novel mechanism by which ATL-1 inhibits growth of lung cancer cells. Thus, targeting PDK1 pinpoints a potential in the lung cancer treatment.
背景/目的：白术是传统中药中的一种重要成分。据报道，其一种主要生物活性化合物苍术素-1（ATL-1）具有抗炎和抗肿瘤活性。然而，与之相关的潜在分子机制尚未完全阐明。
分别使用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）和流式细胞术检测细胞活力和细胞周期分布。进行蛋白质免疫印迹分析以检测细胞外信号调节激酶1/2（ERK1/2）、信号转导和转录激活因子3（Stat3）、3-磷酸肌醇依赖性蛋白激酶-1（PDK1）和转录因子SP1的磷酸化及蛋白表达。采用实时定量聚合酶链反应（QRT-PCR）检测PDK1基因的mRNA水平。通过瞬时转染实验进行Stat3、PDK1和SP1的外源性表达。使用分泌型双荧光素酶检测试剂盒测量PDK1启动子活性。利用裸鼠异种移植模型证实体外实验结果。
我们发现ATL-1抑制人肺癌细胞生长并诱导细胞周期停滞。此外，我们发现ATL-1刺激ERK1/2磷酸化，抑制Stat3和SP1的磷酸化及蛋白表达；在存在MEK/ERK抑制剂PD98059的情况下，后者被消除。此外，ATL-1降低了PDK1的蛋白、mRNA表达及启动子活性。有趣的是，外源性表达的Stat3和SP1分别克服了ATL-1对SP1和Stat3以及PDK1蛋白表达的抑制。此外，PDK1的过表达抵抗了ATL-1对肺癌细胞生长的抑制。与体外实验结果一致，ATL-1在体内抑制肿瘤生长、Stat3、SP1和PDK1的蛋白表达，并诱导ERK1/2磷酸化。
总之，我们的结果表明，ATL-1通过激活ERK1/2，随后抑制SP1蛋白表达来抑制肺癌细胞生长。ATL-1还降低Stat3的磷酸化水平和蛋白水平。这些是受ATL-1影响的Stat3和SP1蛋白之间的相互调节。这最终抑制了PDK1基因表达。本研究揭示了ATL-1抑制肺癌细胞生长的新机制。因此，靶向PDK1为肺癌治疗指明了一个潜在方向。
