School of Basic Medical Science, Shanghai University of Traditional Chinese Medicine, Shanghai 201203,China.
Department of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiaotong University, Shanghai 200092,China.
J Ethnopharmacol. 2019 Apr 24;234:180-188. doi: 10.1016/j.jep.2019.01.007. Epub 2019 Jan 17.
Ze-Qi-Tang (ZQT), a classic Chinese herbal formula, has been for over thousand years used for the treatment of several respiratory ailments like cough, asthma, hydrothorax and lung cancer.
Cumulative literature on ZQT herbal formula reveals that its several constituent components are potent inducer of apoptosis in different cancer cells. However, the activity of ZQT against non-small-cell-lung cancer (NSCLC) has not been previously examined. The aim of the study is to investigate the molecular mechanism of ZQT on NSCLC cells.
Cell growth were determined by CCK-8 and colony formation assay. Induction of cellular apoptosis or arrest of cell cycle were determined by flow cytometric analysis using annexin V/ propidium iodide, Hoechst 33342 or TUNEL staining method. In some assay p53 activity of NSCLC ( A549 and H460) cells were blocked with pifithrin-a, prior to treatment with ZQT. The level of expression of cell cycle and apoptosis related marker proteins were estimated by western blot. The anticancer activity of ZQT in vivo were monitored in nude mice that were induced with tumor by subcutaneous inoculation of A549 cells and then treated by ZQT(100 mg/kg,200 mg/kg,400 mg/kg) gavaging for 30 days. Mice' body weight and tumor volume were measured weekly. The survival carve was recorded. Apoptosis from mice' tissue was observed by TUNEL assay. Pathological histology of liver, kidney and heart were detected by H&E staining, and its functions were tested by ELISA.
Dose- and time-dependent inhibition of proliferation of NSCLC ( A549 and H460) cells by ZQT therapy along with induction of cell cycle arrest at G0⁄G1 phase were observed. The arrest of cell cycle arrest and inhibition of cellular proliferation were associated with up regulation of p53 along with down regulation of Cyclin B1 and Cdk2 indicating a mitochondrial related induction of apoptosis with ZQT. A reversal of ZQT-induced apoptosis and G0⁄G1 arrest was observed with pifithrin-a pretreatment. ZQT was also found to suppress the progression of tumor growth in mouse xenograft models and prolong survival. In addition, no hepato- or nephro- or cardio-toxicity with ZQT treatment were detected in mice.
These findings suggest that the ZQT formula inhibits the growth of NSCLC cells and is a potential agent of complementary and alternative treatment for lung cancer.
泽七汤（ZQT）是一种经典的中草药配方，已有一千多年的历史，用于治疗咳嗽、哮喘、胸腔积液和肺癌等多种呼吸道疾病。
累积的 ZQT 草药配方文献表明，其几种组成成分是不同癌细胞凋亡的有效诱导剂。然而，ZQT 对非小细胞肺癌（NSCLC）的活性尚未得到检验。本研究旨在探讨 ZQT 对 NSCLC 细胞的分子机制。
通过 CCK-8 和集落形成测定法测定细胞生长。通过流式细胞术分析用 Annexin V/碘化丙啶、Hoechst 33342 或 TUNEL 染色法测定细胞凋亡或细胞周期阻滞的诱导。在一些测定中，在用 ZQT 处理之前，用 pifithrin-a 阻断 NSCLC（A549 和 H460）细胞中的 p53 活性。通过 Western blot 估计细胞周期和凋亡相关标记蛋白的表达水平。通过皮下接种 A549 细胞诱导裸鼠肿瘤，然后用 ZQT（100mg/kg、200mg/kg、400mg/kg）灌胃 30 天，监测 ZQT 的体内抗癌活性。每周测量小鼠体重和肿瘤体积。通过 TUNEL 测定观察小鼠组织中的凋亡。通过 H&E 染色检测肝、肾和心脏的组织病理学变化，并通过 ELISA 检测其功能。
ZQT 治疗呈剂量和时间依赖性抑制 NSCLC（A549 和 H460）细胞的增殖，同时观察到细胞周期阻滞在 G0/G1 期。细胞周期阻滞和细胞增殖抑制与 p53 上调以及细胞周期蛋白 B1 和 Cdk2 下调相关，表明 ZQT 诱导与线粒体相关的细胞凋亡。用 pifithrin-a 预处理可观察到 ZQT 诱导的凋亡和 G0/G1 阻滞的逆转。还发现 ZQT 抑制小鼠异种移植模型中的肿瘤生长进展并延长存活时间。此外，在小鼠中未检测到 ZQT 治疗的肝、肾或心脏毒性。
这些发现表明，ZQT 配方可抑制 NSCLC 细胞的生长，是肺癌互补和替代治疗的潜在药物。
