Liu Rangru, Yu Zutao, Chen Zhuo, Liu Danqi, Huang Fengying, Li Qianbin, Hu Gaoyun, Yi Xinan, Li Xi, Zhou Honghao, Liu Zhaoqian
Key Laboratory of Tropical Translational Medicine of the Ministry of Education & Hainan Key Laboratory for Research and Development of Tropical Herbs, School of Pharmacy, Hainan Medical University, Haikou, People's Republic of China.
Department of Clinical Pharmacology, Xiangya Hospital, Central South University, Changsha, People's Republic of China.
PeerJ. 2020 Oct 2;8:e9981. doi: 10.7717/peerj.9981. eCollection 2020.
A novel dual MEK/PDK1 inhibitor named 9za has been synthesized by our research team. Preliminary study showed that 9za possessed potent cytotoxicity and proapoptosis in non-small cell lung cancer (NSCLC) cells. Nevertheless, the precise underlying mechanism is vague.
In this work, we adopted the MTT assay, the Cell Cycle Detection Kit, and the JC-1 staining assay to detect the cell viability, the cell cycle distribution and the mitochondrial membrane potential (MMP), respectively. Cell apoptosis was measured by the morphology observation under a light microscope, Annexin V-FITC/propidium iodide (PI) apoptosis detection and the colorimetric TUNEL assay. Western blot was used to monitor the cell cycle-, apoptosis-related proteins and relevant proteins involved in the signaling pathways.
The MTT assay demonstrated that 9za sharply decreased the viability of NSCLC cells. Cell cycle analysis revealed that low concentrations of 9za arrested the cell cycle at the G/G phase , which was further confirmed by the decreased levels of Cyclin D1, cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6). Additionally, morphological observations, Annexin V-FITC/propidium iodide (PI) apoptosis analysis and TUNEL assays indicated that high concentrations of 9za induced cell apoptosis. Furthermore, the JC-1 staining assay revealed that the mitochondrial membrane potential was downregulated following 9za exposure. Western blot also showed that 9za markedly decreased the expression levels of total Bcl-2, Cytochrome C in the mitochondria and BCL2 associated X (BAX) in the cytoplasm. However, the levels of BAX in the mitochondria, Cytochrome C in the cytoplasm, active caspase-9, active caspase-3 and cleaved-PARP showed the opposite changes. Moreover, the dose-dependent decreased phosphorylation levels of PDK1, protein kinase B (Akt), MEK and extracellular signal regulated kinase 1/2 (ERK1/2) after 9za treatment verified that 9za was indeed a dual MEK/PDK1 inhibitor, as we expected. Compared with a single MEK inhibitor PD0325901 or a single PDK1 inhibitor BX517, the dual MEK/PDK1 inhibitor 9za could strengthen the cytotoxic and proapoptotic effect, indicating that the double blocking of the MEK and PDK1 signaling pathways plays stronger cell growth inhibition and apoptosis induction roles than the single blocking of the MEK or PDK1 signaling pathway in NSCLC cells. Our work elucidated the molecular mechanisms for 9za as a novel drug candidate against NSCLC.
我们的研究团队合成了一种名为9za的新型双重MEK/PDK1抑制剂。初步研究表明，9za在非小细胞肺癌（NSCLC）细胞中具有强大的细胞毒性和促凋亡作用。然而，确切的潜在机制尚不清楚。
在本研究中，我们分别采用MTT法、细胞周期检测试剂盒和JC-1染色法检测细胞活力、细胞周期分布和线粒体膜电位（MMP）。通过光学显微镜下的形态学观察、膜联蛋白V-FITC/碘化丙啶（PI）凋亡检测和比色TUNEL法检测细胞凋亡。采用蛋白质印迹法监测细胞周期、凋亡相关蛋白以及信号通路中相关蛋白的表达。
MTT法表明9za显著降低了NSCLC细胞的活力。细胞周期分析显示，低浓度的9za使细胞周期停滞在G/G期，细胞周期蛋白D1、细胞周期蛋白依赖性激酶4（CDK4）和细胞周期蛋白依赖性激酶6（CDK6）水平的降低进一步证实了这一点。此外，形态学观察、膜联蛋白V-FITC/碘化丙啶（PI）凋亡分析和TUNEL检测表明，高浓度的9za诱导细胞凋亡。此外，JC-1染色法显示，9za处理后线粒体膜电位下调。蛋白质印迹法还显示，9za显著降低了线粒体中总Bcl-2、细胞色素C以及细胞质中BCL2相关X蛋白（BAX）的表达水平。然而，线粒体中BAX、细胞质中细胞色素C、活性半胱天冬酶-9、活性半胱天冬酶-3和裂解的聚（ADP-核糖）聚合酶（PARP）的水平则呈现相反的变化。此外，9za处理后PDK1、蛋白激酶B（Akt）、MEK和细胞外信号调节激酶1/2（ERK1/2）的磷酸化水平呈剂量依赖性降低，证实了9za确实是一种双重MEK/PDK1抑制剂，正如我们所预期的。与单一MEK抑制剂PD0325901或单一PDK1抑制剂BX517相比，双重MEK/PDK1抑制剂9za可增强细胞毒性和促凋亡作用，表明在NSCLC细胞中，双重阻断MEK和PDK1信号通路比单一阻断MEK或PDK1信号通路发挥更强有力的细胞生长抑制和凋亡诱导作用。我们的研究阐明了9za作为一种新型NSCLC候选药物的分子机制。
