A novel dual MEK/PDK1 inhibitor 9za retards the cell cycle at G 0 /G 1 phase and induces mitochondrial apoptosis in non-small cell lung cancer cells

PeerJ

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Rangru Liu¹  Zutao Yu⁴  Zhuo Chen⁴  Danqi Liu⁵  Fengying Huang⁶  Qianbin Li⁴  Gaoyun Hu⁴  Xinan Yi⁷  Xi Li²  Honghao Zhou²  Zhaoqian Liu² 

[1] 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;Department of Clinical Pharmacology, Xiangya Hospital, Central South University;Hunan Key Laboratory of Pharmacogenetics, National Clinical Research Center of Geriatric Disorders, Xiangya Hospital, Central South University;Department of Medicinal Chemistry, Xiangya School of Pharmaceutical Sciences, Central South University;Department of Pharmacy, Xiangya Hospital, Central South University;Key Laboratory of Tropical Diseases and Translational Medicine of the Ministry of Education & Hainan Provincial Key Laboratory of Tropical Medicine, Hainan Medical University;The United Laboratory for Neurosciences of Hainan Medical University and the Fourth Military Medical University

关键词: A dual MEK/PDK1 inhibitor;    9za;    Cytotoxicity;    Cell cycle arrest;    Mitochondrial apoptosis;    Non-small cell lung cancer;   

DOI  :  10.7717/peerj.9981

学科分类：社会科学、人文和艺术（综合）

来源: Inra

【 摘 要 】

BackgroundA 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.MethodsIn 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.ResultsThe 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 G0/G1 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.

【 授权许可】

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