Centipeda minima (Ebushicao) extract inhibits PI3K-Akt-mTOR signaling in nasopharyngeal carcinoma CNE-1 cells

BackgroundCentipeda minima (Ebushicao) has been used for the treatment of various diseases, such as nasal allergies, rhinitis and sinusitis, nasopharyngeal carcinoma, cough, and headache. This study aims to investigate the anticancer activities of Centipeda minima ethanol extracts (CME) against nasopharyngeal carcinoma cell CNE-1 and their underlying mechanism.MethodsCNE-1 cells were treated with different concentrations (15–50 μg/mL) of CME for different time intervals (24, 48, and 72 h). Cytotoxicity of CME was determined by MTT assay. Cell morphological changes were observed by fluorescence microscopy after HO 33258 staining. Cell cycle status was evaluated by flow cytometry following propidium iodide staining. Apoptosis was detected by flow cytometry following annexin V-FITC/PI staining. The levels of apoptosis-associated and PI3K-Akt-mTOR signaling related proteins were measured by western blotting analysis.ResultsCME (15–50 μg/mL) significantly inhibited the proliferation of CNE-1 in a dose- and time-dependent manner (P = 0.026 for 15 μg/mL, P < 0.001 for 25, 30, 40, and 50 μg/mL, respectively); the IC50 values (μg/mL) were 41.57 ± 0.17, 30.34 ± 0.06 and 24.98 ± 0.08 for 24, 48 and 72 h treatments, respectively. Significant morphological changes of CNE-1 cells displaying apoptosis were observed after CME treatment. CME showed low cytotoxicity toward normal LO2 cells. CNE-1 cells were arrested in the G2/M phase while treated with 15, 25, 40 μg/mL of CME, respectively (P = 0.032, P = 0.0053, P < 0.001). CME (15, 25, 40 μg/mL) down-regulated Bcl-2 expression (P = 0.032, P = 0.0074, P < 0.001), and up-regulated Bax (P = 0.026, P = 0.0056, P < 0.001) with activation of caspase-3, caspase-8, caspase-9, and PARP observed in CNE-1 cells (P = 0.015, P = 0.0067, P < 0.001 for caspase 3; P = 0.210, 0.028, < 0.001 for caspase 8; P = 0.152, 0.082, 0.0080 for caspase 9; P = 0.265, 0.0072, < 0.001 for PARP). CME suppressed the activation of the PI3K-AKT-mTOR pathway (P = 0.03, 0.0007, 0.004, 0.006, 0.022 for p-PI3K, p-Akt-Ser473, p-Akt-Thr308, p-mTOR-Ser2448, p-mTOR-Ser2481, respectively after 40 μg/mL of CME treated for 24 h).ConclusionCME inhibited the proliferation of CNE-1 cells and activation of the PI3K-AKT-mTOR signaling pathway.

[1]  M. Peter,et al.  Two CD95 (APO‐1/Fas) signaling pathways , 1998, The EMBO journal.

[2]  Ji Luo,et al.  The evolution of phosphatidylinositol 3-kinases as regulators of growth and metabolism , 2006, Nature Reviews Genetics.

[3]  Huanhai Liu,et al.  Reactive oxygen species-mediated mitochondrial dysfunction is involved in apoptosis in human nasopharyngeal carcinoma CNE cells induced by Selaginella doederleinii extract. , 2011, Journal of ethnopharmacology.

[4]  P. Xia,et al.  Intensity-modulated radiotherapy in the treatment of nasopharyngeal carcinoma: an update of the UCSF experience. , 2001, International journal of radiation oncology, biology, physics.

[5]  N. Liang,et al.  Two new sesquiterpene lactones from the supercritical fluid extract of Centipeda minima , 2012, Journal of Asian natural products research.

[6]  Jian-Min Yuan,et al.  Epidemiology of nasopharyngeal carcinoma. , 2002, Seminars in cancer biology.

[7]  W. Ye,et al.  2β-(Isobutyryloxy)florilenalin, a Sesquiterpene Lactone Isolated from the Medicinal Plant Centipeda minima, Induces Apoptosis in Human Nasopharyngeal Carcinoma CNE Cells , 2009, Molecules.

[8]  W. Fang,et al.  Lentivirus-mediated RNAi silencing targeting ABCC2 increasing the sensitivity of a human nasopharyngeal carcinoma cell line against cisplatin , 2008, Journal of Translational Medicine.

[9]  N. Raab-Traub.  Epstein-Barr virus in the pathogenesis of NPC. , 2002, Seminars in cancer biology.

[10]  D. Jin,et al.  The pathological roles of BART miRNAs in nasopharyngeal carcinoma , 2012, The Journal of pathology.

[11]  W. Cho,et al.  Clinical Efficacy of Traditional Chinese Medicine as a Concomitant Therapy for Nasopharyngeal Carcinoma: A Systematic Review and Meta-Analysis , 2009, Cancer investigation.

[12]  Jianpin Liu,et al.  Correction: Traditional Chinese Medicine in Cancer Care: A Review of Controlled Clinical Studies Published in Chinese , 2013, PLoS ONE.

[13]  N. Hay,et al.  The Akt-mTOR tango and its relevance to cancer. , 2005, Cancer cell.

[14]  Yi-Te Hsu,et al.  Movement of Bax from the Cytosol to Mitochondria during Apoptosis , 1997, The Journal of cell biology.

[15]  L. Hartwell,et al.  Cell cycle arrest of cdc mutants and specificity of the RAD9 checkpoint. , 1993, Genetics.

[16]  H. Chung,et al.  Supercritical fluid extraction assisted isolation of sesquiterpene lactones with antiproliferative effects from Centipeda minima. , 2012, Phytochemistry.

[17]  Q. Tao,et al.  Epstein-Barr virus (EBV) and its associated human cancers--genetics, epigenetics, pathobiology and novel therapeutics. , 2006, Frontiers in bioscience : a journal and virtual library.

[18]  G. He,et al.  14-Thienyl Methylene Matrine (YYJ18), the Derivative from Matrine, Induces Apoptosis of Human Nasopharyngeal Carcinoma Cells by Targeting MAPK and PI3K/Akt Pathways in Vitro , 2014, Cellular Physiology and Biochemistry.

[19]  S. Korsmeyer,et al.  Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c , 2000, Cell Death and Differentiation.

[20]  H. Chung,et al.  Deoxyelephantopin from Elephantopus scaber L. induces cell-cycle arrest and apoptosis in the human nasopharyngeal cancer CNE cells. , 2011, Biochemical and biophysical research communications.

[21]  H. Chung,et al.  Antiproliferative Effects of Volatile Oils from Centipeda minima on Human Nasopharyngeal Cancer CNE Cells , 2010, Natural product communications.

[22]  T. Mok,et al.  Concurrent chemotherapy-radiotherapy compared with radiotherapy alone in locoregionally advanced nasopharyngeal carcinoma: progression-free survival analysis of a phase III randomized trial. , 2002, Journal of clinical oncology : official journal of the American Society of Clinical Oncology.

[23]  Paul S Mischel,et al.  Analysis of the phosphatidylinositol 3'-kinase signaling pathway in glioblastoma patients in vivo. , 2003, Cancer research.

[24]  X. Liu,et al.  An APAF-1·Cytochrome c Multimeric Complex Is a Functional Apoptosome That Activates Procaspase-9* , 1999, The Journal of Biological Chemistry.

[25]  S. H. Cheng,et al.  Long-term survival of nasopharyngeal carcinoma following concomitant radiotherapy and chemotherapy. , 2000, International journal of radiation oncology, biology, physics.

[26]  A. Pandiella,et al.  Activation of the PI3K/mTOR/AKT Pathway and Survival in Solid Tumors: Systematic Review and Meta-Analysis , 2014, PloS one.

[27]  Jacqueline Cloos,et al.  Cell sensitivity assays: the MTT assay. , 2011, Methods in molecular biology.

[28]  J. B. Hutchins,et al.  Bcl‐2 Protects Isolated Plasma and Mitochondrial Membranes Against Lipid Peroxidation Induced by Hydrogen Peroxide and Amyloid β‐Peptide , 1998, Journal of neurochemistry.