title: IRE1α-TRAF2-ASK1 pathway is involved in CSTMP-induced apoptosis and ER stress in human non-small cell lung cancer A549 cells.
abstract: BACKGROUND
  CSTMP, a Tetramethylpyrazine (TMP) analogue, is designed and synthesized based on the pharmacophores of TMP and resveratrol. Recent studies showed that CSTMP had strong protective effects in endothelial cells apoptosis by its anti-oxidant activity. However, the pharmacological function of CSTMP in cancer have not been elucidated to date. The objective of this study was to investigate the anti-cancer effect of CSTMP against human non-small cell lung cancer (NSCLC) A549 cells and the underlying mechanisms.
  
  
  METHODS
  The cell proliferation and apoptosis were detected by MTT assay and flow cytometry. Caspases activity was determined spectrophotometricaly at 405nm using a microtiter plate reader. Western blot and real-time PCR was used to assess the protein and mRNA expression. Immunoprecipitation was used to examine the protein-protein interactions.
  
  
  RESULTS
  CSTMP inhibited the proliferation and induced cell cycle arrest and apoptosis of A549 cells. Caspase3, 8, 9 and PARP-1 activation, and Bax/Bcl-2 ratio analyses demonstrated that the anti-cancer effect of CSTMP in A549 cells was mediated by promoting caspase- and mitochondria-dependent apoptosis. Furthermore, CSTMP induced ER stress in A549 cells as evidenced by elevated levels of GRP78, GRP94, CHOP, IRE1α, TRAF2, p-ASK1 and p-JNK, activation of caspase12 and 4, and enhanced formation of an IRE1α-TRAF2-ASK1 complex. Knockdown of IRE1α by siRNA suppressed activation of IRE1α, TRAF2, p-ASK1 and p-JNK in CSTMP treated A549 cells. In addition, the effects of CSTMP on the formation of an IRE1α-TRAF2-ASK1 complex, caspase- and mitochondria-dependent apoptosis were also reversed by IRE1α siRNA in A549 cells.
  
  
  CONCLUSIONS
  Collectively, we showed that CSTMP induced apoptosis of A549 cells were through IRE1α-TRAF2-ASK1 complex-mediated ER stress, JNK activation, and mitochondrial dysfunction. These insights on this novel compound CSTMP may provide a novel anti-cancer candidate for the treatment of NSCLC.
authors.affiliation: National Clinical Research Center for Respiratory Disease, State Key Laboratory of Respiratory Disease, Department of Medicine, Guangzhou Institute of Respiratory Disease, Guangzhou 510120, China.
authors.forename: Jiexia
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Medical Oncology, Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Guangzhou, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Liang
authors.affiliation: Department of Thoracic Surgery, Sun Yat-sen University Cancer Center; State Key Laboratory of Oncology in South China, Guangzhou, China; Collaborative Innovation Center for Cancer Medicine, Guangzhou, China.
authors.forename: Yongbin
authors.initials: Y
authors.lastname: Lin
authors.affiliation: National Clinical Research Center for Respiratory Disease, State Key Laboratory of Respiratory Disease, Department of Medicine, Guangzhou Institute of Respiratory Disease, Guangzhou 510120, China.
authors.forename: Yuanbin
authors.initials: Y
authors.lastname: Liu
authors.affiliation: National Clinical Research Center for Respiratory Disease, State Key Laboratory of Respiratory Disease, Department of Medicine, Guangzhou Institute of Respiratory Disease, Guangzhou 510120, China.
authors.lastname: YouYou
authors.affiliation: National Clinical Research Center for Respiratory Disease, State Key Laboratory of Respiratory Disease, Department of Medicine, Guangzhou Institute of Respiratory Disease, Guangzhou 510120, China. Electronic address: yinweiqianggz@163.com.
authors.forename: Weiqiang
authors.initials: W
authors.lastname: Yin
mesh_terms: D000072283:A549 Cells; D017209:Apoptosis / Q000187:drug effects*; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism / Q000473:pathology*; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D004722:Endoribonucleases / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology*; D048848:MAP Kinase Kinase Kinase 5 / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011719:Pyrazines / Q000737:chemistry / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D047992:TNF Receptor-Associated Factor 2 / Q000378:metabolism*
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; D011719:Pyrazines; D034741:RNA, Small Interfering; D047992:TNF Receptor-Associated Factor 2; C528229:ERN1 protein, human; D017346:Protein Serine-Threonine Kinases; D048848:MAP Kinase Kinase Kinase 5; C482858:MAP3K5 protein, human; D004722:Endoribonucleases; D020169:Caspases; C017953:tetramethylpyrazine
keywords: Apoptosis; CSTMP; ER stress; IRE1α; JNK; Non-small cell lung cancer