title: β-elemene enhances the radiosensitivity of gastric cancer cells by inhibiting Pak1 activation.
abstract: OBJECTIVE
  To explore the potential of β-elemene as a radiosensitizer for gastric cancer cells and the underlying mechanisms.
  
  
  METHODS
  SGC7901, MKN45, MKN28, N87, and AGS human gastric cancer cell lines were used to screen for radioresistant gastric cancer cell lines. A 3-(4,5-dimeth-ylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay was used to determine the effects of β-elemene and IPA-3 on cell viability in MKN45 and SGC7901 gastric cancer cell lines. A clonogenic survival assay and annexin V-FITC/PI apoptosis detection assay were used to evaluate cellular radiosensitivity and radiation-induced cell death, respectively. A proteomic method, isobaric tags for relative and absolute quantitation (iTRAQ), was employed to screen the proteins regulated by β-elemene pretreatment prior to ionizing radiation (IR) in SGC7901 gastric cancer cell line. IPA-3 was used as a specific small molecule inhibitor of p21-activated protein kinase 1 (Pak1) to target Pak1 signaling. Protein levels of PAK1IP1 (p21-activated protein kinase-interacting protein 1), total Pak1 (t-Pak1), phospho-Pak1 (T423), phospho-ERK1/2 (Thr202/Tyr204), and cleaved caspase-3 (17 kDa) were assessed by western blotting.
  
  
  RESULTS
  MKN45 and SGC7901 gastric cancer cell lines were relatively more resistant to IR. β-elemene pretreatment decreased clonogenic survival following IR in MKN45 and SGC7901 gastric cancer cell lines. Additionally, β-elemene pretreatment prior to IR increased radiation-induced cell death compared with IR alone in MKN45 (10.4% ± 0.9% vs 34.8% ± 2.8%, P < 0.05) and SGC7901 (11.6% ± 0.9% vs 46.7% ± 5.2%, P < 0.05) human gastric cancer cell lines, respectively, consistent with the level of cleaved caspase-3 (17 kDa). Through iTRAQ analysis and western blot validation, we found that β-elemene upregulated PAK1IP1 and downregulated phospho-Pak1 (T423) and phospho-ERK1/2 in SGC7901 gastric cancer cells. IR increased the level of phospho-Pak1 (T423). Pretreatment with β-elemene decreased radiation-induced Pak1 and ERK1/2 phosphorylation. Inhibition of Pak1 using IPA-3 decreased clonogenic survival following IR. In addition, IPA-3 increased radiation-induced cell death in MKN45 (13.4% ± 0.3% vs 26.6% ± 1.0%, P < 0.05) and SGC7901 (16.0% ± 0.6% vs 37.3% ± 1.7%, P < 0.05) gastric cancer cell lines, respectively, consistent with the level of cleaved caspase-3 (17 kDa). Western blotting showed that IPA-3 decreased radiation-induced Pak1 and ERK1/2 phosphorylation.
  
  
  CONCLUSIONS
  This is the first demonstration that β-elemene enhances radiosensitivity of gastric cancer cells, and that the mechanism involves inhibition of Pak1 signaling.
authors.affiliation: Jun-Song Liu, Xiang-Ming Che, Shuai Chang, Guang-Lin Qiu, Shi-Cai He, Lin Fan, Wei Zhao, Zheng-Liang Zhang, Shu-Feng Wang, Department of General Surgery, First Affiliated Hospital of Xi'an Jiaotong University School of Medicine, Xi'an 710061, Shaanxi Province, China.
authors.forename: Jun-Song
authors.initials: JS
authors.lastname: Liu
authors.affiliation: Jun-Song Liu, Xiang-Ming Che, Shuai Chang, Guang-Lin Qiu, Shi-Cai He, Lin Fan, Wei Zhao, Zheng-Liang Zhang, Shu-Feng Wang, Department of General Surgery, First Affiliated Hospital of Xi'an Jiaotong University School of Medicine, Xi'an 710061, Shaanxi Province, China.
authors.forename: Xiang-Ming
authors.initials: XM
authors.lastname: Che
authors.affiliation: Jun-Song Liu, Xiang-Ming Che, Shuai Chang, Guang-Lin Qiu, Shi-Cai He, Lin Fan, Wei Zhao, Zheng-Liang Zhang, Shu-Feng Wang, Department of General Surgery, First Affiliated Hospital of Xi'an Jiaotong University School of Medicine, Xi'an 710061, Shaanxi Province, China.
authors.forename: Shuai
authors.initials: S
authors.lastname: Chang
authors.affiliation: Jun-Song Liu, Xiang-Ming Che, Shuai Chang, Guang-Lin Qiu, Shi-Cai He, Lin Fan, Wei Zhao, Zheng-Liang Zhang, Shu-Feng Wang, Department of General Surgery, First Affiliated Hospital of Xi'an Jiaotong University School of Medicine, Xi'an 710061, Shaanxi Province, China.
authors.forename: Guang-Lin
authors.initials: GL
authors.lastname: Qiu
authors.affiliation: Jun-Song Liu, Xiang-Ming Che, Shuai Chang, Guang-Lin Qiu, Shi-Cai He, Lin Fan, Wei Zhao, Zheng-Liang Zhang, Shu-Feng Wang, Department of General Surgery, First Affiliated Hospital of Xi'an Jiaotong University School of Medicine, Xi'an 710061, Shaanxi Province, China.
authors.forename: Shi-Cai
authors.initials: SC
authors.lastname: He
authors.affiliation: Jun-Song Liu, Xiang-Ming Che, Shuai Chang, Guang-Lin Qiu, Shi-Cai He, Lin Fan, Wei Zhao, Zheng-Liang Zhang, Shu-Feng Wang, Department of General Surgery, First Affiliated Hospital of Xi'an Jiaotong University School of Medicine, Xi'an 710061, Shaanxi Province, China.
authors.forename: Lin
authors.initials: L
authors.lastname: Fan
authors.affiliation: Jun-Song Liu, Xiang-Ming Che, Shuai Chang, Guang-Lin Qiu, Shi-Cai He, Lin Fan, Wei Zhao, Zheng-Liang Zhang, Shu-Feng Wang, Department of General Surgery, First Affiliated Hospital of Xi'an Jiaotong University School of Medicine, Xi'an 710061, Shaanxi Province, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.affiliation: Jun-Song Liu, Xiang-Ming Che, Shuai Chang, Guang-Lin Qiu, Shi-Cai He, Lin Fan, Wei Zhao, Zheng-Liang Zhang, Shu-Feng Wang, Department of General Surgery, First Affiliated Hospital of Xi'an Jiaotong University School of Medicine, Xi'an 710061, Shaanxi Province, China.
authors.forename: Zheng-Liang
authors.initials: ZL
authors.lastname: Zhang
authors.affiliation: Jun-Song Liu, Xiang-Ming Che, Shuai Chang, Guang-Lin Qiu, Shi-Cai He, Lin Fan, Wei Zhao, Zheng-Liang Zhang, Shu-Feng Wang, Department of General Surgery, First Affiliated Hospital of Xi'an Jiaotong University School of Medicine, Xi'an 710061, Shaanxi Province, China.
authors.forename: Shu-Feng
authors.initials: SF
authors.lastname: Wang
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004305:Dose-Response Relationship, Drug; D004307:Dose-Response Relationship, Radiation; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D040901:Proteomics / Q000379:methods; D011836:Radiation Tolerance / Q000187:drug effects*; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology*; D012717:Sesquiterpenes / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D013274:Stomach Neoplasms / Q000201:enzymology / Q000473:pathology / Q000532:radiotherapy*; D013997:Time Factors; D054462:p21-Activated Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C430457:PAK1IP1 protein, human; D047428:Protein Kinase Inhibitors; D011838:Radiation-Sensitizing Agents; D012717:Sesquiterpenes; C445979:beta-elemene; C515903:PAK1 protein, human; D054462:p21-Activated Kinases; D048049:Extracellular Signal-Regulated MAP Kinases
keywords: Apoptosis; Clonogenic survival; Gastric cancer cells; Radiosensitivity; p21-activated protein kinase 1; β-elemene