title: Role of ERK-MAPK signaling pathway in pentagastrin-regulated growth of large intestinal carcinoma.
abstract: OBJECTIVE
  To explore the role and mechanisms of extracellular signal-regulated protein kinase-mitogen-activated protein kinase (ERK-MAPK) signaling in pentagastrin-regulated growth of large intestinal carcinoma.
  
  
  METHODS
  HT-29 cells were incubated in different media and divided into the control group, pentagastrin group, proglumide group, and pentagastrin + proglumide group. No reagent was added to the control group, and other groups were incubated with reagent at different concentrations. Changes in proliferation of HT-29 cells were detected by MTT assay, and the optimal concentrations of pentagastrin and proglumide were determined. The changes in proliferation index (PI) and apoptosis rate (AR) of HT-29 cells were detected by Annexin V-fluorescein isothiocyanate flow cytometry. mRNA expression of pentagastrin receptor/cholecystokinin-B receptor (CCK-BR), ERK1/2 and K-ras were detected by reverse transcriptase polymerase chain reaction. The protein and phosphorylation level of ERK1/2 and K-ras were detected by western blotting. All data were analyzed by analysis of variance and SNK-q test.
  
  
  RESULTS
  The proliferation of HT-29 cells was stimulated by pentagastrin at a concentration of 6.25-100 mg/L, and the optimal concentration of pentagastrin was 25.0 mg/L (F = 31.36, P < 0.05). Proglumide had no obvious effect on the proliferation of HT-29 cells, while it significantly inhibited the proliferation of HT-29 cells stimulated by pentagastrin when the concentration of proglumide was 8.0-128.0 mg/L, and the optimal concentration was 32.0 mg/L (F = 24.31, P < 0.05). The PI of the pentagastrin (25.0 mg/L) group was 37.5% ± 5.2%, which was significantly higher than 27.7% ± 5.0% of the control group and 27.3% ± 5.8% of the pentagastrin (25.0 mg/L) + proglumide (32.0 mg/L) group (Q = 4.56-4.75, P < 0.05). The AR of the pentagastrin (25.0 mg/L) group was 1.9% ± 0.4%, which was significantly lower than 2.5% ± 0.4% of the control group and 2.4% ± 0.3% of the pentagastrin (25.0 mg/L) + proglumide (32.0 mg/L) group (Q = 4.23-4.06, P < 0.05). mRNA expression of CCK-BR was detected in HT-29 cells. The phosphorylation levels of ERK1/2 protein and phosphorylated K-ras protein of the pentagastrin group were 0.43% ± 0.04% and 0.45% ± 0.06%, which were significantly higher than 0.32% ± 0.02% and 0.31% ± 0.05% of the control group (Q = 7.78-4.95, P < 0.05), and 0.36% ± 0.01% and 0.35% ± 0.04% of the pentagastrin + proglumide group (Q = 5.72-4.08, P < 0.05). There were no significant differences in the mRNA and protein expression of ERK1/2 and K-ras among the control, pentagastrin, proglumide and pentagastrin + proglumide groups (F = 0.52, 0.72, 0.78, 0.28; P > 0.05).
  
  
  CONCLUSIONS
  Gastrin stimulates proliferation of HT-29 cells and inhibits apoptosis by upregulating phosphorylation of ERK and K-ras through the Ras-Raf-MEK1/2-ERK1/2 pathway, and this is restrained by proglumide.
authors.affiliation: Jia-Ding Mao, Pei Wu, Jian-Xiong Huang, Jian Wu, Guang Yang, Department of General Surgery, the First Affiliated Yijishan Hospital of Wannan Medical College, Wuhu 241001, Anhui Province, China.
authors.forename: Jia-Ding
authors.initials: JD
authors.lastname: Mao
authors.affiliation: Jia-Ding Mao, Pei Wu, Jian-Xiong Huang, Jian Wu, Guang Yang, Department of General Surgery, the First Affiliated Yijishan Hospital of Wannan Medical College, Wuhu 241001, Anhui Province, China.
authors.forename: Pei
authors.initials: P
authors.lastname: Wu
authors.affiliation: Jia-Ding Mao, Pei Wu, Jian-Xiong Huang, Jian Wu, Guang Yang, Department of General Surgery, the First Affiliated Yijishan Hospital of Wannan Medical College, Wuhu 241001, Anhui Province, China.
authors.forename: Jian-Xiong
authors.initials: JX
authors.lastname: Huang
authors.affiliation: Jia-Ding Mao, Pei Wu, Jian-Xiong Huang, Jian Wu, Guang Yang, Department of General Surgery, the First Affiliated Yijishan Hospital of Wannan Medical College, Wuhu 241001, Anhui Province, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Wu
authors.affiliation: Jia-Ding Mao, Pei Wu, Jian-Xiong Huang, Jian Wu, Guang Yang, Department of General Surgery, the First Affiliated Yijishan Hospital of Wannan Medical College, Wuhu 241001, Anhui Province, China.
authors.forename: Guang
authors.initials: G
authors.lastname: Yang
mesh_terms: D000230:Adenocarcinoma / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D017209:Apoptosis / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects*; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D019073:HT29 Cells; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D019950:Mitogen-Activated Protein Kinase 1 / Q000235:genetics / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000235:genetics / Q000378:metabolism*; D010418:Pentagastrin / Q000494:pharmacology*; D010766:Phosphorylation; D011377:Proglumide / Q000494:pharmacology; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras); D012333:RNA, Messenger / Q000378:metabolism; D043887:Receptor, Cholecystokinin B / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D043887:Receptor, Cholecystokinin B; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins; D010418:Pentagastrin; D011377:Proglumide
keywords: Extracellular signal-regulated protein kinase 1/2; Gastrin; K-ras; Large intestinal carcinoma; Mitogen-activated protein kinase