title: Inhibition of MEK pathway in vestibular schwannoma cell culture.
abstract: OBJECTIVE
  The purpose of this study was to evaluate the Ras GTPase (Ras) to extracellular signal-regulated kinase (ERK) pathway in vestibular schwannoma (VS) cell cultures and patient excised schwannoma tumors. Mitogen-activated protein kinase kinase (MEK) inhibitor CI-1040 (PD184352) was utilized to evaluate the effect of specific MEK inhibition on benign schwannoma cell culture proliferation and apoptosis.
  
  
  METHODS
  Prospective evaluation of human schwannoma cell lines and tumors.
  
  
  METHODS
  Western blotting was completed with phospho-antibodies for proteins in the Ras-ERK pathway. Increasing concentrations of CI-1040 were utilized in schwannoma cell cultures to evaluate cell proliferation and apoptosis. Proliferation was measured with the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide proliferation assay, and apoptosis was monitored with flow cytometry of annexin V/propidium iodide-stained cells.
  
  
  RESULTS
  The most consistent Ras-ERK pathway alterations were found in phospho-MEK and ERK. Phospho-MEK was not overexpressed in the schwannoma cell lines, but six out of 10 VS showed significant increases compared to benign Schwann cell controls. Similarly, nine of 10 VS tumors showed increased phospho-ERK expression. CI-1040 showed significantly reduced schwannoma cell proliferation at the 50 and 100 μM (IC(50)  20 μM and 30 μM) concentrations when compared to carrier only controls in two out three schwannoma cell lines. The remaining schwannoma cell line was relatively refractory to the antiproliferative effects of CI-1040 at doses up to 100 μM (IC(50)  58 μM). Cumulative data of four separate schwannoma cell lines demonstrated that apoptosis was increased in treated schwannoma cells at CI-1040 concentrations of 50 and 100 μM at 72 hours.
  
  
  CONCLUSIONS
  There is overexpression of phosphorylated (activated) proteins in the Ras-ERK pathway in schwannoma cultures and tumors as compared to benign human Schwann cell culture controls. MEK inhibitor, CI-1040, created significantly decreased schwannoma cell proliferation and increased apoptosis in cell culture. These data justify the use of MEK inhibitors in animal treatment studies of VS.
authors.affiliation: Department of Otolaryngology-Head and Neck Surgery, Mayo Clinic School of Medicine, Rochester, Minnesota 55905, USA. neff.brian@mayo.edu
authors.forename: Brian A
authors.initials: BA
authors.lastname: Neff
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Voss
authors.forename: William R
authors.initials: WR
authors.lastname: Schmitt
authors.forename: Colin L W
authors.initials: CL
authors.lastname: Driscoll
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Link
authors.forename: Charles W
authors.initials: CW
authors.lastname: Beatty
authors.forename: Hirohito
authors.initials: H
authors.lastname: Kita
mesh_terms: D017209:Apoptosis / Q000187:drug effects*; D001549:Benzamides / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D049109:Cell Proliferation; D004305:Dose-Response Relationship, Drug; D006801:Humans; D020128:Inhibitory Concentration 50; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D009464:Neuroma, Acoustic / Q000188:drug therapy* / Q000378:metabolism* / Q000473:pathology; D011446:Prospective Studies; D014407:Tumor Cells, Cultured
chemical_list: C120227:2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide; D001549:Benzamides