Inhibition of MEK pathway in vestibular schwannoma cell culture

Brian A. Neff, Stephen G. Voss, William R. Schmitt, Colin L W Driscoll, Michael J. Link, Charles W. Beatty, Hirohito Kita

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

Objectives/Hypothesis: 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. Study Design: 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 (IC50 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 (IC50 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.

Original languageEnglish (US)
Pages (from-to)2269-2278
Number of pages10
JournalLaryngoscope
Volume122
Issue number10
DOIs
StatePublished - Oct 2012

Fingerprint

Acoustic Neuroma
Mitogen-Activated Protein Kinase Kinases
Neurilemmoma
Cell Culture Techniques
Extracellular Signal-Regulated MAP Kinases
ras Proteins
Apoptosis
Cell Proliferation
Cell Line
Schwann Cells
Inhibitory Concentration 50
Neoplasms
Propidium
Annexin A5
2-(2-chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide
Tumor Cell Line
Flow Cytometry
Western Blotting
Prospective Studies

Keywords

  • acoustic neuroma
  • CI-1040
  • MEK inhibitor
  • neurofibromatosis type 2
  • PD-184352
  • Vestibular schwannoma

ASJC Scopus subject areas

  • Otorhinolaryngology

Cite this

Neff, B. A., Voss, S. G., Schmitt, W. R., Driscoll, C. L. W., Link, M. J., Beatty, C. W., & Kita, H. (2012). Inhibition of MEK pathway in vestibular schwannoma cell culture. Laryngoscope, 122(10), 2269-2278. https://doi.org/10.1002/lary.23472

Inhibition of MEK pathway in vestibular schwannoma cell culture. / Neff, Brian A.; Voss, Stephen G.; Schmitt, William R.; Driscoll, Colin L W; Link, Michael J.; Beatty, Charles W.; Kita, Hirohito.

In: Laryngoscope, Vol. 122, No. 10, 10.2012, p. 2269-2278.

Research output: Contribution to journalArticle

Neff, BA, Voss, SG, Schmitt, WR, Driscoll, CLW, Link, MJ, Beatty, CW & Kita, H 2012, 'Inhibition of MEK pathway in vestibular schwannoma cell culture', Laryngoscope, vol. 122, no. 10, pp. 2269-2278. https://doi.org/10.1002/lary.23472
Neff BA, Voss SG, Schmitt WR, Driscoll CLW, Link MJ, Beatty CW et al. Inhibition of MEK pathway in vestibular schwannoma cell culture. Laryngoscope. 2012 Oct;122(10):2269-2278. https://doi.org/10.1002/lary.23472
Neff, Brian A. ; Voss, Stephen G. ; Schmitt, William R. ; Driscoll, Colin L W ; Link, Michael J. ; Beatty, Charles W. ; Kita, Hirohito. / Inhibition of MEK pathway in vestibular schwannoma cell culture. In: Laryngoscope. 2012 ; Vol. 122, No. 10. pp. 2269-2278.
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abstract = "Objectives/Hypothesis: 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. Study Design: 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 (IC50 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 (IC50 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.",
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AU - Voss, Stephen G.

AU - Schmitt, William R.

AU - Driscoll, Colin L W

AU - Link, Michael J.

AU - Beatty, Charles W.

AU - Kita, Hirohito

PY - 2012/10

Y1 - 2012/10

N2 - Objectives/Hypothesis: 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. Study Design: 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 (IC50 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 (IC50 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.

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KW - acoustic neuroma

KW - CI-1040

KW - MEK inhibitor

KW - neurofibromatosis type 2

KW - PD-184352

KW - Vestibular schwannoma

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