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Nicotine enhances proliferation, migration, and radioresistance of human malignant glioma cells through EGFR activation

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Abstract

It has been suggested that continued tobacco use during radiation therapy contributes to maintenance of neoplastic growth despite treatment with radiation. Nicotine is a cigarette component that is an established risk factor for many diseases, neoplastic and otherwise. The hypothesis of this work is that nicotine promotes the proliferation, migration, and radioresistance of human malignant glioma cells. The effect of nicotine on cellular proliferation, migration, signaling, and radiation sensitivity were evaluated for malignant glioma U87 and GBM12 cells by use of the AlamarBlue, scratch healing, and clonogenic survival assays. Signal transduction was assessed by immunoblotting for activated EGFR, ERK, and AKT. At concentrations comparable with those found in chronic smokers, nicotine induced malignant glioma cell migration, growth, colony formation, and radioresistance. Nicotine increased phosphorylation of EGFRtyr992, AKTser473, and ERK. These molecular effects were reduced by pharmacological inhibitors of EGFR, PI3K, and MEK. It was therefore concluded that nicotine stimulates the malignant behavior of glioma cells in vitro by activation of the EGFR and downstream AKT and ERK pathways.

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Authors and Affiliations

  1. Department of Otolaryngology, Head and Neck Surgery, University of Virginia Health System, Jordan Building, Rm. 2221, Charlottesville, VA, 22908, USA

    Ashraf A. Khalil & Mark J. Jameson

  2. Department of Neurosurgery, Virginia Commonwealth University, H. F. Young Neurosurgical Cent, BOX 980631, Richmond, VA, 23298, USA

    William C. Broaddus

  3. Department of Radiation Oncology, Massey Cancer Center, BOX 980058, Richmond, VA, 23298, USA

    Peck Sun Lin

  4. Department of Radiation Oncology, Georgia Health Sciences University, 821 St Sebastian Way, Augusta, GA, 30912, USA

    Theodore D. Chung

Authors
  1. Ashraf A. Khalil
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  2. Mark J. Jameson
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  3. William C. Broaddus
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  4. Peck Sun Lin
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  5. Theodore D. Chung
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Correspondence to Theodore D. Chung.

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10014_2012_101_MOESM1_ESM.eps

Supplementary Fig. 1 AEE788 inhibits phosphorylation of EGFR tyr922 . U87 and GBM12 cells in 6 cm dishes were treated with 0.5, 1, 2.5, or 5 µM AEE788 for 1 h before treatment with 20 ng rh-EGF for 15 min. Control dishes of untreated cells were also included. Cells were collected for western blot. The intensities of the bands were quantified by use of Licor software, normalized to the beta actin band intensities, then expressed relative to the untreated control band intensity. Identical results were obtained in two independent experiments. AEE788 causes dose-dependent inhibition of EGFRtyr922 with the maximum effect at 5 µM. We used 5 µM AEE788 to perform subsequent experiments to ensure EGFR inhibition (EPS 888 kb)

10014_2012_101_MOESM2_ESM.eps

Supplementary Fig. 2 (A) Nicotine enhances cell migration and AEE788 inhibits it. GBM12 cells were subjected to monolayer wound assay. Cells were treated with either nicotine 0.5 µM or with nicotine 0.5 µM and AEE788 5 µM. A control group of untreated cells was also included. All treatments were in media containing 0.5% serum and were in triplicate. After 18 h, cells were fixed and cell migration across the wound was assessed. (B) Nicotine dose response stimulation of cell migration. In similar experiments, GBM12 cells were treated with 0.01, 0.1, 0.5, or 10 µM nicotine either alone (lanes 4–7) or combined with 5 µM AEE788 (lanes 8–11). A control group of cells that did not receive any treatment (lane 1 at 0 h and lane 2 at 18 h) or were treated with AEE788 alone (lane 3) were also included. The density of the cells which migrated across the wounded area was determined by use of Bio-Rad software and normalized to the control untreated cells. (C) The effects of nicotine on cell migration compared with those of rh-EGF. GBM12 cells were treated with 20 ng/ml EGF alone (lane 2), 0.5 µM Nicotine (lane 3), both (lane 4), 5 µM AEE788 alone (lane 5) or with nicotine (lane 6), or with EGF (lane 7). A control group of cells that did not receive any treatment (lane 1) were also included. Data points are relative intensity units (RIU). Error bars indicate SEM, n = 3. Multiplication symbols (×) denote changes in RIU levels as a multiple (“fold”) of control results (no treatment). *p < 0.05. (D) U0126 and LY294002 inhibit nicotine-induced cell growth. GBM12 cells were subjected to the monolayer wound-migration assay. Cells were treated with 0.5 µM nicotine (lane 2); 0.5 µM LY294002 either alone (lane 3) or with 0.5 µM nicotine (lane 5); 5 µM U0126 either alone (lane 4) or combined with 0.5 µM nicotine (lane 6). A control group of cells that did not receive any treatment (lane 1) were also included. All treatments were in 0.5 % serum-containing medium. The density of the cells migrating across the wound was determined by use of imaging software and normalized to untreated control cells. (EPS 1522 kb)

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Khalil, A.A., Jameson, M.J., Broaddus, W.C. et al. Nicotine enhances proliferation, migration, and radioresistance of human malignant glioma cells through EGFR activation. Brain Tumor Pathol 30, 73–83 (2013). https://doi.org/10.1007/s10014-012-0101-5

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  • DOI: https://doi.org/10.1007/s10014-012-0101-5

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