Inhibition of radiation induced migration of human head and neck squamous cell carcinoma cells by blocking of EGF receptor pathways

<p>Abstract</p> <p>Background</p> <p>Recently it has been shown that radiation induces migration of glioma cells and facilitates a further spread of tumor cells locally and systemically. The aim of this study was to evaluate whether radiotherapy induces migration in head and neck squamous cell carcinoma (HNSCC). A further aim was to investigate the effects of blocking the epidermal growth factor receptor (EGFR) and its downstream pathways (Raf/MEK/ERK, PI3K/Akt) on tumor cell migration in vitro.</p> <p>Methods</p> <p>Migration of tumor cells was assessed via a wound healing assay and proliferation by a MTT colorimeritric assay using 3 HNSCC cell lines (BHY, CAL-27, HN). The cells were treated with increasing doses of irradiation (2 Gy, 5 Gy, 8 Gy) in the presence or absence of EGF, EGFR-antagonist (AG1478) or inhibitors of the downstream pathways PI3K (LY294002), mTOR (rapamycin) and MEK1 (PD98059). Biochemical activation of EGFR and the downstream markers Akt and ERK were examined by Western blot analysis.</p> <p>Results</p> <p>In absence of stimulation or inhibition, increasing doses of irradiation induced a dose-dependent enhancement of migrating cells (p < 0.05 for the 3 HNSCC cell lines) and a decrease of cell proliferation (p < 0.05 for the 3 HNSCC cell lines). The inhibition of EGFR or the downstream pathways reduced cell migration significantly (almost all p < 0.05 for the 3 HNSCC cell lines). Stimulation of HNSCC cells with EGF caused a significant increase in migration (p < 0.05 for the 3 HNSCC cell lines). After irradiation alone a pronounced activation of EGFR was observed by Western blot analysis.</p> <p>Conclusion</p> <p>Our results demonstrate that the EGFR is involved in radiation induced migration of HNSCC cells. Therefore EGFR or the downstream pathways might be a target for the treatment of HNSCC to improve the efficacy of radiotherapy.</p

Muscle architectures of GAS, PLA, and SOL of R1 left pelvic limb.

<p>Muscle fascicles of GAS medialis and lateralis are shown in light red and yellow, respectively. The proximodistal axis corresponds to the mean force axis of the calf muscles, running from mean muscle origin at the humerus to the insertion at the calcaneus. The corresponding 3D data of the muscle fascicles are provided in the Supporting Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130985#pone.0130985.s002" target="_blank">S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130985#pone.0130985.s004" target="_blank">S4</a> Datasets).</p

Schematic of the rabbit calf muscles.

<p>(A) Medial view of the left pelvic limb and the calf muscles whose dynamic muscle properties and architecture have been determined (GAS, PLA, SOL). The grey dashed line marks the transversal cross-section of the limb shown in (B). For the grey muscles (FDL, EDL, and TA), only dynamic muscle properties were determined (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130985#sec014" target="_blank">Supporting Information</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130985#pone.0130985.s014" target="_blank">S1 Text</a>). White muscles (**<i>peronaei</i> muscles, * <i>M</i>. <i>extensor hallucis longus</i>) were not examined. The axes are shown for orientation.</p

Specifications of observed muscles.

<p>Muscle and animal mass as well as the muscle-tendon complex length <i>L</i>_{<i>MTC_0</i>} measured at ankle and knee joint angles of 90° (cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130985#pone.0130985.g001" target="_blank">Fig 1</a>). <i>n</i>: number of muscles.</p><p>*Architecture of GAS, PLA, and SOL was determined from the left legs of three rabbits.</p><p>Specifications of observed muscles.</p

Force enhancement (FE) and force depression (FD) experiments.

<p>Typical experiments are shown for one GAS (<i>m</i> = 14.8 g), SOL (<i>m</i> = 3.3 g), and PLA (<i>m</i> = 7.5 g), respectively. Exemplary isokinetic ramps are depicted for GAS in the top row; numbers without units indicate velocity in mean fascicle lengths per second. FE (difference between black triangles) and FD (difference between white triangles) are the force difference between ramp experiment (black) and isometric reference contraction (grey) determined 500ms (GAS, PLA) and 1300ms (SOL) after the end of the ramp, shown exemplarily for the slowest (0.35 <i>l</i>_<i>fm</i>/s) ramp.</p

Muscle properties of GAS, PLA, and SOL.

<p>The black curves indicate mean values, whereas the grey areas depict the standard deviations. First row: force–length (<i>f</i>_<i>l</i>) relation. <i>F</i>_<i>im</i> is the maximum isometric muscle force, <i>l</i>_<i>CC</i> and <i>l</i>_<i>CCopt</i> are the length and the optimal length of the contractile component, respectively. To avoid muscle damage, the muscles were lengthened until passive forces reached about 0.2 <i>F</i>_<i>im</i> (marked with a white circle). Second row: force–velocity (<i>f</i>_<i>v</i>) relation. <i>v</i>_<i>CCmax</i> is the maximal shortening velocity of the contractile component. Third row: Force–strain relation of the series elastic component (SEC). <i>Δl</i>_<i>SEC</i> and <i>l</i>_<i>SEC0</i> are the length change and the slack length of the series elastic component, respectively. Last row: Force–strain relation of the parallel elastic component (PEC). <i>Δl</i>_<i>PEC</i> and <i>l</i>_<i>PEC0</i> are the length change and the slack length of the parallel elastic component, respectively.</p

Hill-type muscle model and associated muscle properties.

<p>The muscle model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130985#pone.0130985.ref028" target="_blank">28</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130985#pone.0130985.ref029" target="_blank">29</a>] for which the parameters are determined in this study consists of a contractile component (CC), a serial elastic component (SEC) and a parallel elastic component (PEC). Muscle components and associated muscle properties (force-velocity relation, force-length relation, activation-time relation, force-elongation relation of SEC and PEC) are marked with the same background color. Corresponding model parameters are explained in section 2.3.</p