Inhibition of cellular proliferation and induction of cell cycle arrest by PEG-8000 in SCC-25 cells- SCC-25 cells were treated with different concentrations of PEG-8000 for 24 h and then assayed for proliferation using standard WST-1 assay.

<p>As shown (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038047#pone-0038047-g004" target="_blank">Figure 4A</a>), there was a dose dependent decrease in the cell growth, with maximal decrease of 43% obtained at 10% PEG-8000. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038047#pone-0038047-g004" target="_blank">Figure 4B</a> shows a ∼50% decrease on the expression of proliferation marker PCNA by PEG. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038047#pone-0038047-g004" target="_blank">Figure 4C</a> shows the effect of PEG on cell cycle distribution. The 24 h PEG treated cells were stained with propidium iodide and analyzed by flow cytometry. PEG blocked cells in the S-phase (by 62%) and correspondingly increases the cells in G2-M phase (by 38%).</p

Effect of topical oral application of PEG-8000 on the initiation and progression of 4NQO-induced oral cancer –Fisher rats were provided 4NQO (20 ppm) in drinking water for 14 weeks before switching to regular water and randomizing into two groups.

<p>The first group received a daily (3–4 minute) topical application of 10% PEG-8000 via oral painting and the second group was sham painted (PEG-control group). This regimen was continued for 14 additional weeks before euthanization. The rats were euthanized after 14 weeks and the oral cavity subjected to macroscopic tumor assessment of total tumors (≥0.2 cm). As shown, 4NQO-treated rats developed multiple large tumors in the oral cavity mostly originating from tongue and few from the wall of the oral cavity. PEG reduced the overall tumor number (tumors/tumor bearing rat) (p = 0.05) and the growth of tumors (tumor volume) compared to their age-matched counterparts (p = 0.02). The tumor volume (size) was measured according to the formula width × length × height × π/6.</p

Effect of PEG-8000 on EGFR, cyclin D1 and p21 expression in SCC-25 cells-For these studies, western blot analysis was performed on lysates obtained from SCC-25 cell treated for 24 h with PEG-8000.

<p>As shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038047#pone-0038047-g005" target="_blank">Figure 5A</a>, consonant with the <i>in vivo</i> experiments PEG caused a significant decrease EGFR (∼45% compared to vehicle control; p<002) and cyclin D1 (∼57% compared to control vehicle; p<0.005) expression. In a similar experimental protocol (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038047#pone-0038047-g005" target="_blank">Figure 5B</a>), PEG-8000 caused ∼54% increase in p21 expression (p<0.001).</p

Effect of topical oral application of PEG-8000 on the premalignant epithelial hyperproliferation – To evaluate histopathological grading of the oral/tongue tissue, the formalin fixed sections were paraffin embedded, sectioned and subjected to H&E staining.

<p>As shown (top Panel; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038047#pone-0038047-g002" target="_blank">Figure 2A</a>), 4NQO-treated rat sections revealed many localized regions of mild to moderate epithelial dysplasia in morphologically normal appearing mucosa that was normalized by PEG [higher magnification (63×; cropped image) insets demonstrate the presence of mitotic figures in the 4 NQO-treated group alone]. To further study the effects of PEG on the mucosal hyper-proliferation, we performed the immunohistochemical analysis of nuclear antigen Ki67, a well-defined marker of proliferation. A total of 1000 epithelial cells were evaluated from 6–7 fields. As shown (bottom Panel; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038047#pone-0038047-g002" target="_blank">Figure 2A–B</a>), 4NQO-treatment increased the proliferation in morphologically normal tongue mucosa as depicted by increased number of Ki67-labelled epithelial cells/per optical field compared to age matched carcinogen free controls. (p<0.01). Topical application of PEG on the other hand dramatically reduced the number of Ki67-labelled epithelial cells/per optical field (p<0.01).</p