Dietary Administration of Scallion Extract Effectively Inhibits Colorectal Tumor Growth: Cellular and Molecular Mechanisms in Mice

<div><p>Colorectal cancer is a common malignancy and a leading cause of cancer death worldwide. Diet is known to play an important role in the etiology of colon cancer and dietary chemoprevention is receiving increasing attention for prevention and/or alternative treatment of colon cancers. <em>Allium fistulosum</em> L., commonly known as scallion, is popularly used as a spice or vegetable worldwide, and as a traditional medicine in Asian cultures for treating a variety of diseases. In this study we evaluated the possible beneficial effects of dietary scallion on chemoprevention of colon cancer using a mouse model of colon carcinoma (CT-26 cells subcutaneously inoculated into BALB/c mice). Tumor lysates were subjected to western blotting for analysis of key inflammatory markers, ELISA for analysis of cytokines, and immunohistochemistry for analysis of inflammatory markers. Metabolite profiles of scallion extracts were analyzed by LC-MS/MS. Scallion extracts, particularly hot-water extract, orally fed to mice at 50 mg (dry weight)/kg body weight resulted in significant suppression of tumor growth and enhanced the survival rate of test mice. At the molecular level, scallion extracts inhibited the key inflammatory markers COX-2 and iNOS, and suppressed the expression of various cellular markers known to be involved in tumor apoptosis (apoptosis index), proliferation (cyclin D1 and c-Myc), angiogenesis (VEGF and HIF-1α), and tumor invasion (MMP-9 and ICAM-1) when compared with vehicle control-treated mice. Our findings may warrant further investigation of the use of common scallion as a chemopreventive dietary agent to lower the risk of colon cancer.</p> </div

Pro-apoptotic and anti-proliferative effects of scallion extracts on growth of CT-26 tumor cells in vivo.

<p>At the end of the experiment a portion of each test tumor from the control and scallion extract-treated groups was collected and subjected to TUNEL assay and immunohistochemical analysis of cyclin D1 expression. Representative images of TUNEL staining (A), and cyclin D1 (C) immunoreactivity are shown (400× magnification). Quantification of apoptotic index and proliferation are shown as number of TUNEL-positive cells (B) and positive scores of cyclin D1-expressing cells (D), respectively. A portion of two selected tumors from each test group were analyzed for cyclin D1 and c-Myc protein expression by western blotting. Densitometric data are shown as fold-change versus the control under each band (E).</p

Effect of scallion extracts on MMP9 and ICAM-1 expression in CT-26 tumor-bearing mice.

<p>Immunohistochemical staining and western blot analyses for MMP9 and ICAM-1 are performed as described above in Fig. 4. (A) Representative images of immunohistochemical staining of MMP9 expression (400× magnification). (B) Quantification of MMP9-expressing cells shown as number of positive cells. (C) A portion of two selected tumors from each group were analyzed for MMP9 and ICAM-1 protein expression using western blotting.</p

Effect of scallion extracts on expression of VEGF and HIF-1α in CT-26 tumor-bearing mice.

<p>At the end of the experiment portions of each tumor from the control and scallion extract-treated groups were collected and subjected to immunohistochemical, western blot and marke<b>r</b> protein analyses. (A) Representative images of immunohistochemical staining of VEGF (400× magnification). (B) Quantification of VEGF-expressing cells shown as number of positive cells. (C) A portion of two selected tumors from each group were analyzed for VEGF and HIF-1α protein expression using western blotting. (D) Overexpression of transgenic VEGF effectively rescued the cell viability of test CT-26 cells when treated with SCE extracts.</p

Effect of scallion extracts on expression of iNOS, COX2 and cytokines in test mice.

<p>At the end of the experiment portions of each tumor from the control and scallion extract-treated groups were collected and subjected to immunohistochemical and western blot analyses of test marker proteins. (A) Representative images (400× magnification) of immunohistochemical staining of COX2 expression. (B) Quantification of COX2-expressing cells as positive scores. (C) Western blot analysis of iNOS and COX2 protein expression in two independent tumors from each test group. (D) Levels of IL-6 and (E) TNF-α in tumor lysates analyzed by ELISA.</p

Scallion hot water extract may act by targeting multiple signaling pathways.

<p>Schematic representation of proposed anti-cancer mechanisms for the inhibition of colorectal tumor growth by dietary uptake of Allium fistulosum hot water extract.</p

Effect of three different scallion extract preparations on CT-26 tumor-bearing mice.

<p>(A) Schematic diagram of the experimental design for the in vivo antitumor effect of scallion extracts. (B) Mean body weight. (C) The differential cytotoxic effect of SCE treatments on colon tumor and normal colorectal cells. (D) Final tumor volume. (E) Survival rate of test mice. (F) Weight of excised tumors. (G) Tumor inhibition rate. (H) Representative photographs of tumors from tumor-bearing mice of all test groups.</p

HPLC chromatograms of hot water extracts of five <i>Wedelia</i> species.

<p>Apigenin was supplemented only as an internal standard to calibrate the HPLC data. The colors of signals, blue, red, green and pink, denote different absorbance levels of 254 nm, 280 nm, 300 nm and 350 nm, respectively. The peaks of a, b, c, d, e, f and g represent the different peaks detected between WC and WT extracts.</p

Summary of macroscopic and microscopic characteristics observed for stem and leaf tissues of <i>Wedelia</i> species.

<p>Abbreviations, including WC, WT, WB, WP, WBR and WPR, are described as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129067#pone.0129067.t002" target="_blank">Table 2</a>.</p><p>Summary of macroscopic and microscopic characteristics observed for stem and leaf tissues of <i>Wedelia</i> species.</p

Kimura’s two-parameter’s genetic distances as determined based on total ITS sequence.

<p>Kimura’s two-parameter’s genetic distances as determined based on total ITS sequence.</p