Multivariate analysis of OS.

<p>Multivariate analysis of OS.</p

Multivariate analysis of PFS.

<p>Multivariate analysis of PFS.</p

Immunohistochemical assessment of ERCC1 and correlation between ERCC1 and survival.

<p>A: Expression of ERCC1 was assessed by immunohistochemistry. Expression of ERCC1 protein was detected in the nuclei of cancer cells (x400). B: Kaplan-Meier survival curves according to ERCC1 expression score. C: ERCC1-negative patients with exon 19 deletion had longer PFS than the others, and ERCC1-positive patients without exon 19 deletion had shorter PFS than the others.</p

Multivariate analysis of PFS.

<p>Multivariate analysis of PFS.</p

Multivariate analysis of OS.

<p>Multivariate analysis of OS.</p

Relation between expression score for ERCC1 and various patient characteristics.

a<p>Determined by Fisher’s exact test.</p

Relation between expression score for EGFR mutants and various patient characteristics.

a<p>Determined by Fisher’s exact test.</p

Comparison of protein and mRNA expression levels of various factors between low and highly metastatic gastric cancer cell lines.

<p>(A) Western blot analysis of total cell lysates shows protein expression levels of NDRG1, growth factor receptor, EMT-related proteins, Wnt/β-catenin-related proteins, and other factors in HSC-58, 58As1 and 58As9 cells. (B) Comparison of mRNA expression levels of NDRG1, E-cadherin, vimentin, Snail, MMP-1 and β-catenin in HSC-58, 58As1 and 58As9 cells by qRT-PCR analysis. (C) Immunocytochemical analysis of E-cadherin and β-catenin in HSC-58 and 58As9, using specific antibodies against E-cadherin, β-catenin and DAP1. Magnification×200. (D) Western blot analysis shows expression of β-catenin and Snail in nucleus and cytosol fraction. CREB, a nuclear marker, and α-tubulin, a cytosol marker. (E,F) Comparison of luciferase activity driven by E-cadhrin promoter and β-catenin (TopFlash) driven promoter between HSC-58 and its highly metastatic cell lines. The relative promoter activity is presented when normalized by the activity in HSC-58. *p<0.01.</p

Altered expression of EMT-related factors by NDRG1 knockdown in highly metastatic 58As1.

<p>(A) Microarray analysis for the effect of NDRG1 knockdown on expression of genes that are up- or down- regulated in Asl/Sic50 versus As1/Mock3. Relative expression rates are presented on genes belonging to three biological functions. (B) Comparison of protein expression levels of NDRG1, EMT-related proteins, β-catenin, Akt, p-Akt, ERK1/2, p-ERK1/2, GSK-3β, p-GSK-3β and EGFR by western blot analysis with total cell lysate. (C) The mRNA expression of NDRG1, E-cadherin, vimentin, Snail, MMP-1 and β-catenin was determined by qRT-PCR analysis. (D) Comparison of luciferase activity driven by β-catenin (TopFlash) between As1/Mock3 and its NDRG1 knockdowned cell lines. Relative luminescence fold is presented when normalized by the value in As1/Mock3. Each column is average of triplicate trials±SD.</p

Suppression of peritoneal dissemination by NDRG1 knockdown.

<p>(A) Macroscopic images show enlarged peritoneal cavity and metastatic nodules by As1/Mock3 and As1/Sic50. Arrowheads show nodules. (B) Number of metastatic nodules in the mesenterium was 51±16 (As1/Mock3) and 35±14 (As1/Sic50) (<i>p</i> = 0.21), but the As1/Mock3 nodule size was 3–4 times larger than those of As1/Sic50. (C) Comparison of the volume of ascites between As1/Mock3 (3.9±1.0 ml) and As1/Sic50 cells (0.5±0.6 ml) following orthotopic implantation (n = 7) (* <i>p</i><0.01). (D) Survival curves show that survival rate in As1/Sic50 tumor-bearing mice was significantly (* <i>p</i><0.01) longer than that of As1/Mock3 tumor-bearing mice (n = 6). (E) Our hypothetic model how NDRG1 overexpression promotes metastasis including peritoneal dissemination through alteration of EMT by scirrhous gastric cancer cells, possibly through modification of Snail expression.</p