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

Enhancement of E-cadherin promoter activity by NDRG1 knockdown in highly metastatic gastric cancer cell.

<p>(A) Comparison of protein expression levels of E-cadherin, β-catenin, NDRG1 and vimentin when transiently treated with Snail siRNA for 0, 48 and 96 hr by western blot analysis in HSC-58 cell. (B) E-cadherin promoter-driven luciferase activity in the absence or presence of Snail expression in HSC-58 and BxPC-3 cells. E-cadherin-luc was transfected with or without pcDNA3-Snail, and the luciferase activity was measured. Each column is average of triplicate trials (*<i>p</i><0.05). (C) Comparison of E-cadherin promoter-driven luciferase activity (E-cadherin-luc) in As1/Mock3, As1/Sic50 and As1/Sic54. Each column is average of triplicate trials (*<i>p</i><0.05). (D) The effect of CT99021 on protein expression of NDRG1 and various EMT-related molecules by Western blot analysis. 58Asl cells were treated with indicated doses of the drug for 24 hr. (E) The effect of β-catenin knockdown by its siRNAs on expression of E-cadherin. HSC-58 cells were transfected with siRNAs for 24 hr, and total cell lysates were analyzed by Western blot analysis.</p

Biological properties of low and highly metastatic gastric cancer cell lines <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Comparison of cell proliferation rates <i>in vitro</i>. Cells were seeded on day 0 (5×10⁴ cells/dish) and proliferation was measured in RPMI 1640 containing 10% FBS. Doubling times were 34, 27, and 23 hr for HSC-58 (black), 58As1 (gray) and 58As9 cells (white), respectively. (B) Morphology of gastric cancer cell line <i>in vitro</i>. HSC-58 and 58As9 cell growth was visible as attached layers with fibroblastic morphology. 58As1 cells showed suspension-type cell growth with round morphology. (C) Tumor growth of gastric cancer cell lines at day 14 and 28. HSC-58 (black), 58As1 (light gray), and 58As9 (dark gray) were subcutaneously implanted with 1×10⁷ cells at day 0. 58As1 or 58As9 cells showed significantly (*<i>p</i><0.01) higher tumor growth rates than HSC-58 cells. (D, E) Peritoneal dissemination and ascites formation <i>in vivo</i>. Typical figures show high and low peritoneal dissemination and ascites accumulation at day 55 after inoculation of 1×10⁶ cells into peritoneal cavity. Each mouse showed ascites accumulation of 0.7–2.5 ml and 5–12 nodules on the mesenterium following 58As1 inoculation, but this was not apparent with HSC-58 cells. N.D., not detectable. (F) Microarray analysis on expression of genes that are up- or down- regulated in highly metastatic cell line, 58Asl, as compared with HSC-58. Relative expression rates are presented on genes belonging to three biological functions.</p