20 research outputs found
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 (5×10<sup>4</sup> 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 1×10<sup>7</sup> 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 1×10<sup>6</sup> 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
Effect of IL-1Ra on lymphangiogenesis and the accumulation of M2-type macrophages by Matrigel plug assay.
<p>(A) Matrigel plugs containing N15 and LNM35 with or without anakinra (n = 4 each). (B) Tumor angiogenesis, lymphangiogenesis, and infiltrated macrophages in Matrigel plugs (n = 6 per group) were determined immunohistochemically in frozen sections using CD31 (red), LYVE-1 (red), and F4/80 (green) as specific markers for microvessels, lymphatic vessels, and infiltrated macrophages, respectively. (C) VEGF-A and VEGF-C expression in macrophages purified from Matrigel plugs was determined by qRT-PCR. *<i>p</i><0.05 and ** <i>p</i><0.01. (D) Expression of M1- (iNOS and IL-12) and M2-type (arginase and IL-10) specific biomarkers in macrophages purified from Matrigel plugs was determined by qRT-PCR. ** <i>p</i><0.01.</p
Our hypothetical model how macrophages and cancer cells mutually promote lymph node metastasis through IL-1/IL-1R.
<p>Lung cancer cells form lymph node metastases, promote the development of lymphatic vessels, exhibit invasive tumor growth, and constitutively express IL-1 and CXC chemokines. Highly metastatic cancer cells intrinsically produce larger amounts of IL-1 and CXC chemokines. IL-1α and, to a lesser extent, IL-1β induce the up-regulation of CXC chemokines, which in turn promotes the recruitment and activation of macrophage in the tumor microenvironment. Direct interaction of these cancer cells with these macrophages markedly enhances the production of potent angiogenic and lymphangiogenic factors, leading to further malignant progression including lymph node metastasis and lymphangiogenesis. This sequence of events is suppressed by the IL-1R antagonist anakinra.</p
Highly metastatic LNM35 tumors are highly angiogenic and lymphangiogenic and rich in macrophages.
<p>(A) Comparison of tumor growth rates between N15 and LNM35 cells inoculated subcutaneously at day 0. Tumor growth rates were significantly different (**<i>p</i><0.01, n = 6). (B) IHC analysis using antibodies against vascular endothelium (CD31) and lymphatic vessels (LYVE-1), a macrophage-specific antibody (F4/80), and a neutrophil-specific antibody (Gr-1). N15 and LNM35 tumors were quantitatively analyzed, scoring five areas in each tumor section for microvascular density (MVD), lymphatic vessel density (LVD), F4/80-positive cells, and Gr-1-positive cells; *<i>p</i><0.05 and ** <i>p</i><0.01. (C) Comparison of lymph node weights between N15 and LNM35 tumors on day 35 after subcutaneous inoculation (**<i>p</i><0.01, n = 6). (D) H&E staining of lymph node samples from mice subcutaneously injected with N15 and LNM35 cells (n = 2 each); T: tumor, LN: lymph node. (E) Comparison of tumor growth rates following the subcutaneous inoculation of N15 and LNM35 cells at day 0. N15 tumors at day 38 and LNM35 tumors at day19 were of similar size and were further analyzed. (F) IHC analysis using antibodies against vascular endothelium (CD31), lymphatic vessels (LYVE-1), macrophages (F4/80), and neutrophils (Gr-1). Five N15 and LNM35 tumors were quantitatively analyzed, scoring five areas in each tumor section for MVD, LVD, F4/80-positive cells, and Gr-1-positive cells. ** <i>p</i><0.01.</p
Effect of IL-1Ra on tumor growth and lymph node metastasis by highly metastatic cancer cells.
<p>(A) Anti-tumor effect of anakinra on the growth of LNM35 xenografts. LNM35 cells were subcutaneously inoculated at day 0, after which tumor growth with or without a daily subcutaneous injection of anakinra was followed until day 35; *<i>p</i><0.05 between non-treated and treated groups (n = 5 mice per group). (B) Inhibition of lymph node metastasis by anakinra. Three metastatic lymph nodes representative of each group at day 35 are shown. (C) Effects of anakinra on angiogenesis, lymphangiogenesis, and macrophage and neutrophil infiltration by LNM35 tumors, analyzed on day 35 by IHC staining using specific antibodies for vascular endothelium (CD31), lymphatic vessels (LYVE-1), infiltrated macrophages (F4/80), and infiltrated neutrophils (Gr-1). Five areas of each tumor section from five tumor samples were quantitatively analyzed; *<i>p</i><0.05 and **<i>p</i><0.01. (D) Effect of anakinra on mouse VEGF-A, VEGF-C, and VEGF-D mRNA levels in LNM35 tumors, determined by qRT-PCR analysis of five tumors at day 35; *<i>p</i><0.05 and **<i>p</i><0.01. (E) Effect of anakinra on human CXCL8/IL-8 protein levels in LNM35 tumors, determined by ELISA analysis of five tumors at day 35; *<i>p</i><0.05. (F) Inhibitory effect of human COX2 expression in anakinra-treated tumors. COX2 expression in two representative tumors from mice treated or not with anakinra (5 mg) was analyzed by western blotting.</p
Effect of macrophage depletion on tumor growth, lymph node metastasis, and VEGF-A and VEGF-C expression.
<p>(A) Anti-tumor effect of Cl<sub>2</sub>MDP-LIP on tumor growth by LNM35 xenografts. Mice were subcutaneously inoculated with LNM35 cells at day 0, and tumor growth was followed until day 35 in animals intravenously injected twice weekly with PBS-LIP or Cl<sub>2</sub>MDP-LIP. *<i>p</i><0.05 between PBS-LIP and Cl<sub>2</sub>MDP-LIP-treated groups (n = 5 mice per group). (B) Inhibitory effect of Cl<sub>2</sub>MDP-LIP on lymph node metastasis. The area occupied by cancer cells in the lymph node was determined by H&E staining. Relative tumor area is expressed as the percent of the total lymph node area (n = 5 mice per group); **p<0.01 compared with the PBS-LIP-treated group. (C) Effect of Cl<sub>2</sub>MDP-LIP on angiogenesis, lymphangiogenesis, and macrophage and neutrophil infiltration by LNM35 tumors. IHC analysis was performed on day 35 using specific antibodies for vascular endothelium (CD31), lymphatic vessels (LYVE-1), infiltrated macrophages (F4/80), and infiltrated neutrophils (Gr-1). Five areas of each tumor section from five tumor samples were quantitatively analyzed; **<i>p</i><0.01. (D) Effect of Cl<sub>2</sub>MDP-LIP on the expression of mouse VEGF-A, VEGF-C, and VEGF-D mRNA in LNM35 tumors, determined by qRT-PCR analysis of five tumors on day 35; *<i>p</i><0.05 compared with the PBS-LIP-treated group.</p