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Macrophage Infiltration Induces Gastric Cancer Invasiveness by Activating the β-Catenin Pathway.

Wu MH, Lee WJ, Hua KT, Kuo ML, Lin MT - PLoS ONE (2015)

Bottom Line: Increased density of macrophages was associated with advanced stage and poor survival.AKT but not ERK regulated β-catenin translocation.Macrophages may induce invasiveness by activating the β-catenin pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan.

ABSTRACT

Background: Despite evidence that activated macrophages act in an inflammatory microenvironment to promote gastric tumorigenesis via β-catenin signaling, the effects of β-catenin signaling on gastric cancer cell metastasis and the relationship of these cells with surrounding tumor associated macrophages have not been directly studied.

Methods: Immunohistochemical staining was employed to analyze 103 patients. An invasion assay was used to evaluate the relationship between macrophages and gastric cancer cells. β-catenin gain-of-function and loss-of-function approaches were performed. To assess the β-catenin regulation mechanism in gastric cancer cells, Western blotting and reverse-transcription polymerase chain reaction were used.

Results: Increased density of macrophages was associated with advanced stage and poor survival. Gastric cancer cell lines co-cultured with macrophages conditioned medium showed increased nuclear accumulation of β-catenin and increased invading ability. AKT but not ERK regulated β-catenin translocation. MMP7 and CD44, both β-catenin downstream genes, were involved in macrophage-activated gastric cancer cell invasion.

Conclusion(s): Collectively, the clinical data suggest that macrophage infiltration is correlated with increased grade and poor prognosis for gastric cancer patients who underwent radical resection. Macrophages may induce invasiveness by activating the β-catenin pathway.

No MeSH data available.


Related in: MedlinePlus

Effect of macrophage CM on β-catenin pathway.(A) β-catenin accumulates in nucleus after treatment with macrophage CM for 30 minutes in N87 cells. (B) Immunofiuorescent images were observed with a confocal microscope. β-catenin positive cells were analyzed by using an anti-β-catenin antibody, which is recognized by secondary rabbit antibody conjugated with FITC, depicted by green fluorescence; Nuclear staining was detected by counterstaining cells with 4', 6-Diamidino-2-phenylindole (DAPI), represented as blue fluorescence. Co-culturing with macrophage CM, immunofiuorescence staining showed β-catenin-FITC complexes translocated into nucleus. (C) Invasion ability of GC cells treated by macrophage CM.
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pone.0134122.g005: Effect of macrophage CM on β-catenin pathway.(A) β-catenin accumulates in nucleus after treatment with macrophage CM for 30 minutes in N87 cells. (B) Immunofiuorescent images were observed with a confocal microscope. β-catenin positive cells were analyzed by using an anti-β-catenin antibody, which is recognized by secondary rabbit antibody conjugated with FITC, depicted by green fluorescence; Nuclear staining was detected by counterstaining cells with 4', 6-Diamidino-2-phenylindole (DAPI), represented as blue fluorescence. Co-culturing with macrophage CM, immunofiuorescence staining showed β-catenin-FITC complexes translocated into nucleus. (C) Invasion ability of GC cells treated by macrophage CM.

Mentions: We conducted an experiment in vitro to understand whether β-catenin signaling was involved in macrophage-activated GC cell invasion. As shown in Fig 5A, β-catenin immunoreactivity was found in the nuclear fraction of N87 cells as early as 15 minutes after macrophage CM treatment, and the amount of nuclear β-catenin protein increased in a time-dependent manner. In line with these findings, immunofluorescence showed accumulation and nuclear localization of β-catenin in GC cells co-cultured with macrophage CM as compared to controls cocultured in N87 (Fig 5B). These results strongly suggest that the soluble factors derived from macrophages help mediate the accumulation and nuclear translocation of β-catenin in GC cells. We transiently knocked down β-catenin by shRNA and found decreased β-catenin protein expression within 24 h. Genetic ablation of β-catenin in N87 cells fails to increase their invasive ability after macrophage CM treatment. By contrast, non-transfected and control shRNA had no change in the invasive capability in N87 cells under macrophage CM treatment (Fig 5C). The amount of nuclear β-catenin protein was also increased in the AGS and MKN45 cells after macrophage CM treatment (S1 Fig).


Macrophage Infiltration Induces Gastric Cancer Invasiveness by Activating the β-Catenin Pathway.

Wu MH, Lee WJ, Hua KT, Kuo ML, Lin MT - PLoS ONE (2015)

Effect of macrophage CM on β-catenin pathway.(A) β-catenin accumulates in nucleus after treatment with macrophage CM for 30 minutes in N87 cells. (B) Immunofiuorescent images were observed with a confocal microscope. β-catenin positive cells were analyzed by using an anti-β-catenin antibody, which is recognized by secondary rabbit antibody conjugated with FITC, depicted by green fluorescence; Nuclear staining was detected by counterstaining cells with 4', 6-Diamidino-2-phenylindole (DAPI), represented as blue fluorescence. Co-culturing with macrophage CM, immunofiuorescence staining showed β-catenin-FITC complexes translocated into nucleus. (C) Invasion ability of GC cells treated by macrophage CM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520459&req=5

pone.0134122.g005: Effect of macrophage CM on β-catenin pathway.(A) β-catenin accumulates in nucleus after treatment with macrophage CM for 30 minutes in N87 cells. (B) Immunofiuorescent images were observed with a confocal microscope. β-catenin positive cells were analyzed by using an anti-β-catenin antibody, which is recognized by secondary rabbit antibody conjugated with FITC, depicted by green fluorescence; Nuclear staining was detected by counterstaining cells with 4', 6-Diamidino-2-phenylindole (DAPI), represented as blue fluorescence. Co-culturing with macrophage CM, immunofiuorescence staining showed β-catenin-FITC complexes translocated into nucleus. (C) Invasion ability of GC cells treated by macrophage CM.
Mentions: We conducted an experiment in vitro to understand whether β-catenin signaling was involved in macrophage-activated GC cell invasion. As shown in Fig 5A, β-catenin immunoreactivity was found in the nuclear fraction of N87 cells as early as 15 minutes after macrophage CM treatment, and the amount of nuclear β-catenin protein increased in a time-dependent manner. In line with these findings, immunofluorescence showed accumulation and nuclear localization of β-catenin in GC cells co-cultured with macrophage CM as compared to controls cocultured in N87 (Fig 5B). These results strongly suggest that the soluble factors derived from macrophages help mediate the accumulation and nuclear translocation of β-catenin in GC cells. We transiently knocked down β-catenin by shRNA and found decreased β-catenin protein expression within 24 h. Genetic ablation of β-catenin in N87 cells fails to increase their invasive ability after macrophage CM treatment. By contrast, non-transfected and control shRNA had no change in the invasive capability in N87 cells under macrophage CM treatment (Fig 5C). The amount of nuclear β-catenin protein was also increased in the AGS and MKN45 cells after macrophage CM treatment (S1 Fig).

Bottom Line: Increased density of macrophages was associated with advanced stage and poor survival.AKT but not ERK regulated β-catenin translocation.Macrophages may induce invasiveness by activating the β-catenin pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, National Taiwan University Hospital, Taipei, Taiwan.

ABSTRACT

Background: Despite evidence that activated macrophages act in an inflammatory microenvironment to promote gastric tumorigenesis via β-catenin signaling, the effects of β-catenin signaling on gastric cancer cell metastasis and the relationship of these cells with surrounding tumor associated macrophages have not been directly studied.

Methods: Immunohistochemical staining was employed to analyze 103 patients. An invasion assay was used to evaluate the relationship between macrophages and gastric cancer cells. β-catenin gain-of-function and loss-of-function approaches were performed. To assess the β-catenin regulation mechanism in gastric cancer cells, Western blotting and reverse-transcription polymerase chain reaction were used.

Results: Increased density of macrophages was associated with advanced stage and poor survival. Gastric cancer cell lines co-cultured with macrophages conditioned medium showed increased nuclear accumulation of β-catenin and increased invading ability. AKT but not ERK regulated β-catenin translocation. MMP7 and CD44, both β-catenin downstream genes, were involved in macrophage-activated gastric cancer cell invasion.

Conclusion(s): Collectively, the clinical data suggest that macrophage infiltration is correlated with increased grade and poor prognosis for gastric cancer patients who underwent radical resection. Macrophages may induce invasiveness by activating the β-catenin pathway.

No MeSH data available.


Related in: MedlinePlus