Helicobacter pylori-infected MSCs acquire a pro-inflammatory phenotype and induce human gastric cancer migration by promoting EMT in gastric cancer cells

Abstract

Accumulating clinical and experimental evidence has suggested that Helicobacter pylori (H. pylori) infection-associated gastric cancer (GC) is associated with high rates of mortality and serious health effects. The majority of patients succumb to H. pylori infection-associated GC due to metastasis. Mesenchymal stem cells (MSCs), which have multipotent differentiation potential, may be recruited into the tumor-associated stroma. MSCs are crucial components of the H. pylori infection-associated GC microenvironment, and may be critical for GC cell migration. In this study, an MSCs/H. pylori co-culture model was designed, and the effect of H. pylori-infected MSCs on the migration of GC cells was evaluated using a Transwell migration assay. H. pylori-infected MSC cytokine expression was evaluated using Luminex/ELISA. The expression of epithelial-mesenchymal transition (EMT) markers in the GC cells treated with supernatants from H. pylori-infected MSCs were detected by western blot analysis. The results demonstrated that the interaction between MSCs and H. pylori may induce GC cell migration, through secretion of a combination of cytokines that promote EMT in GC cells. The expression of phosphorylated forms of nuclear factor-κB (NF-κB) was observed to be increased in MSCs by H. pylori. Inhibition of NF-κB activation by pyrrolidine dithiocarbamate blocked the effects of H. pylori-infected MSCs on SGC-7901 human stomach adenocarcinoma cell migration. Overall, the results of the present study suggest that H. pylori-infected MSCs acquire a pro-inflammatory phenotype through secretion of a combination of multiple cytokines, a number of which are NF-κB-dependent. These cytokines enhance H. pylori infection-associated GC cell migration by promoting EMT in GC cells. The results of the present study provide novel evidence for the modulatory effect of MSCs in the tumor microenvironment and provide insight into the significance of stromal cell involvement in GC progression.

Keywords: Helicobacter pylori; epithelial-mesenchymal transition; gastric cancer; mesenchymal stem cells; migration.

Figure 1.

H. pylori-infected hucMSCs promote the migration and epithelial-mesenchymal transition of SGC-7901 cells. (A) The ability of hucMSCs to induce SCG-7901 gastric cancer cell migration following H. pylori-infection was evaluated by Transwell migration assay. (B) Western blot analysis of E-cadherin, N-cadherin and vimentin protein levels. (C) Representative images of Transwell migration assay (magnification, ×100) and (D) histogram of the numbers of migrated SGC-7901 cells in each treatment group. *P<0.01 and **P<0.05 (n=3). Values are presented as the mean ± standard deviation and are representative of three independent experiments. (E) The capacity of cells to migrate to fill a scratched area devoid of cells was assessed in co-cultures. H. pylori-infected hucMSCs more effectively enhanced SGC-7901 cell membrane penetration and migration. (a) SCG-7901 cells alone and with (b) H. pylori or the supernatants from (c) hucMSCs and (d) hucMSCs infected with H. pylori. H. pylori, Helicobacter pylori; hucMSCs, human umbilical cord mesenchymal stem cells.

Figure 2.

Expression of inflammatory cytokines in hucMSCs is upregulated following co-culture with H. pylori, and the cytokines produced during co-culture promote migration of SGC-7901 cells. (A) Luminex assay for the expression of cytokines in the supernatants of hucMSCs and H. pylori-infected hucMSCs. (B) The fold-changes in cytokine expression in H. pylori-infected humMSCs relative to uninfected cells were as follows: IL-8, 2.14; PDGF-B, 2.61; IL-6, 3.9; MCP-1, 2.17; GM-1, 8.0; VEGF, 2.8; EGF, 2.4; TNF-α, 4.4; IL-1β, 2.35. Fold-change = concentration of cytokine in H. pylori-infected humMSCs/uninfected humMSCs. (C) ELISA assay to determine IL-6, IL-8 and PDGF-B levels in the supernatants of hucMSCs and H. pylori-infected hucMSCs. (D) SGC-7901 cells were stimulated by 50 ng/ml concentrations of purified cytokines and migration assays were then performed. *P<0.05 and ^#P<0.01 compared with migration medium alone. Values are presented as the mean ± standard deviation. Data are representative of three independent experiments. H. pylori, Helicobacter pylori; hucMSCs, human umbilical cord mesenchymal stem cells; IL, interleukin; PDGF, platelet-derived growth factor; MCP, monocyte chemoattractant protein; GM-CSF, granulocyte macrophage colony-stimulating factor; VEGF, vascular endothelial growth factor; EGF, epidermal growth factor; TNF, tumor necrosis factor; MOI, multiplicity of infection.

Figure 3.

hucMSCs infected with H. pylori enhance SCG-7901 gastric cancer cell migration via NF-κB activation. (A) H. pylori-induced NF-κB-p65 phosphorylation and IκB-a change in hucMSCs for 24 h. (B and C) Time-course (0, 30, 60, 120, 180 min) of H. pylori induced NF-κB-p65 phosphorylation and IκB-a change in hucMSCs treated with H. pylori in the presence or absence of PDTC (100nM). (D) Western blot analysis of (a) cytoplasmic protein and (b) nucleoprotein expression levels. (E) hucMSCs were pre-incubated with PDTC for 90 min, stimulated with H. pylori for 24 h and then the concentration of cytokines in the cultured medium was determined by ELISA. The NF-κB inhibitor PDTC was able to inhibit the expression of numerous cytokines (IL-6, IL-8 and PDGF-B) in the hucMSCs infected with H. pylori. Data are expressed as the mean ± standard deviation; *P<0.05, **P<0.01; data are representative of three independent experiments. H. pylori, Helicobacter pylori; hucMSCs, human umbilical cord mesenchymal stem cells, PDTC, pyrrolidine dithiocarbamate; IL, interleukin; PDGF, platelet derived growth factor; IκB-a, nuclear factor-κB polypeptide gene enhancer; NF-κB, nuclear factor-κB; p-NF-κB, phosphorylated NF-κB; t-NF-κB, total NF-κB.

Figure 4.

Inhibition of NF-κB activation by PDTC abrogates the effect of H. pylori-infected MSCs on gastric cancer cells. (A) The ability of H. pylori-infected MSCs to induce migration following pretreatment with PDTC was evaluated using cell culture inserts; magnification, ×100. (B) Histogram indicating the number of migrated SGC-7901 cells. Data are expressed as the mean ± standard deviation; *P<0.05 and **P<0.01. Data are representative of three independent experiments. (C) Capacity of cells to migrate to fill a scratched area devoid of cells was assessed in co-cultures of SCG-7901 cells with supernatants from (a) hucMSCs, (b) H. pylori-infected hucMSCs and (c) H. pylori-infected hucMSCs pretreated with PDTC for 48 h, respectively. (D) Western blot analyses of E-cadherin, N-cadherin and vimentin protein levels in SGC-7901 cells treated with the supernatants from hucMSCs, H. pylori-infected hucMSCs and the infected hucMSCs pretreated with PDTC, respectively. H. pylori, Helicobacter pylori; hucMSCs, human umbilical cord mesenchymal stem cells, PDTC, pyrrolidine dithiocarbamate; NF-κB, nuclear factor-κB.