Interleukin-6 mediates epithelial-stromal interactions and promotes gastric tumorigenesis - PubMed (original) (raw)

. 2013 Apr 12;8(4):e60914.

doi: 10.1371/journal.pone.0060914. Print 2013.

Yoshihiro Hirata, Hayato Nakagawa, Kei Sakamoto, Yoku Hayakawa, Ryota Takahashi, Wachiko Nakata, Kosuke Sakitani, Takako Serizawa, Yohko Hikiba, Masao Akanuma, Wataru Shibata, Shin Maeda, Kazuhiko Koike

Affiliations

Interleukin-6 mediates epithelial-stromal interactions and promotes gastric tumorigenesis

Hiroto Kinoshita et al. PLoS One. 2013.

Abstract

Interleukin-6 (IL-6) is a pleiotropic cytokine that affects various functions, including tumor development. Although the importance of IL-6 in gastric cancer has been documented in experimental and clinical studies, the mechanism by which IL-6 promotes gastric cancer remains unclear. In this study, we investigated the role of IL-6 in the epithelial-stromal interaction in gastric tumorigenesis. Immunohistochemical analysis of human gastritis, gastric adenoma, and gastric cancer tissues revealed that IL-6 was frequently detected in the stroma. IL-6-positive cells in the stroma showed positive staining for the fibroblast marker α-smooth muscle actin, suggesting that stromal fibroblasts produce IL-6. We compared IL-6 knockout (IL-6(-/-)) mice with wild-type (WT) mice in a model of gastric tumorigenesis induced by the chemical carcinogen N-methyl-N-nitrosourea. The stromal fibroblasts expressed IL-6 in tumors from WT mice. Gastric tumorigenesis was attenuated in IL-6(-/-) mice, compared with WT mice. Impaired tumor development in IL-6(-/-) mice was correlated with the decreased activation of STAT3, a factor associated with gastric cancer cell proliferation. In vitro, when gastric cancer cell line was co-cultured with primary human gastric fibroblast, STAT3-related genes including COX-2 and iNOS were induced in gastric cancer cells and this response was attenuated with neutralizing anti-IL-6 receptor antibody. IL-6 production from fibroblasts was increased when fibroblasts were cultured in the presence of gastric cancer cell-conditioned media. IL-6 production from fibroblasts was suppressed by an interleukin-1 (IL-1) receptor antagonist and siRNA inhibition of IL-1α in the fibroblasts. IL-1α mRNA and protein were increased in fibroblast lysate, suggesting that cell-associated IL-1α in fibroblasts may be involved. Our results suggest the importance of IL-6 mediated stromal-epithelial cell interaction in gastric tumorigenesis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Interleukin-6 (IL-6) expression in gastric cancer tissues.

(A) Representative pictures of immunohistochemical analyses of IL-6 in gastric cancer and _Helicobacter pylori_–negative healthy control (original magnification, ×200). (B) Enzyme-linked immunosorbent assay analysis of IL-6 expression in human gastric cancer tissues and normal gastric mucosa. (C) Representative pictures of immunohistochemical analyses of IL-6, α-smooth muscle actin (αSMA), CD11b, and CD68 in _Helicobacter pylori_–positive gastritis, gastric adenoma, and gastric cancer (original magnification, ×200). (D) Representative double staining of IL-6 (red) and αSMA (green) in human gastric cancer tissue (original magnification, ×200). (E) Representative double staining of IL-6 (red) and CD68 (green) in human gastric cancer tissue (original magnification, ×200).

Figure 2

Figure 2. Interleukin-6 (IL-6) deficiency attenuated N-methyl-N-nitrosourea (MNU)-induced gastric tumorigenesis in mice.

(A) Representative images of hematoxylin & eosin (H&E) staining of MNU-induced gastric tumors from wild-type (WT) mice [original magnification, ×40 (left), ×200 (right)]. (B) Tumor incidence rates, numbers of tumors, and maximal diameters of tumors. Values represent the means (± standard deviation) for WT (n = 14) and IL-6−/− (n = 16) mice. *P<0.05 compared with WT mice. (C) Representative images of IL-6 (left) and α-smooth muscle actin (right) staining in a gastric tumor from a WT mouse (original magnification, ×200).

Figure 3

Figure 3. Interleukin-6 (IL-6) promoted gastric tumor development through STAT3 activation.

(A) Immunoblot analysis of phosphorylated and total STAT3 in non-tumor tissues and tumors from wild-type (WT) and IL-6−/− mice in the N-methyl-N-nitrosourea (MNU)-induced gastric tumorigenesis model. (B) Immunohistochemical analysis of phosphorylated STAT3 in WT (left) and IL-6−/− (right) mice in the MNU-induced gastric tumorigenesis model. (C) Immunohistochemical analysis of phosphorylated STAT3 in _Helicobacter pylori_–negative healthy control, _Helicobacter pylori_–positive gastritis, gastric adenoma, and gastric cancer (original magnification, ×200). (D) Immunoblot analysis of phosphorylated and total STAT3 in SH101 and AGS cells treated with pyridone 6 (P6; 1 µM) for the indicated times. (E) SH101 and AGS cells were cultured with or without P6 (1 µM), and cell numbers were determined at the indicated times. Data are plotted as means (± standard deviations). *P<0.05 compared with cells without P6. (F) qRT-PCR for the indicated genes in gastric cancer cell line NUGC4 when co-cultured with fibroblasts with or without neutralizing anti-IL-6 receptor antibody (MRA). *P<0.05 compared with co-culture without MRA. (G) Immunohistochemical analysis of CD44 in stomach tissues of WT and IL-6−/− mice in chemically-induced gastric tumorigenesis model. (original magnification, ×200).

Figure 4

Figure 4. Gastric fibroblasts produced interleukin-6 (IL-6) in response to gastric cancer cell–conditioned media.

(A) IL-6 concentrations in the supernatants of fibroblasts (black bars) and in the indicated conditioned media (gray bars). Fibroblasts were cultured with the indicated cancer cell–conditioned media or standard media. St. Med, standard media. CM, conditioned media. Sup. of Fibroblasts, supernatants of fibroblasts cultured with indicated media. ND, not detected. (B) qRT-PCR analysis of IL-6 expression in fibroblasts treated with conditioned media from SH101 cells. *P<0.05 compared with standard media. (C) Immunoblot analysis of the indicated proteins from the fibroblasts treated with SH101 cell–conditioned media for the indicated times. (D) IL-6 concentrations in the supernatants of fibroblasts treated with the indicated cancer cell–conditioned media with or without IL-1 receptor antagonist (100 ng/mL) or anti–tumor necrosis factor (TNF)α neutralizing antibody (100 µg/mL). *P<0.05 compared with conditioned media alone. (E) qRT-PCR (left) and enzyme-linked immunosorbent assay (right) for IL-1α in fibroblasts treated with the indicated cancer cell–conditioned media. *P<0.05 compared with standard media. (F) IL-6 concentrations in the supernatants of control or IL-1α siRNA transfected fibroblasts treated with the cancer cell–conditioned media. *P<0.05. (G) Immunohistochemical analysis of phosph-IκBα in stromal–IL-6 positive gastric cancer and _Helicobacter pylori_–negative healthy control (original magnification, ×200).

Figure 5

Figure 5. The proposed model of interaction between fibroblasts and tumor cells in gastric tumorigenesis.

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