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Reciprocal activation between IL-6/STAT3 and NOX4/Akt signalings promotes proliferation and survival of non-small cell lung cancer cells.

Li J, Lan T, Zhang C, Zeng C, Hou J, Yang Z, Zhang M, Liu J, Liu B - Oncotarget (2015)

Bottom Line: Inflammatory cytokines and oxidative stress are two critical mediators in inflammation-associated cancer.Interleukin-6 (IL-6) is one of the most critical tumor-promoting cytokines in non-small cell lung cancer (NSCLC).The in vivo results were similar to those obtained in vitro.

View Article: PubMed Central - PubMed

Affiliation: Clinical Pharmacy Department, Guangdong Pharmaceutical University, Guangzhou 510006, China.

ABSTRACT
Inflammatory cytokines and oxidative stress are two critical mediators in inflammation-associated cancer. Interleukin-6 (IL-6) is one of the most critical tumor-promoting cytokines in non-small cell lung cancer (NSCLC). In our recent study, we confirmed that NADPH oxidase 4 (NOX4), an important source of reactive oxygen species (ROS) production in NSCLC cells, promotes malignant progression of NSCLC. However, whether the crosstalk of NOX4 and IL-6 signalings exists in NSCLC remains undentified. In this study, we show that NOX4 expression is positively correlated with IL-6 expression in NSCLC tissues. Exogenous IL-6 treatment significantly enhances NOX4/ROS/Akt signaling in NSCLC cells. NOX4 also enhances IL-6 production and activates IL-6/STAT3 signaling in NSCLC cells. Specifically, NOX4 is confirmed to functionally interplay with IL-6 to promote NSCLC cell proliferation and survival. The in vivo results were similar to those obtained in vitro. These data indicate a novel NOX4-dependent link among IL-6 in the NSCLC microenvironment, oxidative stress in NSCLC cells and autocrined IL-6 in NSCLC cells. NOX4/Akt and IL-6/STAT3 signalings can reciprocally and positively regulate each other, leading to enhanced NSCLC cell proliferation and survival. Therefore, NOX4 may serve as a promising target against NSCLC alone with IL-6 signaling.

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NOX4 stimulates IL-6 expression and JAK1/STAT3 activity in A549 cells via activation of PI3K/Akt pathway(A) Western blotting analysis of NOX4 expression in normal lung epithelial cells and cultured NSCLC cell lines. (B) A549 cells were stably transfected with control vector, NOX4 plasmid, respectively. Overexpression of NOX4 was confirmed by western blotting. (C-E) The effects of NOX4 overexpression on ROS production, IL-6 production and JAK1/STAT3 activity after 24 hours. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P < 0.05. (F) A549 cells were stably transfected with scramble shRNA, NOX4 shRNA, respectively. Knockdown of NOX4 was analyzed by western blotting. (G-I) Silencing NOX4 significantly inhibited ROS production, IL-6 production and JAK1/STAT3 activity after 24 hours. Bars are mean ± SD from five independent experiments. *Significantly different from vector control, P < 0.05. (J-K) Stably NOX4 overexpressing A549 cells were incubated with two common ROS scavengers including NAC (25 μM) and DPI (10 μM, an inhibitor of NADPH oxidase) for 24 hours, and then IL-6 levels and JAK1/STAT3 activity were determined. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P < 0.05. (L-M) Stably NOX4 overexpressing A549 cells were incubated with 30 μM of LY294002 or 10 μM of Wortmannin and control solvent for 24 hours, and then IL-6 levels and JAK1/STAT3 activity were determined. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P < 0.05.
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Figure 3: NOX4 stimulates IL-6 expression and JAK1/STAT3 activity in A549 cells via activation of PI3K/Akt pathway(A) Western blotting analysis of NOX4 expression in normal lung epithelial cells and cultured NSCLC cell lines. (B) A549 cells were stably transfected with control vector, NOX4 plasmid, respectively. Overexpression of NOX4 was confirmed by western blotting. (C-E) The effects of NOX4 overexpression on ROS production, IL-6 production and JAK1/STAT3 activity after 24 hours. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P < 0.05. (F) A549 cells were stably transfected with scramble shRNA, NOX4 shRNA, respectively. Knockdown of NOX4 was analyzed by western blotting. (G-I) Silencing NOX4 significantly inhibited ROS production, IL-6 production and JAK1/STAT3 activity after 24 hours. Bars are mean ± SD from five independent experiments. *Significantly different from vector control, P < 0.05. (J-K) Stably NOX4 overexpressing A549 cells were incubated with two common ROS scavengers including NAC (25 μM) and DPI (10 μM, an inhibitor of NADPH oxidase) for 24 hours, and then IL-6 levels and JAK1/STAT3 activity were determined. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P < 0.05. (L-M) Stably NOX4 overexpressing A549 cells were incubated with 30 μM of LY294002 or 10 μM of Wortmannin and control solvent for 24 hours, and then IL-6 levels and JAK1/STAT3 activity were determined. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P < 0.05.

Mentions: To explore whether NOX4 enhances IL-6 expression in NSCLC cells as well, we first sought to determine the NOX4 expression phenotype in NSCLC cell lines (A549, H460, H358, H441 and HCC827) and normal lung epithelial BEAS2B cells. The results of western blotting assay revealed that NOX4 expression was markedly higher in NSCLC cell lines than that in the normal lung epithelial cells (Fig. 3A).


Reciprocal activation between IL-6/STAT3 and NOX4/Akt signalings promotes proliferation and survival of non-small cell lung cancer cells.

Li J, Lan T, Zhang C, Zeng C, Hou J, Yang Z, Zhang M, Liu J, Liu B - Oncotarget (2015)

NOX4 stimulates IL-6 expression and JAK1/STAT3 activity in A549 cells via activation of PI3K/Akt pathway(A) Western blotting analysis of NOX4 expression in normal lung epithelial cells and cultured NSCLC cell lines. (B) A549 cells were stably transfected with control vector, NOX4 plasmid, respectively. Overexpression of NOX4 was confirmed by western blotting. (C-E) The effects of NOX4 overexpression on ROS production, IL-6 production and JAK1/STAT3 activity after 24 hours. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P < 0.05. (F) A549 cells were stably transfected with scramble shRNA, NOX4 shRNA, respectively. Knockdown of NOX4 was analyzed by western blotting. (G-I) Silencing NOX4 significantly inhibited ROS production, IL-6 production and JAK1/STAT3 activity after 24 hours. Bars are mean ± SD from five independent experiments. *Significantly different from vector control, P < 0.05. (J-K) Stably NOX4 overexpressing A549 cells were incubated with two common ROS scavengers including NAC (25 μM) and DPI (10 μM, an inhibitor of NADPH oxidase) for 24 hours, and then IL-6 levels and JAK1/STAT3 activity were determined. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P < 0.05. (L-M) Stably NOX4 overexpressing A549 cells were incubated with 30 μM of LY294002 or 10 μM of Wortmannin and control solvent for 24 hours, and then IL-6 levels and JAK1/STAT3 activity were determined. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P < 0.05.
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Figure 3: NOX4 stimulates IL-6 expression and JAK1/STAT3 activity in A549 cells via activation of PI3K/Akt pathway(A) Western blotting analysis of NOX4 expression in normal lung epithelial cells and cultured NSCLC cell lines. (B) A549 cells were stably transfected with control vector, NOX4 plasmid, respectively. Overexpression of NOX4 was confirmed by western blotting. (C-E) The effects of NOX4 overexpression on ROS production, IL-6 production and JAK1/STAT3 activity after 24 hours. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P < 0.05. (F) A549 cells were stably transfected with scramble shRNA, NOX4 shRNA, respectively. Knockdown of NOX4 was analyzed by western blotting. (G-I) Silencing NOX4 significantly inhibited ROS production, IL-6 production and JAK1/STAT3 activity after 24 hours. Bars are mean ± SD from five independent experiments. *Significantly different from vector control, P < 0.05. (J-K) Stably NOX4 overexpressing A549 cells were incubated with two common ROS scavengers including NAC (25 μM) and DPI (10 μM, an inhibitor of NADPH oxidase) for 24 hours, and then IL-6 levels and JAK1/STAT3 activity were determined. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P < 0.05. (L-M) Stably NOX4 overexpressing A549 cells were incubated with 30 μM of LY294002 or 10 μM of Wortmannin and control solvent for 24 hours, and then IL-6 levels and JAK1/STAT3 activity were determined. Bars are mean ± SD from four independent experiments. *Significantly different from vector control, P < 0.05.
Mentions: To explore whether NOX4 enhances IL-6 expression in NSCLC cells as well, we first sought to determine the NOX4 expression phenotype in NSCLC cell lines (A549, H460, H358, H441 and HCC827) and normal lung epithelial BEAS2B cells. The results of western blotting assay revealed that NOX4 expression was markedly higher in NSCLC cell lines than that in the normal lung epithelial cells (Fig. 3A).

Bottom Line: Inflammatory cytokines and oxidative stress are two critical mediators in inflammation-associated cancer.Interleukin-6 (IL-6) is one of the most critical tumor-promoting cytokines in non-small cell lung cancer (NSCLC).The in vivo results were similar to those obtained in vitro.

View Article: PubMed Central - PubMed

Affiliation: Clinical Pharmacy Department, Guangdong Pharmaceutical University, Guangzhou 510006, China.

ABSTRACT
Inflammatory cytokines and oxidative stress are two critical mediators in inflammation-associated cancer. Interleukin-6 (IL-6) is one of the most critical tumor-promoting cytokines in non-small cell lung cancer (NSCLC). In our recent study, we confirmed that NADPH oxidase 4 (NOX4), an important source of reactive oxygen species (ROS) production in NSCLC cells, promotes malignant progression of NSCLC. However, whether the crosstalk of NOX4 and IL-6 signalings exists in NSCLC remains undentified. In this study, we show that NOX4 expression is positively correlated with IL-6 expression in NSCLC tissues. Exogenous IL-6 treatment significantly enhances NOX4/ROS/Akt signaling in NSCLC cells. NOX4 also enhances IL-6 production and activates IL-6/STAT3 signaling in NSCLC cells. Specifically, NOX4 is confirmed to functionally interplay with IL-6 to promote NSCLC cell proliferation and survival. The in vivo results were similar to those obtained in vitro. These data indicate a novel NOX4-dependent link among IL-6 in the NSCLC microenvironment, oxidative stress in NSCLC cells and autocrined IL-6 in NSCLC cells. NOX4/Akt and IL-6/STAT3 signalings can reciprocally and positively regulate each other, leading to enhanced NSCLC cell proliferation and survival. Therefore, NOX4 may serve as a promising target against NSCLC alone with IL-6 signaling.

Show MeSH
Related in: MedlinePlus