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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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IL-6 stimulates NOX4/Akt pathway in A549 cells(A) ELISA analysis of IL-6 production in normal lung epithelial cells and cultured NSCLC cell lines. Bars are mean ± SD from four independent experiments. *Significantly different from BEAS2B cell group, P < 0.05. (B-D) The effects of IL-6 administration on NOX4 expression, ROS production and Akt activity in A549 cells at the indicated times, respectively. Bars are mean ± SD from four independent experiments. *Significantly different from control, P < 0.05. (E) The effect of IL-6 on STAT3 activity, and the influence of IL-6 neutralizing antibody siltuximab, JAKs inhibitor P6 (2.5 μM) or a selective JAK2 inhibitor of AG490 on IL-6-mediated STAT3 activation in A549 cells. (F-H) P6 or siltuximab could efficiently block the enhancement effects of IL-6 on NOX4 expression, ROS production and Akt activity in A549 cells after 48-hour incubation. Bars are mean ± SD from four independent experiments. *Significantly different from control, P < 0.05.
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Figure 2: IL-6 stimulates NOX4/Akt pathway in A549 cells(A) ELISA analysis of IL-6 production in normal lung epithelial cells and cultured NSCLC cell lines. Bars are mean ± SD from four independent experiments. *Significantly different from BEAS2B cell group, P < 0.05. (B-D) The effects of IL-6 administration on NOX4 expression, ROS production and Akt activity in A549 cells at the indicated times, respectively. Bars are mean ± SD from four independent experiments. *Significantly different from control, P < 0.05. (E) The effect of IL-6 on STAT3 activity, and the influence of IL-6 neutralizing antibody siltuximab, JAKs inhibitor P6 (2.5 μM) or a selective JAK2 inhibitor of AG490 on IL-6-mediated STAT3 activation in A549 cells. (F-H) P6 or siltuximab could efficiently block the enhancement effects of IL-6 on NOX4 expression, ROS production and Akt activity in A549 cells after 48-hour incubation. Bars are mean ± SD from four independent experiments. *Significantly different from control, P < 0.05.

Mentions: To dissect whether IL-6 stimulates NOX4/Akt signaling, we first examined the IL-6 production in NSCLC cell lines (A549, H460, H358, H441 and HCC827) and normal lung epithelial BEAS2B cells. The results showed that all the NSCLC cell lines and BEAS2B cells produced their own IL-6, and IL-6 production was markedly higher in NSCLC cell lines than that in the normal lung epithelial cells (Fig. 2A). Fig. 2B showed that IL-6 (10 ng/mL) treatment led to a time-dependent increase in NOX4 level in A549 cells. Besides, IL-6 could also enhance ROS production analyzed by DCF assay, as well as the preduction of superoxide and hydrogen peroxide analyzed by amplex red assay, respectively (Fig. 2C) and stimulate Akt activity (Fig. 2D) in a time-dependent manner in these cells.


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)

IL-6 stimulates NOX4/Akt pathway in A549 cells(A) ELISA analysis of IL-6 production in normal lung epithelial cells and cultured NSCLC cell lines. Bars are mean ± SD from four independent experiments. *Significantly different from BEAS2B cell group, P < 0.05. (B-D) The effects of IL-6 administration on NOX4 expression, ROS production and Akt activity in A549 cells at the indicated times, respectively. Bars are mean ± SD from four independent experiments. *Significantly different from control, P < 0.05. (E) The effect of IL-6 on STAT3 activity, and the influence of IL-6 neutralizing antibody siltuximab, JAKs inhibitor P6 (2.5 μM) or a selective JAK2 inhibitor of AG490 on IL-6-mediated STAT3 activation in A549 cells. (F-H) P6 or siltuximab could efficiently block the enhancement effects of IL-6 on NOX4 expression, ROS production and Akt activity in A549 cells after 48-hour incubation. Bars are mean ± SD from four independent experiments. *Significantly different from control, P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4359215&req=5

Figure 2: IL-6 stimulates NOX4/Akt pathway in A549 cells(A) ELISA analysis of IL-6 production in normal lung epithelial cells and cultured NSCLC cell lines. Bars are mean ± SD from four independent experiments. *Significantly different from BEAS2B cell group, P < 0.05. (B-D) The effects of IL-6 administration on NOX4 expression, ROS production and Akt activity in A549 cells at the indicated times, respectively. Bars are mean ± SD from four independent experiments. *Significantly different from control, P < 0.05. (E) The effect of IL-6 on STAT3 activity, and the influence of IL-6 neutralizing antibody siltuximab, JAKs inhibitor P6 (2.5 μM) or a selective JAK2 inhibitor of AG490 on IL-6-mediated STAT3 activation in A549 cells. (F-H) P6 or siltuximab could efficiently block the enhancement effects of IL-6 on NOX4 expression, ROS production and Akt activity in A549 cells after 48-hour incubation. Bars are mean ± SD from four independent experiments. *Significantly different from control, P < 0.05.
Mentions: To dissect whether IL-6 stimulates NOX4/Akt signaling, we first examined the IL-6 production in NSCLC cell lines (A549, H460, H358, H441 and HCC827) and normal lung epithelial BEAS2B cells. The results showed that all the NSCLC cell lines and BEAS2B cells produced their own IL-6, and IL-6 production was markedly higher in NSCLC cell lines than that in the normal lung epithelial cells (Fig. 2A). Fig. 2B showed that IL-6 (10 ng/mL) treatment led to a time-dependent increase in NOX4 level in A549 cells. Besides, IL-6 could also enhance ROS production analyzed by DCF assay, as well as the preduction of superoxide and hydrogen peroxide analyzed by amplex red assay, respectively (Fig. 2C) and stimulate Akt activity (Fig. 2D) in a time-dependent manner in these cells.

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