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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 and IL-6 promotes proliferation and survival of A549 cells(A) The effect of NOX4 overexpression on A549 cell proliferation, and effects of inhibition of PI3K/Akt pathway by LY294002 or Wortmannin on NOX4-promoted cell proliferation. The proliferation of cells was evaluated using colony formation assay. Bars are mean ± SD from three independent experiments. *Significantly different from vector control, P < 0.05. (B-C) A549 cells were transfected with NOX4 shRNA, Akt siRNA or NOX4 shRNA plus Akt siRNA, respectively. The proliferation of cells was evaluated using colony formation assay. Cell apoptosis was confirmed by flow cytometry analysis. Bars are mean ± SD from four to five independent experiments. *Significantly different from vector control, P < 0.05. (D) The effects of NOX4 overexpression or knockdown on Akt activity after 24 hours determined by western blotting. (E) The effect of IL-6 on cell proliferation, and the influence of IL-6 neutralizing antibody siltuximab or JAKs inhibitor P6 on IL-6-mediated proliferation in A549 cells. Bars are mean ± SD from four independent experiments. *Significantly different from control, P < 0.05. (F) The effects of siltuximab and P6 on cell apoptosis after 48-hour incubation. Bars are mean ± SD from five independent experiments. *Significantly different from control, P < 0.05. (G) The effects of IL-6 on JAK1/STAT3 activity determined by western blotting.
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Figure 4: NOX4 and IL-6 promotes proliferation and survival of A549 cells(A) The effect of NOX4 overexpression on A549 cell proliferation, and effects of inhibition of PI3K/Akt pathway by LY294002 or Wortmannin on NOX4-promoted cell proliferation. The proliferation of cells was evaluated using colony formation assay. Bars are mean ± SD from three independent experiments. *Significantly different from vector control, P < 0.05. (B-C) A549 cells were transfected with NOX4 shRNA, Akt siRNA or NOX4 shRNA plus Akt siRNA, respectively. The proliferation of cells was evaluated using colony formation assay. Cell apoptosis was confirmed by flow cytometry analysis. Bars are mean ± SD from four to five independent experiments. *Significantly different from vector control, P < 0.05. (D) The effects of NOX4 overexpression or knockdown on Akt activity after 24 hours determined by western blotting. (E) The effect of IL-6 on cell proliferation, and the influence of IL-6 neutralizing antibody siltuximab or JAKs inhibitor P6 on IL-6-mediated proliferation in A549 cells. Bars are mean ± SD from four independent experiments. *Significantly different from control, P < 0.05. (F) The effects of siltuximab and P6 on cell apoptosis after 48-hour incubation. Bars are mean ± SD from five independent experiments. *Significantly different from control, P < 0.05. (G) The effects of IL-6 on JAK1/STAT3 activity determined by western blotting.

Mentions: As Fig. 4A showed, NOX4 overexpression could substantially increase the anchorage-dependent growth of A549 cells. Treatment of NOX4-transduced A549 cells with a highly selective PI3K/Akt pathway inhibitor, LY294002 (30 μM), could block the enhancement effect of NOX4 on cell growth. Comparable data were also obtained from cells treated with another PI3K/Akt pathway inhibitor, wartmannin (10 μM). However, inhibition of MEK/Erk pathway by PD98509 (30 μM) had no significant effects on NOX4-mediated growth of A549 cells (data not shown). Further studies indicated that either depletion of NOX4 by its specific shRNA or knockdown of Akt by Akt siRNA substantially suppressed A549 cell growth, and when Akt expression was depleted, NOX4 shRNA had no additional inhibitory effect on A549 cell growth (Fig. 4B). Next we performed flow cytometry assay to determine the role of NOX4 in cell apoptosis. The results indicated that knockdown of either NOX4 or Akt increased A549 cell apoptosis after 48 hours, and NOX4 depletion had no additional enhancement effect on cell apoptosis compared with Akt knockdown alone (Fig. 4C). Fig. 4D showed that NOX4 overexpression significantly stimulated Akt activity, while NOX4 depletion caused a reduction in Akt activity in A549 cells. Therefore, these results demonstrate that NOX4 promotes A549 cell growth and survival via PI3K/Akt pathway-dependent manner.


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 and IL-6 promotes proliferation and survival of A549 cells(A) The effect of NOX4 overexpression on A549 cell proliferation, and effects of inhibition of PI3K/Akt pathway by LY294002 or Wortmannin on NOX4-promoted cell proliferation. The proliferation of cells was evaluated using colony formation assay. Bars are mean ± SD from three independent experiments. *Significantly different from vector control, P < 0.05. (B-C) A549 cells were transfected with NOX4 shRNA, Akt siRNA or NOX4 shRNA plus Akt siRNA, respectively. The proliferation of cells was evaluated using colony formation assay. Cell apoptosis was confirmed by flow cytometry analysis. Bars are mean ± SD from four to five independent experiments. *Significantly different from vector control, P < 0.05. (D) The effects of NOX4 overexpression or knockdown on Akt activity after 24 hours determined by western blotting. (E) The effect of IL-6 on cell proliferation, and the influence of IL-6 neutralizing antibody siltuximab or JAKs inhibitor P6 on IL-6-mediated proliferation in A549 cells. Bars are mean ± SD from four independent experiments. *Significantly different from control, P < 0.05. (F) The effects of siltuximab and P6 on cell apoptosis after 48-hour incubation. Bars are mean ± SD from five independent experiments. *Significantly different from control, P < 0.05. (G) The effects of IL-6 on JAK1/STAT3 activity determined by western blotting.
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Figure 4: NOX4 and IL-6 promotes proliferation and survival of A549 cells(A) The effect of NOX4 overexpression on A549 cell proliferation, and effects of inhibition of PI3K/Akt pathway by LY294002 or Wortmannin on NOX4-promoted cell proliferation. The proliferation of cells was evaluated using colony formation assay. Bars are mean ± SD from three independent experiments. *Significantly different from vector control, P < 0.05. (B-C) A549 cells were transfected with NOX4 shRNA, Akt siRNA or NOX4 shRNA plus Akt siRNA, respectively. The proliferation of cells was evaluated using colony formation assay. Cell apoptosis was confirmed by flow cytometry analysis. Bars are mean ± SD from four to five independent experiments. *Significantly different from vector control, P < 0.05. (D) The effects of NOX4 overexpression or knockdown on Akt activity after 24 hours determined by western blotting. (E) The effect of IL-6 on cell proliferation, and the influence of IL-6 neutralizing antibody siltuximab or JAKs inhibitor P6 on IL-6-mediated proliferation in A549 cells. Bars are mean ± SD from four independent experiments. *Significantly different from control, P < 0.05. (F) The effects of siltuximab and P6 on cell apoptosis after 48-hour incubation. Bars are mean ± SD from five independent experiments. *Significantly different from control, P < 0.05. (G) The effects of IL-6 on JAK1/STAT3 activity determined by western blotting.
Mentions: As Fig. 4A showed, NOX4 overexpression could substantially increase the anchorage-dependent growth of A549 cells. Treatment of NOX4-transduced A549 cells with a highly selective PI3K/Akt pathway inhibitor, LY294002 (30 μM), could block the enhancement effect of NOX4 on cell growth. Comparable data were also obtained from cells treated with another PI3K/Akt pathway inhibitor, wartmannin (10 μM). However, inhibition of MEK/Erk pathway by PD98509 (30 μM) had no significant effects on NOX4-mediated growth of A549 cells (data not shown). Further studies indicated that either depletion of NOX4 by its specific shRNA or knockdown of Akt by Akt siRNA substantially suppressed A549 cell growth, and when Akt expression was depleted, NOX4 shRNA had no additional inhibitory effect on A549 cell growth (Fig. 4B). Next we performed flow cytometry assay to determine the role of NOX4 in cell apoptosis. The results indicated that knockdown of either NOX4 or Akt increased A549 cell apoptosis after 48 hours, and NOX4 depletion had no additional enhancement effect on cell apoptosis compared with Akt knockdown alone (Fig. 4C). Fig. 4D showed that NOX4 overexpression significantly stimulated Akt activity, while NOX4 depletion caused a reduction in Akt activity in A549 cells. Therefore, these results demonstrate that NOX4 promotes A549 cell growth and survival via PI3K/Akt pathway-dependent manner.

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