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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 interplays with IL-6 to regulate A549 cell proliferation and survival in vivo(A-B) A549 cells stably expressing NOX4 or its control vector, or stably transfecting NOX4 shRNA or its scramble shRNA were transplanted into nude mice (n = 10 per group), respectively. After seven days, siltuximab (10 mg/kg, twice a week, i. p.) or IL-6 (25 μg/Kg, three times a week, sc) was administrated, respectively. Tumor size was measured every 2 days for indicated period. The representative tumors and growth curves of tumor are shown. (C) The positive staining of Ki67 expression per field from paraffin-embedded sections of NOX-transfected A549 cells or those treated with siltuximab determined by immunohistochemistry and morphometric quantification (n = 10 per group). All scale bars represent 50 μm. *Significantly different from control, P < 0.05. #Significantly different from NOX4 or NOX4 shRNA group, respectively, P < 0.05. (D) Apoptotic nuclei with fragmented DNA were detected by TUNEL staining, and further analyzed by morphometric quantification. The final statistical graphs represent 10 non-overlapping images of each tumor specimens (n = 10 per group). *Significantly different from control, P < 0.05. # Significantly different from NOX4 shRNA group, P < 0.05.
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Figure 8: NOX4 interplays with IL-6 to regulate A549 cell proliferation and survival in vivo(A-B) A549 cells stably expressing NOX4 or its control vector, or stably transfecting NOX4 shRNA or its scramble shRNA were transplanted into nude mice (n = 10 per group), respectively. After seven days, siltuximab (10 mg/kg, twice a week, i. p.) or IL-6 (25 μg/Kg, three times a week, sc) was administrated, respectively. Tumor size was measured every 2 days for indicated period. The representative tumors and growth curves of tumor are shown. (C) The positive staining of Ki67 expression per field from paraffin-embedded sections of NOX-transfected A549 cells or those treated with siltuximab determined by immunohistochemistry and morphometric quantification (n = 10 per group). All scale bars represent 50 μm. *Significantly different from control, P < 0.05. #Significantly different from NOX4 or NOX4 shRNA group, respectively, P < 0.05. (D) Apoptotic nuclei with fragmented DNA were detected by TUNEL staining, and further analyzed by morphometric quantification. The final statistical graphs represent 10 non-overlapping images of each tumor specimens (n = 10 per group). *Significantly different from control, P < 0.05. # Significantly different from NOX4 shRNA group, P < 0.05.

Mentions: We further confirmed the functional interplay of NOX4 and IL-6 in the tumorigenecity of NSCLC cells in vivo. A549 cells (approximately 1 × 106 cells) were subcutaneously inoculated into the right flank of 6-week-old female nude mice. As shown in Fig. 8A, tumors formed by NOX4-transduced A549 cells grew faster than vector-control tumors, and treatment with siltuximab partially reversed such effect. Conversely, NOX4 shRNA-transfected A549 cells produced smaller tumors compared with scramble, and additional IL-6 administration could significantly rescue tumor growth (Fig. 8B). Furthermore, the functional interplay of NOX4 and IL-6 in enhancing growth and survival of NSCLC cells in vivo was also confirmed by Ki67 (the biomarker to evaluate tumor proliferation) (Fig. 8C) and TUNEL staining analysis (evaluation of tumor apoptosis) (Fig. 8D).


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 interplays with IL-6 to regulate A549 cell proliferation and survival in vivo(A-B) A549 cells stably expressing NOX4 or its control vector, or stably transfecting NOX4 shRNA or its scramble shRNA were transplanted into nude mice (n = 10 per group), respectively. After seven days, siltuximab (10 mg/kg, twice a week, i. p.) or IL-6 (25 μg/Kg, three times a week, sc) was administrated, respectively. Tumor size was measured every 2 days for indicated period. The representative tumors and growth curves of tumor are shown. (C) The positive staining of Ki67 expression per field from paraffin-embedded sections of NOX-transfected A549 cells or those treated with siltuximab determined by immunohistochemistry and morphometric quantification (n = 10 per group). All scale bars represent 50 μm. *Significantly different from control, P < 0.05. #Significantly different from NOX4 or NOX4 shRNA group, respectively, P < 0.05. (D) Apoptotic nuclei with fragmented DNA were detected by TUNEL staining, and further analyzed by morphometric quantification. The final statistical graphs represent 10 non-overlapping images of each tumor specimens (n = 10 per group). *Significantly different from control, P < 0.05. # Significantly different from NOX4 shRNA group, P < 0.05.
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Figure 8: NOX4 interplays with IL-6 to regulate A549 cell proliferation and survival in vivo(A-B) A549 cells stably expressing NOX4 or its control vector, or stably transfecting NOX4 shRNA or its scramble shRNA were transplanted into nude mice (n = 10 per group), respectively. After seven days, siltuximab (10 mg/kg, twice a week, i. p.) or IL-6 (25 μg/Kg, three times a week, sc) was administrated, respectively. Tumor size was measured every 2 days for indicated period. The representative tumors and growth curves of tumor are shown. (C) The positive staining of Ki67 expression per field from paraffin-embedded sections of NOX-transfected A549 cells or those treated with siltuximab determined by immunohistochemistry and morphometric quantification (n = 10 per group). All scale bars represent 50 μm. *Significantly different from control, P < 0.05. #Significantly different from NOX4 or NOX4 shRNA group, respectively, P < 0.05. (D) Apoptotic nuclei with fragmented DNA were detected by TUNEL staining, and further analyzed by morphometric quantification. The final statistical graphs represent 10 non-overlapping images of each tumor specimens (n = 10 per group). *Significantly different from control, P < 0.05. # Significantly different from NOX4 shRNA group, P < 0.05.
Mentions: We further confirmed the functional interplay of NOX4 and IL-6 in the tumorigenecity of NSCLC cells in vivo. A549 cells (approximately 1 × 106 cells) were subcutaneously inoculated into the right flank of 6-week-old female nude mice. As shown in Fig. 8A, tumors formed by NOX4-transduced A549 cells grew faster than vector-control tumors, and treatment with siltuximab partially reversed such effect. Conversely, NOX4 shRNA-transfected A549 cells produced smaller tumors compared with scramble, and additional IL-6 administration could significantly rescue tumor growth (Fig. 8B). Furthermore, the functional interplay of NOX4 and IL-6 in enhancing growth and survival of NSCLC cells in vivo was also confirmed by Ki67 (the biomarker to evaluate tumor proliferation) (Fig. 8C) and TUNEL staining analysis (evaluation of tumor apoptosis) (Fig. 8D).

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