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NOX1 to NOX2 switch deactivates AMPK and induces invasive phenotype in colon cancer cells through overexpression of MMP-7.

Banskota S, Regmi SC, Kim JA - Mol. Cancer (2015)

Bottom Line: TPA-induced invasive phenotype in HT29 cells was abolished by treatment with Vit.E, DPI, apocynin, and NOX2 siRNA but not NOX1 siRNA, indicating NOX2-derived ROS production induced an invasive phenotype.TPA-induced induction of MMP-7 expression was suppressed by AP-1, NF-κB, and MAPK (ERK, p38, and JNK) inhibitors, whereas TPA-induced expression of NOX2 and its regulators, p47phox and p67phox, was blocked by p38 and NF-κB inhibitors.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Yeungnam University, Gyeongsan, 712-749, South Korea. suhrid@ynu.ac.kr.

ABSTRACT

Background: Although matrix metalloproteinase (MMP)-7 expression is correlated with increased metastatic potential in human colon cancer cells, the underlying molecular mechanism of invasive phenotype remains unknown. In the current study, we investigated the regulatory effects of membrane NADPH oxidase (NOX) and AMP activated protein kinase (AMPK) on MMP-7 expression and invasive phenotype change in colon cancer cells.

Methods: Production of superoxide anion was measured by lucigenin chemiluminescence assay using whole cells and protein extracts (NADPH oxidase activity), and intracellular reactive oxygen species (ROS) by fluorescence microscopy using 2',7'-dichlorofluorescein diacetate (DCF-DA). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to measure mRNA and protein levels, respectively. siRNA transfection was used to assess involvement of genes in cancer invasion, which were identified by Matrigel transwell invasion assay. Luciferase reporter assay was performed to identify transcription factors linked to gene expression.

Results: Under basal conditions, less invasive human colon cancer cells (HT29 and Caco-2) showed low MMP-7 expression but high NOX1 expression and AMPK phosphorylation. Treatment of HT29 and Caco-2 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced an invasive phenotype response along with corresponding increases in ROS production and NOX2 and MMP-7 expression as well as reduced AMPK phosphorylation, which resemble basal conditions of highly invasive human colon cancer cells (SW620 and HCT116). In addition, inverse regulation between AMPK phosphorylation and NOX2 and MMP-7 expression was observed in HT29 cells treated with different concentrations of exogenous hydrogen peroxide. TPA-induced invasive phenotype in HT29 cells was abolished by treatment with Vit. E, DPI, apocynin, and NOX2 siRNA but not NOX1 siRNA, indicating NOX2-derived ROS production induced an invasive phenotype. TPA-induced induction of MMP-7 expression was suppressed by AP-1, NF-κB, and MAPK (ERK, p38, and JNK) inhibitors, whereas TPA-induced expression of NOX2 and its regulators, p47phox and p67phox, was blocked by p38 and NF-κB inhibitors.

Conclusions: Molecular switch from NOX1 to NOX2 in colon cancer cells induces ROS production and subsequently enhances MMP-7 expression by deactivating AMPK, which otherwise inhibits stimulus-induced autoregulation of ROS and NOX2 gene expression.

No MeSH data available.


Related in: MedlinePlus

Reduced AMPK phosphorylation corresponds to increased ROS production and invasion of TPA-treated HT29 cells, which is reversed by AMPK activators. a Basal AMPK phosphorylation was compared between HT29 and SW620 cells. The bar graph represents the mean ± SEM of phospho-AMPK/AMPK from three independent experiments. *P < 0.05 compared to HT29 cells. b Protein extracts from TPA-treated HT29 cells were examined for phospho-AMPK by Western blotting. c HT29 cells were treated with AMPK activators, AICAR (250 μM) and D942 (10 μM), in the presence or absence of TPA for 1 h. Cells were then examined for ROS production (upper panel) by DCF fluorescence microscopy and invasion (lower panel). d Superoxide anion production was measured by lucigenin chemiluminescence assay, and invasion was quantitated by counting cells on the bottom of the insert. The bar graph represents the mean ± SEM from three independent experiments. *P < 0.05 compared to vehicle-treated control group. #P < 0.05 compared to TPA-treated cells
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Fig5: Reduced AMPK phosphorylation corresponds to increased ROS production and invasion of TPA-treated HT29 cells, which is reversed by AMPK activators. a Basal AMPK phosphorylation was compared between HT29 and SW620 cells. The bar graph represents the mean ± SEM of phospho-AMPK/AMPK from three independent experiments. *P < 0.05 compared to HT29 cells. b Protein extracts from TPA-treated HT29 cells were examined for phospho-AMPK by Western blotting. c HT29 cells were treated with AMPK activators, AICAR (250 μM) and D942 (10 μM), in the presence or absence of TPA for 1 h. Cells were then examined for ROS production (upper panel) by DCF fluorescence microscopy and invasion (lower panel). d Superoxide anion production was measured by lucigenin chemiluminescence assay, and invasion was quantitated by counting cells on the bottom of the insert. The bar graph represents the mean ± SEM from three independent experiments. *P < 0.05 compared to vehicle-treated control group. #P < 0.05 compared to TPA-treated cells

Mentions: We also investigated AMPK activity in response to ROS during colon cancer cell invasion. Under serum-starved conditions, AMPK phosphorylation was higher in HT29 cells than in SW620 cells (Fig. 5a). TPA treatment inhibited AMPK phosphorylation in HT-29 cells (Fig. 5b) starting from 5 min after treatment, which is in line with increased ROS production (Fig. 2a and g) and subsequent invasion. In SW620 cells, reduction of AMPK phosphorylation by TPA occurred in a similar pattern compared to that in HT29 cells. In addition, pretreatment with AICAR and D942 (AMPK activators) significantly blocked TPA-induced ROS production and cell invasion (Fig. 5c and d). On the other hand, treatment with AICAR or D942 alone without TPA did not cause any change in ROS production or cell invasion.Fig. 5


NOX1 to NOX2 switch deactivates AMPK and induces invasive phenotype in colon cancer cells through overexpression of MMP-7.

Banskota S, Regmi SC, Kim JA - Mol. Cancer (2015)

Reduced AMPK phosphorylation corresponds to increased ROS production and invasion of TPA-treated HT29 cells, which is reversed by AMPK activators. a Basal AMPK phosphorylation was compared between HT29 and SW620 cells. The bar graph represents the mean ± SEM of phospho-AMPK/AMPK from three independent experiments. *P < 0.05 compared to HT29 cells. b Protein extracts from TPA-treated HT29 cells were examined for phospho-AMPK by Western blotting. c HT29 cells were treated with AMPK activators, AICAR (250 μM) and D942 (10 μM), in the presence or absence of TPA for 1 h. Cells were then examined for ROS production (upper panel) by DCF fluorescence microscopy and invasion (lower panel). d Superoxide anion production was measured by lucigenin chemiluminescence assay, and invasion was quantitated by counting cells on the bottom of the insert. The bar graph represents the mean ± SEM from three independent experiments. *P < 0.05 compared to vehicle-treated control group. #P < 0.05 compared to TPA-treated cells
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4482031&req=5

Fig5: Reduced AMPK phosphorylation corresponds to increased ROS production and invasion of TPA-treated HT29 cells, which is reversed by AMPK activators. a Basal AMPK phosphorylation was compared between HT29 and SW620 cells. The bar graph represents the mean ± SEM of phospho-AMPK/AMPK from three independent experiments. *P < 0.05 compared to HT29 cells. b Protein extracts from TPA-treated HT29 cells were examined for phospho-AMPK by Western blotting. c HT29 cells were treated with AMPK activators, AICAR (250 μM) and D942 (10 μM), in the presence or absence of TPA for 1 h. Cells were then examined for ROS production (upper panel) by DCF fluorescence microscopy and invasion (lower panel). d Superoxide anion production was measured by lucigenin chemiluminescence assay, and invasion was quantitated by counting cells on the bottom of the insert. The bar graph represents the mean ± SEM from three independent experiments. *P < 0.05 compared to vehicle-treated control group. #P < 0.05 compared to TPA-treated cells
Mentions: We also investigated AMPK activity in response to ROS during colon cancer cell invasion. Under serum-starved conditions, AMPK phosphorylation was higher in HT29 cells than in SW620 cells (Fig. 5a). TPA treatment inhibited AMPK phosphorylation in HT-29 cells (Fig. 5b) starting from 5 min after treatment, which is in line with increased ROS production (Fig. 2a and g) and subsequent invasion. In SW620 cells, reduction of AMPK phosphorylation by TPA occurred in a similar pattern compared to that in HT29 cells. In addition, pretreatment with AICAR and D942 (AMPK activators) significantly blocked TPA-induced ROS production and cell invasion (Fig. 5c and d). On the other hand, treatment with AICAR or D942 alone without TPA did not cause any change in ROS production or cell invasion.Fig. 5

Bottom Line: TPA-induced invasive phenotype in HT29 cells was abolished by treatment with Vit.E, DPI, apocynin, and NOX2 siRNA but not NOX1 siRNA, indicating NOX2-derived ROS production induced an invasive phenotype.TPA-induced induction of MMP-7 expression was suppressed by AP-1, NF-κB, and MAPK (ERK, p38, and JNK) inhibitors, whereas TPA-induced expression of NOX2 and its regulators, p47phox and p67phox, was blocked by p38 and NF-κB inhibitors.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Yeungnam University, Gyeongsan, 712-749, South Korea. suhrid@ynu.ac.kr.

ABSTRACT

Background: Although matrix metalloproteinase (MMP)-7 expression is correlated with increased metastatic potential in human colon cancer cells, the underlying molecular mechanism of invasive phenotype remains unknown. In the current study, we investigated the regulatory effects of membrane NADPH oxidase (NOX) and AMP activated protein kinase (AMPK) on MMP-7 expression and invasive phenotype change in colon cancer cells.

Methods: Production of superoxide anion was measured by lucigenin chemiluminescence assay using whole cells and protein extracts (NADPH oxidase activity), and intracellular reactive oxygen species (ROS) by fluorescence microscopy using 2',7'-dichlorofluorescein diacetate (DCF-DA). Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting were used to measure mRNA and protein levels, respectively. siRNA transfection was used to assess involvement of genes in cancer invasion, which were identified by Matrigel transwell invasion assay. Luciferase reporter assay was performed to identify transcription factors linked to gene expression.

Results: Under basal conditions, less invasive human colon cancer cells (HT29 and Caco-2) showed low MMP-7 expression but high NOX1 expression and AMPK phosphorylation. Treatment of HT29 and Caco-2 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced an invasive phenotype response along with corresponding increases in ROS production and NOX2 and MMP-7 expression as well as reduced AMPK phosphorylation, which resemble basal conditions of highly invasive human colon cancer cells (SW620 and HCT116). In addition, inverse regulation between AMPK phosphorylation and NOX2 and MMP-7 expression was observed in HT29 cells treated with different concentrations of exogenous hydrogen peroxide. TPA-induced invasive phenotype in HT29 cells was abolished by treatment with Vit. E, DPI, apocynin, and NOX2 siRNA but not NOX1 siRNA, indicating NOX2-derived ROS production induced an invasive phenotype. TPA-induced induction of MMP-7 expression was suppressed by AP-1, NF-κB, and MAPK (ERK, p38, and JNK) inhibitors, whereas TPA-induced expression of NOX2 and its regulators, p47phox and p67phox, was blocked by p38 and NF-κB inhibitors.

Conclusions: Molecular switch from NOX1 to NOX2 in colon cancer cells induces ROS production and subsequently enhances MMP-7 expression by deactivating AMPK, which otherwise inhibits stimulus-induced autoregulation of ROS and NOX2 gene expression.

No MeSH data available.


Related in: MedlinePlus