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Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism

View Article: PubMed Central - PubMed

ABSTRACT

Arsenic is a well-documented human carcinogen. The present study explored the role of the onco-miR, miR-21 and its target protein, programmed cell death 4 (PDCD4) in arsenic induced malignant cell transformation and tumorigenesis. Our results showed that treatment of human bronchial epithelial (BEAS-2B) cells with arsenic induces ROS through p47phox, one of the NOX subunits that is the key source of arsenic-induced ROS. Arsenic exposure induced an upregulation of miR-21 expression associated with inhibition of PDCD4, and caused malignant cell transformation and tumorigenesis of BEAS-2B cells. Indispensably, STAT3 transcriptional activation by IL-6 is crucial for the arsenic induced miR-21 increase. Upregulated miR-21 levels and suppressed PDCD4 expression was also observed in xenograft tumors generated with chronic arsenic exposed BEAS-2B cells. Stable shut down of miR-21, p47phox or STAT3 and overexpression of PDCD4 or catalase in BEAS-2B cells markedly inhibited the arsenic induced malignant transformation and tumorigenesis. Similarly, silencing of miR-21 or STAT3 and forced expression of PDCD4 in arsenic transformed cells (AsT) also inhibited cell proliferation and tumorigenesis. Furthermore, arsenic suppressed the downstream protein E-cadherin expression and induced β-catenin/TCF-dependent transcription of uPAR and c-Myc. These results indicate that the ROS-STAT3-miR-21-PDCD4 signaling axis plays an important role in arsenic -induced carcinogenesis.

No MeSH data available.


Related in: MedlinePlus

Arsenic increases ROS generation and induces an miR-21-PDCD4 signaling cascade.BEAS-2B cells were exposed to arsenic (0 to 10 μM) for 12 h. Arsenic induced generation of the ROS radicals O2− and H2O2 were identified by DHE (A) and DCFDA (B) staining, respectively. Upper panels show representative images obtained by fluorescence microscopy and the graphs (lower panels) demonstrate fluorescent intensity determined by flow cytometry. (C) Generation of •OH as determined by electron spin resonance. The generation of a 1:2:2:1 quartet ESR signal is shown. (D) NOX activity, measured after 6, 12 and 24 h arsenic exposure, utilized the lucigenin chemiluminescence assay. Activity increased in a time- and dose-dependent manner. (E) Western blot demonstrates an apparent increase in protein levels of the NOX subunit, p47phox with arsenic exposure. (F–H) BEAS-2B cells exposed to increasing concentrations (0–10 μm) of arsenic for 24 h. (F) The relative miR-21 level was determined by Taqman real-time PCR. (G) Immunoblot analysis of PDCD4 protein levels after acute arsenic treatment. Arsenic induced increases in miR-21 and decreases in PDCD4 levels. (H) Representative images of fluorescence immunostaining demonstrate decreased PDCD4 expression with arsenic treatment. Dapi: blue-nuclear; Phalloid: red-cytoplasmic actin; PDCD4, green (I) BEAS-2B cells were transfected with the renilla reporter construct (pGL3-PDCD4_3′-UTR), miR-21 inhibitor (100 nM), negative control (100 nM), or pGL3-promoters and treated with 10 μM arsenic for 6 h. Cellular lysates were subjected to a luciferase reporter analysis as described in Materials and Methods and results are expressed as a relative activity (relative luminescence units, (RLU) normalized to the luciferase activity in the vector control cells without treatment. Arsenic increased the binding of miR-21 to the 3′-UTR of PDCD4. Exogenous addition of ROS inhibitors catalase or NAC inhibited the acute arsenic-induced (J) miR-21 increase and (K) PDCD4 suppression. Data presented in the bar graphs are the mean ± SD of three independent experiments. *Indicates a statistically significant difference from control cells with p < 0.05.
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f1: Arsenic increases ROS generation and induces an miR-21-PDCD4 signaling cascade.BEAS-2B cells were exposed to arsenic (0 to 10 μM) for 12 h. Arsenic induced generation of the ROS radicals O2− and H2O2 were identified by DHE (A) and DCFDA (B) staining, respectively. Upper panels show representative images obtained by fluorescence microscopy and the graphs (lower panels) demonstrate fluorescent intensity determined by flow cytometry. (C) Generation of •OH as determined by electron spin resonance. The generation of a 1:2:2:1 quartet ESR signal is shown. (D) NOX activity, measured after 6, 12 and 24 h arsenic exposure, utilized the lucigenin chemiluminescence assay. Activity increased in a time- and dose-dependent manner. (E) Western blot demonstrates an apparent increase in protein levels of the NOX subunit, p47phox with arsenic exposure. (F–H) BEAS-2B cells exposed to increasing concentrations (0–10 μm) of arsenic for 24 h. (F) The relative miR-21 level was determined by Taqman real-time PCR. (G) Immunoblot analysis of PDCD4 protein levels after acute arsenic treatment. Arsenic induced increases in miR-21 and decreases in PDCD4 levels. (H) Representative images of fluorescence immunostaining demonstrate decreased PDCD4 expression with arsenic treatment. Dapi: blue-nuclear; Phalloid: red-cytoplasmic actin; PDCD4, green (I) BEAS-2B cells were transfected with the renilla reporter construct (pGL3-PDCD4_3′-UTR), miR-21 inhibitor (100 nM), negative control (100 nM), or pGL3-promoters and treated with 10 μM arsenic for 6 h. Cellular lysates were subjected to a luciferase reporter analysis as described in Materials and Methods and results are expressed as a relative activity (relative luminescence units, (RLU) normalized to the luciferase activity in the vector control cells without treatment. Arsenic increased the binding of miR-21 to the 3′-UTR of PDCD4. Exogenous addition of ROS inhibitors catalase or NAC inhibited the acute arsenic-induced (J) miR-21 increase and (K) PDCD4 suppression. Data presented in the bar graphs are the mean ± SD of three independent experiments. *Indicates a statistically significant difference from control cells with p < 0.05.

Mentions: Arsenic -induced ROS production was quantified by flow cytometry using the fluorescent probes DHE and DCFDA. Arsenic exposure dramatically stimulated O2.− and H2O2 generation in BEAS-2B cells, as indicated by an increase of DHE (Fig. 1A) and DCFDA (Fig. 1B) fluorescence intensity, respectively, when levels were compared to those generated from untreated control cells. Our previous studies show that pretreatment with MnTMPyP decreased O2.− generation and catalase decreased H2O2 production, respectively induced by arsenic37. Furthermore, the arsenic-induced •OH generation in BEAS-2B cells was detected by Electron spin resonance (ESR) (Fig. 1C). NOX activity was significantly (p < 0.05) increased in arsenic-treated cells within 6 h and the effect lasted up to 24 h (Fig. 1D). Moreover we found that acute arsenic treatment also increased the expression of p47phox, one of the NOX subunits (Fig. 1E). Previous studies from our group showed that pretreatment with apocynin (NOX inhibitor) decreased arsenic-induced O2.− and H2O2 generation in BEAS-2B cells37. Taken together, these results suggest that arsenic exposure induces ROS production in BEAS-2B cells, and that activation of NOX is required for this ROS generation.


Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism
Arsenic increases ROS generation and induces an miR-21-PDCD4 signaling cascade.BEAS-2B cells were exposed to arsenic (0 to 10 μM) for 12 h. Arsenic induced generation of the ROS radicals O2− and H2O2 were identified by DHE (A) and DCFDA (B) staining, respectively. Upper panels show representative images obtained by fluorescence microscopy and the graphs (lower panels) demonstrate fluorescent intensity determined by flow cytometry. (C) Generation of •OH as determined by electron spin resonance. The generation of a 1:2:2:1 quartet ESR signal is shown. (D) NOX activity, measured after 6, 12 and 24 h arsenic exposure, utilized the lucigenin chemiluminescence assay. Activity increased in a time- and dose-dependent manner. (E) Western blot demonstrates an apparent increase in protein levels of the NOX subunit, p47phox with arsenic exposure. (F–H) BEAS-2B cells exposed to increasing concentrations (0–10 μm) of arsenic for 24 h. (F) The relative miR-21 level was determined by Taqman real-time PCR. (G) Immunoblot analysis of PDCD4 protein levels after acute arsenic treatment. Arsenic induced increases in miR-21 and decreases in PDCD4 levels. (H) Representative images of fluorescence immunostaining demonstrate decreased PDCD4 expression with arsenic treatment. Dapi: blue-nuclear; Phalloid: red-cytoplasmic actin; PDCD4, green (I) BEAS-2B cells were transfected with the renilla reporter construct (pGL3-PDCD4_3′-UTR), miR-21 inhibitor (100 nM), negative control (100 nM), or pGL3-promoters and treated with 10 μM arsenic for 6 h. Cellular lysates were subjected to a luciferase reporter analysis as described in Materials and Methods and results are expressed as a relative activity (relative luminescence units, (RLU) normalized to the luciferase activity in the vector control cells without treatment. Arsenic increased the binding of miR-21 to the 3′-UTR of PDCD4. Exogenous addition of ROS inhibitors catalase or NAC inhibited the acute arsenic-induced (J) miR-21 increase and (K) PDCD4 suppression. Data presented in the bar graphs are the mean ± SD of three independent experiments. *Indicates a statistically significant difference from control cells with p < 0.05.
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f1: Arsenic increases ROS generation and induces an miR-21-PDCD4 signaling cascade.BEAS-2B cells were exposed to arsenic (0 to 10 μM) for 12 h. Arsenic induced generation of the ROS radicals O2− and H2O2 were identified by DHE (A) and DCFDA (B) staining, respectively. Upper panels show representative images obtained by fluorescence microscopy and the graphs (lower panels) demonstrate fluorescent intensity determined by flow cytometry. (C) Generation of •OH as determined by electron spin resonance. The generation of a 1:2:2:1 quartet ESR signal is shown. (D) NOX activity, measured after 6, 12 and 24 h arsenic exposure, utilized the lucigenin chemiluminescence assay. Activity increased in a time- and dose-dependent manner. (E) Western blot demonstrates an apparent increase in protein levels of the NOX subunit, p47phox with arsenic exposure. (F–H) BEAS-2B cells exposed to increasing concentrations (0–10 μm) of arsenic for 24 h. (F) The relative miR-21 level was determined by Taqman real-time PCR. (G) Immunoblot analysis of PDCD4 protein levels after acute arsenic treatment. Arsenic induced increases in miR-21 and decreases in PDCD4 levels. (H) Representative images of fluorescence immunostaining demonstrate decreased PDCD4 expression with arsenic treatment. Dapi: blue-nuclear; Phalloid: red-cytoplasmic actin; PDCD4, green (I) BEAS-2B cells were transfected with the renilla reporter construct (pGL3-PDCD4_3′-UTR), miR-21 inhibitor (100 nM), negative control (100 nM), or pGL3-promoters and treated with 10 μM arsenic for 6 h. Cellular lysates were subjected to a luciferase reporter analysis as described in Materials and Methods and results are expressed as a relative activity (relative luminescence units, (RLU) normalized to the luciferase activity in the vector control cells without treatment. Arsenic increased the binding of miR-21 to the 3′-UTR of PDCD4. Exogenous addition of ROS inhibitors catalase or NAC inhibited the acute arsenic-induced (J) miR-21 increase and (K) PDCD4 suppression. Data presented in the bar graphs are the mean ± SD of three independent experiments. *Indicates a statistically significant difference from control cells with p < 0.05.
Mentions: Arsenic -induced ROS production was quantified by flow cytometry using the fluorescent probes DHE and DCFDA. Arsenic exposure dramatically stimulated O2.− and H2O2 generation in BEAS-2B cells, as indicated by an increase of DHE (Fig. 1A) and DCFDA (Fig. 1B) fluorescence intensity, respectively, when levels were compared to those generated from untreated control cells. Our previous studies show that pretreatment with MnTMPyP decreased O2.− generation and catalase decreased H2O2 production, respectively induced by arsenic37. Furthermore, the arsenic-induced •OH generation in BEAS-2B cells was detected by Electron spin resonance (ESR) (Fig. 1C). NOX activity was significantly (p < 0.05) increased in arsenic-treated cells within 6 h and the effect lasted up to 24 h (Fig. 1D). Moreover we found that acute arsenic treatment also increased the expression of p47phox, one of the NOX subunits (Fig. 1E). Previous studies from our group showed that pretreatment with apocynin (NOX inhibitor) decreased arsenic-induced O2.− and H2O2 generation in BEAS-2B cells37. Taken together, these results suggest that arsenic exposure induces ROS production in BEAS-2B cells, and that activation of NOX is required for this ROS generation.

View Article: PubMed Central - PubMed

ABSTRACT

Arsenic is a well-documented human carcinogen. The present study explored the role of the onco-miR, miR-21 and its target protein, programmed cell death 4 (PDCD4) in arsenic induced malignant cell transformation and tumorigenesis. Our results showed that treatment of human bronchial epithelial (BEAS-2B) cells with arsenic induces ROS through p47phox, one of the NOX subunits that is the key source of arsenic-induced ROS. Arsenic exposure induced an upregulation of miR-21 expression associated with inhibition of PDCD4, and caused malignant cell transformation and tumorigenesis of BEAS-2B cells. Indispensably, STAT3 transcriptional activation by IL-6 is crucial for the arsenic induced miR-21 increase. Upregulated miR-21 levels and suppressed PDCD4 expression was also observed in xenograft tumors generated with chronic arsenic exposed BEAS-2B cells. Stable shut down of miR-21, p47phox or STAT3 and overexpression of PDCD4 or catalase in BEAS-2B cells markedly inhibited the arsenic induced malignant transformation and tumorigenesis. Similarly, silencing of miR-21 or STAT3 and forced expression of PDCD4 in arsenic transformed cells (AsT) also inhibited cell proliferation and tumorigenesis. Furthermore, arsenic suppressed the downstream protein E-cadherin expression and induced &beta;-catenin/TCF-dependent transcription of uPAR and c-Myc. These results indicate that the ROS-STAT3-miR-21-PDCD4 signaling axis plays an important role in arsenic -induced carcinogenesis.

No MeSH data available.


Related in: MedlinePlus