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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.


Arsenic-induced ROS generation and STAT3 activation are essential for the miR-21 increase and PDCD4 suppression.(A–D) BEAS-2B cells with stable overexpression of catalase or their corresponding vehicle vector were exposed to arsenic (0 or 0.5 μM) for 6 months. (A) Catalase overexpression was verified by western blot analysis. (B) The relative miR-21 level was determined by Taqman real-time PCR and decreased with catalase overexpression. (C) Cell lysates were prepared to determine the protein level of PDCD4 by western blot analysis. Apparent PDCD4 levels increased with catalase overexpression. (D) Anchorage-independent colony growth, assessed as previously described, decreased with catalase overexpression. (E–H) p47phox expression was stably knocked down in BEAS-2B cells and exposed to arsenic (0 or 0.5 μM) for 6 months. (E) P47phox knockdown was confirmed by western blot analysis. (F) Relative miR-21 level, determined by Taqman real-time PCR was decreased. (G) Cell lysates were prepared to evaluate PDCD4 protein levels by western blot analysis. An apparent restoration in PDCD4 levels was observed. (H) Anchorage-independent colony growth, assessed as previously described, was also decreased compared with respective vector controls. (I–K) BEAS-2B cells with stable knockdown of STAT3 or their corresponding vehicle vector were exposed to arsenic (0 or 0.5 μM) for 6 months. (I) The Relative miR-21 level, determined by Taqman real-time PCR, decreased compared with respective vector controls. (J) Cell lysates were prepared to determine the protein level of PDCD4 by western blot analysis. Apparent PDCD4 levels increased. (K) Anchorage-independent colony growth, assessed as previously described, decreased compared with respective vector controls. Data presented in the bar graphs are the mean ± SD of three independent experiments. *Indicates a statistically significant difference compared to control with p < 0.05.
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f4: Arsenic-induced ROS generation and STAT3 activation are essential for the miR-21 increase and PDCD4 suppression.(A–D) BEAS-2B cells with stable overexpression of catalase or their corresponding vehicle vector were exposed to arsenic (0 or 0.5 μM) for 6 months. (A) Catalase overexpression was verified by western blot analysis. (B) The relative miR-21 level was determined by Taqman real-time PCR and decreased with catalase overexpression. (C) Cell lysates were prepared to determine the protein level of PDCD4 by western blot analysis. Apparent PDCD4 levels increased with catalase overexpression. (D) Anchorage-independent colony growth, assessed as previously described, decreased with catalase overexpression. (E–H) p47phox expression was stably knocked down in BEAS-2B cells and exposed to arsenic (0 or 0.5 μM) for 6 months. (E) P47phox knockdown was confirmed by western blot analysis. (F) Relative miR-21 level, determined by Taqman real-time PCR was decreased. (G) Cell lysates were prepared to evaluate PDCD4 protein levels by western blot analysis. An apparent restoration in PDCD4 levels was observed. (H) Anchorage-independent colony growth, assessed as previously described, was also decreased compared with respective vector controls. (I–K) BEAS-2B cells with stable knockdown of STAT3 or their corresponding vehicle vector were exposed to arsenic (0 or 0.5 μM) for 6 months. (I) The Relative miR-21 level, determined by Taqman real-time PCR, decreased compared with respective vector controls. (J) Cell lysates were prepared to determine the protein level of PDCD4 by western blot analysis. Apparent PDCD4 levels increased. (K) Anchorage-independent colony growth, assessed as previously described, decreased compared with respective vector controls. Data presented in the bar graphs are the mean ± SD of three independent experiments. *Indicates a statistically significant difference compared to control with p < 0.05.

Mentions: To determine whether ROS plays an important role in chronic arsenic-induced miR-21 increase and PDCD4 suppression, catalase was overexpressed in BEAS-2B cells (Fig. 4A) and treated with arsenic (0.5 μM) for six months. Forced expression of catalase markedly inhibited miR-21 expression (Fig. 4B) and suppressed the PDCD4 reduction (Fig. 4C) induced by chronic arsenic treatment. Importantly, catalase overexpression also decreased the chronic arsenic-induced malignant cell transformation (Fig. 4D). Knockdown of p47phox in BEAS-2B cells (Fig. 4E) noticeably decreased the arsenic-induced increase in miR-21 expression (Fig. 4F) and suppressed the PDCD4 reduction (Fig. 4G). Interestingly, silencing p47phox significantly inhibited chronic arsenic-induced oncogenic transformation (Fig. 4H). These results clearly demonstrate the indispensable role of ROS in regulating miR-21-PDCD4 signaling during chronic arsenic-induced malignant cell transformation.


Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism
Arsenic-induced ROS generation and STAT3 activation are essential for the miR-21 increase and PDCD4 suppression.(A–D) BEAS-2B cells with stable overexpression of catalase or their corresponding vehicle vector were exposed to arsenic (0 or 0.5 μM) for 6 months. (A) Catalase overexpression was verified by western blot analysis. (B) The relative miR-21 level was determined by Taqman real-time PCR and decreased with catalase overexpression. (C) Cell lysates were prepared to determine the protein level of PDCD4 by western blot analysis. Apparent PDCD4 levels increased with catalase overexpression. (D) Anchorage-independent colony growth, assessed as previously described, decreased with catalase overexpression. (E–H) p47phox expression was stably knocked down in BEAS-2B cells and exposed to arsenic (0 or 0.5 μM) for 6 months. (E) P47phox knockdown was confirmed by western blot analysis. (F) Relative miR-21 level, determined by Taqman real-time PCR was decreased. (G) Cell lysates were prepared to evaluate PDCD4 protein levels by western blot analysis. An apparent restoration in PDCD4 levels was observed. (H) Anchorage-independent colony growth, assessed as previously described, was also decreased compared with respective vector controls. (I–K) BEAS-2B cells with stable knockdown of STAT3 or their corresponding vehicle vector were exposed to arsenic (0 or 0.5 μM) for 6 months. (I) The Relative miR-21 level, determined by Taqman real-time PCR, decreased compared with respective vector controls. (J) Cell lysates were prepared to determine the protein level of PDCD4 by western blot analysis. Apparent PDCD4 levels increased. (K) Anchorage-independent colony growth, assessed as previously described, decreased compared with respective vector controls. Data presented in the bar graphs are the mean ± SD of three independent experiments. *Indicates a statistically significant difference compared to control with p < 0.05.
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f4: Arsenic-induced ROS generation and STAT3 activation are essential for the miR-21 increase and PDCD4 suppression.(A–D) BEAS-2B cells with stable overexpression of catalase or their corresponding vehicle vector were exposed to arsenic (0 or 0.5 μM) for 6 months. (A) Catalase overexpression was verified by western blot analysis. (B) The relative miR-21 level was determined by Taqman real-time PCR and decreased with catalase overexpression. (C) Cell lysates were prepared to determine the protein level of PDCD4 by western blot analysis. Apparent PDCD4 levels increased with catalase overexpression. (D) Anchorage-independent colony growth, assessed as previously described, decreased with catalase overexpression. (E–H) p47phox expression was stably knocked down in BEAS-2B cells and exposed to arsenic (0 or 0.5 μM) for 6 months. (E) P47phox knockdown was confirmed by western blot analysis. (F) Relative miR-21 level, determined by Taqman real-time PCR was decreased. (G) Cell lysates were prepared to evaluate PDCD4 protein levels by western blot analysis. An apparent restoration in PDCD4 levels was observed. (H) Anchorage-independent colony growth, assessed as previously described, was also decreased compared with respective vector controls. (I–K) BEAS-2B cells with stable knockdown of STAT3 or their corresponding vehicle vector were exposed to arsenic (0 or 0.5 μM) for 6 months. (I) The Relative miR-21 level, determined by Taqman real-time PCR, decreased compared with respective vector controls. (J) Cell lysates were prepared to determine the protein level of PDCD4 by western blot analysis. Apparent PDCD4 levels increased. (K) Anchorage-independent colony growth, assessed as previously described, decreased compared with respective vector controls. Data presented in the bar graphs are the mean ± SD of three independent experiments. *Indicates a statistically significant difference compared to control with p < 0.05.
Mentions: To determine whether ROS plays an important role in chronic arsenic-induced miR-21 increase and PDCD4 suppression, catalase was overexpressed in BEAS-2B cells (Fig. 4A) and treated with arsenic (0.5 μM) for six months. Forced expression of catalase markedly inhibited miR-21 expression (Fig. 4B) and suppressed the PDCD4 reduction (Fig. 4C) induced by chronic arsenic treatment. Importantly, catalase overexpression also decreased the chronic arsenic-induced malignant cell transformation (Fig. 4D). Knockdown of p47phox in BEAS-2B cells (Fig. 4E) noticeably decreased the arsenic-induced increase in miR-21 expression (Fig. 4F) and suppressed the PDCD4 reduction (Fig. 4G). Interestingly, silencing p47phox significantly inhibited chronic arsenic-induced oncogenic transformation (Fig. 4H). These results clearly demonstrate the indispensable role of ROS in regulating miR-21-PDCD4 signaling during chronic arsenic-induced malignant cell transformation.

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.