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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.

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The arsenic-induced miR-21 increase and PDCD4 suppression contribute to malignant cell transformation.BEAS-2B cells were maintained in a medium containing various concentrations of arsenic (0.1, 0.25 and 0.5 μM) for 6 months. (A,B) Cells were cultured in 0.35% soft agar for 5 weeks and number of colonies in the entire dish counted. (A) Representative images of control (left panel) and arsenic-treated (right panel) colonies. (B) Colony number increased in a dose-dependent manner. (C) The relative miR-21 level, determined by Taqman real-time PCR, increased in a time- and dose-dependent manner. (D) Total cell lysates were prepared for western blot analysis after 2, 4 and 6 months exposure to arsenic using specific antibodies against PDCD4, p47phox, pSTAT3 and STAT3. Apparent protein levels for PDCD4 decreased and P47phox and pSTAT3 increased in a time- and dose-dependent manner. (E) Representative images of fluorescence immunostaining for PDCD4 and pSTAT3 after 2, 4 and 6 months exposure to arsenic, and confirm results from western blot analysis. 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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f3: The arsenic-induced miR-21 increase and PDCD4 suppression contribute to malignant cell transformation.BEAS-2B cells were maintained in a medium containing various concentrations of arsenic (0.1, 0.25 and 0.5 μM) for 6 months. (A,B) Cells were cultured in 0.35% soft agar for 5 weeks and number of colonies in the entire dish counted. (A) Representative images of control (left panel) and arsenic-treated (right panel) colonies. (B) Colony number increased in a dose-dependent manner. (C) The relative miR-21 level, determined by Taqman real-time PCR, increased in a time- and dose-dependent manner. (D) Total cell lysates were prepared for western blot analysis after 2, 4 and 6 months exposure to arsenic using specific antibodies against PDCD4, p47phox, pSTAT3 and STAT3. Apparent protein levels for PDCD4 decreased and P47phox and pSTAT3 increased in a time- and dose-dependent manner. (E) Representative images of fluorescence immunostaining for PDCD4 and pSTAT3 after 2, 4 and 6 months exposure to arsenic, and confirm results from western blot analysis. 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: Malignant cell transformation was assessed by anchorage-independent growth in soft agar39. BEAS-2B cells were treated with selected concentrations (0.125, 0.25 and 0.5 μM) of arsenic for six months. Chronic exposure (6 months) to low concentrations of arsenic (0.1, 0.25 and 0.5 μM) induced malignant transformation of BEAS-2B cells as shown by the marked increase in size and number of colonies compared to untreated control (Fig. 3A,B). The chronic treatment of arsenic significantly (p < 0.05) increased miR-21 levels in a time and dose dependent manner (Fig. 3C). We also found a dose-dependent and drastic decrease in the PDCD4 expression and increase in the p47phox expression and STAT3 phosphorylation with chronic arsenic exposure (Fig. 3D). Similar results were observed by immunofluorescence; BEAS-2B cells treated with arsenic (0.5 μM) for six months showed a marked decrease in the PDCD4 level and increase in the STAT3 phosphorylation (Fig. 3E). The above results demonstrate a role for miR-21-PDCD4 signaling in arsenic-induced transformation.


Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism
The arsenic-induced miR-21 increase and PDCD4 suppression contribute to malignant cell transformation.BEAS-2B cells were maintained in a medium containing various concentrations of arsenic (0.1, 0.25 and 0.5 μM) for 6 months. (A,B) Cells were cultured in 0.35% soft agar for 5 weeks and number of colonies in the entire dish counted. (A) Representative images of control (left panel) and arsenic-treated (right panel) colonies. (B) Colony number increased in a dose-dependent manner. (C) The relative miR-21 level, determined by Taqman real-time PCR, increased in a time- and dose-dependent manner. (D) Total cell lysates were prepared for western blot analysis after 2, 4 and 6 months exposure to arsenic using specific antibodies against PDCD4, p47phox, pSTAT3 and STAT3. Apparent protein levels for PDCD4 decreased and P47phox and pSTAT3 increased in a time- and dose-dependent manner. (E) Representative images of fluorescence immunostaining for PDCD4 and pSTAT3 after 2, 4 and 6 months exposure to arsenic, and confirm results from western blot analysis. 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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Related In: Results  -  Collection

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f3: The arsenic-induced miR-21 increase and PDCD4 suppression contribute to malignant cell transformation.BEAS-2B cells were maintained in a medium containing various concentrations of arsenic (0.1, 0.25 and 0.5 μM) for 6 months. (A,B) Cells were cultured in 0.35% soft agar for 5 weeks and number of colonies in the entire dish counted. (A) Representative images of control (left panel) and arsenic-treated (right panel) colonies. (B) Colony number increased in a dose-dependent manner. (C) The relative miR-21 level, determined by Taqman real-time PCR, increased in a time- and dose-dependent manner. (D) Total cell lysates were prepared for western blot analysis after 2, 4 and 6 months exposure to arsenic using specific antibodies against PDCD4, p47phox, pSTAT3 and STAT3. Apparent protein levels for PDCD4 decreased and P47phox and pSTAT3 increased in a time- and dose-dependent manner. (E) Representative images of fluorescence immunostaining for PDCD4 and pSTAT3 after 2, 4 and 6 months exposure to arsenic, and confirm results from western blot analysis. 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: Malignant cell transformation was assessed by anchorage-independent growth in soft agar39. BEAS-2B cells were treated with selected concentrations (0.125, 0.25 and 0.5 μM) of arsenic for six months. Chronic exposure (6 months) to low concentrations of arsenic (0.1, 0.25 and 0.5 μM) induced malignant transformation of BEAS-2B cells as shown by the marked increase in size and number of colonies compared to untreated control (Fig. 3A,B). The chronic treatment of arsenic significantly (p < 0.05) increased miR-21 levels in a time and dose dependent manner (Fig. 3C). We also found a dose-dependent and drastic decrease in the PDCD4 expression and increase in the p47phox expression and STAT3 phosphorylation with chronic arsenic exposure (Fig. 3D). Similar results were observed by immunofluorescence; BEAS-2B cells treated with arsenic (0.5 μM) for six months showed a marked decrease in the PDCD4 level and increase in the STAT3 phosphorylation (Fig. 3E). The above results demonstrate a role for miR-21-PDCD4 signaling in arsenic-induced 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.


Related in: MedlinePlus