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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

Stable knockdown of miR-21 or STAT3 and overexpression of PDCD4 in AsT cells suppresses tumorigenesis.BEAS-2B cells were maintained in a medium containing arsenic (0.5 μM) for 6 months, and then cells were cultured in 0.35% soft agar for 5 weeks. (A) The arsenic-transformed cells (AsT) from anchorage-independent colonies were selected and maintained in DMEM. Passage-matched cells without arsenic treatment were used as the control. (B) The relative miR-21 level, determined by Taqman real-time PCR, was increased. (C) Total cell lysates were prepared for western blot analysis; apparent PDCD4 protein levels decreased. (D–E) Clonogenic assays were used to determine tumor cell proliferation. Cells (300) from indicated treatments were seeded into each of three dishes (60 mm diameter), and grown for an additional 10 days, then stained with crystal violet. Colony numbers in the entire dish were counted. (D) Images of representative plates for each treatment showing clonogenic activity, and (E) graphic representation of colony counts. Cells from indicated treatments were injected into the flanks of 6-week old athymic nude mice (2 × 106 cells per mouse) and tumor volume was measured after 30 days. (F) Images of representative tumors excised from mice from each treatment group and (G) the associated tumor volumes were obtained. Knockdown of miR-21 or STAT3 and overexpression of PDCD4 inhibited tumor growth. (H) The relative miR-21 level for each treatment, determined by Taqman real-time PCR, was decreased relative to levels obtained from vector control tissues. (I) Representative images immunohistochemical staining show increased PDCD4 protein expression and (J) STAT3 phosphorylation. Data presented in the bar graphs are the mean ± SD of three independent experiments. *#Indicates a statistically significant difference from respective control cells with p < 0.05.
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f8: Stable knockdown of miR-21 or STAT3 and overexpression of PDCD4 in AsT cells suppresses tumorigenesis.BEAS-2B cells were maintained in a medium containing arsenic (0.5 μM) for 6 months, and then cells were cultured in 0.35% soft agar for 5 weeks. (A) The arsenic-transformed cells (AsT) from anchorage-independent colonies were selected and maintained in DMEM. Passage-matched cells without arsenic treatment were used as the control. (B) The relative miR-21 level, determined by Taqman real-time PCR, was increased. (C) Total cell lysates were prepared for western blot analysis; apparent PDCD4 protein levels decreased. (D–E) Clonogenic assays were used to determine tumor cell proliferation. Cells (300) from indicated treatments were seeded into each of three dishes (60 mm diameter), and grown for an additional 10 days, then stained with crystal violet. Colony numbers in the entire dish were counted. (D) Images of representative plates for each treatment showing clonogenic activity, and (E) graphic representation of colony counts. Cells from indicated treatments were injected into the flanks of 6-week old athymic nude mice (2 × 106 cells per mouse) and tumor volume was measured after 30 days. (F) Images of representative tumors excised from mice from each treatment group and (G) the associated tumor volumes were obtained. Knockdown of miR-21 or STAT3 and overexpression of PDCD4 inhibited tumor growth. (H) The relative miR-21 level for each treatment, determined by Taqman real-time PCR, was decreased relative to levels obtained from vector control tissues. (I) Representative images immunohistochemical staining show increased PDCD4 protein expression and (J) STAT3 phosphorylation. Data presented in the bar graphs are the mean ± SD of three independent experiments. *#Indicates a statistically significant difference from respective control cells with p < 0.05.

Mentions: The arsenic-transformed cells (AsT) from anchorage-independent colonies were selected and grown in DMEM (Fig. 8A). Passage-matched cells without arsenic treatment were used as the control. Increased miR-21 levels (Fig. 8B) and decreased PDCD4 expression (Fig. 8C) in AsT cells compared to passage-matched cells was observed. The clonogenic assay (Fig. 8D,E) showed that AsT cells formed more colonies compared to passage-match cells, and that knockdown of miR-21 or STAT3 and that overexpression of PDCD4 in AsT cells significantly (p < 0.05) reduced the colony number. (F–J) We observed a visible tumor formation that progressively increased in size in mice injected with AsT cells compared to passage-match cells, while the tumors were small from mice injected with AsT cells stably knockdown with miR-21 or STAT3 and overexpressing of PDCD4 (Fig. 8F,G). Furthermore, tumors developed from AsT cells with stable knockdown of miR-21 or STAT3 and those overexpressing PDCD4 showed decreased miR-21 levels (Fig. 8H), increased PDCD4 expression (Fig. 8I) and decreased levels of phosphorylated STAT3 (Fig. 8J) compared to those tumors developed from AsT-vector cells.


Oncogenic transformation of human lung bronchial epithelial cells induced by arsenic involves ROS-dependent activation of STAT3-miR-21-PDCD4 mechanism
Stable knockdown of miR-21 or STAT3 and overexpression of PDCD4 in AsT cells suppresses tumorigenesis.BEAS-2B cells were maintained in a medium containing arsenic (0.5 μM) for 6 months, and then cells were cultured in 0.35% soft agar for 5 weeks. (A) The arsenic-transformed cells (AsT) from anchorage-independent colonies were selected and maintained in DMEM. Passage-matched cells without arsenic treatment were used as the control. (B) The relative miR-21 level, determined by Taqman real-time PCR, was increased. (C) Total cell lysates were prepared for western blot analysis; apparent PDCD4 protein levels decreased. (D–E) Clonogenic assays were used to determine tumor cell proliferation. Cells (300) from indicated treatments were seeded into each of three dishes (60 mm diameter), and grown for an additional 10 days, then stained with crystal violet. Colony numbers in the entire dish were counted. (D) Images of representative plates for each treatment showing clonogenic activity, and (E) graphic representation of colony counts. Cells from indicated treatments were injected into the flanks of 6-week old athymic nude mice (2 × 106 cells per mouse) and tumor volume was measured after 30 days. (F) Images of representative tumors excised from mice from each treatment group and (G) the associated tumor volumes were obtained. Knockdown of miR-21 or STAT3 and overexpression of PDCD4 inhibited tumor growth. (H) The relative miR-21 level for each treatment, determined by Taqman real-time PCR, was decreased relative to levels obtained from vector control tissues. (I) Representative images immunohistochemical staining show increased PDCD4 protein expression and (J) STAT3 phosphorylation. Data presented in the bar graphs are the mean ± SD of three independent experiments. *#Indicates a statistically significant difference from respective control cells with p < 0.05.
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Related In: Results  -  Collection

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f8: Stable knockdown of miR-21 or STAT3 and overexpression of PDCD4 in AsT cells suppresses tumorigenesis.BEAS-2B cells were maintained in a medium containing arsenic (0.5 μM) for 6 months, and then cells were cultured in 0.35% soft agar for 5 weeks. (A) The arsenic-transformed cells (AsT) from anchorage-independent colonies were selected and maintained in DMEM. Passage-matched cells without arsenic treatment were used as the control. (B) The relative miR-21 level, determined by Taqman real-time PCR, was increased. (C) Total cell lysates were prepared for western blot analysis; apparent PDCD4 protein levels decreased. (D–E) Clonogenic assays were used to determine tumor cell proliferation. Cells (300) from indicated treatments were seeded into each of three dishes (60 mm diameter), and grown for an additional 10 days, then stained with crystal violet. Colony numbers in the entire dish were counted. (D) Images of representative plates for each treatment showing clonogenic activity, and (E) graphic representation of colony counts. Cells from indicated treatments were injected into the flanks of 6-week old athymic nude mice (2 × 106 cells per mouse) and tumor volume was measured after 30 days. (F) Images of representative tumors excised from mice from each treatment group and (G) the associated tumor volumes were obtained. Knockdown of miR-21 or STAT3 and overexpression of PDCD4 inhibited tumor growth. (H) The relative miR-21 level for each treatment, determined by Taqman real-time PCR, was decreased relative to levels obtained from vector control tissues. (I) Representative images immunohistochemical staining show increased PDCD4 protein expression and (J) STAT3 phosphorylation. Data presented in the bar graphs are the mean ± SD of three independent experiments. *#Indicates a statistically significant difference from respective control cells with p < 0.05.
Mentions: The arsenic-transformed cells (AsT) from anchorage-independent colonies were selected and grown in DMEM (Fig. 8A). Passage-matched cells without arsenic treatment were used as the control. Increased miR-21 levels (Fig. 8B) and decreased PDCD4 expression (Fig. 8C) in AsT cells compared to passage-matched cells was observed. The clonogenic assay (Fig. 8D,E) showed that AsT cells formed more colonies compared to passage-match cells, and that knockdown of miR-21 or STAT3 and that overexpression of PDCD4 in AsT cells significantly (p < 0.05) reduced the colony number. (F–J) We observed a visible tumor formation that progressively increased in size in mice injected with AsT cells compared to passage-match cells, while the tumors were small from mice injected with AsT cells stably knockdown with miR-21 or STAT3 and overexpressing of PDCD4 (Fig. 8F,G). Furthermore, tumors developed from AsT cells with stable knockdown of miR-21 or STAT3 and those overexpressing PDCD4 showed decreased miR-21 levels (Fig. 8H), increased PDCD4 expression (Fig. 8I) and decreased levels of phosphorylated STAT3 (Fig. 8J) compared to those tumors developed from AsT-vector cells.

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