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NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract.

Liu X, Togo S, Al-Mugotir M, Kim H, Fang Q, Kobayashi T, Wang X, Mao L, Bitterman P, Rennard S - Respir. Res. (2008)

Bottom Line: Inhibition of NF-kappaB by the pharmacologic inhibitor curcumin (20 microM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure.The current study demonstrates that CSE activates NF-kappaB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65.These results suggest that activation of NF-kappaB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA. srennard@unmc.edu.

ABSTRACT

Background: We have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-kappaB is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-kappaB in mediating cell survival in response to cigarette smoke exposure in HBECs.

Methods: Both the pharmacologic inhibitor of NF-kappaB, curcumin, and RNA interference targeting p65 were used to block NF-kappaB signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay.

Results: Cigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-kappaB -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-kappaB by the pharmacologic inhibitor curcumin (20 microM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium.

Conclusion: The current study demonstrates that CSE activates NF-kappaB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-kappaB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.

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Related in: MedlinePlus

Effect of NF-κB inhibitor on the viability of HBEC exposed to cigarette smoke extract. Cells were pre-treated with 20 μM curcumin for 30 minutes followed by exposure to 10% cigarette smoke extract for 24 hours in the presence or absence of curcumin. Cell viability and cytotoxicity were determined by LIVE/DEAD assay. Panel A: One representative micro-photograph of LIVE/DEAD staining. Green: live cells; Red: dead cells. Magnification: 200×. Panel B: Quantitative data from 3 separate experiments. Vertical axis: percent of dead cells = red cells/(green cells + read cells) * 100%. Horizontal axis: treatment.
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Figure 3: Effect of NF-κB inhibitor on the viability of HBEC exposed to cigarette smoke extract. Cells were pre-treated with 20 μM curcumin for 30 minutes followed by exposure to 10% cigarette smoke extract for 24 hours in the presence or absence of curcumin. Cell viability and cytotoxicity were determined by LIVE/DEAD assay. Panel A: One representative micro-photograph of LIVE/DEAD staining. Green: live cells; Red: dead cells. Magnification: 200×. Panel B: Quantitative data from 3 separate experiments. Vertical axis: percent of dead cells = red cells/(green cells + read cells) * 100%. Horizontal axis: treatment.

Mentions: To investigate the role of NF-κB in modulating cell survival, both the pharmacological inhibitor of NF-κB, curcumin, and RNA interference targeting p65 were used in the current study. Neither 10% CSE nor curcumin (up to 20 μM) alone affected cell viability as examined by LIVE/DEAD Cytotoxicity and Viability assay (Figure 3). When cigarette smoke extract and curcumin were added together, however, the number of dead cells was significantly increased (41.4 ± 1.2% of CSE plus curcumin vs 11.9 ± 1.8% of control, p < 0.01, Figure 3B).


NF-kappaB mediates the survival of human bronchial epithelial cells exposed to cigarette smoke extract.

Liu X, Togo S, Al-Mugotir M, Kim H, Fang Q, Kobayashi T, Wang X, Mao L, Bitterman P, Rennard S - Respir. Res. (2008)

Effect of NF-κB inhibitor on the viability of HBEC exposed to cigarette smoke extract. Cells were pre-treated with 20 μM curcumin for 30 minutes followed by exposure to 10% cigarette smoke extract for 24 hours in the presence or absence of curcumin. Cell viability and cytotoxicity were determined by LIVE/DEAD assay. Panel A: One representative micro-photograph of LIVE/DEAD staining. Green: live cells; Red: dead cells. Magnification: 200×. Panel B: Quantitative data from 3 separate experiments. Vertical axis: percent of dead cells = red cells/(green cells + read cells) * 100%. Horizontal axis: treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2567966&req=5

Figure 3: Effect of NF-κB inhibitor on the viability of HBEC exposed to cigarette smoke extract. Cells were pre-treated with 20 μM curcumin for 30 minutes followed by exposure to 10% cigarette smoke extract for 24 hours in the presence or absence of curcumin. Cell viability and cytotoxicity were determined by LIVE/DEAD assay. Panel A: One representative micro-photograph of LIVE/DEAD staining. Green: live cells; Red: dead cells. Magnification: 200×. Panel B: Quantitative data from 3 separate experiments. Vertical axis: percent of dead cells = red cells/(green cells + read cells) * 100%. Horizontal axis: treatment.
Mentions: To investigate the role of NF-κB in modulating cell survival, both the pharmacological inhibitor of NF-κB, curcumin, and RNA interference targeting p65 were used in the current study. Neither 10% CSE nor curcumin (up to 20 μM) alone affected cell viability as examined by LIVE/DEAD Cytotoxicity and Viability assay (Figure 3). When cigarette smoke extract and curcumin were added together, however, the number of dead cells was significantly increased (41.4 ± 1.2% of CSE plus curcumin vs 11.9 ± 1.8% of control, p < 0.01, Figure 3B).

Bottom Line: Inhibition of NF-kappaB by the pharmacologic inhibitor curcumin (20 microM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure.The current study demonstrates that CSE activates NF-kappaB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65.These results suggest that activation of NF-kappaB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Pulmonary, Critical Care and Sleep Medicine, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska, USA. srennard@unmc.edu.

ABSTRACT

Background: We have previously reported that low concentrations of cigarette smoke extract induce DNA damage without leading to apoptosis or necrosis in human bronchial epithelial cells (HBECs), and that IL-6/STAT3 signaling contributes to the cell survival. Since NF-kappaB is also involved in regulating apoptosis and cell survival, the current study was designed to investigate the role of NF-kappaB in mediating cell survival in response to cigarette smoke exposure in HBECs.

Methods: Both the pharmacologic inhibitor of NF-kappaB, curcumin, and RNA interference targeting p65 were used to block NF-kappaB signaling in HBECs. Apoptosis and cell survival were then assessed by various methods including COMET assay, LIVE/DEAD Cytotoxicity/Viability assay and colony formation assay.

Results: Cigarette smoke extract (CSE) caused DNA damage and cell cycle arrest in S phase without leading to apoptosis in HBECs as evidenced by TUNEL assay, COMET assay and DNA content assay. CSE stimulated NF-kappaB -DNA binding activity and up-regulated Bcl-XL protein in HBECs. Inhibition of NF-kappaB by the pharmacologic inhibitor curcumin (20 microM) or suppression of p65 by siRNA resulted in a significant increase in cell death in response to cigarette smoke exposure. Furthermore, cells lacking p65 were incapable of forming cellular colonies when these cells were exposed to CSE, while they behaved normally in the regular culture medium.

Conclusion: The current study demonstrates that CSE activates NF-kappaB and up-regulates Bcl-XL through NF-kB activation in HBECs, and that CSE induces cell death in cells lacking p65. These results suggest that activation of NF-kappaB regulates cell survival following DNA damage by cigarette smoke in human bronchial epithelial cells.

Show MeSH
Related in: MedlinePlus