Limits...
Nicotine-Induced Apoptosis in Human Renal Proximal Tubular Epithelial Cells.

Kim CS, Choi JS, Joo SY, Bae EH, Ma SK, Lee J, Kim SW - PLoS ONE (2016)

Bottom Line: Nicotine treatment reduced cell viability dose dependently, increased ROS levels, and increased extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK expression.While nicotine treatment increased the expression of phosphorylated cdc2 and histone H3, a marker of G2/M phase arrest, hexamethonium and Bay 11-7082 pretreatment reduced their expression.Nicotine-induced apoptosis in HK-2 cells involves the nAChRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Chonnam National University Medical School, Gwangju, Korea.

ABSTRACT

Background: Nicotine is, to a large extent, responsible for smoking-mediated renal dysfunction. This study investigated nicotine's effects on renal tubular epithelial cell apoptosis in vitro and it explored the mechanisms underlying its effects.

Methods: Human proximal tubular epithelial (HK-2) cells were treated with nicotine. Cell viability was examined by using the WST-1 assay. Intracellular levels of reactive oxygen species (ROS) and the expression of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) proteins were determined. The messenger ribonucleic acid and the protein expression associated with the nicotine acetylcholine receptors (nAChRs) in HK-2 cells was examined, and apoptosis was detected using flow cytometry, cell cycle analysis, and immunoblot analysis.

Results: The HK-2 cells were endowed with nAChRs. Nicotine treatment reduced cell viability dose dependently, increased ROS levels, and increased extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK expression. Nicotine increased NF-κB activation, which was attenuated by N-acetyl-L-cysteine, and ERK and JNK inhibitors, but was not affected by a p38 MAPK inhibitor. Nicotine increased the Bax/Bcl-2 ratio, which was attenuated by N-acetyl-L-cysteine, the NF-κB inhibitor, Bay 11-7082, and hexamethonium, a non-specific nAChR blocker. Flow cytometry revealed nicotine-induced G2/M phase arrest. While nicotine treatment increased the expression of phosphorylated cdc2 and histone H3, a marker of G2/M phase arrest, hexamethonium and Bay 11-7082 pretreatment reduced their expression.

Conclusions: Nicotine caused apoptosis in HK-2 cells by inducing ROS generation that activated the NF-κB signaling pathway via the MAPK pathway and it arrested the cell cycle at the G2/M phase. Nicotine-induced apoptosis in HK-2 cells involves the nAChRs.

Show MeSH

Related in: MedlinePlus

The effect of nicotine on cell cycle arrest in human tubular epithelial cells.The HK-2 cells were exposed to nicotine (200 μM for 16 h) with or without pretreatment with (a) hexamethonium chloride (1 mM) or (b) Bay 11–7082 for 3 h, then the levels of expression of the proteins cyclin B1, phosphorylated cdc2, and histone H3 were determined. *P < 0.05 or **P < 0.01 compared with the controls. †P < 0.05 or ††P < 0.01 compared with nicotine treatment. Each column represents the mean ± the standard error of the mean. The data are representative of at least three independent experiments. (c) To elucidate the distribution of the cell cycle phases, cell cycle analysis was performed using flow cytometry. The HK-2 cells were treated with nicotine (0, 200, and 400 μM for 16 h).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4814027&req=5

pone.0152591.g005: The effect of nicotine on cell cycle arrest in human tubular epithelial cells.The HK-2 cells were exposed to nicotine (200 μM for 16 h) with or without pretreatment with (a) hexamethonium chloride (1 mM) or (b) Bay 11–7082 for 3 h, then the levels of expression of the proteins cyclin B1, phosphorylated cdc2, and histone H3 were determined. *P < 0.05 or **P < 0.01 compared with the controls. †P < 0.05 or ††P < 0.01 compared with nicotine treatment. Each column represents the mean ± the standard error of the mean. The data are representative of at least three independent experiments. (c) To elucidate the distribution of the cell cycle phases, cell cycle analysis was performed using flow cytometry. The HK-2 cells were treated with nicotine (0, 200, and 400 μM for 16 h).

Mentions: Tubular cell cycle arrest mediates apoptosis in a variety of renal diseases. Therefore, we investigated whether nicotine influences the arrest of the cell cycle in HK-2 cells. The levels of expression of phosphorylated cdc2 (Tyr 15) and histone H3 (Ser 10), which are markers of the G2/M phase of the cell cycle, increased after treatment with nicotine, and these increases were reduced when the cells had been incubated with the non-specific nAChR blocker, hexamethonium chloride (Fig 5A). The nicotine-induced G2/M phase arrest was reversed by Bay 11–7082 treatment (Fig 5B). When we examined the distribution of the cell cycle phases among the cells using flow cytometry, we found that the proportion of the cell population that was in the G2/M phase increased in a dose-dependent manner following nicotine treatment. The percentage of cells in the G2/M phase of the cycle increased from 5.8% to 13.1% following nicotine treatment (400 μM) (Fig 5C).


Nicotine-Induced Apoptosis in Human Renal Proximal Tubular Epithelial Cells.

Kim CS, Choi JS, Joo SY, Bae EH, Ma SK, Lee J, Kim SW - PLoS ONE (2016)

The effect of nicotine on cell cycle arrest in human tubular epithelial cells.The HK-2 cells were exposed to nicotine (200 μM for 16 h) with or without pretreatment with (a) hexamethonium chloride (1 mM) or (b) Bay 11–7082 for 3 h, then the levels of expression of the proteins cyclin B1, phosphorylated cdc2, and histone H3 were determined. *P < 0.05 or **P < 0.01 compared with the controls. †P < 0.05 or ††P < 0.01 compared with nicotine treatment. Each column represents the mean ± the standard error of the mean. The data are representative of at least three independent experiments. (c) To elucidate the distribution of the cell cycle phases, cell cycle analysis was performed using flow cytometry. The HK-2 cells were treated with nicotine (0, 200, and 400 μM for 16 h).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814027&req=5

pone.0152591.g005: The effect of nicotine on cell cycle arrest in human tubular epithelial cells.The HK-2 cells were exposed to nicotine (200 μM for 16 h) with or without pretreatment with (a) hexamethonium chloride (1 mM) or (b) Bay 11–7082 for 3 h, then the levels of expression of the proteins cyclin B1, phosphorylated cdc2, and histone H3 were determined. *P < 0.05 or **P < 0.01 compared with the controls. †P < 0.05 or ††P < 0.01 compared with nicotine treatment. Each column represents the mean ± the standard error of the mean. The data are representative of at least three independent experiments. (c) To elucidate the distribution of the cell cycle phases, cell cycle analysis was performed using flow cytometry. The HK-2 cells were treated with nicotine (0, 200, and 400 μM for 16 h).
Mentions: Tubular cell cycle arrest mediates apoptosis in a variety of renal diseases. Therefore, we investigated whether nicotine influences the arrest of the cell cycle in HK-2 cells. The levels of expression of phosphorylated cdc2 (Tyr 15) and histone H3 (Ser 10), which are markers of the G2/M phase of the cell cycle, increased after treatment with nicotine, and these increases were reduced when the cells had been incubated with the non-specific nAChR blocker, hexamethonium chloride (Fig 5A). The nicotine-induced G2/M phase arrest was reversed by Bay 11–7082 treatment (Fig 5B). When we examined the distribution of the cell cycle phases among the cells using flow cytometry, we found that the proportion of the cell population that was in the G2/M phase increased in a dose-dependent manner following nicotine treatment. The percentage of cells in the G2/M phase of the cycle increased from 5.8% to 13.1% following nicotine treatment (400 μM) (Fig 5C).

Bottom Line: Nicotine treatment reduced cell viability dose dependently, increased ROS levels, and increased extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK expression.While nicotine treatment increased the expression of phosphorylated cdc2 and histone H3, a marker of G2/M phase arrest, hexamethonium and Bay 11-7082 pretreatment reduced their expression.Nicotine-induced apoptosis in HK-2 cells involves the nAChRs.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, Chonnam National University Medical School, Gwangju, Korea.

ABSTRACT

Background: Nicotine is, to a large extent, responsible for smoking-mediated renal dysfunction. This study investigated nicotine's effects on renal tubular epithelial cell apoptosis in vitro and it explored the mechanisms underlying its effects.

Methods: Human proximal tubular epithelial (HK-2) cells were treated with nicotine. Cell viability was examined by using the WST-1 assay. Intracellular levels of reactive oxygen species (ROS) and the expression of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) proteins were determined. The messenger ribonucleic acid and the protein expression associated with the nicotine acetylcholine receptors (nAChRs) in HK-2 cells was examined, and apoptosis was detected using flow cytometry, cell cycle analysis, and immunoblot analysis.

Results: The HK-2 cells were endowed with nAChRs. Nicotine treatment reduced cell viability dose dependently, increased ROS levels, and increased extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK expression. Nicotine increased NF-κB activation, which was attenuated by N-acetyl-L-cysteine, and ERK and JNK inhibitors, but was not affected by a p38 MAPK inhibitor. Nicotine increased the Bax/Bcl-2 ratio, which was attenuated by N-acetyl-L-cysteine, the NF-κB inhibitor, Bay 11-7082, and hexamethonium, a non-specific nAChR blocker. Flow cytometry revealed nicotine-induced G2/M phase arrest. While nicotine treatment increased the expression of phosphorylated cdc2 and histone H3, a marker of G2/M phase arrest, hexamethonium and Bay 11-7082 pretreatment reduced their expression.

Conclusions: Nicotine caused apoptosis in HK-2 cells by inducing ROS generation that activated the NF-κB signaling pathway via the MAPK pathway and it arrested the cell cycle at the G2/M phase. Nicotine-induced apoptosis in HK-2 cells involves the nAChRs.

Show MeSH
Related in: MedlinePlus