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Paraquat Induces Apoptosis through Cytochrome C Release and ERK Activation.

Seo HJ, Choi SJ, Lee JH - Biomol Ther (Seoul) (2014)

Bottom Line: Paraquat treatment resulted in activation of ERK, and U0126, inhibitors of the MEK/ERK signaling pathway, prevented apoptosis.Moreover, paraquat-induced apoptosis was associated with cytochrome C release, which could be prevented by treatment with the MEK inhibitors.Taken together, our findings suggest that ERK activation plays an active role in mediating paraquat-induced apoptosis of NIH3T3 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic and Cardiovascular Surgery, Chosun University School of Medicine, Gwangju 501-759, Republic of Korea.

ABSTRACT
Paraquat has been suggested to induce apoptosis by generation of reactive oxygen species (ROS). However, little is known about the mechanism of paraquat-induced apoptosis. Here, we demonstrate that extracellular signal-regulated protein kinase (ERK) is required for paraquat-induced apoptosis in NIH3T3 cells. Paraquat treatment resulted in activation of ERK, and U0126, inhibitors of the MEK/ERK signaling pathway, prevented apoptosis. Moreover, paraquat-induced apoptosis was associated with cytochrome C release, which could be prevented by treatment with the MEK inhibitors. Taken together, our findings suggest that ERK activation plays an active role in mediating paraquat-induced apoptosis of NIH3T3 cells.

No MeSH data available.


Related in: MedlinePlus

Suppression of cytochrome C release by MEK inhibitor. Cells were pretreated with the U0126 (20 μM) for 30 min and then treated with 1 mM paraquat for 24 h. Upper panel, the cytosolic fraction was separated from mitochondria-enriched fraction and subjected to Western blot analysis with a monoclonal antibody to cytochrome C. Lower panel, the amount of cytochrome C release was quantified by densitometry and corrected for the amount of α-Tubulin in the corresponding lysate. Results are shown as means ± S.D. (n=3). *<0.05.
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f5-bt-22-503: Suppression of cytochrome C release by MEK inhibitor. Cells were pretreated with the U0126 (20 μM) for 30 min and then treated with 1 mM paraquat for 24 h. Upper panel, the cytosolic fraction was separated from mitochondria-enriched fraction and subjected to Western blot analysis with a monoclonal antibody to cytochrome C. Lower panel, the amount of cytochrome C release was quantified by densitometry and corrected for the amount of α-Tubulin in the corresponding lysate. Results are shown as means ± S.D. (n=3). *<0.05.

Mentions: Apoptosis have been described in two major pathways. One pathway is extrinsic apoptosis pathway by external receptor-dependent stimuli. The ligands, such as FASL, TRAIL or TNF, interact with their receptors, and then adaptor molecule, FADD (Fas-associated death domain protein), is associated with death receptors, and subsequently activates caspase-8 leading to activation of downstream effector caspases, thus inducing apoptosis (Lavrik and Krammer, 2012; Nikoletopoulou et al., 2013). The second pathway is intrinsic apoptosis pathway that is mitochondria-dependent and results from release of cytochrome C leading to caspase-9 activation through the apoptotic protease-activating factor-1 (Apaf-1) (Huttemann et al., 2011; Landes and Martinou, 2011; Monian and Jiang, 2012). Therefore, we sought to investigate whether cytochrome C release occurred in response to paraquat treatment, and if so, to determine whether it was dependent on ERK activation. We isolated cytosolic fractions from lysates of NIH3T3 cells treated with paraquat for 24 h in the presence or absence of U0126 (20 μM). Western blot analysis revealed accumulation of cytosolic cytochrome C in paraquat-induced apoptosis (Fig. 5). Importantly, this process was markedly inhibited in the presence of the MEK inhibitors, U0126, suggesting that ERK activation is required for paraquat-induced cytochrome C release (Fig. 5). In the present studies, we did not observe any change in either Fas or FasL expression in paraquat-treated NIH3T3 cells (data not shown). We did, however, observe increased levels of cytochrome C in the cytoplasm of paraquat-treated cells relative to untreated cells. These results suggest that cytochrome C release play a role in mediating paraquat-induced apoptosis. The ability of the MEK inhibitors to diminish this effect suggests that the ERK signaling pathway functions upstream of cytochrome C release in the paraquat-induced apoptosis.


Paraquat Induces Apoptosis through Cytochrome C Release and ERK Activation.

Seo HJ, Choi SJ, Lee JH - Biomol Ther (Seoul) (2014)

Suppression of cytochrome C release by MEK inhibitor. Cells were pretreated with the U0126 (20 μM) for 30 min and then treated with 1 mM paraquat for 24 h. Upper panel, the cytosolic fraction was separated from mitochondria-enriched fraction and subjected to Western blot analysis with a monoclonal antibody to cytochrome C. Lower panel, the amount of cytochrome C release was quantified by densitometry and corrected for the amount of α-Tubulin in the corresponding lysate. Results are shown as means ± S.D. (n=3). *<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256029&req=5

f5-bt-22-503: Suppression of cytochrome C release by MEK inhibitor. Cells were pretreated with the U0126 (20 μM) for 30 min and then treated with 1 mM paraquat for 24 h. Upper panel, the cytosolic fraction was separated from mitochondria-enriched fraction and subjected to Western blot analysis with a monoclonal antibody to cytochrome C. Lower panel, the amount of cytochrome C release was quantified by densitometry and corrected for the amount of α-Tubulin in the corresponding lysate. Results are shown as means ± S.D. (n=3). *<0.05.
Mentions: Apoptosis have been described in two major pathways. One pathway is extrinsic apoptosis pathway by external receptor-dependent stimuli. The ligands, such as FASL, TRAIL or TNF, interact with their receptors, and then adaptor molecule, FADD (Fas-associated death domain protein), is associated with death receptors, and subsequently activates caspase-8 leading to activation of downstream effector caspases, thus inducing apoptosis (Lavrik and Krammer, 2012; Nikoletopoulou et al., 2013). The second pathway is intrinsic apoptosis pathway that is mitochondria-dependent and results from release of cytochrome C leading to caspase-9 activation through the apoptotic protease-activating factor-1 (Apaf-1) (Huttemann et al., 2011; Landes and Martinou, 2011; Monian and Jiang, 2012). Therefore, we sought to investigate whether cytochrome C release occurred in response to paraquat treatment, and if so, to determine whether it was dependent on ERK activation. We isolated cytosolic fractions from lysates of NIH3T3 cells treated with paraquat for 24 h in the presence or absence of U0126 (20 μM). Western blot analysis revealed accumulation of cytosolic cytochrome C in paraquat-induced apoptosis (Fig. 5). Importantly, this process was markedly inhibited in the presence of the MEK inhibitors, U0126, suggesting that ERK activation is required for paraquat-induced cytochrome C release (Fig. 5). In the present studies, we did not observe any change in either Fas or FasL expression in paraquat-treated NIH3T3 cells (data not shown). We did, however, observe increased levels of cytochrome C in the cytoplasm of paraquat-treated cells relative to untreated cells. These results suggest that cytochrome C release play a role in mediating paraquat-induced apoptosis. The ability of the MEK inhibitors to diminish this effect suggests that the ERK signaling pathway functions upstream of cytochrome C release in the paraquat-induced apoptosis.

Bottom Line: Paraquat treatment resulted in activation of ERK, and U0126, inhibitors of the MEK/ERK signaling pathway, prevented apoptosis.Moreover, paraquat-induced apoptosis was associated with cytochrome C release, which could be prevented by treatment with the MEK inhibitors.Taken together, our findings suggest that ERK activation plays an active role in mediating paraquat-induced apoptosis of NIH3T3 cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Thoracic and Cardiovascular Surgery, Chosun University School of Medicine, Gwangju 501-759, Republic of Korea.

ABSTRACT
Paraquat has been suggested to induce apoptosis by generation of reactive oxygen species (ROS). However, little is known about the mechanism of paraquat-induced apoptosis. Here, we demonstrate that extracellular signal-regulated protein kinase (ERK) is required for paraquat-induced apoptosis in NIH3T3 cells. Paraquat treatment resulted in activation of ERK, and U0126, inhibitors of the MEK/ERK signaling pathway, prevented apoptosis. Moreover, paraquat-induced apoptosis was associated with cytochrome C release, which could be prevented by treatment with the MEK inhibitors. Taken together, our findings suggest that ERK activation plays an active role in mediating paraquat-induced apoptosis of NIH3T3 cells.

No MeSH data available.


Related in: MedlinePlus