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Withaferin A-caused production of intracellular reactive oxygen species modulates apoptosis via PI3K/Akt and JNKinase in rabbit articular chondrocytes.

Yu SM, Kim SJ - J. Korean Med. Sci. (2014)

Bottom Line: We also found that WFA causes the activation of PI3K/Akt and JNKinase.Inhibition of PI3K/Akt and JNKinase with LY294002 (LY)/triciribine (TB) or SP600125 (SP) in WFA-treated cells reduced accumulation of p53 and inhibited fragmented DNA.Our findings suggested that apoptosis caused by WFA-induced intracellular ROS generation is regulated through PI3K/Akt and JNKinase in rabbit articular chondrocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Kongju National University, Gongju, Korea.

ABSTRACT
Withaferin A (WFA) is known as a constituent of Ayurvedic medicinal plant, Withania somnifera, and has been used for thousands of years. Although WFA has been used for the treatment of osteoarthritis (OA) and has a wide range of biochemical and pharmacologic activities, there are no findings suggesting its properties on chondrocytes or cartilage. The aim of the present study is to investigate the effects of WFA on apoptosis with focus on generation of intracellular reactive oxygen species (ROS). Here we showed that WFA significantly increased the generation of intracellular ROS in a dose-dependent manner. We also determined that WFA markedly leads to apoptosis as evidenced by accumulation of p53 by Western blot analysis. N-Acetyl-L-Cystein (NAC), an antioxidant, prevented WFA-caused expression of p53 and inhibited apoptosis of chondrocytes. We also found that WFA causes the activation of PI3K/Akt and JNKinase. Inhibition of PI3K/Akt and JNKinase with LY294002 (LY)/triciribine (TB) or SP600125 (SP) in WFA-treated cells reduced accumulation of p53 and inhibited fragmented DNA. Our findings suggested that apoptosis caused by WFA-induced intracellular ROS generation is regulated through PI3K/Akt and JNKinase in rabbit articular chondrocytes.

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Inhibition of PI3K/Akt and JNK with inhibitors, LY/TB or SP, or activator, 740 Y-P is blocked the expression of p53 and p21. (A) Chondrocytes were untreated or treated with 5 µM WFA for 24 hr in the absence or presence of the indicated concentrations of SP600125 (SP). (B) Articular chondrocytes were untreated or treated with 5 µM WFA in the absence or presence of the indicated concentrations of LY294002 (LY). (C) Chondrocytes were untreated or treated with 5 µM WFA in the absence or presence of the indicated concentrations of triciribine (TB, an inhibitor of Akt). (D) Chondrocytes were untreated or treated with 5 µM WFA in the absence or presence of the indicated concentrations of 740 Y-P (an activator of PI3K). (A-D) Expressions of p53 (24 hr), p21 (24 hr), pJNK (10 min), pc-Jun (3 hr), pAkt (6 hr), and actin (24 hr) and actin were detected by Western blot analysis. Expression of actin was used as a loading control. The data are typical results from four independent experiments with similar results.
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Figure 6: Inhibition of PI3K/Akt and JNK with inhibitors, LY/TB or SP, or activator, 740 Y-P is blocked the expression of p53 and p21. (A) Chondrocytes were untreated or treated with 5 µM WFA for 24 hr in the absence or presence of the indicated concentrations of SP600125 (SP). (B) Articular chondrocytes were untreated or treated with 5 µM WFA in the absence or presence of the indicated concentrations of LY294002 (LY). (C) Chondrocytes were untreated or treated with 5 µM WFA in the absence or presence of the indicated concentrations of triciribine (TB, an inhibitor of Akt). (D) Chondrocytes were untreated or treated with 5 µM WFA in the absence or presence of the indicated concentrations of 740 Y-P (an activator of PI3K). (A-D) Expressions of p53 (24 hr), p21 (24 hr), pJNK (10 min), pc-Jun (3 hr), pAkt (6 hr), and actin (24 hr) and actin were detected by Western blot analysis. Expression of actin was used as a loading control. The data are typical results from four independent experiments with similar results.

Mentions: To provide further evidence for the critical role of PI3K/Akt and JNK/c-jun in the apoptotic effect of WFA in chondrocytes, the PI3K-specific inhibitor LY294002, the Akt-direct inhibitor triciribine, the JNK-specific inhibitor SP600125, and the PI3-kinase activator 740Y-P were used (Fig. 6). Chondrocytes were treated with the specific inhibitor of JNK, SP600125, or with the specific inhibitor of PI3K/Akt, LY294002/triciribine for 1 hr (Fig. 6A-C). Inhibition of JNK and PI3K/Akt with SP600125 or LY294002/triciribine were abolished the expression of p53 and p21WAF-1, and inhibition of PI3K/Akt with LY294002/triciribine were prevented the phosphorylation at JNK and c-jun (Fig. 6A-C). The activation of JNK and c-jun was reduced by pre-treating LY294002 or triciribine, but the phosphorylation of Akt was not changed by treatment of SP600125 (Fig. 6A-C). The observed findings indicate that JNK/c-jun and PI3K/Akt pathways are a crucial role in WFA-caused apoptosis and the activation of JNK is a downstream of PI3K/Akt in WFA-induced apoptosis (Fig. 6). To gain further insight into the mechanism of PI3K/Akt activated by WFA, we investigated whether 740Y-P, a cell-permeable phospho-peptide capable of binding to the p85 regulatory subunit of PI3K to stimulate enzyme activity, could block the expression of p53 and p21WAF-1. Pretreatment of chondrocytes with different doses of 740Y-P, followed by 24 hr exposure to WFA continued to maintain p53 and p21WAF-1 and an increase of phosphorylation in Akt, JNK, and c-jun, suggesting the Akt pathway involved in WFA-induced apoptosis (Fig. 6D). These findings suggest that PI3K/Akt and JNK/c-jun play a crucial role in WFA induces apoptosis and also intracellular ROS caused by WFA are sufficient to activate PI3K/Akt and JNKinase pathways (Fig. 6).


Withaferin A-caused production of intracellular reactive oxygen species modulates apoptosis via PI3K/Akt and JNKinase in rabbit articular chondrocytes.

Yu SM, Kim SJ - J. Korean Med. Sci. (2014)

Inhibition of PI3K/Akt and JNK with inhibitors, LY/TB or SP, or activator, 740 Y-P is blocked the expression of p53 and p21. (A) Chondrocytes were untreated or treated with 5 µM WFA for 24 hr in the absence or presence of the indicated concentrations of SP600125 (SP). (B) Articular chondrocytes were untreated or treated with 5 µM WFA in the absence or presence of the indicated concentrations of LY294002 (LY). (C) Chondrocytes were untreated or treated with 5 µM WFA in the absence or presence of the indicated concentrations of triciribine (TB, an inhibitor of Akt). (D) Chondrocytes were untreated or treated with 5 µM WFA in the absence or presence of the indicated concentrations of 740 Y-P (an activator of PI3K). (A-D) Expressions of p53 (24 hr), p21 (24 hr), pJNK (10 min), pc-Jun (3 hr), pAkt (6 hr), and actin (24 hr) and actin were detected by Western blot analysis. Expression of actin was used as a loading control. The data are typical results from four independent experiments with similar results.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 6: Inhibition of PI3K/Akt and JNK with inhibitors, LY/TB or SP, or activator, 740 Y-P is blocked the expression of p53 and p21. (A) Chondrocytes were untreated or treated with 5 µM WFA for 24 hr in the absence or presence of the indicated concentrations of SP600125 (SP). (B) Articular chondrocytes were untreated or treated with 5 µM WFA in the absence or presence of the indicated concentrations of LY294002 (LY). (C) Chondrocytes were untreated or treated with 5 µM WFA in the absence or presence of the indicated concentrations of triciribine (TB, an inhibitor of Akt). (D) Chondrocytes were untreated or treated with 5 µM WFA in the absence or presence of the indicated concentrations of 740 Y-P (an activator of PI3K). (A-D) Expressions of p53 (24 hr), p21 (24 hr), pJNK (10 min), pc-Jun (3 hr), pAkt (6 hr), and actin (24 hr) and actin were detected by Western blot analysis. Expression of actin was used as a loading control. The data are typical results from four independent experiments with similar results.
Mentions: To provide further evidence for the critical role of PI3K/Akt and JNK/c-jun in the apoptotic effect of WFA in chondrocytes, the PI3K-specific inhibitor LY294002, the Akt-direct inhibitor triciribine, the JNK-specific inhibitor SP600125, and the PI3-kinase activator 740Y-P were used (Fig. 6). Chondrocytes were treated with the specific inhibitor of JNK, SP600125, or with the specific inhibitor of PI3K/Akt, LY294002/triciribine for 1 hr (Fig. 6A-C). Inhibition of JNK and PI3K/Akt with SP600125 or LY294002/triciribine were abolished the expression of p53 and p21WAF-1, and inhibition of PI3K/Akt with LY294002/triciribine were prevented the phosphorylation at JNK and c-jun (Fig. 6A-C). The activation of JNK and c-jun was reduced by pre-treating LY294002 or triciribine, but the phosphorylation of Akt was not changed by treatment of SP600125 (Fig. 6A-C). The observed findings indicate that JNK/c-jun and PI3K/Akt pathways are a crucial role in WFA-caused apoptosis and the activation of JNK is a downstream of PI3K/Akt in WFA-induced apoptosis (Fig. 6). To gain further insight into the mechanism of PI3K/Akt activated by WFA, we investigated whether 740Y-P, a cell-permeable phospho-peptide capable of binding to the p85 regulatory subunit of PI3K to stimulate enzyme activity, could block the expression of p53 and p21WAF-1. Pretreatment of chondrocytes with different doses of 740Y-P, followed by 24 hr exposure to WFA continued to maintain p53 and p21WAF-1 and an increase of phosphorylation in Akt, JNK, and c-jun, suggesting the Akt pathway involved in WFA-induced apoptosis (Fig. 6D). These findings suggest that PI3K/Akt and JNK/c-jun play a crucial role in WFA induces apoptosis and also intracellular ROS caused by WFA are sufficient to activate PI3K/Akt and JNKinase pathways (Fig. 6).

Bottom Line: We also found that WFA causes the activation of PI3K/Akt and JNKinase.Inhibition of PI3K/Akt and JNKinase with LY294002 (LY)/triciribine (TB) or SP600125 (SP) in WFA-treated cells reduced accumulation of p53 and inhibited fragmented DNA.Our findings suggested that apoptosis caused by WFA-induced intracellular ROS generation is regulated through PI3K/Akt and JNKinase in rabbit articular chondrocytes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Kongju National University, Gongju, Korea.

ABSTRACT
Withaferin A (WFA) is known as a constituent of Ayurvedic medicinal plant, Withania somnifera, and has been used for thousands of years. Although WFA has been used for the treatment of osteoarthritis (OA) and has a wide range of biochemical and pharmacologic activities, there are no findings suggesting its properties on chondrocytes or cartilage. The aim of the present study is to investigate the effects of WFA on apoptosis with focus on generation of intracellular reactive oxygen species (ROS). Here we showed that WFA significantly increased the generation of intracellular ROS in a dose-dependent manner. We also determined that WFA markedly leads to apoptosis as evidenced by accumulation of p53 by Western blot analysis. N-Acetyl-L-Cystein (NAC), an antioxidant, prevented WFA-caused expression of p53 and inhibited apoptosis of chondrocytes. We also found that WFA causes the activation of PI3K/Akt and JNKinase. Inhibition of PI3K/Akt and JNKinase with LY294002 (LY)/triciribine (TB) or SP600125 (SP) in WFA-treated cells reduced accumulation of p53 and inhibited fragmented DNA. Our findings suggested that apoptosis caused by WFA-induced intracellular ROS generation is regulated through PI3K/Akt and JNKinase in rabbit articular chondrocytes.

Show MeSH
Related in: MedlinePlus