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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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Treatment of NAC inhibits WFA-induced apoptosis. (A-C) Articular chondrocytes were untreated or treated with 5 µM WFA for 24 hr in the absence or presence of 5 mM NAC or 1 mM L-NMMA. (A) Cell viability was determined by MTT assay. (B) Flow cytometric analysis of apoptotic cells was performed using propidium iodide (PI). (C) Fragmented nucleus of cells was stained with 4'-6-diamidino-2-phenylindole (DAPI) using fluorescence microscope (magnification, ×400). The data are representative result of three similar experiments. Values are means ± SEM of three experiments (n = 3); statistically significant: *P < 0.01 (versus the corresponding untreated group).
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Figure 3: Treatment of NAC inhibits WFA-induced apoptosis. (A-C) Articular chondrocytes were untreated or treated with 5 µM WFA for 24 hr in the absence or presence of 5 mM NAC or 1 mM L-NMMA. (A) Cell viability was determined by MTT assay. (B) Flow cytometric analysis of apoptotic cells was performed using propidium iodide (PI). (C) Fragmented nucleus of cells was stained with 4'-6-diamidino-2-phenylindole (DAPI) using fluorescence microscope (magnification, ×400). The data are representative result of three similar experiments. Values are means ± SEM of three experiments (n = 3); statistically significant: *P < 0.01 (versus the corresponding untreated group).

Mentions: Previous studies presented that a decrease of DCF intensity was regulated by NAC but not by L-NMMA. To test the protective effect of NAC and L-NMMA on WFA-induced apoptosis, cells were treated with WFA in the presence of the indicated concentrations of NAC or L-NMMA (Fig. 3). The results indicated that WFA inhibited proliferation of chondrocytes, but these inhibitory effects were abolished by NAC (Fig. 3A). To further verify the extent of apoptosis induced by WFA, we performed PI staining to detect apoptotic cells by flow cytometry analysis (Fig. 3B). We confirmed that NAC inhibits WFA-caused apoptosis but L-NMMA does not (Fig. 3B). To confirm this, we further performed DAPI staining (Fig. 3C). Compared to the control cells, WFA-treated cells had condensed and fragmented DNA in nucleus (Fig. 3C). NAC completely blocked WFA-mediated fragmented DNA, showing that intracellular ROS production is required for the induction of apoptosis (Fig. 3). These data suggested that WFA-induced apoptosis was triggered by ROS (Fig. 3).


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)

Treatment of NAC inhibits WFA-induced apoptosis. (A-C) Articular chondrocytes were untreated or treated with 5 µM WFA for 24 hr in the absence or presence of 5 mM NAC or 1 mM L-NMMA. (A) Cell viability was determined by MTT assay. (B) Flow cytometric analysis of apoptotic cells was performed using propidium iodide (PI). (C) Fragmented nucleus of cells was stained with 4'-6-diamidino-2-phenylindole (DAPI) using fluorescence microscope (magnification, ×400). The data are representative result of three similar experiments. Values are means ± SEM of three experiments (n = 3); statistically significant: *P < 0.01 (versus the corresponding untreated group).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4129194&req=5

Figure 3: Treatment of NAC inhibits WFA-induced apoptosis. (A-C) Articular chondrocytes were untreated or treated with 5 µM WFA for 24 hr in the absence or presence of 5 mM NAC or 1 mM L-NMMA. (A) Cell viability was determined by MTT assay. (B) Flow cytometric analysis of apoptotic cells was performed using propidium iodide (PI). (C) Fragmented nucleus of cells was stained with 4'-6-diamidino-2-phenylindole (DAPI) using fluorescence microscope (magnification, ×400). The data are representative result of three similar experiments. Values are means ± SEM of three experiments (n = 3); statistically significant: *P < 0.01 (versus the corresponding untreated group).
Mentions: Previous studies presented that a decrease of DCF intensity was regulated by NAC but not by L-NMMA. To test the protective effect of NAC and L-NMMA on WFA-induced apoptosis, cells were treated with WFA in the presence of the indicated concentrations of NAC or L-NMMA (Fig. 3). The results indicated that WFA inhibited proliferation of chondrocytes, but these inhibitory effects were abolished by NAC (Fig. 3A). To further verify the extent of apoptosis induced by WFA, we performed PI staining to detect apoptotic cells by flow cytometry analysis (Fig. 3B). We confirmed that NAC inhibits WFA-caused apoptosis but L-NMMA does not (Fig. 3B). To confirm this, we further performed DAPI staining (Fig. 3C). Compared to the control cells, WFA-treated cells had condensed and fragmented DNA in nucleus (Fig. 3C). NAC completely blocked WFA-mediated fragmented DNA, showing that intracellular ROS production is required for the induction of apoptosis (Fig. 3). These data suggested that WFA-induced apoptosis was triggered by ROS (Fig. 3).

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