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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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Blockage of PI3K/Akt and JNKinase pathways inhibits WFA-induced apoptosis. (A-D) Rabbit articular chondrocytes were untreated or treated with 5 µM WFA for 24 hr in the absence or presence of NAC, SP600125 (SP), or LY294002 (LY). (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). (D) The production of intracellular ROS was evidenced by an increase in intensity of the DCF signal using the FLx8000 fluorometer. 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 7: Blockage of PI3K/Akt and JNKinase pathways inhibits WFA-induced apoptosis. (A-D) Rabbit articular chondrocytes were untreated or treated with 5 µM WFA for 24 hr in the absence or presence of NAC, SP600125 (SP), or LY294002 (LY). (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). (D) The production of intracellular ROS was evidenced by an increase in intensity of the DCF signal using the FLx8000 fluorometer. 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: To establish the effects of blockage of PI3K and JNK/c-jun kinase pathways on WFA-caused apoptosis, chondrocytes were treated with SP600125 and LY294002 in the presence or absence of WFA (Fig. 7) and performed MTT assay, flow cytometry analysis, and DAPI staining. As shown in Fig. 7, by treatment of SP600125 or LY294002, decreased WFA-caused cell apoptosis and a diminished sub-G1 population recovered (Fig. 7A, B). In consistent with the result of flow cytometry analysis, nuclear morphology and DNA fragmentation of chondrocytes were reversed by treating inhibitors, as determined by DAPI staining (Fig. 7C).


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)

Blockage of PI3K/Akt and JNKinase pathways inhibits WFA-induced apoptosis. (A-D) Rabbit articular chondrocytes were untreated or treated with 5 µM WFA for 24 hr in the absence or presence of NAC, SP600125 (SP), or LY294002 (LY). (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). (D) The production of intracellular ROS was evidenced by an increase in intensity of the DCF signal using the FLx8000 fluorometer. 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 7: Blockage of PI3K/Akt and JNKinase pathways inhibits WFA-induced apoptosis. (A-D) Rabbit articular chondrocytes were untreated or treated with 5 µM WFA for 24 hr in the absence or presence of NAC, SP600125 (SP), or LY294002 (LY). (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). (D) The production of intracellular ROS was evidenced by an increase in intensity of the DCF signal using the FLx8000 fluorometer. 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: To establish the effects of blockage of PI3K and JNK/c-jun kinase pathways on WFA-caused apoptosis, chondrocytes were treated with SP600125 and LY294002 in the presence or absence of WFA (Fig. 7) and performed MTT assay, flow cytometry analysis, and DAPI staining. As shown in Fig. 7, by treatment of SP600125 or LY294002, decreased WFA-caused cell apoptosis and a diminished sub-G1 population recovered (Fig. 7A, B). In consistent with the result of flow cytometry analysis, nuclear morphology and DNA fragmentation of chondrocytes were reversed by treating inhibitors, as determined by DAPI staining (Fig. 7C).

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