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Characterization of apoptosis induced by emodin and related regulatory mechanisms in human neuroblastoma cells.

Huang FJ, Hsuuw YD, Chan WH - Int J Mol Sci (2013)

Bottom Line: Recent studies have shown that emodin can induce or prevent cell apoptosis, although the precise molecular mechanisms underlying these effects are unknown.Our results collectively indicate that emodin at concentrations of 10-20 μM triggers apoptosis of IMR-32 cells via a mechanism involving both ROS and NO.Based on the collective results, we propose a model for an emodin-triggered apoptotic signaling cascade that sequentially involves ROS, Ca²⁺, NO, p53, caspase-9 and caspase-3.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung 83301, Taiwan. whchan@cycu.edu.tw.

ABSTRACT
Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Recent studies have shown that emodin can induce or prevent cell apoptosis, although the precise molecular mechanisms underlying these effects are unknown. Experiments from the current study revealed that emodin (10-20 μM) induces apoptotic processes in the human neuroblastoma cell line, IMR-32, but exerts no injury effects at treatment doses below 10 μM. Treatment with emodin at concentrations of 10-20 μM led to a direct increase in the reactive oxygen species (ROS) content in IMR-32 cells, along with significant elevation of cytoplasmic free calcium and nitric oxide (NO) levels, loss of mitochondrial membrane potential (MMP), activation of caspases-9 and -3, and cell death. Pretreatment with nitric oxide (NO) scavengers suppressed the apoptotic biochemical changes induced by 20 μM emodin, and attenuated emodin-induced p53 and p21 expression involved in apoptotic signaling. Our results collectively indicate that emodin at concentrations of 10-20 μM triggers apoptosis of IMR-32 cells via a mechanism involving both ROS and NO. Based on the collective results, we propose a model for an emodin-triggered apoptotic signaling cascade that sequentially involves ROS, Ca²⁺, NO, p53, caspase-9 and caspase-3.

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Emodin promotes oxidative stress in IMR-32 cells. (A) IMR-32 cells were treated with 0–20 μM emodin for 24 h, and ROS generation assayed using DCF-DA (20 μM) or dihydrorhodamine 123 (DHR 123; 20 μM); (B) IMR-32 cells were pre-incubated with N-acetyl cysteine (NAC; 300 μM) for 30 min, followed by treatment with or without emodin, as indicated. Reactive oxygen species (ROS) generation was assayed using DCF-DA. Data are representative of eight independent experiments. *p < 0.05, ** p < 0.01 and *** p < 0.001 versus the untreated control group. # p < 0.001 versus the emodin-treated group.
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f2-ijms-14-20139: Emodin promotes oxidative stress in IMR-32 cells. (A) IMR-32 cells were treated with 0–20 μM emodin for 24 h, and ROS generation assayed using DCF-DA (20 μM) or dihydrorhodamine 123 (DHR 123; 20 μM); (B) IMR-32 cells were pre-incubated with N-acetyl cysteine (NAC; 300 μM) for 30 min, followed by treatment with or without emodin, as indicated. Reactive oxygen species (ROS) generation was assayed using DCF-DA. Data are representative of eight independent experiments. *p < 0.05, ** p < 0.01 and *** p < 0.001 versus the untreated control group. # p < 0.001 versus the emodin-treated group.

Mentions: In view of our previous finding that numerous chemical stimuli trigger apoptosis via ROS generation [28,29], we employed DCF-DA and DHR 123 to examine ROS formation in IMR-32 cells treated with emodin. Emodin (10–20 μM) stimulated ROS generation about 2.8–5.4-fold, compared with the untreated control group (Figure 2A). Notably, pretreatment with N-acetyl cysteine (NAC), a commonly used ROS scavenger, effectively prevented ROS production in the presence of 10–20 μM of emodin (Figure 2B). Our results clearly indicate that the mechanism of emodin-induced apoptosis of IMR-32 cells involves ROS generation.


Characterization of apoptosis induced by emodin and related regulatory mechanisms in human neuroblastoma cells.

Huang FJ, Hsuuw YD, Chan WH - Int J Mol Sci (2013)

Emodin promotes oxidative stress in IMR-32 cells. (A) IMR-32 cells were treated with 0–20 μM emodin for 24 h, and ROS generation assayed using DCF-DA (20 μM) or dihydrorhodamine 123 (DHR 123; 20 μM); (B) IMR-32 cells were pre-incubated with N-acetyl cysteine (NAC; 300 μM) for 30 min, followed by treatment with or without emodin, as indicated. Reactive oxygen species (ROS) generation was assayed using DCF-DA. Data are representative of eight independent experiments. *p < 0.05, ** p < 0.01 and *** p < 0.001 versus the untreated control group. # p < 0.001 versus the emodin-treated group.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3821607&req=5

f2-ijms-14-20139: Emodin promotes oxidative stress in IMR-32 cells. (A) IMR-32 cells were treated with 0–20 μM emodin for 24 h, and ROS generation assayed using DCF-DA (20 μM) or dihydrorhodamine 123 (DHR 123; 20 μM); (B) IMR-32 cells were pre-incubated with N-acetyl cysteine (NAC; 300 μM) for 30 min, followed by treatment with or without emodin, as indicated. Reactive oxygen species (ROS) generation was assayed using DCF-DA. Data are representative of eight independent experiments. *p < 0.05, ** p < 0.01 and *** p < 0.001 versus the untreated control group. # p < 0.001 versus the emodin-treated group.
Mentions: In view of our previous finding that numerous chemical stimuli trigger apoptosis via ROS generation [28,29], we employed DCF-DA and DHR 123 to examine ROS formation in IMR-32 cells treated with emodin. Emodin (10–20 μM) stimulated ROS generation about 2.8–5.4-fold, compared with the untreated control group (Figure 2A). Notably, pretreatment with N-acetyl cysteine (NAC), a commonly used ROS scavenger, effectively prevented ROS production in the presence of 10–20 μM of emodin (Figure 2B). Our results clearly indicate that the mechanism of emodin-induced apoptosis of IMR-32 cells involves ROS generation.

Bottom Line: Recent studies have shown that emodin can induce or prevent cell apoptosis, although the precise molecular mechanisms underlying these effects are unknown.Our results collectively indicate that emodin at concentrations of 10-20 μM triggers apoptosis of IMR-32 cells via a mechanism involving both ROS and NO.Based on the collective results, we propose a model for an emodin-triggered apoptotic signaling cascade that sequentially involves ROS, Ca²⁺, NO, p53, caspase-9 and caspase-3.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics and Gynecology, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung 83301, Taiwan. whchan@cycu.edu.tw.

ABSTRACT
Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a major constituent of rhubarb, has a wide range of therapeutic applications. Recent studies have shown that emodin can induce or prevent cell apoptosis, although the precise molecular mechanisms underlying these effects are unknown. Experiments from the current study revealed that emodin (10-20 μM) induces apoptotic processes in the human neuroblastoma cell line, IMR-32, but exerts no injury effects at treatment doses below 10 μM. Treatment with emodin at concentrations of 10-20 μM led to a direct increase in the reactive oxygen species (ROS) content in IMR-32 cells, along with significant elevation of cytoplasmic free calcium and nitric oxide (NO) levels, loss of mitochondrial membrane potential (MMP), activation of caspases-9 and -3, and cell death. Pretreatment with nitric oxide (NO) scavengers suppressed the apoptotic biochemical changes induced by 20 μM emodin, and attenuated emodin-induced p53 and p21 expression involved in apoptotic signaling. Our results collectively indicate that emodin at concentrations of 10-20 μM triggers apoptosis of IMR-32 cells via a mechanism involving both ROS and NO. Based on the collective results, we propose a model for an emodin-triggered apoptotic signaling cascade that sequentially involves ROS, Ca²⁺, NO, p53, caspase-9 and caspase-3.

Show MeSH
Related in: MedlinePlus