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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 induces changes in the intracellular calcium and nitric oxide (NO) content of IMR-32 cells. (A) IMR-32 cells were incubated with 0–20 μM emodin for 24 h. Intracellular Fluo-3 fluorescence intensity was measured in the presence/absence of extracellular Ca2+; (B) Intracellular Ca2+ level changes following treatment with 20 μM emodin and effects of ROS and NO inhibitors (NAC: 300 μM; L-NMMA: 400 μM; PTIO: 20 μM) were examined; (C) IMR-32 cells were pretreated with L-NMMA (400 μM), PTIO (20 μM) or BAPTA-AM (10 μM) for 30 min, followed by incubation in the presence or absence of 20 μM emodin. Intracellular NO generation was measured using DAF-2DA fluorescence dye. Data are presented as a percentage of the control group. * 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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f3-ijms-14-20139: Emodin induces changes in the intracellular calcium and nitric oxide (NO) content of IMR-32 cells. (A) IMR-32 cells were incubated with 0–20 μM emodin for 24 h. Intracellular Fluo-3 fluorescence intensity was measured in the presence/absence of extracellular Ca2+; (B) Intracellular Ca2+ level changes following treatment with 20 μM emodin and effects of ROS and NO inhibitors (NAC: 300 μM; L-NMMA: 400 μM; PTIO: 20 μM) were examined; (C) IMR-32 cells were pretreated with L-NMMA (400 μM), PTIO (20 μM) or BAPTA-AM (10 μM) for 30 min, followed by incubation in the presence or absence of 20 μM emodin. Intracellular NO generation was measured using DAF-2DA fluorescence dye. Data are presented as a percentage of the control group. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the untreated control group. # p < 0.001 versus the “emodin-treated” group.

Mentions: Changes in [Ca2+]i in emodin-treated IMR-32 cells were detected using Fluo-3AM fluorescence dye. Treatment with 10–20 μM emodin elicited an increase in [Ca2+]i (Figure 3A). Furthermore, cells cultured in Ca2+-containing medium displayed a ~3.1-fold increase in [Ca2+]i following treatment with 20 μM emodin, while those cultured in Ca2+-free medium showed no effect (Figure 3A). These findings indicate that the increase in [Ca2+]i is primarily attributed to release of internal Ca2+, similar to that observed in endoplasmic reticulum, mitochondria, nucleus and/or calcium-binding proteins (Figure 3A).


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 induces changes in the intracellular calcium and nitric oxide (NO) content of IMR-32 cells. (A) IMR-32 cells were incubated with 0–20 μM emodin for 24 h. Intracellular Fluo-3 fluorescence intensity was measured in the presence/absence of extracellular Ca2+; (B) Intracellular Ca2+ level changes following treatment with 20 μM emodin and effects of ROS and NO inhibitors (NAC: 300 μM; L-NMMA: 400 μM; PTIO: 20 μM) were examined; (C) IMR-32 cells were pretreated with L-NMMA (400 μM), PTIO (20 μM) or BAPTA-AM (10 μM) for 30 min, followed by incubation in the presence or absence of 20 μM emodin. Intracellular NO generation was measured using DAF-2DA fluorescence dye. Data are presented as a percentage of the control group. * 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

f3-ijms-14-20139: Emodin induces changes in the intracellular calcium and nitric oxide (NO) content of IMR-32 cells. (A) IMR-32 cells were incubated with 0–20 μM emodin for 24 h. Intracellular Fluo-3 fluorescence intensity was measured in the presence/absence of extracellular Ca2+; (B) Intracellular Ca2+ level changes following treatment with 20 μM emodin and effects of ROS and NO inhibitors (NAC: 300 μM; L-NMMA: 400 μM; PTIO: 20 μM) were examined; (C) IMR-32 cells were pretreated with L-NMMA (400 μM), PTIO (20 μM) or BAPTA-AM (10 μM) for 30 min, followed by incubation in the presence or absence of 20 μM emodin. Intracellular NO generation was measured using DAF-2DA fluorescence dye. Data are presented as a percentage of the control group. * p < 0.05, ** p < 0.01 and *** p < 0.001 versus the untreated control group. # p < 0.001 versus the “emodin-treated” group.
Mentions: Changes in [Ca2+]i in emodin-treated IMR-32 cells were detected using Fluo-3AM fluorescence dye. Treatment with 10–20 μM emodin elicited an increase in [Ca2+]i (Figure 3A). Furthermore, cells cultured in Ca2+-containing medium displayed a ~3.1-fold increase in [Ca2+]i following treatment with 20 μM emodin, while those cultured in Ca2+-free medium showed no effect (Figure 3A). These findings indicate that the increase in [Ca2+]i is primarily attributed to release of internal Ca2+, similar to that observed in endoplasmic reticulum, mitochondria, nucleus and/or calcium-binding proteins (Figure 3A).

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