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Inhibition of citrinin-induced apoptotic biochemical signaling in human hepatoma G2 cells by resveratrol.

Chen CC, Chan WH - Int J Mol Sci (2009)

Bottom Line: Resveratrol, a member of the phytoalexin family found in grapes and other dietary plants, possesses antioxidant and anti-tumor properties.In the present study, we examined the effects of resveratrol on apoptotic biochemical events in Hep G2 cells induced by CTN.These results collectively demonstrate that CTN stimulates ROS generation and JNK activation for mitochondria-dependent apoptotic signaling in Hep G2 cells, and these apoptotic biochemical events are blocked by pretreatment with resveratrol, which exerts antioxidant effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience Technology and Center for Nanotechnology, Chung Yuan Christian University, Chung Li, Taiwan. cha_chi_chen@yahoo.com.tw

ABSTRACT
The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. CTN causes cell injury, including apoptosis, but its precise regulatory mechanisms of action are currently unclear. Resveratrol, a member of the phytoalexin family found in grapes and other dietary plants, possesses antioxidant and anti-tumor properties. In the present study, we examined the effects of resveratrol on apoptotic biochemical events in Hep G2 cells induced by CTN. Resveratrol inhibited CTN-induced ROS generation, activation of JNK, loss of mitochondrial membrane potential (MMP), as well as activation of caspase-9, caspase-3 and PAK2. Moreover, resveratrol and the ROS scavengers, NAC and alpha-tocopherol, abolished CTN-stimulated intracellular oxidative stress and apoptosis. Active JNK was required for CTN-induced mitochondria-dependent apoptotic biochemical changes, including loss of MMP, and activation of caspases and PAK2. Activation of PAK2 was essential for apoptosis triggered by CTN. These results collectively demonstrate that CTN stimulates ROS generation and JNK activation for mitochondria-dependent apoptotic signaling in Hep G2 cells, and these apoptotic biochemical events are blocked by pretreatment with resveratrol, which exerts antioxidant effects.

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Resveratrol attenuates CTN-induced oxidative stress.Hep G2 cells were preloaded with 20 μM DCF-DA for 1 h. Cells were left untreated or treated with 20 μM resveratrol (RSVL), 400 μM N-acetyl cysteine (NAC), or 300 μM α-tocopherol (Toc) as indicated for 1 h, followed by incubation of 10–30 μM CTN for another 24 h. (A) ROS generation was assayed by DCF-DA, and expressed as absorbance/mg of protein. (B) Apoptosis was detected with the ELISA kit. Values are presented as means ± SD of eight determinations. ***P<0.001 versus value of the group treated with CTN.
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f3-ijms-10-03338: Resveratrol attenuates CTN-induced oxidative stress.Hep G2 cells were preloaded with 20 μM DCF-DA for 1 h. Cells were left untreated or treated with 20 μM resveratrol (RSVL), 400 μM N-acetyl cysteine (NAC), or 300 μM α-tocopherol (Toc) as indicated for 1 h, followed by incubation of 10–30 μM CTN for another 24 h. (A) ROS generation was assayed by DCF-DA, and expressed as absorbance/mg of protein. (B) Apoptosis was detected with the ELISA kit. Values are presented as means ± SD of eight determinations. ***P<0.001 versus value of the group treated with CTN.

Mentions: Earlier studies report that CTN promotes oxidative stress in cells [7,20], and ROS are effective apoptotic inducers. Accordingly, we examined ROS formation in CTN-treated Hep G2 cells and the effects of resveratrol on this process, using DCF-DA as the detection reagent. As shown in Figure 3A, CTN augmented the intracellular ROS content in Hep G2 cells, which was significantly attenuated upon pre-treatment with resveratrol. Two frequently used ROS scavengers, N-acetyl cysteine (NAC) and α-tocopherol, were tested in parallel. Pretreatment with NAC (400 μM) and α-tocopherol (300 μM) additionally led to suppression of CTN-stimulated intracellular ROS levels and apoptosis (Figures 3A and 3B). Interestingly, under our assay conditions, resveratrol appeared to be the most potent blocker of CTN-induced oxidative stress and apoptosis (Figures 3A and 3B). Based on these results, we propose that resveratrol effectively prevents CTN-induced apoptosis via its ability to act as a ROS scavenger.


Inhibition of citrinin-induced apoptotic biochemical signaling in human hepatoma G2 cells by resveratrol.

Chen CC, Chan WH - Int J Mol Sci (2009)

Resveratrol attenuates CTN-induced oxidative stress.Hep G2 cells were preloaded with 20 μM DCF-DA for 1 h. Cells were left untreated or treated with 20 μM resveratrol (RSVL), 400 μM N-acetyl cysteine (NAC), or 300 μM α-tocopherol (Toc) as indicated for 1 h, followed by incubation of 10–30 μM CTN for another 24 h. (A) ROS generation was assayed by DCF-DA, and expressed as absorbance/mg of protein. (B) Apoptosis was detected with the ELISA kit. Values are presented as means ± SD of eight determinations. ***P<0.001 versus value of the group treated with CTN.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812821&req=5

f3-ijms-10-03338: Resveratrol attenuates CTN-induced oxidative stress.Hep G2 cells were preloaded with 20 μM DCF-DA for 1 h. Cells were left untreated or treated with 20 μM resveratrol (RSVL), 400 μM N-acetyl cysteine (NAC), or 300 μM α-tocopherol (Toc) as indicated for 1 h, followed by incubation of 10–30 μM CTN for another 24 h. (A) ROS generation was assayed by DCF-DA, and expressed as absorbance/mg of protein. (B) Apoptosis was detected with the ELISA kit. Values are presented as means ± SD of eight determinations. ***P<0.001 versus value of the group treated with CTN.
Mentions: Earlier studies report that CTN promotes oxidative stress in cells [7,20], and ROS are effective apoptotic inducers. Accordingly, we examined ROS formation in CTN-treated Hep G2 cells and the effects of resveratrol on this process, using DCF-DA as the detection reagent. As shown in Figure 3A, CTN augmented the intracellular ROS content in Hep G2 cells, which was significantly attenuated upon pre-treatment with resveratrol. Two frequently used ROS scavengers, N-acetyl cysteine (NAC) and α-tocopherol, were tested in parallel. Pretreatment with NAC (400 μM) and α-tocopherol (300 μM) additionally led to suppression of CTN-stimulated intracellular ROS levels and apoptosis (Figures 3A and 3B). Interestingly, under our assay conditions, resveratrol appeared to be the most potent blocker of CTN-induced oxidative stress and apoptosis (Figures 3A and 3B). Based on these results, we propose that resveratrol effectively prevents CTN-induced apoptosis via its ability to act as a ROS scavenger.

Bottom Line: Resveratrol, a member of the phytoalexin family found in grapes and other dietary plants, possesses antioxidant and anti-tumor properties.In the present study, we examined the effects of resveratrol on apoptotic biochemical events in Hep G2 cells induced by CTN.These results collectively demonstrate that CTN stimulates ROS generation and JNK activation for mitochondria-dependent apoptotic signaling in Hep G2 cells, and these apoptotic biochemical events are blocked by pretreatment with resveratrol, which exerts antioxidant effects.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience Technology and Center for Nanotechnology, Chung Yuan Christian University, Chung Li, Taiwan. cha_chi_chen@yahoo.com.tw

ABSTRACT
The mycotoxin citrinin (CTN), a natural contaminant in foodstuffs and animal feeds, exerts cytotoxic and genotoxic effects on various mammalian cells. CTN causes cell injury, including apoptosis, but its precise regulatory mechanisms of action are currently unclear. Resveratrol, a member of the phytoalexin family found in grapes and other dietary plants, possesses antioxidant and anti-tumor properties. In the present study, we examined the effects of resveratrol on apoptotic biochemical events in Hep G2 cells induced by CTN. Resveratrol inhibited CTN-induced ROS generation, activation of JNK, loss of mitochondrial membrane potential (MMP), as well as activation of caspase-9, caspase-3 and PAK2. Moreover, resveratrol and the ROS scavengers, NAC and alpha-tocopherol, abolished CTN-stimulated intracellular oxidative stress and apoptosis. Active JNK was required for CTN-induced mitochondria-dependent apoptotic biochemical changes, including loss of MMP, and activation of caspases and PAK2. Activation of PAK2 was essential for apoptosis triggered by CTN. These results collectively demonstrate that CTN stimulates ROS generation and JNK activation for mitochondria-dependent apoptotic signaling in Hep G2 cells, and these apoptotic biochemical events are blocked by pretreatment with resveratrol, which exerts antioxidant effects.

Show MeSH
Related in: MedlinePlus