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Curcumin-induced fibroblast apoptosis and in vitro wound contraction are regulated by antioxidants and heme oxygenase: implications for scar formation.

Scharstuhl A, Mutsaers HA, Pennings SW, Szarek WA, Russel FG, Wagener FA - J. Cell. Mol. Med. (2008)

Bottom Line: NAC-mediated inhibition of 25-microM curcumin-induced apoptosis was demonstrated to act in part via restored HO-1-induction, since the rescuing effect of NAC could be reduced by inhibiting HO-activity.On a functional level, fibroblast-mediated collagen gel contraction, an in vitro wound contraction model, was completely prevented by 25-microM curcumin, while this could be reversed by co-incubation with NAC, an effect that was also partially HO-mediated.In conclusion, curcumin treatment in high doses (>25 microM) may provide a novel way to modulate pathological scar formation through the induction of fibroblast apoptosis, while antioxidants, HO-activity and its effector molecules act as a possible fine-tuning regulator.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, The Netherlands.

ABSTRACT
Fibroblast apoptosis plays a crucial role in normal and pathological scar formation and therefore we studied whether the putative apoptosis-inducing factor curcumin affects fibroblast apoptosis and may function as a novel therapeutic. We show that 25-microM curcumin causes fibroblast apoptosis and that this could be inhibited by co-administration of antioxidants N-acetyl-l-cysteine (NAC), biliverdin or bilirubin, suggesting that reactive oxygen species (ROS) are involved. This is supported by our observation that 25-microM curcumin caused the generation of ROS, which could be completely blocked by addition of NAC or bilirubin. Since biliverdin and bilirubin are downstream products of heme degradation by heme oxygenase (HO), it has been suggested that HO-activity protects against curcumin-induced apoptosis. Interestingly, exposure to curcumin maximally induced HO-1 protein and HO-activity at 10-15 microM, whereas, at a concentration of >20-microM curcumin HO-1-expression and HO-activity was negligible. NAC-mediated inhibition of 25-microM curcumin-induced apoptosis was demonstrated to act in part via restored HO-1-induction, since the rescuing effect of NAC could be reduced by inhibiting HO-activity. Moreover pre-induction of HO-1 using 5-microM curcumin protected fibroblasts against 25-microM curcumin-induced apoptosis. On a functional level, fibroblast-mediated collagen gel contraction, an in vitro wound contraction model, was completely prevented by 25-microM curcumin, while this could be reversed by co-incubation with NAC, an effect that was also partially HO-mediated. In conclusion, curcumin treatment in high doses (>25 microM) may provide a novel way to modulate pathological scar formation through the induction of fibroblast apoptosis, while antioxidants, HO-activity and its effector molecules act as a possible fine-tuning regulator.

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HO-1 protein expression and HO-activity in fibroblasts treated with curcumin, NAC or the combination. Adherent cells were treated with 25-μM curcumin, 6-mM NAC or the combination for 24 hrs. The HO-1 level or activity in the untreated group was set at 1. (A) Quantification of Western blot analysis for HO-1, and (B) HO-activity assay. For details on the procedures, see ‘materials and methods’ section. Shown are the mean ± S.D. from 5 independent experiments. ***P < 0.001 compared to untreated.
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fig04: HO-1 protein expression and HO-activity in fibroblasts treated with curcumin, NAC or the combination. Adherent cells were treated with 25-μM curcumin, 6-mM NAC or the combination for 24 hrs. The HO-1 level or activity in the untreated group was set at 1. (A) Quantification of Western blot analysis for HO-1, and (B) HO-activity assay. For details on the procedures, see ‘materials and methods’ section. Shown are the mean ± S.D. from 5 independent experiments. ***P < 0.001 compared to untreated.

Mentions: We further investigated how NAC prevented curcumin-induced fibroblast apoptosis and studied HO-1 expression and HO-activity. Figure 4A and B shows that either 25-μM curcumin or 6-mM NAC did not induce HO-1 protein expression or activity. Interestingly, the combination of curcumin with NAC strongly induced HO-1 expression, and, furthermore, the induced HO-1 was shown to be active. These results suggest a role for HO-1 in the protection against curcumin-induced apoptosis by NAC. Indeed when we blocked HO-activity using the specific HO-activity inhibitor QC-15 in the presence of the curcumin-NAC combination, we observed a significant inhibition of the rescuing effects of NAC on curcumin-induced apoptosis (Fig. 5A–C). Combined treatment of curcumin and NAC increased the fraction of living cells by 35% compared to curcumin alone (P < 0.001). When we inhibited HO-1-activity in the curcumin-NAC group by QC-15, the fraction of living cells was significantly decreased by 17% (P < 0.01). Moreover, application of the HO-activity inhibitor together with curcumin and NAC significantly increased the fraction of early apoptotic cells by 59%, compared to treatment with the curcumin-NAC combination (P < 0.05). Similarly, also the fraction of late apoptotic cells is significantly increased in the curcumin-NAC-QC group, 229 ± 1.3% compared to a normalized fraction of 100 ± 10.4% in the curcumin-NAC treated group (P < 0.001). These results show that HO-activity, at least partially, mediates the rescuing effects of NAC on curcumin-induced apoptosis.


Curcumin-induced fibroblast apoptosis and in vitro wound contraction are regulated by antioxidants and heme oxygenase: implications for scar formation.

Scharstuhl A, Mutsaers HA, Pennings SW, Szarek WA, Russel FG, Wagener FA - J. Cell. Mol. Med. (2008)

HO-1 protein expression and HO-activity in fibroblasts treated with curcumin, NAC or the combination. Adherent cells were treated with 25-μM curcumin, 6-mM NAC or the combination for 24 hrs. The HO-1 level or activity in the untreated group was set at 1. (A) Quantification of Western blot analysis for HO-1, and (B) HO-activity assay. For details on the procedures, see ‘materials and methods’ section. Shown are the mean ± S.D. from 5 independent experiments. ***P < 0.001 compared to untreated.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3822878&req=5

fig04: HO-1 protein expression and HO-activity in fibroblasts treated with curcumin, NAC or the combination. Adherent cells were treated with 25-μM curcumin, 6-mM NAC or the combination for 24 hrs. The HO-1 level or activity in the untreated group was set at 1. (A) Quantification of Western blot analysis for HO-1, and (B) HO-activity assay. For details on the procedures, see ‘materials and methods’ section. Shown are the mean ± S.D. from 5 independent experiments. ***P < 0.001 compared to untreated.
Mentions: We further investigated how NAC prevented curcumin-induced fibroblast apoptosis and studied HO-1 expression and HO-activity. Figure 4A and B shows that either 25-μM curcumin or 6-mM NAC did not induce HO-1 protein expression or activity. Interestingly, the combination of curcumin with NAC strongly induced HO-1 expression, and, furthermore, the induced HO-1 was shown to be active. These results suggest a role for HO-1 in the protection against curcumin-induced apoptosis by NAC. Indeed when we blocked HO-activity using the specific HO-activity inhibitor QC-15 in the presence of the curcumin-NAC combination, we observed a significant inhibition of the rescuing effects of NAC on curcumin-induced apoptosis (Fig. 5A–C). Combined treatment of curcumin and NAC increased the fraction of living cells by 35% compared to curcumin alone (P < 0.001). When we inhibited HO-1-activity in the curcumin-NAC group by QC-15, the fraction of living cells was significantly decreased by 17% (P < 0.01). Moreover, application of the HO-activity inhibitor together with curcumin and NAC significantly increased the fraction of early apoptotic cells by 59%, compared to treatment with the curcumin-NAC combination (P < 0.05). Similarly, also the fraction of late apoptotic cells is significantly increased in the curcumin-NAC-QC group, 229 ± 1.3% compared to a normalized fraction of 100 ± 10.4% in the curcumin-NAC treated group (P < 0.001). These results show that HO-activity, at least partially, mediates the rescuing effects of NAC on curcumin-induced apoptosis.

Bottom Line: NAC-mediated inhibition of 25-microM curcumin-induced apoptosis was demonstrated to act in part via restored HO-1-induction, since the rescuing effect of NAC could be reduced by inhibiting HO-activity.On a functional level, fibroblast-mediated collagen gel contraction, an in vitro wound contraction model, was completely prevented by 25-microM curcumin, while this could be reversed by co-incubation with NAC, an effect that was also partially HO-mediated.In conclusion, curcumin treatment in high doses (>25 microM) may provide a novel way to modulate pathological scar formation through the induction of fibroblast apoptosis, while antioxidants, HO-activity and its effector molecules act as a possible fine-tuning regulator.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, The Netherlands.

ABSTRACT
Fibroblast apoptosis plays a crucial role in normal and pathological scar formation and therefore we studied whether the putative apoptosis-inducing factor curcumin affects fibroblast apoptosis and may function as a novel therapeutic. We show that 25-microM curcumin causes fibroblast apoptosis and that this could be inhibited by co-administration of antioxidants N-acetyl-l-cysteine (NAC), biliverdin or bilirubin, suggesting that reactive oxygen species (ROS) are involved. This is supported by our observation that 25-microM curcumin caused the generation of ROS, which could be completely blocked by addition of NAC or bilirubin. Since biliverdin and bilirubin are downstream products of heme degradation by heme oxygenase (HO), it has been suggested that HO-activity protects against curcumin-induced apoptosis. Interestingly, exposure to curcumin maximally induced HO-1 protein and HO-activity at 10-15 microM, whereas, at a concentration of >20-microM curcumin HO-1-expression and HO-activity was negligible. NAC-mediated inhibition of 25-microM curcumin-induced apoptosis was demonstrated to act in part via restored HO-1-induction, since the rescuing effect of NAC could be reduced by inhibiting HO-activity. Moreover pre-induction of HO-1 using 5-microM curcumin protected fibroblasts against 25-microM curcumin-induced apoptosis. On a functional level, fibroblast-mediated collagen gel contraction, an in vitro wound contraction model, was completely prevented by 25-microM curcumin, while this could be reversed by co-incubation with NAC, an effect that was also partially HO-mediated. In conclusion, curcumin treatment in high doses (>25 microM) may provide a novel way to modulate pathological scar formation through the induction of fibroblast apoptosis, while antioxidants, HO-activity and its effector molecules act as a possible fine-tuning regulator.

Show MeSH
Related in: MedlinePlus