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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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Pre-treatment with low doses of curcumin protects against apoptosis induced by 25 μM curcumin. Adherent fibroblasts were treated for 24 hrs with 5 μM curcumin alone or in combination with 20 μM SnMP. Control cells were not pre-treated. Subsequently, medium was changed and the cells were incubated with 25 μM curcumin for 48 hrs. All cells were collected and stained with Annexin V-FITC and PI. Using flow cytometry, the staining intensity of the cells for Annexin V-FITC and PI was determined. The effect of 25 μM curcumin without pre-treatment was stated as 100%. Shown is the quantification of quadrant analysis of 5 independent experiments (mean ± S.D.). Asterisks indicate statistically significant different from control cells, *P < 0.05, **P < 0.01, ***P < 0.001 and #P < 0.05 compared to 5 μM curcumin as pre-treatment.
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fig06: Pre-treatment with low doses of curcumin protects against apoptosis induced by 25 μM curcumin. Adherent fibroblasts were treated for 24 hrs with 5 μM curcumin alone or in combination with 20 μM SnMP. Control cells were not pre-treated. Subsequently, medium was changed and the cells were incubated with 25 μM curcumin for 48 hrs. All cells were collected and stained with Annexin V-FITC and PI. Using flow cytometry, the staining intensity of the cells for Annexin V-FITC and PI was determined. The effect of 25 μM curcumin without pre-treatment was stated as 100%. Shown is the quantification of quadrant analysis of 5 independent experiments (mean ± S.D.). Asterisks indicate statistically significant different from control cells, *P < 0.05, **P < 0.01, ***P < 0.001 and #P < 0.05 compared to 5 μM curcumin as pre-treatment.

Mentions: HO has been described as an enzyme with cytoprotective functions. Therefore, we studied whether pre-induction of HO-1 would protect against apoptosis induced by 25-μM curcumin. In Figure 6, it is shown that 5-μM curcumin pre-treatment followed by 25-μM curcumin treatment indeed causes a significant increase in the percentage of living fibroblasts, compared to cells that received no pre-treatment (P < 0.001). This observation was accompanied by a significant decrease in the percentage dead cells after pre-treatment with 5-μM curcumin (P < 0.01). Pre-treatment with 2.5-μM curcumin had similar but milder effects (data not shown). Importantly, the protective effect of preconditioning was blocked significantly when co-treated with the HO-activity inhibitor SnMP (P < 0.05). Taken together, these data indicate that a modest up-regulation of HO-1 protein expression and HO-activity by preconditioning with curcumin, subsequently, had significant cytoprotective effects in fibroblasts.


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)

Pre-treatment with low doses of curcumin protects against apoptosis induced by 25 μM curcumin. Adherent fibroblasts were treated for 24 hrs with 5 μM curcumin alone or in combination with 20 μM SnMP. Control cells were not pre-treated. Subsequently, medium was changed and the cells were incubated with 25 μM curcumin for 48 hrs. All cells were collected and stained with Annexin V-FITC and PI. Using flow cytometry, the staining intensity of the cells for Annexin V-FITC and PI was determined. The effect of 25 μM curcumin without pre-treatment was stated as 100%. Shown is the quantification of quadrant analysis of 5 independent experiments (mean ± S.D.). Asterisks indicate statistically significant different from control cells, *P < 0.05, **P < 0.01, ***P < 0.001 and #P < 0.05 compared to 5 μM curcumin as pre-treatment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3822878&req=5

fig06: Pre-treatment with low doses of curcumin protects against apoptosis induced by 25 μM curcumin. Adherent fibroblasts were treated for 24 hrs with 5 μM curcumin alone or in combination with 20 μM SnMP. Control cells were not pre-treated. Subsequently, medium was changed and the cells were incubated with 25 μM curcumin for 48 hrs. All cells were collected and stained with Annexin V-FITC and PI. Using flow cytometry, the staining intensity of the cells for Annexin V-FITC and PI was determined. The effect of 25 μM curcumin without pre-treatment was stated as 100%. Shown is the quantification of quadrant analysis of 5 independent experiments (mean ± S.D.). Asterisks indicate statistically significant different from control cells, *P < 0.05, **P < 0.01, ***P < 0.001 and #P < 0.05 compared to 5 μM curcumin as pre-treatment.
Mentions: HO has been described as an enzyme with cytoprotective functions. Therefore, we studied whether pre-induction of HO-1 would protect against apoptosis induced by 25-μM curcumin. In Figure 6, it is shown that 5-μM curcumin pre-treatment followed by 25-μM curcumin treatment indeed causes a significant increase in the percentage of living fibroblasts, compared to cells that received no pre-treatment (P < 0.001). This observation was accompanied by a significant decrease in the percentage dead cells after pre-treatment with 5-μM curcumin (P < 0.01). Pre-treatment with 2.5-μM curcumin had similar but milder effects (data not shown). Importantly, the protective effect of preconditioning was blocked significantly when co-treated with the HO-activity inhibitor SnMP (P < 0.05). Taken together, these data indicate that a modest up-regulation of HO-1 protein expression and HO-activity by preconditioning with curcumin, subsequently, had significant cytoprotective effects in fibroblasts.

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