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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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Dose-dependent induction of HO-1 protein expression and HO-activity by curcumin. HO-1 protein expression was studied in fibroblasts treated for 24 hrs with different doses of curcumin. As a positive control, cells were treated with 10-μM FePP or CoPP. Cell lysates were prepared and proteins were separated by SDS-PAGE and subsequently blotted onto a nitrocellulose membrane. HO-1 protein and beta-actin (as a loading control) were stained with specific antibodies. Detection was done with the appropriate fluorescently labelled secondary antibodies and the Odyssey Infrared Imaging System. (A) Results of the Western blot showing bands corresponding to HO-1 and beta-actin protein. (B) Relative quantification of HO-1 proteins bands corrected for beta-actin from 5 independent experiments (mean ± S.D.) (C) Determination of HO-activity by quantifying bilirubin levels via HPLC. For details on the HO-activity assay, see ‘materials and methods’ section. The mean ± S.D. of the area under the curve of the peak are shown corresponding to bilirubin of 5 independent experiments. The bilirubin peak in the untreated group was set at 1 and the bilirubin peaks in the other groups were normalized accordingly. *P < 0.05, **P < 0.01 and **P < 0.001 compared to the untreated group.
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fig03: Dose-dependent induction of HO-1 protein expression and HO-activity by curcumin. HO-1 protein expression was studied in fibroblasts treated for 24 hrs with different doses of curcumin. As a positive control, cells were treated with 10-μM FePP or CoPP. Cell lysates were prepared and proteins were separated by SDS-PAGE and subsequently blotted onto a nitrocellulose membrane. HO-1 protein and beta-actin (as a loading control) were stained with specific antibodies. Detection was done with the appropriate fluorescently labelled secondary antibodies and the Odyssey Infrared Imaging System. (A) Results of the Western blot showing bands corresponding to HO-1 and beta-actin protein. (B) Relative quantification of HO-1 proteins bands corrected for beta-actin from 5 independent experiments (mean ± S.D.) (C) Determination of HO-activity by quantifying bilirubin levels via HPLC. For details on the HO-activity assay, see ‘materials and methods’ section. The mean ± S.D. of the area under the curve of the peak are shown corresponding to bilirubin of 5 independent experiments. The bilirubin peak in the untreated group was set at 1 and the bilirubin peaks in the other groups were normalized accordingly. *P < 0.05, **P < 0.01 and **P < 0.001 compared to the untreated group.

Mentions: Since biliverdin and bilirubin are down-stream products of heme degradation by the rate-limiting enzyme HO, we investigated whether pre-induction of HO-1 could protect against curcumin-induced apoptosis in fibroblasts. Therefore, we first examined whether doses of curcumin, not affecting fibroblast viability, could induce HO-1 expression in fibroblasts. Figure 3 demonstrates that, as expected, both 10-μM heme and 10-μM CoPP cause a clear induction in the expression of HO-1. Differential effects of curcumin (0–27.5 μM) on HO-1 expression are clearly visible; from 5 μM up to a dose of 17.5 μM, a significant induction of HO-1 protein can be observed, whereas from 20 to 27.5 μM no significant difference from basal levels could be detected. The maximal induction of ∼50-fold compared to untreated cells was observed between 10 and 15 μM curcumin (P < 0.001). HO-1 expression was at basal levels after treatment with 25-μM curcumin.


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)

Dose-dependent induction of HO-1 protein expression and HO-activity by curcumin. HO-1 protein expression was studied in fibroblasts treated for 24 hrs with different doses of curcumin. As a positive control, cells were treated with 10-μM FePP or CoPP. Cell lysates were prepared and proteins were separated by SDS-PAGE and subsequently blotted onto a nitrocellulose membrane. HO-1 protein and beta-actin (as a loading control) were stained with specific antibodies. Detection was done with the appropriate fluorescently labelled secondary antibodies and the Odyssey Infrared Imaging System. (A) Results of the Western blot showing bands corresponding to HO-1 and beta-actin protein. (B) Relative quantification of HO-1 proteins bands corrected for beta-actin from 5 independent experiments (mean ± S.D.) (C) Determination of HO-activity by quantifying bilirubin levels via HPLC. For details on the HO-activity assay, see ‘materials and methods’ section. The mean ± S.D. of the area under the curve of the peak are shown corresponding to bilirubin of 5 independent experiments. The bilirubin peak in the untreated group was set at 1 and the bilirubin peaks in the other groups were normalized accordingly. *P < 0.05, **P < 0.01 and **P < 0.001 compared to the untreated group.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3822878&req=5

fig03: Dose-dependent induction of HO-1 protein expression and HO-activity by curcumin. HO-1 protein expression was studied in fibroblasts treated for 24 hrs with different doses of curcumin. As a positive control, cells were treated with 10-μM FePP or CoPP. Cell lysates were prepared and proteins were separated by SDS-PAGE and subsequently blotted onto a nitrocellulose membrane. HO-1 protein and beta-actin (as a loading control) were stained with specific antibodies. Detection was done with the appropriate fluorescently labelled secondary antibodies and the Odyssey Infrared Imaging System. (A) Results of the Western blot showing bands corresponding to HO-1 and beta-actin protein. (B) Relative quantification of HO-1 proteins bands corrected for beta-actin from 5 independent experiments (mean ± S.D.) (C) Determination of HO-activity by quantifying bilirubin levels via HPLC. For details on the HO-activity assay, see ‘materials and methods’ section. The mean ± S.D. of the area under the curve of the peak are shown corresponding to bilirubin of 5 independent experiments. The bilirubin peak in the untreated group was set at 1 and the bilirubin peaks in the other groups were normalized accordingly. *P < 0.05, **P < 0.01 and **P < 0.001 compared to the untreated group.
Mentions: Since biliverdin and bilirubin are down-stream products of heme degradation by the rate-limiting enzyme HO, we investigated whether pre-induction of HO-1 could protect against curcumin-induced apoptosis in fibroblasts. Therefore, we first examined whether doses of curcumin, not affecting fibroblast viability, could induce HO-1 expression in fibroblasts. Figure 3 demonstrates that, as expected, both 10-μM heme and 10-μM CoPP cause a clear induction in the expression of HO-1. Differential effects of curcumin (0–27.5 μM) on HO-1 expression are clearly visible; from 5 μM up to a dose of 17.5 μM, a significant induction of HO-1 protein can be observed, whereas from 20 to 27.5 μM no significant difference from basal levels could be detected. The maximal induction of ∼50-fold compared to untreated cells was observed between 10 and 15 μM curcumin (P < 0.001). HO-1 expression was at basal levels after treatment with 25-μM curcumin.

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