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Viscum album-mediated COX-2 inhibition implicates destabilization of COX-2 mRNA.

Saha C, Hegde P, Friboulet A, Bayry J, Kaveri SV - PLoS ONE (2015)

Bottom Line: We have recently demonstrated that, VA preparations exert a potent anti-inflammatory effect by selectively down-regulating the COX-2-mediated cytokine-induced secretion of prostaglandin E2 (PGE2), one of the important molecular signatures of inflammatory reactions.Using cyclohexamide pulse chase experiment, we demonstrate that, COX-2 protein degradation is not affected by the treatment with VA whereas experiments on transcriptional blockade with actinomycin D, revealed a marked reduction in the half life of COX-2 mRNA due to its rapid degradation in the cells treated with VA compared to that in IL-1β-stimulated cells.These results thus demonstrate that VA-mediated inhibition of PGE2 implicates destabilization of COX-2 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, Unité 1138, Paris, France; Université de Technologie de Compiègne, UMR CNRS 6022, Compiègne, France; Centre de Recherche des Cordeliers, Equipe-Immunopathology and therapeutic immunointervention, Paris, France.

ABSTRACT
Extensive use of Viscum album (VA) preparations in the complementary therapy of cancer and in several other human pathologies has led to an increasing number of cellular and molecular approaches to explore the mechanisms of action of VA. We have recently demonstrated that, VA preparations exert a potent anti-inflammatory effect by selectively down-regulating the COX-2-mediated cytokine-induced secretion of prostaglandin E2 (PGE2), one of the important molecular signatures of inflammatory reactions. In this study, we observed a significant down-regulation of COX-2 protein expression in VA-treated A549 cells however COX-2 mRNA levels were unaltered. Therefore, we hypothesized that VA induces destabilisation of COX-2 mRNA, thereby depleting the available functional COX-2 mRNA for the protein synthesis and for the subsequent secretion of PGE2. To address this question, we analyzed the molecular degradation of COX-2 protein and its corresponding mRNA in A549 cell line. Using cyclohexamide pulse chase experiment, we demonstrate that, COX-2 protein degradation is not affected by the treatment with VA whereas experiments on transcriptional blockade with actinomycin D, revealed a marked reduction in the half life of COX-2 mRNA due to its rapid degradation in the cells treated with VA compared to that in IL-1β-stimulated cells. These results thus demonstrate that VA-mediated inhibition of PGE2 implicates destabilization of COX-2 mRNA.

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Effect of Viscum album on the stability of COX-2 protein as determined by western blot.Confluent A549 cells were treated with IL-1β in the presence and absence of VA Qu Spez in dose dependent concentrations in μg/ml. Cells were harvested at different time intervals after blocking the protein synthesis with cyclohexamide (10 μg/ml) for 90 minutes till 11 hours. COX-2 expression was measured by western blot using the cytosolic extracts. (A), inhibition of COX2 protein synthesis by VA at 18 hours. (B) (C) (D) are the representative western blots after 90 minutes, 5 hours and 11 hours respectively showing level of COX-2 expression after cyclohexamide treatment with or without Viscum album. β-actin was used as an internal control. All blots are representative of three independent experiments and the densitometry values for each band are mentioned below the representative blots.
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pone.0114965.g003: Effect of Viscum album on the stability of COX-2 protein as determined by western blot.Confluent A549 cells were treated with IL-1β in the presence and absence of VA Qu Spez in dose dependent concentrations in μg/ml. Cells were harvested at different time intervals after blocking the protein synthesis with cyclohexamide (10 μg/ml) for 90 minutes till 11 hours. COX-2 expression was measured by western blot using the cytosolic extracts. (A), inhibition of COX2 protein synthesis by VA at 18 hours. (B) (C) (D) are the representative western blots after 90 minutes, 5 hours and 11 hours respectively showing level of COX-2 expression after cyclohexamide treatment with or without Viscum album. β-actin was used as an internal control. All blots are representative of three independent experiments and the densitometry values for each band are mentioned below the representative blots.

Mentions: In order to address the effect of VA on the molecular stability of COX-2, which could be a potential contributing factor for the observed reduction in COX-2 protein expression, we analyzed the stability of COX-2 protein. A549 cells were stimulated with a pro-inflammatory cytokine IL-1β in the presence and absence of VA Qu Spez. At 18 hours, we observed a significant reduction in COX-2 protein level treated with VA Qu Spez. Further, cells were harvested at different time intervals after blocking the protein synthesis by treating the cells with cyclohexamide and analyzed for COX-2. Flow cytometric analysis of COX-2 protein has revealed that, there is no significant difference in the protein degradation profile of COX-2 in VA-treated and untreated cells after 90 minutes of blocking the protein synthesis (Fig. 2A and Fig. 2B). Further, western blot analysis of COX-2 protein expression at different time intervals showed that despite the clear inhibition in the protein expression after 18 hours of exposure to cytokine followed by VA treatment (Fig. 3A), upon blocking the protein synthesis, there is no remarkable difference in the COX-2 degradation profile in cells treated with cytokine irrespective of VA treatment (Fig. 3B, 3C and 3D). Fig. 3B indicates the level of COX-2 expression immediately after 90 minutes of cyclohexamide addition (0 hour). Figs. 3C and 3D indicate the level of COX-2 expression upon blocking the protein synthesis after 5 and 11 hours respectively. These results may indicate that the regulation of COX-2 by VA may occur in an early phase of COX-2 expression but not at the later stages of protein expression and stabilization.


Viscum album-mediated COX-2 inhibition implicates destabilization of COX-2 mRNA.

Saha C, Hegde P, Friboulet A, Bayry J, Kaveri SV - PLoS ONE (2015)

Effect of Viscum album on the stability of COX-2 protein as determined by western blot.Confluent A549 cells were treated with IL-1β in the presence and absence of VA Qu Spez in dose dependent concentrations in μg/ml. Cells were harvested at different time intervals after blocking the protein synthesis with cyclohexamide (10 μg/ml) for 90 minutes till 11 hours. COX-2 expression was measured by western blot using the cytosolic extracts. (A), inhibition of COX2 protein synthesis by VA at 18 hours. (B) (C) (D) are the representative western blots after 90 minutes, 5 hours and 11 hours respectively showing level of COX-2 expression after cyclohexamide treatment with or without Viscum album. β-actin was used as an internal control. All blots are representative of three independent experiments and the densitometry values for each band are mentioned below the representative blots.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321838&req=5

pone.0114965.g003: Effect of Viscum album on the stability of COX-2 protein as determined by western blot.Confluent A549 cells were treated with IL-1β in the presence and absence of VA Qu Spez in dose dependent concentrations in μg/ml. Cells were harvested at different time intervals after blocking the protein synthesis with cyclohexamide (10 μg/ml) for 90 minutes till 11 hours. COX-2 expression was measured by western blot using the cytosolic extracts. (A), inhibition of COX2 protein synthesis by VA at 18 hours. (B) (C) (D) are the representative western blots after 90 minutes, 5 hours and 11 hours respectively showing level of COX-2 expression after cyclohexamide treatment with or without Viscum album. β-actin was used as an internal control. All blots are representative of three independent experiments and the densitometry values for each band are mentioned below the representative blots.
Mentions: In order to address the effect of VA on the molecular stability of COX-2, which could be a potential contributing factor for the observed reduction in COX-2 protein expression, we analyzed the stability of COX-2 protein. A549 cells were stimulated with a pro-inflammatory cytokine IL-1β in the presence and absence of VA Qu Spez. At 18 hours, we observed a significant reduction in COX-2 protein level treated with VA Qu Spez. Further, cells were harvested at different time intervals after blocking the protein synthesis by treating the cells with cyclohexamide and analyzed for COX-2. Flow cytometric analysis of COX-2 protein has revealed that, there is no significant difference in the protein degradation profile of COX-2 in VA-treated and untreated cells after 90 minutes of blocking the protein synthesis (Fig. 2A and Fig. 2B). Further, western blot analysis of COX-2 protein expression at different time intervals showed that despite the clear inhibition in the protein expression after 18 hours of exposure to cytokine followed by VA treatment (Fig. 3A), upon blocking the protein synthesis, there is no remarkable difference in the COX-2 degradation profile in cells treated with cytokine irrespective of VA treatment (Fig. 3B, 3C and 3D). Fig. 3B indicates the level of COX-2 expression immediately after 90 minutes of cyclohexamide addition (0 hour). Figs. 3C and 3D indicate the level of COX-2 expression upon blocking the protein synthesis after 5 and 11 hours respectively. These results may indicate that the regulation of COX-2 by VA may occur in an early phase of COX-2 expression but not at the later stages of protein expression and stabilization.

Bottom Line: We have recently demonstrated that, VA preparations exert a potent anti-inflammatory effect by selectively down-regulating the COX-2-mediated cytokine-induced secretion of prostaglandin E2 (PGE2), one of the important molecular signatures of inflammatory reactions.Using cyclohexamide pulse chase experiment, we demonstrate that, COX-2 protein degradation is not affected by the treatment with VA whereas experiments on transcriptional blockade with actinomycin D, revealed a marked reduction in the half life of COX-2 mRNA due to its rapid degradation in the cells treated with VA compared to that in IL-1β-stimulated cells.These results thus demonstrate that VA-mediated inhibition of PGE2 implicates destabilization of COX-2 mRNA.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et de la Recherche Médicale, Unité 1138, Paris, France; Université de Technologie de Compiègne, UMR CNRS 6022, Compiègne, France; Centre de Recherche des Cordeliers, Equipe-Immunopathology and therapeutic immunointervention, Paris, France.

ABSTRACT
Extensive use of Viscum album (VA) preparations in the complementary therapy of cancer and in several other human pathologies has led to an increasing number of cellular and molecular approaches to explore the mechanisms of action of VA. We have recently demonstrated that, VA preparations exert a potent anti-inflammatory effect by selectively down-regulating the COX-2-mediated cytokine-induced secretion of prostaglandin E2 (PGE2), one of the important molecular signatures of inflammatory reactions. In this study, we observed a significant down-regulation of COX-2 protein expression in VA-treated A549 cells however COX-2 mRNA levels were unaltered. Therefore, we hypothesized that VA induces destabilisation of COX-2 mRNA, thereby depleting the available functional COX-2 mRNA for the protein synthesis and for the subsequent secretion of PGE2. To address this question, we analyzed the molecular degradation of COX-2 protein and its corresponding mRNA in A549 cell line. Using cyclohexamide pulse chase experiment, we demonstrate that, COX-2 protein degradation is not affected by the treatment with VA whereas experiments on transcriptional blockade with actinomycin D, revealed a marked reduction in the half life of COX-2 mRNA due to its rapid degradation in the cells treated with VA compared to that in IL-1β-stimulated cells. These results thus demonstrate that VA-mediated inhibition of PGE2 implicates destabilization of COX-2 mRNA.

Show MeSH
Related in: MedlinePlus