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The role of reactive oxygen species and subsequent DNA-damage response in the emergence of resistance towards resveratrol in colon cancer models.

Colin DJ, Limagne E, Ragot K, Lizard G, Ghiringhelli F, Solary É, Chauffert B, Latruffe N, Delmas D - Cell Death Dis (2014)

Bottom Line: Ultimately, stable resistant populations towards RSV displaying higher degrees of ploidy and macronucleation as compared to parental cells emerged.We linked these transient effects and resistance emergence to the abilities of these cells to progressively escape RSV-induced DNA damage.This study provides a pre-clinical analysis of the long-term effects of RSV and highlights ROS as main agents in RSV's indirect DNA-damaging properties and consequences in terms of anticancer response and potent resistance emergence.

View Article: PubMed Central - PubMed

Affiliation: 1] Université de Bourgogne, Dijon F-21000, France [2] Centre de Recherche Inserm U866-Lipids, Nutrition, Cancers, Dijon F-21000, France [3] EA7270-Bio-PeroxIL Biochimie du peroxysome, inflammation et métabolisme lipidique, Dijon F-21000, France.

ABSTRACT
In spite of the novel strategies to treat colon cancer, mortality rates associated with this disease remain consistently high. Tumour recurrence has been linked to the induction of resistance towards chemotherapy that involves cellular events that enable cancer cells to escape cell death. Treatment of colon cancer mainly implicates direct or indirect DNA-damaging agents and increased repair or tolerances towards subsequent lesions contribute to generate resistant populations. Resveratrol (RSV), a potent chemosensitising polyphenol, might share common properties with chemotherapeutic drugs through its indirect DNA-damaging effects reported in vitro. In this study, we investigated how RSV exerts its anticancer effects in models of colon cancer with a particular emphasis on the DNA-damage response (DDR; PIKKs-Chks-p53 signalling cascade) and its cellular consequences. We showed in vitro and in vivo that colon cancer models could progressively escape the repeated pharmacological treatments with RSV. We observed for the first time that this response was correlated with transient activation of the DDR, of apoptosis and senescence. In vitro, a single treatment with RSV induced a DDR correlated with S-phase delay and apoptosis, but prolonged treatments led to transient micronucleations and senescence phenotypes associated with polyploidisation. Ultimately, stable resistant populations towards RSV displaying higher degrees of ploidy and macronucleation as compared to parental cells emerged. We linked these transient effects and resistance emergence to the abilities of these cells to progressively escape RSV-induced DNA damage. Finally, we demonstrated that this DNA damage was triggered by an overproduction of reactive oxygen species (ROS) against which cancer cells could adapt under prolonged exposure to RSV. This study provides a pre-clinical analysis of the long-term effects of RSV and highlights ROS as main agents in RSV's indirect DNA-damaging properties and consequences in terms of anticancer response and potent resistance emergence.

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Resveratrol promotes the phosphorylation of the DNA-damage marker histone H2AX.(a) Flow cytometric analyses of γH2AX/PI staining onPROb and SW620 cells after 1 day of treatment (d1) with 30 μMof RSV (R30) or mock-treated (Ctl); the percentages represent the amounts ofγH2AX positive cells. (b) Microscopic analysis ofγH2AX immunostainings of PROb cells grown on coverslips treatedwith R30 during 1 (d1) or 3 days (d3). Arrows show macronucleated cells (M) andapoptotic ones (A); bars, on the left 100 μm, on theright 20 μm. (c, d) Time course of expression andphosphorylation of the components of the ATR, ATM and DNA-PK pathways wereanalysed by immunoblotting using the specified antibodies. PROb and SW620 cells aswell as their resistant counterpart, R2PROb and R2SW620populations, were treated with R30 for indicated times in days (d) and comparedwith the untreated cells (Ctl)
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fig6: Resveratrol promotes the phosphorylation of the DNA-damage marker histone H2AX.(a) Flow cytometric analyses of γH2AX/PI staining onPROb and SW620 cells after 1 day of treatment (d1) with 30 μMof RSV (R30) or mock-treated (Ctl); the percentages represent the amounts ofγH2AX positive cells. (b) Microscopic analysis ofγH2AX immunostainings of PROb cells grown on coverslips treatedwith R30 during 1 (d1) or 3 days (d3). Arrows show macronucleated cells (M) andapoptotic ones (A); bars, on the left 100 μm, on theright 20 μm. (c, d) Time course of expression andphosphorylation of the components of the ATR, ATM and DNA-PK pathways wereanalysed by immunoblotting using the specified antibodies. PROb and SW620 cells aswell as their resistant counterpart, R2PROb and R2SW620populations, were treated with R30 for indicated times in days (d) and comparedwith the untreated cells (Ctl)

Mentions: Resistance emergence towards RSV in CCR appeared to be related to a loss ofsensitivity towards its pro-apoptotic and pro-senescence activities associatedwith a transient DDR induction in vivo. Thereafter, we took advantage ofthis new in vitro sensitive/resistant model to further characterisethe DNA-damaging effects of RSV. To decipher the sequence of these events, weassessed the induction of γH2AX in a cell cycle-dependent manner.After 24 h of treatment, ~70% of RSV-treated cells exhibited highlevels of γH2AX (Figure 6a). Most ofthe γH2AX positive cells had diploid or tetraploid S-phase DNAcontents, which is a hallmark of cells under replication stress. Microscopicanalyses demonstrated typical γH2AX foci after treatments andhighly positive apoptotic cells as γH2AX is known to be involved inthis process (Figure 6b). Finally, as theγH2AX induction appeared to be highly transient in RSV-treatedresistant populations (Supplementary Figure S3),we hypothesised that RSV could generate a replication stress in sensitive cellswhich would ultimately adapt to this damaging effect.


The role of reactive oxygen species and subsequent DNA-damage response in the emergence of resistance towards resveratrol in colon cancer models.

Colin DJ, Limagne E, Ragot K, Lizard G, Ghiringhelli F, Solary É, Chauffert B, Latruffe N, Delmas D - Cell Death Dis (2014)

Resveratrol promotes the phosphorylation of the DNA-damage marker histone H2AX.(a) Flow cytometric analyses of γH2AX/PI staining onPROb and SW620 cells after 1 day of treatment (d1) with 30 μMof RSV (R30) or mock-treated (Ctl); the percentages represent the amounts ofγH2AX positive cells. (b) Microscopic analysis ofγH2AX immunostainings of PROb cells grown on coverslips treatedwith R30 during 1 (d1) or 3 days (d3). Arrows show macronucleated cells (M) andapoptotic ones (A); bars, on the left 100 μm, on theright 20 μm. (c, d) Time course of expression andphosphorylation of the components of the ATR, ATM and DNA-PK pathways wereanalysed by immunoblotting using the specified antibodies. PROb and SW620 cells aswell as their resistant counterpart, R2PROb and R2SW620populations, were treated with R30 for indicated times in days (d) and comparedwith the untreated cells (Ctl)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260744&req=5

fig6: Resveratrol promotes the phosphorylation of the DNA-damage marker histone H2AX.(a) Flow cytometric analyses of γH2AX/PI staining onPROb and SW620 cells after 1 day of treatment (d1) with 30 μMof RSV (R30) or mock-treated (Ctl); the percentages represent the amounts ofγH2AX positive cells. (b) Microscopic analysis ofγH2AX immunostainings of PROb cells grown on coverslips treatedwith R30 during 1 (d1) or 3 days (d3). Arrows show macronucleated cells (M) andapoptotic ones (A); bars, on the left 100 μm, on theright 20 μm. (c, d) Time course of expression andphosphorylation of the components of the ATR, ATM and DNA-PK pathways wereanalysed by immunoblotting using the specified antibodies. PROb and SW620 cells aswell as their resistant counterpart, R2PROb and R2SW620populations, were treated with R30 for indicated times in days (d) and comparedwith the untreated cells (Ctl)
Mentions: Resistance emergence towards RSV in CCR appeared to be related to a loss ofsensitivity towards its pro-apoptotic and pro-senescence activities associatedwith a transient DDR induction in vivo. Thereafter, we took advantage ofthis new in vitro sensitive/resistant model to further characterisethe DNA-damaging effects of RSV. To decipher the sequence of these events, weassessed the induction of γH2AX in a cell cycle-dependent manner.After 24 h of treatment, ~70% of RSV-treated cells exhibited highlevels of γH2AX (Figure 6a). Most ofthe γH2AX positive cells had diploid or tetraploid S-phase DNAcontents, which is a hallmark of cells under replication stress. Microscopicanalyses demonstrated typical γH2AX foci after treatments andhighly positive apoptotic cells as γH2AX is known to be involved inthis process (Figure 6b). Finally, as theγH2AX induction appeared to be highly transient in RSV-treatedresistant populations (Supplementary Figure S3),we hypothesised that RSV could generate a replication stress in sensitive cellswhich would ultimately adapt to this damaging effect.

Bottom Line: Ultimately, stable resistant populations towards RSV displaying higher degrees of ploidy and macronucleation as compared to parental cells emerged.We linked these transient effects and resistance emergence to the abilities of these cells to progressively escape RSV-induced DNA damage.This study provides a pre-clinical analysis of the long-term effects of RSV and highlights ROS as main agents in RSV's indirect DNA-damaging properties and consequences in terms of anticancer response and potent resistance emergence.

View Article: PubMed Central - PubMed

Affiliation: 1] Université de Bourgogne, Dijon F-21000, France [2] Centre de Recherche Inserm U866-Lipids, Nutrition, Cancers, Dijon F-21000, France [3] EA7270-Bio-PeroxIL Biochimie du peroxysome, inflammation et métabolisme lipidique, Dijon F-21000, France.

ABSTRACT
In spite of the novel strategies to treat colon cancer, mortality rates associated with this disease remain consistently high. Tumour recurrence has been linked to the induction of resistance towards chemotherapy that involves cellular events that enable cancer cells to escape cell death. Treatment of colon cancer mainly implicates direct or indirect DNA-damaging agents and increased repair or tolerances towards subsequent lesions contribute to generate resistant populations. Resveratrol (RSV), a potent chemosensitising polyphenol, might share common properties with chemotherapeutic drugs through its indirect DNA-damaging effects reported in vitro. In this study, we investigated how RSV exerts its anticancer effects in models of colon cancer with a particular emphasis on the DNA-damage response (DDR; PIKKs-Chks-p53 signalling cascade) and its cellular consequences. We showed in vitro and in vivo that colon cancer models could progressively escape the repeated pharmacological treatments with RSV. We observed for the first time that this response was correlated with transient activation of the DDR, of apoptosis and senescence. In vitro, a single treatment with RSV induced a DDR correlated with S-phase delay and apoptosis, but prolonged treatments led to transient micronucleations and senescence phenotypes associated with polyploidisation. Ultimately, stable resistant populations towards RSV displaying higher degrees of ploidy and macronucleation as compared to parental cells emerged. We linked these transient effects and resistance emergence to the abilities of these cells to progressively escape RSV-induced DNA damage. Finally, we demonstrated that this DNA damage was triggered by an overproduction of reactive oxygen species (ROS) against which cancer cells could adapt under prolonged exposure to RSV. This study provides a pre-clinical analysis of the long-term effects of RSV and highlights ROS as main agents in RSV's indirect DNA-damaging properties and consequences in terms of anticancer response and potent resistance emergence.

Show MeSH
Related in: MedlinePlus