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The Cytoprotective Effects of E-α-(4-Methoxyphenyl)-2',3,4,4'-Tetramethoxychalcone (E-α-p-OMe-C6H4-TMC)--A Novel and Non-Cytotoxic HO-1 Inducer.

Kaufmann KB, Al-Rifai N, Ulbrich F, Schallner N, Rücker H, Enzinger M, Petkes H, Pitzl S, Goebel U, Amslinger S - PLoS ONE (2015)

Bottom Line: We found that amongst all tested chalcones only E-α-(4-methoxyphenyl)-2',3,4,4'-tetramethoxychalcone (E-α-p-OMe-C6H4-TMC) demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C6H4-TMC displayed no significant activity.The only weakly electrophilic chalcone E-α-p-OMe-C6H4-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus.The observed cytoprotective effect may partly be related to both, the activation of the Nrf2- and inhibition of the NF-κB pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology and Intensive Care Medicine, University Medical Center Freiburg, Freiburg, Germany.

ABSTRACT
Cell protection against different noxious stimuli like oxidative stress or chemical toxins plays a central role in the treatment of many diseases. The inducible heme oxygenase isoform, heme oxygenase-1 (HO-1), is known to protect cells against a variety of harmful conditions including apoptosis. Because a number of medium strong electrophiles from a series of α-X-substituted 2',3,4,4'-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH) had proven to activate Nrf2 resulting in HO-1 induction and inhibit NF-κB downstream target genes, their protective effect against staurosporine induced apoptosis and reactive oxygen species (ROS) production was investigated. RAW264.7 macrophages treated with 19 different chalcones (15 α-X-TMCs, chalcone, 2'-hydroxychalcone, calythropsin and 2'-hydroxy-3,4,4'-trimethoxychalcone) prior to staurosporine treatment were analyzed for apoptosis and ROS production, as well as HO-1 protein expression and enzyme activity. Additionally, Nrf2 and NF-κB activity was assessed. We found that amongst all tested chalcones only E-α-(4-methoxyphenyl)-2',3,4,4'-tetramethoxychalcone (E-α-p-OMe-C6H4-TMC) demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C6H4-TMC displayed no significant activity. Also, E-α-p-OMe-C6H4-TMC induced HO-1 protein expression and increased HO-1 activity, whilst inhibition of HO-1 by SnPP-IX abolished its antiapoptotic effect. The only weakly electrophilic chalcone E-α-p-OMe-C6H4-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus. Furthermore, staurosporine induced NF-κB activity was attenuated following E-α-p-OMe-C6H4-TMC treatment. Overall, E-α-p-OMe-C6H4-TMC demonstrated its effective cytoprotective potential via a non-toxic induction of HO-1 in RAW264.7 macrophages. The observed cytoprotective effect may partly be related to both, the activation of the Nrf2- and inhibition of the NF-κB pathway.

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Flow cytometric analysis.Effect of different chalcones on staurosporine-induced apoptosis in RAW264.7 macrophages. Cells were pretreated with the indicated concentrations of chalcones for 3 h, before apoptosis was induced by staurosporine (1 μM for 2 h). Cells were harvested and stained with FITC annexin-V and propidium iodide. 1 × 104 cells were analyzed in each experiment. (n = 4; mean ± S.E.M.; *** = p < 0.001 untreated vs. staurosporine and staurosporine vs. 10, 100, 500 and 1000 μM E-α-p-OMe-C6H4-TMC + staurosporine and * = p < 0.05 staurosporine vs. 1 μM E-α-p-OMe-C6H4-TMC + staurosporine). k2 values for the reaction with the model thiol cysteamine are taken from [21]. N, native; S, staurosporine.
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pone.0142932.g001: Flow cytometric analysis.Effect of different chalcones on staurosporine-induced apoptosis in RAW264.7 macrophages. Cells were pretreated with the indicated concentrations of chalcones for 3 h, before apoptosis was induced by staurosporine (1 μM for 2 h). Cells were harvested and stained with FITC annexin-V and propidium iodide. 1 × 104 cells were analyzed in each experiment. (n = 4; mean ± S.E.M.; *** = p < 0.001 untreated vs. staurosporine and staurosporine vs. 10, 100, 500 and 1000 μM E-α-p-OMe-C6H4-TMC + staurosporine and * = p < 0.05 staurosporine vs. 1 μM E-α-p-OMe-C6H4-TMC + staurosporine). k2 values for the reaction with the model thiol cysteamine are taken from [21]. N, native; S, staurosporine.

Mentions: We used our library of α-X-TMCs which possesses a wide range of electrophilicity together with the medium strong to moderate electrophilic chalcones 2’-hydroxychalcone, calythropsin, 2’-hydroxy-3,4,4’-trimethoxychalcone (α-H-HC) and chalcone itself. The second-order rate constants k2 for the reaction with the model thiol cysteamine are shown in Figs 1 and 2 [20, 21]. By applying these compounds we can utilize a chemical reactivity range of more than 6 orders of magnitude. When including the previously not described geometric isomer Z-α-p-OMe-C6H4-TMC the difference in the k2 values between the most electrophilic compound α-CN-TMC and Z-α-p-OMe-C6H4-TMC is even 300 million fold, since we determined the k2 value for Z-α-p-OMe-C6H4-TMC to be 0.0000196 ± 0.0000021 M-1s-1.


The Cytoprotective Effects of E-α-(4-Methoxyphenyl)-2',3,4,4'-Tetramethoxychalcone (E-α-p-OMe-C6H4-TMC)--A Novel and Non-Cytotoxic HO-1 Inducer.

Kaufmann KB, Al-Rifai N, Ulbrich F, Schallner N, Rücker H, Enzinger M, Petkes H, Pitzl S, Goebel U, Amslinger S - PLoS ONE (2015)

Flow cytometric analysis.Effect of different chalcones on staurosporine-induced apoptosis in RAW264.7 macrophages. Cells were pretreated with the indicated concentrations of chalcones for 3 h, before apoptosis was induced by staurosporine (1 μM for 2 h). Cells were harvested and stained with FITC annexin-V and propidium iodide. 1 × 104 cells were analyzed in each experiment. (n = 4; mean ± S.E.M.; *** = p < 0.001 untreated vs. staurosporine and staurosporine vs. 10, 100, 500 and 1000 μM E-α-p-OMe-C6H4-TMC + staurosporine and * = p < 0.05 staurosporine vs. 1 μM E-α-p-OMe-C6H4-TMC + staurosporine). k2 values for the reaction with the model thiol cysteamine are taken from [21]. N, native; S, staurosporine.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643879&req=5

pone.0142932.g001: Flow cytometric analysis.Effect of different chalcones on staurosporine-induced apoptosis in RAW264.7 macrophages. Cells were pretreated with the indicated concentrations of chalcones for 3 h, before apoptosis was induced by staurosporine (1 μM for 2 h). Cells were harvested and stained with FITC annexin-V and propidium iodide. 1 × 104 cells were analyzed in each experiment. (n = 4; mean ± S.E.M.; *** = p < 0.001 untreated vs. staurosporine and staurosporine vs. 10, 100, 500 and 1000 μM E-α-p-OMe-C6H4-TMC + staurosporine and * = p < 0.05 staurosporine vs. 1 μM E-α-p-OMe-C6H4-TMC + staurosporine). k2 values for the reaction with the model thiol cysteamine are taken from [21]. N, native; S, staurosporine.
Mentions: We used our library of α-X-TMCs which possesses a wide range of electrophilicity together with the medium strong to moderate electrophilic chalcones 2’-hydroxychalcone, calythropsin, 2’-hydroxy-3,4,4’-trimethoxychalcone (α-H-HC) and chalcone itself. The second-order rate constants k2 for the reaction with the model thiol cysteamine are shown in Figs 1 and 2 [20, 21]. By applying these compounds we can utilize a chemical reactivity range of more than 6 orders of magnitude. When including the previously not described geometric isomer Z-α-p-OMe-C6H4-TMC the difference in the k2 values between the most electrophilic compound α-CN-TMC and Z-α-p-OMe-C6H4-TMC is even 300 million fold, since we determined the k2 value for Z-α-p-OMe-C6H4-TMC to be 0.0000196 ± 0.0000021 M-1s-1.

Bottom Line: We found that amongst all tested chalcones only E-α-(4-methoxyphenyl)-2',3,4,4'-tetramethoxychalcone (E-α-p-OMe-C6H4-TMC) demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C6H4-TMC displayed no significant activity.The only weakly electrophilic chalcone E-α-p-OMe-C6H4-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus.The observed cytoprotective effect may partly be related to both, the activation of the Nrf2- and inhibition of the NF-κB pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Anesthesiology and Intensive Care Medicine, University Medical Center Freiburg, Freiburg, Germany.

ABSTRACT
Cell protection against different noxious stimuli like oxidative stress or chemical toxins plays a central role in the treatment of many diseases. The inducible heme oxygenase isoform, heme oxygenase-1 (HO-1), is known to protect cells against a variety of harmful conditions including apoptosis. Because a number of medium strong electrophiles from a series of α-X-substituted 2',3,4,4'-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH) had proven to activate Nrf2 resulting in HO-1 induction and inhibit NF-κB downstream target genes, their protective effect against staurosporine induced apoptosis and reactive oxygen species (ROS) production was investigated. RAW264.7 macrophages treated with 19 different chalcones (15 α-X-TMCs, chalcone, 2'-hydroxychalcone, calythropsin and 2'-hydroxy-3,4,4'-trimethoxychalcone) prior to staurosporine treatment were analyzed for apoptosis and ROS production, as well as HO-1 protein expression and enzyme activity. Additionally, Nrf2 and NF-κB activity was assessed. We found that amongst all tested chalcones only E-α-(4-methoxyphenyl)-2',3,4,4'-tetramethoxychalcone (E-α-p-OMe-C6H4-TMC) demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C6H4-TMC displayed no significant activity. Also, E-α-p-OMe-C6H4-TMC induced HO-1 protein expression and increased HO-1 activity, whilst inhibition of HO-1 by SnPP-IX abolished its antiapoptotic effect. The only weakly electrophilic chalcone E-α-p-OMe-C6H4-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus. Furthermore, staurosporine induced NF-κB activity was attenuated following E-α-p-OMe-C6H4-TMC treatment. Overall, E-α-p-OMe-C6H4-TMC demonstrated its effective cytoprotective potential via a non-toxic induction of HO-1 in RAW264.7 macrophages. The observed cytoprotective effect may partly be related to both, the activation of the Nrf2- and inhibition of the NF-κB pathway.

Show MeSH
Related in: MedlinePlus