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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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Total ROS detection by flow cytometric analysis.RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (short: E-pOMe) for 3 h, ROS production was induced using staurosporine (1 μM for 2 h); A. Representative experiment after intracellular ROS staining. 1 × 104 cells were analyzed in each experiment; B. E-α-p-OMe-C6H4-TMC mediated effect on intracellular ROS production (n = 6; mean ± S.E.M.; * = p < 0.05).
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pone.0142932.g007: Total ROS detection by flow cytometric analysis.RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (short: E-pOMe) for 3 h, ROS production was induced using staurosporine (1 μM for 2 h); A. Representative experiment after intracellular ROS staining. 1 × 104 cells were analyzed in each experiment; B. E-α-p-OMe-C6H4-TMC mediated effect on intracellular ROS production (n = 6; mean ± S.E.M.; * = p < 0.05).

Mentions: ROS production was induced using staurosporine. Compared to untreated cells application of staurosporine lead to a significant increase in ROS production (Fig 7A, and 7B, untreated 30.5 ± 0.9 vs. staurosporine 73.7 ± 6.9% ROS positive cells; *** = p < 0.001) Pretreatment with E-α-p-OMe-C6H4-TMC reduced ROS formation significantly (Fig 7A and 7B, staurosporine 73.7 ± 6.9 vs. staurosporine + 30 μM E-α-p-OMe-C6H4-TMC 40.9 ± 1.5% ROS positive cells; * = p < 0.05). To exclude the possibility that this effect is caused by DMSO we compared ROS production induced by staurosporine in cells pretreated with DMSO alone to cells pretreated with E-α-p-OMe-C6H4-TMC dissolved in DMSO (Fig 7A and 7B, staurosporine + DMSO 54.4 ± 2.5 vs. staurosporine + 30 μM E-α-p-OMe-C6H4-TMC 40.9 ± 1.5% ROS positive cells; * = p < 0.05).


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)

Total ROS detection by flow cytometric analysis.RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (short: E-pOMe) for 3 h, ROS production was induced using staurosporine (1 μM for 2 h); A. Representative experiment after intracellular ROS staining. 1 × 104 cells were analyzed in each experiment; B. E-α-p-OMe-C6H4-TMC mediated effect on intracellular ROS production (n = 6; mean ± S.E.M.; * = p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142932.g007: Total ROS detection by flow cytometric analysis.RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (short: E-pOMe) for 3 h, ROS production was induced using staurosporine (1 μM for 2 h); A. Representative experiment after intracellular ROS staining. 1 × 104 cells were analyzed in each experiment; B. E-α-p-OMe-C6H4-TMC mediated effect on intracellular ROS production (n = 6; mean ± S.E.M.; * = p < 0.05).
Mentions: ROS production was induced using staurosporine. Compared to untreated cells application of staurosporine lead to a significant increase in ROS production (Fig 7A, and 7B, untreated 30.5 ± 0.9 vs. staurosporine 73.7 ± 6.9% ROS positive cells; *** = p < 0.001) Pretreatment with E-α-p-OMe-C6H4-TMC reduced ROS formation significantly (Fig 7A and 7B, staurosporine 73.7 ± 6.9 vs. staurosporine + 30 μM E-α-p-OMe-C6H4-TMC 40.9 ± 1.5% ROS positive cells; * = p < 0.05). To exclude the possibility that this effect is caused by DMSO we compared ROS production induced by staurosporine in cells pretreated with DMSO alone to cells pretreated with E-α-p-OMe-C6H4-TMC dissolved in DMSO (Fig 7A and 7B, staurosporine + DMSO 54.4 ± 2.5 vs. staurosporine + 30 μM E-α-p-OMe-C6H4-TMC 40.9 ± 1.5% ROS positive cells; * = p < 0.05).

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