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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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Related in: MedlinePlus

Effects of E-α-p-OMe-C6H4-TMC (short: E-pOMe) on Nrf2 expression after staurosporine treatment.RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (30 μM) for 3 h, followed by staurosporine (1 μM for 2 h). Nuclear and cytosolic cell extracts were prepared and Nrf2 expression was measured by Western blot analysis. A. The image is representative of four independent experiments that showed similar results; B. Densitometric analysis of nuclear extracts, optical density of Nrf2 normalized against Lamin B1 (n = 4; mean ± S.E.M.; ** = p < 0.005); C. Densitometric analysis of cytosolic extracts, optical density of Nrf2 normalized against β-actin (n = 4; mean ± S.E.M.); D. Densitometric analysis of nuclear Nrf2 normalized to cytosolic Nrf2 (n = 4; mean ± S.E.M.; * = p < 0.05); E. Effects of E-α-p-OMe-C6H4-TMC on NF-κB DNA binding activity after staurosporine treatment. RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (30 μM) for 3 h, followed by staurosporine (1 μM for 2 h). Nuclear extracts were prepared and NF-κB DNA binding activity was measured (n = 4; mean ± S.E.M.; * = p < 0.05).
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pone.0142932.g008: Effects of E-α-p-OMe-C6H4-TMC (short: E-pOMe) on Nrf2 expression after staurosporine treatment.RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (30 μM) for 3 h, followed by staurosporine (1 μM for 2 h). Nuclear and cytosolic cell extracts were prepared and Nrf2 expression was measured by Western blot analysis. A. The image is representative of four independent experiments that showed similar results; B. Densitometric analysis of nuclear extracts, optical density of Nrf2 normalized against Lamin B1 (n = 4; mean ± S.E.M.; ** = p < 0.005); C. Densitometric analysis of cytosolic extracts, optical density of Nrf2 normalized against β-actin (n = 4; mean ± S.E.M.); D. Densitometric analysis of nuclear Nrf2 normalized to cytosolic Nrf2 (n = 4; mean ± S.E.M.; * = p < 0.05); E. Effects of E-α-p-OMe-C6H4-TMC on NF-κB DNA binding activity after staurosporine treatment. RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (30 μM) for 3 h, followed by staurosporine (1 μM for 2 h). Nuclear extracts were prepared and NF-κB DNA binding activity was measured (n = 4; mean ± S.E.M.; * = p < 0.05).

Mentions: To elucidate a possible mechanism leading to the proposed cytoprotective effects via HO-1 induction by E-α-p-OMe-C6H4-TMC, we analyzed the translocation of the transcription factor Nrf2. Untreated cells compared to cells treated with E-α-p-OMe-C6H4-TMC alone showed a significant translocation of Nrf2 into the nucleus (Fig 8A, Lane 1 vs. 2; Fig 8B, untreated normalized to 1 vs. 30 μM E-α-p-OMe-C6H4-TMC 3.3 ± 2.8 fold change Nrf2/Lamin B1, * = p < 0.05). We did not detect an increased Nrf2 translocation after staurosporine induction alone, whereas pretreatment with E-α-p-OMe-C6H4-TMC before induction of apoptosis caused a significant nuclear translocation of Nrf2 (Fig 8A and 8B, staurosporine 1.6 ± 0.5 vs. staurosporine + 30 μM E-α-p-OMe-C6H4-TMC 3.2 ± 0.4 fold change Nrf2/Lamin B1; ** = p < 0.01). The cytosolic extracts did not show any significant differences regarding Nrf2 expression levels (Fig 8A and 8C). Fig 8D demonstrates the nuclear to cytosolic ratio of Nrf2 and in this way combines the results highlighted in Fig 8B and 8C. Macrophages treated with E-α-p-OMe-C6H4-TMC revealed a significantly increased nuclear to cytosolic ratio of Nrf2 compared to untreated cells, whereas E-α-p-OMe-C6H4-TMC pretreatment before induction of apoptosis lead to a significant increased ratio compared to staurosporine alone (Fig 8D, untreated normalized to 1 vs. 30 μM E-α-p-OMe-C6H4-TMC 2.4 ± 1.5; staurosporine 1.5 ± 0.6 vs. staurosporine + 30 μM E-α-p-OMe-C6H4-TMC 4.3 ± 1.9 Nrf2 ratio nuclear/cytosolic, * = 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)

Effects of E-α-p-OMe-C6H4-TMC (short: E-pOMe) on Nrf2 expression after staurosporine treatment.RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (30 μM) for 3 h, followed by staurosporine (1 μM for 2 h). Nuclear and cytosolic cell extracts were prepared and Nrf2 expression was measured by Western blot analysis. A. The image is representative of four independent experiments that showed similar results; B. Densitometric analysis of nuclear extracts, optical density of Nrf2 normalized against Lamin B1 (n = 4; mean ± S.E.M.; ** = p < 0.005); C. Densitometric analysis of cytosolic extracts, optical density of Nrf2 normalized against β-actin (n = 4; mean ± S.E.M.); D. Densitometric analysis of nuclear Nrf2 normalized to cytosolic Nrf2 (n = 4; mean ± S.E.M.; * = p < 0.05); E. Effects of E-α-p-OMe-C6H4-TMC on NF-κB DNA binding activity after staurosporine treatment. RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (30 μM) for 3 h, followed by staurosporine (1 μM for 2 h). Nuclear extracts were prepared and NF-κB DNA binding activity was measured (n = 4; mean ± S.E.M.; * = p < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142932.g008: Effects of E-α-p-OMe-C6H4-TMC (short: E-pOMe) on Nrf2 expression after staurosporine treatment.RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (30 μM) for 3 h, followed by staurosporine (1 μM for 2 h). Nuclear and cytosolic cell extracts were prepared and Nrf2 expression was measured by Western blot analysis. A. The image is representative of four independent experiments that showed similar results; B. Densitometric analysis of nuclear extracts, optical density of Nrf2 normalized against Lamin B1 (n = 4; mean ± S.E.M.; ** = p < 0.005); C. Densitometric analysis of cytosolic extracts, optical density of Nrf2 normalized against β-actin (n = 4; mean ± S.E.M.); D. Densitometric analysis of nuclear Nrf2 normalized to cytosolic Nrf2 (n = 4; mean ± S.E.M.; * = p < 0.05); E. Effects of E-α-p-OMe-C6H4-TMC on NF-κB DNA binding activity after staurosporine treatment. RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (30 μM) for 3 h, followed by staurosporine (1 μM for 2 h). Nuclear extracts were prepared and NF-κB DNA binding activity was measured (n = 4; mean ± S.E.M.; * = p < 0.05).
Mentions: To elucidate a possible mechanism leading to the proposed cytoprotective effects via HO-1 induction by E-α-p-OMe-C6H4-TMC, we analyzed the translocation of the transcription factor Nrf2. Untreated cells compared to cells treated with E-α-p-OMe-C6H4-TMC alone showed a significant translocation of Nrf2 into the nucleus (Fig 8A, Lane 1 vs. 2; Fig 8B, untreated normalized to 1 vs. 30 μM E-α-p-OMe-C6H4-TMC 3.3 ± 2.8 fold change Nrf2/Lamin B1, * = p < 0.05). We did not detect an increased Nrf2 translocation after staurosporine induction alone, whereas pretreatment with E-α-p-OMe-C6H4-TMC before induction of apoptosis caused a significant nuclear translocation of Nrf2 (Fig 8A and 8B, staurosporine 1.6 ± 0.5 vs. staurosporine + 30 μM E-α-p-OMe-C6H4-TMC 3.2 ± 0.4 fold change Nrf2/Lamin B1; ** = p < 0.01). The cytosolic extracts did not show any significant differences regarding Nrf2 expression levels (Fig 8A and 8C). Fig 8D demonstrates the nuclear to cytosolic ratio of Nrf2 and in this way combines the results highlighted in Fig 8B and 8C. Macrophages treated with E-α-p-OMe-C6H4-TMC revealed a significantly increased nuclear to cytosolic ratio of Nrf2 compared to untreated cells, whereas E-α-p-OMe-C6H4-TMC pretreatment before induction of apoptosis lead to a significant increased ratio compared to staurosporine alone (Fig 8D, untreated normalized to 1 vs. 30 μM E-α-p-OMe-C6H4-TMC 2.4 ± 1.5; staurosporine 1.5 ± 0.6 vs. staurosporine + 30 μM E-α-p-OMe-C6H4-TMC 4.3 ± 1.9 Nrf2 ratio nuclear/cytosolic, * = 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