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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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A. HO-1 activity assay. RAW264.7 cells were pretreated with SnPP-IX (80 μM for 1 h) to inhibit HO-1 activity prior to the treatment E-α-p-OMe-C6H4-TMC (short: E-pOMe) for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Whole-cell protein extracts were prepared and HO activity was measured by ELISA-based bilirubin quantification; B. Flow cytometric analysis. RAW264.7 cells were pretreated with SnPP-IX (80 μM for 1 h) to inhibit HO-1 activity prior to the treatment with E-α-p-OMe-C6H4-TMC for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Cells were stained with FITC annexin-V and propidium iodide to mark apoptotic cells. 1 × 104 cells were analyzed in each experiment (n = 4; mean ± S.E.M.; * = p < 0.05).
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pone.0142932.g006: A. HO-1 activity assay. RAW264.7 cells were pretreated with SnPP-IX (80 μM for 1 h) to inhibit HO-1 activity prior to the treatment E-α-p-OMe-C6H4-TMC (short: E-pOMe) for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Whole-cell protein extracts were prepared and HO activity was measured by ELISA-based bilirubin quantification; B. Flow cytometric analysis. RAW264.7 cells were pretreated with SnPP-IX (80 μM for 1 h) to inhibit HO-1 activity prior to the treatment with E-α-p-OMe-C6H4-TMC for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Cells were stained with FITC annexin-V and propidium iodide to mark apoptotic cells. 1 × 104 cells were analyzed in each experiment (n = 4; mean ± S.E.M.; * = p < 0.05).

Mentions: Apart from the HO-1 protein expression, HO-1 activity was analyzed. Cells treated with 30 μM E-α-p-OMe-C6H4-TMC without induction of apoptosis demonstrated a significant increase in HO-1 activity compared to untreated cells (Fig 6A, untreated 1050 ± 339 vs. 30 μM E-α-p-OMe-C6H4-TMC 1780 ± 153 HO activity [pmol BR h-1mg-1 protein]; * = 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)

A. HO-1 activity assay. RAW264.7 cells were pretreated with SnPP-IX (80 μM for 1 h) to inhibit HO-1 activity prior to the treatment E-α-p-OMe-C6H4-TMC (short: E-pOMe) for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Whole-cell protein extracts were prepared and HO activity was measured by ELISA-based bilirubin quantification; B. Flow cytometric analysis. RAW264.7 cells were pretreated with SnPP-IX (80 μM for 1 h) to inhibit HO-1 activity prior to the treatment with E-α-p-OMe-C6H4-TMC for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Cells were stained with FITC annexin-V and propidium iodide to mark apoptotic cells. 1 × 104 cells were analyzed in each experiment (n = 4; 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.g006: A. HO-1 activity assay. RAW264.7 cells were pretreated with SnPP-IX (80 μM for 1 h) to inhibit HO-1 activity prior to the treatment E-α-p-OMe-C6H4-TMC (short: E-pOMe) for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Whole-cell protein extracts were prepared and HO activity was measured by ELISA-based bilirubin quantification; B. Flow cytometric analysis. RAW264.7 cells were pretreated with SnPP-IX (80 μM for 1 h) to inhibit HO-1 activity prior to the treatment with E-α-p-OMe-C6H4-TMC for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Cells were stained with FITC annexin-V and propidium iodide to mark apoptotic cells. 1 × 104 cells were analyzed in each experiment (n = 4; mean ± S.E.M.; * = p < 0.05).
Mentions: Apart from the HO-1 protein expression, HO-1 activity was analyzed. Cells treated with 30 μM E-α-p-OMe-C6H4-TMC without induction of apoptosis demonstrated a significant increase in HO-1 activity compared to untreated cells (Fig 6A, untreated 1050 ± 339 vs. 30 μM E-α-p-OMe-C6H4-TMC 1780 ± 153 HO activity [pmol BR h-1mg-1 protein]; * = 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