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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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Western Blot analysis.A. RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (short: E-pOMe) for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Whole-cell extracts were prepared and HO-1 protein expression levels were analyzed. The image is representative of five independent experiments that showed similar results. B. Densitometric analysis of A, optical density of HO-1 normalized against β-actin (n = 5; mean ± S.E.M.; *** = p < 0.001; * = p < 0.05); C. RAW264.7 cells were pretreated with SnPP-IX (80 μM for 1 h) to inhibit HO-1 expression prior to the treatment with E-α-p-OMe-C6H4-TMC for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Whole-cell extracts were prepared and HO-1 protein expression levels were analyzed. The image is representative of five independent experiments that showed similar results. D. Densitometric analysis of C, optical density of HO-1 normalized against β-actin (n = 5; mean ± S.E.M.; *** = p < 0.001).
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pone.0142932.g005: Western Blot analysis.A. RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (short: E-pOMe) for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Whole-cell extracts were prepared and HO-1 protein expression levels were analyzed. The image is representative of five independent experiments that showed similar results. B. Densitometric analysis of A, optical density of HO-1 normalized against β-actin (n = 5; mean ± S.E.M.; *** = p < 0.001; * = p < 0.05); C. RAW264.7 cells were pretreated with SnPP-IX (80 μM for 1 h) to inhibit HO-1 expression prior to the treatment with E-α-p-OMe-C6H4-TMC for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Whole-cell extracts were prepared and HO-1 protein expression levels were analyzed. The image is representative of five independent experiments that showed similar results. D. Densitometric analysis of C, optical density of HO-1 normalized against β-actin (n = 5; mean ± S.E.M.; *** = p < 0.001).

Mentions: RAW264.7 macrophages treated with 30 μM E-α-p-OMe-C6H4-TMC without induction of apoptosis showed a significant increase in HO-1 protein expression compared to cells treated with DMSO alone (Fig 5B: DMSO 1.01 ± 0.13 vs. 30 μM E-α-p-OMe-C6H4-TMC 1.76 ± 0.33 fold change HO-1/β-actin; n = 5; * = p < 0.05). Treating cells only with staurosporine or in combination with DMSO had no effect on HO-1 protein expression (Fig 5B; staurosporine 1.01 ± 0.19 vs. staurosporine + DMSO 1.3 ± 0.4 fold change HO-1/β-actin; not significant). Since E-α-p-OMe-C6H4-TMC is dissolved in DMSO, pretreatment of macrophages with DMSO before induction of apoptosis served as a negative control. Pretreatment of cells with indicated concentrations of E-α-p-OMe-C6H4-TMC for 3 h prior to induction of apoptosis resulted in a dose dependent and significant increase of HO-1 protein expression (Fig 5A, lanes 3 and 5 vs. 6, 7 and 8 and Fig 5B, staurosporine 1.01 ± 0.19 and staurosporine + DMSO 1.3 ± 0.4 vs. staurosporine + 10/20/30 M E-α-p-OMe-C6H4-TMC: 1.8 ± 0.6; 2.2 ± 0.54 and 2.26 ± 0.8 fold change HO-1/β-actin, * = p < 0.05; *** = p < 0.001).


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)

Western Blot analysis.A. RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (short: E-pOMe) for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Whole-cell extracts were prepared and HO-1 protein expression levels were analyzed. The image is representative of five independent experiments that showed similar results. B. Densitometric analysis of A, optical density of HO-1 normalized against β-actin (n = 5; mean ± S.E.M.; *** = p < 0.001; * = p < 0.05); C. RAW264.7 cells were pretreated with SnPP-IX (80 μM for 1 h) to inhibit HO-1 expression prior to the treatment with E-α-p-OMe-C6H4-TMC for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Whole-cell extracts were prepared and HO-1 protein expression levels were analyzed. The image is representative of five independent experiments that showed similar results. D. Densitometric analysis of C, optical density of HO-1 normalized against β-actin (n = 5; mean ± S.E.M.; *** = p < 0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142932.g005: Western Blot analysis.A. RAW264.7 cells were pretreated with E-α-p-OMe-C6H4-TMC (short: E-pOMe) for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Whole-cell extracts were prepared and HO-1 protein expression levels were analyzed. The image is representative of five independent experiments that showed similar results. B. Densitometric analysis of A, optical density of HO-1 normalized against β-actin (n = 5; mean ± S.E.M.; *** = p < 0.001; * = p < 0.05); C. RAW264.7 cells were pretreated with SnPP-IX (80 μM for 1 h) to inhibit HO-1 expression prior to the treatment with E-α-p-OMe-C6H4-TMC for 3 h, apoptosis was induced using staurosporine (1 μM for 2 h). Whole-cell extracts were prepared and HO-1 protein expression levels were analyzed. The image is representative of five independent experiments that showed similar results. D. Densitometric analysis of C, optical density of HO-1 normalized against β-actin (n = 5; mean ± S.E.M.; *** = p < 0.001).
Mentions: RAW264.7 macrophages treated with 30 μM E-α-p-OMe-C6H4-TMC without induction of apoptosis showed a significant increase in HO-1 protein expression compared to cells treated with DMSO alone (Fig 5B: DMSO 1.01 ± 0.13 vs. 30 μM E-α-p-OMe-C6H4-TMC 1.76 ± 0.33 fold change HO-1/β-actin; n = 5; * = p < 0.05). Treating cells only with staurosporine or in combination with DMSO had no effect on HO-1 protein expression (Fig 5B; staurosporine 1.01 ± 0.19 vs. staurosporine + DMSO 1.3 ± 0.4 fold change HO-1/β-actin; not significant). Since E-α-p-OMe-C6H4-TMC is dissolved in DMSO, pretreatment of macrophages with DMSO before induction of apoptosis served as a negative control. Pretreatment of cells with indicated concentrations of E-α-p-OMe-C6H4-TMC for 3 h prior to induction of apoptosis resulted in a dose dependent and significant increase of HO-1 protein expression (Fig 5A, lanes 3 and 5 vs. 6, 7 and 8 and Fig 5B, staurosporine 1.01 ± 0.19 and staurosporine + DMSO 1.3 ± 0.4 vs. staurosporine + 10/20/30 M E-α-p-OMe-C6H4-TMC: 1.8 ± 0.6; 2.2 ± 0.54 and 2.26 ± 0.8 fold change HO-1/β-actin, * = p < 0.05; *** = p < 0.001).

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