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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.RAW264.7 cells were pretreated with either E-α-p-OMe-C6H4-TMC or Z-α-p-OMe-C6H4-TMC (short: E-pOMe, Z-pOMe) for 3 h. Apoptosis was induced using staurosporine (1 μM for 2 h). A. Representative experiment after FITC annexin-V and propidium iodide (PI) staining. 1 × 104 cells were analyzed in each experiment. B. Effect of E-α-p-OMe-C6H4-TMC and Z-α-p-OMe-C6H4-TMC on cytoprotection (n = 6; mean ± S.E.M.; *** = p < 0.001).
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pone.0142932.g003: Flow cytometric analysis.RAW264.7 cells were pretreated with either E-α-p-OMe-C6H4-TMC or Z-α-p-OMe-C6H4-TMC (short: E-pOMe, Z-pOMe) for 3 h. Apoptosis was induced using staurosporine (1 μM for 2 h). A. Representative experiment after FITC annexin-V and propidium iodide (PI) staining. 1 × 104 cells were analyzed in each experiment. B. Effect of E-α-p-OMe-C6H4-TMC and Z-α-p-OMe-C6H4-TMC on cytoprotection (n = 6; mean ± S.E.M.; *** = p < 0.001).

Mentions: Compared to untreated cells, neither DMSO nor the chalcones E-α-p-OMe-C6H4-TMC or Z-α-p-OMe-C6H4-TMC (each 30 μM for 5 h) showed any increase in annexin-V positive cells compared to untreated cells (Fig 3, untreated 7.6 ± 1.3; DMSO 7.9 ± 0.04; 30 μM E-α-p-OMe-C6H4-TMC 7.8 ± 1.7; 30 μM Z-α-p-OMe-C6H4-TMC 6.8 ± 0.7% annexin-V positive / PI negative cells). Exposure of RAW264.7 cells to 1 μM staurosporine for 2 h increased the amount of annexin-V positive and propidium iodide (PI) negative cells (Fig 3 A and B, staurosporine 32.9 ± 1.7%) [27]. Pretreatment of RAW264.7 macrophages with E-α-p-OMe-C6H4-TMC shows a significant and dose dependent reduction in annexin-V positive / PI negative cells in the context of staurosporine-induced apoptosis (Fig 3A and 3B, staurosporine 32.9 ± 1.7 vs. staurosporine + 10 μM E-α-p-OMe-C6H4-TMC 10.4 ± 0.7; staurosporine + 20 μM E-α-p-OMe-C6H4-TMC 8.2 ± 0.9; staurosporine + 30 μμM E-α-p-OMe-C6H4-TMC 7.1 ± 1.2% annexin-V positive / PI negative cells, *** = p < 0.001). Although pretreatment with DMSO lead to a reduction of annexin-V positive / PI negative cells compared to staurosporine (Fig 3B, staurosporine 32.9 ± 1.7 vs. staurosporine + DMSO 21.7 ± 1.4% annexin-V positive / PI negative cells, *** = p < 0.001), this reduction differed significantly from the reduction achieved by the pretreatment with E-α-p-OMe-C6H4-TMC (Fig 3A and 3B, staurosporine + DMSO 21.7 ± 1.4 vs. staurosporine + 10/20/30 μM E-α-p-OMe-C6H4-TMC: 10.4 ± 0.7; 8.2 ± 0.9; 7.1 ± 1.2% annexin-V positive / PI negative cells, all *** = 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)

Flow cytometric analysis.RAW264.7 cells were pretreated with either E-α-p-OMe-C6H4-TMC or Z-α-p-OMe-C6H4-TMC (short: E-pOMe, Z-pOMe) for 3 h. Apoptosis was induced using staurosporine (1 μM for 2 h). A. Representative experiment after FITC annexin-V and propidium iodide (PI) staining. 1 × 104 cells were analyzed in each experiment. B. Effect of E-α-p-OMe-C6H4-TMC and Z-α-p-OMe-C6H4-TMC on cytoprotection (n = 6; mean ± S.E.M.; *** = p < 0.001).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142932.g003: Flow cytometric analysis.RAW264.7 cells were pretreated with either E-α-p-OMe-C6H4-TMC or Z-α-p-OMe-C6H4-TMC (short: E-pOMe, Z-pOMe) for 3 h. Apoptosis was induced using staurosporine (1 μM for 2 h). A. Representative experiment after FITC annexin-V and propidium iodide (PI) staining. 1 × 104 cells were analyzed in each experiment. B. Effect of E-α-p-OMe-C6H4-TMC and Z-α-p-OMe-C6H4-TMC on cytoprotection (n = 6; mean ± S.E.M.; *** = p < 0.001).
Mentions: Compared to untreated cells, neither DMSO nor the chalcones E-α-p-OMe-C6H4-TMC or Z-α-p-OMe-C6H4-TMC (each 30 μM for 5 h) showed any increase in annexin-V positive cells compared to untreated cells (Fig 3, untreated 7.6 ± 1.3; DMSO 7.9 ± 0.04; 30 μM E-α-p-OMe-C6H4-TMC 7.8 ± 1.7; 30 μM Z-α-p-OMe-C6H4-TMC 6.8 ± 0.7% annexin-V positive / PI negative cells). Exposure of RAW264.7 cells to 1 μM staurosporine for 2 h increased the amount of annexin-V positive and propidium iodide (PI) negative cells (Fig 3 A and B, staurosporine 32.9 ± 1.7%) [27]. Pretreatment of RAW264.7 macrophages with E-α-p-OMe-C6H4-TMC shows a significant and dose dependent reduction in annexin-V positive / PI negative cells in the context of staurosporine-induced apoptosis (Fig 3A and 3B, staurosporine 32.9 ± 1.7 vs. staurosporine + 10 μM E-α-p-OMe-C6H4-TMC 10.4 ± 0.7; staurosporine + 20 μM E-α-p-OMe-C6H4-TMC 8.2 ± 0.9; staurosporine + 30 μμM E-α-p-OMe-C6H4-TMC 7.1 ± 1.2% annexin-V positive / PI negative cells, *** = p < 0.001). Although pretreatment with DMSO lead to a reduction of annexin-V positive / PI negative cells compared to staurosporine (Fig 3B, staurosporine 32.9 ± 1.7 vs. staurosporine + DMSO 21.7 ± 1.4% annexin-V positive / PI negative cells, *** = p < 0.001), this reduction differed significantly from the reduction achieved by the pretreatment with E-α-p-OMe-C6H4-TMC (Fig 3A and 3B, staurosporine + DMSO 21.7 ± 1.4 vs. staurosporine + 10/20/30 μM E-α-p-OMe-C6H4-TMC: 10.4 ± 0.7; 8.2 ± 0.9; 7.1 ± 1.2% annexin-V positive / PI negative cells, all *** = 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