The anti-inflammatory effects of E-α-(p-methoxyphenyl)-2′,3,4,4′-tetramethoxychalcone are mediated via HO-1 induction

Inflammation plays a central role in the pathophysiology of many diseases. The inducible enzyme heme oxygenase-1 (HO-1) protects cells against inflammation and can be induced by electrophilic compounds like the chalcones (1,3-diphenylprop-2-enones) from the class of alpha,beta-unsaturated carbonyl compounds. We hypothesized that the synthetic chalcone E-alpha-(p-methoxyphenyl)-2',3,4,4'-tetramethoxychalcone (E-alpha-p-OMe-C6H4-TMC) exerts anti-inflammatory effects in RAW264.7, Jurkat lymphocytes and HK-2 cells via HO-1 induction. RAW264.7 cells were treated with lipopolysaccharide prior to E-alpha-p-OMe-C6H4-TMC treatment. Subsequently, HO-1 protein induction and activity were analyzed, as well as expression of pro- and anti-inflammatory mediators, transcription factors and mitogen-activated protein kinases to evaluate the possible molecular mechanism. These results were confirmed in human cell lines (Jurkat T-lymphocytes and HK-2 epithelial cells). We found that the E-alpha-p-OMe-C6H4-TMC exerts significant anti-inflammatory effects in a dose dependent manner, showing no toxic effects in LPS-treated RAW264.7 macrophages. E-alpha-p-OMe-C6H4-TMC induced HO-1 and SOD-1 protein expression and HO-1 enzyme activity, reduced the upregulation of COX-2 and 1NOS, while inducing the translocation of Nrf2. NF-kappa B activity was attenuated following E-alpha-p-OMe-C6H4-TMC treatment accompanied by the downregulation of proinflammatory cytokines IL-1 beta, IL-6 and MCP-1. Pretreatment with E-alpha-p-OMe-C6H4-TMC revealed significant changes in phosphorylation of ERK and p38, but not JNK. These anti-inflammatory effects of E-alpha-p-OMe-C6H4-TMC were approved in Jurkat and HK-2 cells, furthermore revealing a downregulation of IL-8 and IL-10. In conclusion, it is tempting to speculate about E-alpha-p-OMe-C6H4-TMC as a new and non-toxic agent, inducing HO-1 in cells. This opens up new opportunities regarding the development of therapeutic agents using beneficial effects of HO-1 and its products. (C) 2016 Elsevier B.V. All rights reserved

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

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

Effects of <i>E</i>-α-<i>p</i>-OMe-C₆H₄-TMC (short: <i>E</i>-<i>p</i>OMe) on Nrf2 expression after staurosporine treatment.

<p>RAW264.7 cells were pretreated with <i>E</i>-α-<i>p</i>-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 <i>E</i>-α-<i>p</i>-OMe-C₆H₄-TMC on NF-κB DNA binding activity after staurosporine treatment. RAW264.7 cells were pretreated with <i>E</i>-α-<i>p</i>-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).</p

Flow cytometric analysis.

<p>Effect of different chalcones on staurosporine-induced apoptosis in RAW264.7 macrophages. Cells were pretreated with the indicated concentrations of chalcones for 3 h, before apoptosis was induced by staurosporine (1 μM for 2 h). Cells were harvested and stained with FITC annexin-V and propidium iodide. 1 × 10⁴ cells were analyzed in each experiment. (n = 4; mean ± S.E.M.; *** = p < 0.001 untreated vs. staurosporine and staurosporine vs. 10, 100, 500 and 1000 μM <i>E</i>-α-<i>p</i>-OMe-C₆H₄-TMC + staurosporine and * = p < 0.05 staurosporine vs. 1 μM <i>E</i>-α-<i>p</i>-OMe-C₆H₄-TMC + staurosporine). k₂ values for the reaction with the model thiol cysteamine are taken from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142932#pone.0142932.ref021" target="_blank">21</a>]. N, native; S, staurosporine.</p

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 <i>E</i>-α-<i>p</i>-OMe-C₆H₄-TMC (short: <i>E</i>-<i>p</i>OMe) 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 <i>E</i>-α-<i>p</i>-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).

<p>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 <i>E</i>-α-<i>p</i>-OMe-C₆H₄-TMC (short: <i>E</i>-<i>p</i>OMe) 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 <i>E</i>-α-<i>p</i>-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).</p

Western Blot analysis.

<p>A. RAW264.7 cells were pretreated with <i>E</i>-α-<i>p</i>-OMe-C₆H₄-TMC (short: <i>E</i>-<i>p</i>OMe) 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 <i>E</i>-α-<i>p</i>-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).</p

Total ROS detection by flow cytometric analysis.

<p>RAW264.7 cells were pretreated with <i>E</i>-α-<i>p</i>-OMe-C₆H₄-TMC (short: <i>E</i>-<i>p</i>OMe) 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. <i>E</i>-α-<i>p</i>-OMe-C₆H₄-TMC mediated effect on intracellular ROS production (n = 6; mean ± S.E.M.; * = p < 0.05).</p