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.
Highlights
While heme oxygenase-2 (HO-2) is constitutively expressed in most tissues, the inducible isoform of heme oxygenases heme oxygenase-1 (HO-1) represents a powerful response to a variety of adverse stimuli including ischemia-reperfusion injury, hypoxia or toxicity, leading to cellular protection [1]
We examined whether the chalcones exert cytoprotective effects in a model of staurosporine-induced apoptosis in RAW264.7 macrophages
Pretreatment of RAW264.7 macrophages using αX-TMCs with X = CN, Br, p-NO2-C6H4, I, H F, Me, Ph, NO2, CF3, COOEt, COOH; α-H-HC, calythropsin and 2’-hydroxychalcone had no diminishing influence on the staurosporine induced increase in annexin-V positive cells. α-Cl-TMC administration revealed a tendency to a reduced number of annexin-V positive cells, but failed to reach significance as was found for chalcone
Summary
While heme oxygenase-2 (HO-2) is constitutively expressed in most tissues, the inducible isoform of heme oxygenases HO-1 represents a powerful response to a variety of adverse stimuli including ischemia-reperfusion injury, hypoxia or toxicity, leading to cellular protection [1]. These cytoprotective effects of HO-1 are caused by various factors. HO-1 eliminates free heme which would otherwise lead to apoptosis in increased concentrations [2, 3]. Apart from that, HO-1 may play an important role in sepsis, where patients suffer from immune suppression as a result of an increased amount of apoptotic immune cells [7, 8]
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