Dimethoxycurcumin, a Synthetic Curcumin Analogue, Induces Heme Oxygenase-1 Expression through Nrf2 Activation in RAW264.7 Macrophages

Curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione] induces heme oxygenase-1 (HO-1) expression via activation of the nuclear factor-erythroid-2-related factor 2 (Nrf2), whereas tetrahydrocurcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-3,5-heptanedione], one of curcumin in vivo metabolites, has no effect on HO-1 expression and Nrf2 activation. The aim of this study was to investigate whether dimethoxycurcumin [1,7-bis(4,3-dimethoxyphenyl)-1,6-heptadiene-3,5-dione], a synthetic curcumin analogue with higher metabolic stability over curcumin, could induce HO-1 expression to the same extent as curcumin in RAW264.7 macrophages. Dimethoxycurcumin and curcumin, but not tetrahydrocurcumin, induced HO-1 expression and Nrf2 nuclear translocation, suggesting that the unsaturated nature of the diarylheptanoid chain of the compounds are crucial for HO-1 expression and Nrf2 activation. Blockage of Nrf2 synthesis by small interfering RNA abolished HO-1 expression by dimethoxycurcumin, indicating that dimethoxycurcumin may induce HO-1 expression via Nrf2 activation. In comparison, dimethoxycurcumin and curcumin had about the same effect on HO-1 expression, suggesting that dimethoxycurcumin retains the HO-1-inducing activity of its parent compound curcumin in RAW264.7 macrophages.


Introduction
Heme oxygenase-1 (HO-1) that catalyzes the first and rate-limiting step in the oxidative degradation of free heme is now recognized as a fundamental endogenous cytoprotec-tive system [1]. This enzyme can be expressed primarily by its substrate, free heme, and also by a wide variety of endogenous and exogenous stimuli, suggesting that the molecular mechanisms that regulate HO-1 expression are complex. The nuclear factor-erythroid-2-related factor 2 (Nrf2) has been shown to mediate HO-1 expression by certain phytochemicals [2].
The dietary phytochemical curcumin (chemical structure shown in Fig. 1) has a long history of medicinal use in India and Southeast Asia for a wide variety of medical conditions [3]. Extensive investigations on the pharmacological activities of curcumin have demonstrated that curcumin can induce HO-1 expression via Nrf2 activation [4], thus being considered a non-cytotoxic HO-1 inducer. Unfortunately, curcumin is rapidly metabolized in vivo into tetrahydrocurcumin (THC; chemical structure shown in Fig. 1) and other reduced forms [5]. Moreover, the antiinflammatory property of curcumin is lost when curcumin is reduced to THC or others [6]. Thus, there is a need to develop curcumin analogues with higher metabolic stability than the original curcumin. Dimethoxycurcumin (DiMC; chemical structure shown in Fig. 1), one of several synthetic curcumin analogues, has been reported to exert an anticancer activity comparable to curcumin and to have increased metabolic stability in comparison with curcumin [7]. However, whether DiMC could exert other biological effects similar to those of curcumin remains to be investigated. Moreover, whether DiMC would induce HO-1 expression in RAW264.7 macrophages is currently unknown, and was thus investigated in this study.

Cell viability assay
Cell viability was determined by a modified MTT reduction assay. MTT is a pale yellow substance that is reduced by living cells to yield a dark blue formazan product. This process requires active mitochondria, and even fresh dead cells do not reduce significant amounts of MTT. RAW264.7 macrophages were cultured in a 96-well flat-bottom plate at concentration of 5 × 10 5 cells/ml. After 12 h of preconditioning, the cells were treated with various concentrations of DiMC or other agents for 18 h. Thereafter, culture medium was aspirated and 100 μl of MTT dye (1 mg/ml in phosphate-buffered saline) was added; the cultures were incubated for 4 h at 37°C. The formazan crystals produced through dye reduction by viable cells were dissolved using acidified isopropanol (0.1 N HCl). Index of cell viability was calculated by measuring the optical density of color produced by MTT dye reduction at 570 nm.

Heme oxygenase activity assay
Heme oxygenase activity was determined at the end of each treatment as described previously [11]. Briefly, harvested cells were subjected to three cycles of freezethawing before addition to a reaction mixture consisting of phosphate buffer (1 ml final volume, pH 7.4) containing magnesium chloride (2 mM), NADPH (0.8 mM), glucose-6phosphate (2 mM), glucose-6-phosphate dehydrogenase (0.2 Units), rat liver cytosol as a source of biliverdin reductase, and the substrate hemin (20 μM). The reaction mixture was incubated in the dark at 37°C for 1 h and was terminated by the addition of 1 ml of chloroform. After being vigorously vortexed and centrifuged, the extracted bilirubin in the chloroform layer was measured by the difference in absorbance between 464 and 530 nm (ε = 40 mM −1 ·cm −1 ).
Western blot analysis RAW264.7 macrophages were incubated with or without reagents. They were harvested, washed ice-cold phosphatebuffered saline (PBS) and kept on ice for 1 min. The suspension was mixed with buffer A (10 mM HEPES, pH 7.5, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, 1 mM DTT, 0.5 mM PMSF, 5 μg/ml aprotinin, 5 μg/ml pepstatin, and 10 μg/ml leupeptin) and lysed by three freeze-thaw cycles. Cytosolic fraction was obtained by centrifugation at 12,000 × g for 20 min at 4°C. The pellets were re-suspended in buffer C (20 mM HEPES, pH 7.5, 0.4 M NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, 5 μg/ml aprotinin, 5 μg/ml pepstatin, and 10 μg/ml leupeptin) on ice for 40 min and centrifuged at 14,000 × g for 20 min at 4°C. The resulting supernatant was used as soluble nuclear fraction. Protein content was determined with BCA protein assay reagent (Pierce, Rockford, IL). Total cellular or nuclear fractions were separated on 10% SDS-polyacrylamide gels, and transferred to the nitrocellulose membranes (Amersham Biosciences, Inc., Piscataway, NJ). The membrane was then blocked in blocking buffer containing 20 mM sodium phosphate buffer, pH 7.6, 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk for 1 h at room temperature. Thereafter, the membrane was incubated with antibodies against HO-1 (1:1000 dilution), Nrf2 (1: 500 dilution) or β-actin (1:1000 dilution) at 4°C overnight. The membrane was then washed four times with PBS-Tween 20 buffer and further incubated with secondary antibody for 1 h at room temperature. Specific bands were detected using enhanced chemiluminescence detection system (Amersham Biosciences), and the membrane was exposed to X-ray film.
Nrf2 siRNA transfection RAW264.7 macrophages were grown in 6-or 12-well plates and transiently transfected with Nrf2 siRNA (Santa Cruz Biotechnology) mixed with siRNA transfection reagent (Santa Cruz Biotechnology) according to the manufacturer's instructions. After incubation at 37°C and 5% CO2 for 30 h, cells were treated with DiMC and curcumin. The samples were then prepared for Western blot analysis.

Statistical analysis
Data were analyzed using Student's t test, one-way analysis of variance or Newman-Keuls multiple comparison test. Differences were considered significant when p<0.05.

Results
The chemical structures of curcumin analogues tested in this study are shown in Fig. 1. While the original form of curcumin contains two methoxy groups at two aromatic rings, DiMC contains four. In comparison with curcumin, THC contains two methoxy groups but lacks conjugated double bonds in the central seven-carbon chain. Curcumin and THC were used to explore possible mechanism(s) of action of DiMC. In RAW264.7 macrophages, DiMC and curcumin, but not THC, exhibited cytotoxicity at more than 20 μM (data not shown).
Treatment of RAW264.7 macrophages with different concentrations of DiMC (2-10 μM) for 6 h resulted in a significant increase in heme oxygenase activity ( Fig. 2A); this enzymatic activation was strongly associated with a marked increase in HO-1 expression, as confirmed by Western blot analysis (Fig. 2B). Similar to the effects evoked by DiMC, treatment with curcumin resulted in a substantial increase in heme oxygenase activity (Fig. 3A) and HO-1 protein levels (Fig. 3B). In contrast, THC failed to increase heme oxygenase activity and HO-1 expression ( Fig. 3).
In other experimental sets, we examined the effects of DiMC and curcumin on Nrf2 activation in RAW264.7 macrophages, and found that both DiMC and curcumin induced the nuclear accumulation of the transcription factor Nrf2 (Fig. 4A). However, THC had no significant effect on Nrf2 nuclear accumulation (not shown). The role of Nrf2 in HO-1 expression by DiMC and curcumin was studied using siRNA against Nrf2. As shown in Fig. 4B, transient transfection with Nrf2 siRNA completely abolished HO-1 expression by DiMC as well as curcumin.

Discussion
As HO-1 is widely recognized as an effective cellular strategy to counteract a variety of stressful events [12], the induction of HO-1 by pharmacological modulators may represent a novel target for therapeutic intervention. The naturally occurring curcumin has been reported to induce HO-1 expression [13][14][15][16], but whether DiMC, a modified curcumin, could also induce HO-1 expression to the same extent as the original form curcumin has not been investigated so far. The present study demonstrates, for the first time, that DiMC can induce HO-1 expression and Nrf2 activation in RAW264.7 macrophages.
The present study determined which structural features of the DiMC molecule could contribute to its ability to serve as an inducer of HO-1. For this purpose, we compared the potency of DiMC and curcumin for HO-1 expression, because DiMC is structurally related to curcumin. DiMC affected the inducer potency only very slightly as compared with that of curcumin (Fig. 3), suggesting that the substituted methoxy groups in the DiMC are not essential for HO-1inducing activity. On the contrary, the α,β-unsaturated carbonyl group may be an important structure of DiMC, because THC, lacking this functional group, was virtually inactive in inducing HO-1 expression. Compounds carrying this reactive group have been reported to induce HO-1 expression through activation of Nrf2 nuclear translocation [17,18]. The cyclopentenone 15-deoxy-Δ 12,14 -prostaglandin J2, possessing the α,β-unsaturated carbonyl group, has been shown to induce HO-1 expression through Nrf2-dependent pathway [19]. Moreover, curcumin which possesses two α,β-unsaturated carbonyl groups has been already reported to induce HO-1 expression by activating Nrf2 nuclear translocation [15]. By structural analogy, DiMC can be recognized to have a chemical property resembling that of  curcumin; this structural similarity prompted us to examine whether DiMC could also activate Nrf2 nuclear translocation. Indeed, DiMC induced the activation of Nrf2 nuclear translocation, and this activation was obviously associated with DiMC-induced HO-1 expression (Fig. 4B). We, therefore, speculate that DiMC is effective in inducing HO-1 expression, at least in part, because it bears the α,βunsaturated carbonyl group. DiMC and curcumin were found to have about the same effect at least partially on HO-1 expression in RAW264.7 macrophages; this may be because they have the same functional group playing a crucial role in HO-1 expression. Thus, our results confirm that DiMC, a synthetic curcumin analogue with higher metabolic stability over curcumin [7], retains the HO-1-inducing activity of its parent compound curcumin.

Conclusion
The results of the present study demonstrate that: (i) the synthetic DiMC induces HO-1 expression through Nrf2dependent pathway in RAW264.7 macrophages; (ii) the α,β-unsaturated carbonyl group of DiMC are crucial for Nrf2-dependent HO-1 expression; and (iii) the HO-1inducing activities of DiMC and curcumin are almost identical.