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Hydroxychalcones exhibit differential effects on XRE transactivation
Toxicology 207 (2005) 303–313
Hydroxychalcones exhibit differential effects on XRE transactivation Huan Wangb,1 , Yun Wangb,1 , Zhen-yu Chena,b , Franky L. Chanc , Lai K. Leunga,b,∗ b
a Food and Nutritional Sciences Programme, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong Department of Biochemistry, The Chinese University of Hong Kong, Rm. 507C MMW Bldg., Shatin, N.T., Hong Kong c Anatomy Department, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong
Received 19 May 2004; received in revised form 12 October 2004; accepted 12 October 2004
Abstract Chalcones are phenolic compounds that can be isolated from plants. Previous studies have described some pharmacological applications for these compounds. Making use of our established reporter gene system, we determined the effect of five hydroxychalcones—2-hydroxychalcone, 2 -hydroxychalcone, 4-hydroxychalcone, 4,2 ,4 -trihydroxychalcone, and 3,4,2 ,4 tetrahydroxychalcone—on the cellular xenobiotic responsive element (XRE)-transactivation. The interference of chalcones acting against polycyclic aromatic hydrocarbon (PAH)–DNA binding was also examined. Enzyme inhibition assays of cytochrome P450 (CYP) 1A1 and CYP1B1 were initially performed on recombinant protein expressed in insect microsomes. 2 -Hydroxychalcone and 2-hydroxychalcone were the most effective among the tested hydroxychalcones. The two hydroxychalcones had comparable IC50 values for CYP1A1 and CYP1B1, which were determined to be at the micromolar and submicromolar range, respectively. However, reporter gene assays indicated that 2 -hydroxychalcone suppressed XRE-transactivation, whereas 2-hydroxychalcone induced it when 7,12-dimethylbenz[a]anthracene (DMBA) was co-administered. In the absence of DMBA, 10 M 2-hydroxychalcone and 2 -hydroxychalcone increased XRE-transactivation by 18- and 2.5-fold, respectively, while other chalcones did not significantly alter the response. Cultures treated with the two hydroxychalcones also displayed separate trends in ethoxyresorufin-O-deethylase (EROD) activity and DMBA–DNA covalent binding. In summary, the present study illustrated that the inhibition of hydroxychalcone on CYP1 enzymes and XRE-transactivation was affected by the position and number of hydroxyl groups in its structure. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Hydroxychalcones; XRE-transactivation; DMBA–DNA binding; CYP1A1/1B1
Abbreviations: XRE, xenobiotic responsive element; B[a]P, benzo[a]pyrene; PAH, polycyclic aromatic hydrocarbon; AHR, aryl hydrocarbon receptor; DMBA, 7,12-dimethylbenz[a]anthracene; GSH, glutathione; EROD, ethoxyresorufin-O-deethylase; EDTA, ethylenediaminetetraacetic acid; PCR, polymerase chain reaction; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; RT-PCR, reverse transcription-polymerase chain reaction; PBS, phosphate buffer saline ∗ Corresponding author. Tel.: +852 2609 8137; fax: +852 2603 7732. E-mail address: [email protected] (L.K. Leung). 1 Contributed equally to this work. 0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.10.005
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1. Introduction Cytochrome P450 (CYP) enzymes belong to a multi-gene super family which is made up of constitutive and inducible haem-containing oxidative enzymes in 17 families in the human. Among them the CYP1-3 families are responsible for 70–80% of all phase 1-dependent metabolism of prescription drugs (Bertz and Granneman, 1997; Evans and Relling, 1999) and are also key players in the metabolism of other xenobiotics (Shimada and Fujii-Kuriyama, 2004). Their primary role is to metabolize drugs and chemicals through hydroxylation, but the process may generate some toxic intermediate in some cases. CYP1 family proteins consist of 1A1, 1A2, and 1B1, and they are capable of activating procarcinogens. CYP1A1 and 1B1 are both involved in the biotransformation of polycyclic aromatic hydrocarbons (PAHs), while CYP1A2 metabolizes aryl- and heterocyclic amines. Two animal models have implicated their significance on PAH-induced tumorigenesis. In aryl hydrocarbon receptor (AHR) null mice, the PAH benzo[a]pyrene (B[a]P) cannot upregulate cyp1a1 expression and tumors are not induced (Shimizu et al., 2000). A separate mouse model has also illustrated that 7,12-dimethylbenz[a]anthracene (DMBA) administration cannot induce lymphomas in cyp1b1 knockout mice (Buters et al., 1999). Because of the ubiquitous distribution of PAHs in the environment, the regulation of CYP1A1 and 1B1 has attracted much interest. Xenobiotic responsive elements (XRE) are cisacting enhancer elements located in the promoter regions of xenobiotic responsive genes, which include genes encoded for cytochrome P450 1A1 and 1B1. The expressions of these xenobiotic responsive genes can be regulated through a pathway involving AHR, which is a cytosolic protein that can be activated by PAH. The activated AHR then translocates to the nucleus, dimerizes with AHR nuclear translocator (ARNT), and interacts with XRE (Kronenberg et al., 2000). Cyp1a1 and 1b1 genes that encode for PAH-biotransformation enzymes can be upregulated in this pathway (Safe, 2001; Dertinger et al., 2000). Our laboratory has previously illustrated that XRE transactivation can be an indicator for the expressions of cyp1a1 and cyp1b1 (Chan et al., 2003).
Fig. 1. Basic structure of chalcone.
Chalcones belong to the flavonoid family and can be extracted from plants or synthesized in laboratory with a basic structure shown in Fig. 1. Naturally occurring chalcones are mostly hydroxylated (Vincenzo et al., 2000), and many reports have documented their biologically active properties (Haraguchi et al., 1998; Hsieh et al., 1998). Among them, 2 -hydroxyl substituted chalcones have attracted much attention because they can prevent platelet aggregation (Lin et al., 1997), polymixin B-induced edema (Hsieh et al., 1998), LPSinduced septic shock (Batt et al., 1993), and glomerulonephritis (Hayashi et al., 1996) in animal models. 2 -Hydroxychalcone can also suppress the adhesion of activated neutrophils to HUVEC cell cultures (Madan et al., 2000), and its derivatives are inhibitors of TPAinduced COX-2 expression in rat macrophages (Kim et al., 2001). Previous studies have shown that some prenylated forms (Henderson et al., 2000) and analogues (Monostory et al., 2003) of chalcones are competitive inhibitors of the CYP1A enzyme. Because of the structural resemblance to some CYP1-inhibiting flavonoids, this study evaluated the potential inhibitory effect of hydroxychalcones on two CYP1 enzymes and the pathway involving XRE-transactivation.
2. Materials and methods 2.1. Chemicals 2 -Hydroxychalcone, 2-hydroxychalcone, 4hydroxychalcone, 4,2 ,4 -trihydroxychalcone, butein (3,4,2 ,4 -tetrahydroxychalcone), ethoxyresorufin, and DMBA were purchased from Sigma Chemicals (St.
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Louis, MO, USA). All other chemicals, if not stated, were acquired from Sigma Chemicals. 2.2. Cell culture MCF-7 cells (gift from Dr. V.C. Jordan) were cultured in RPMI—1640 phenol red free media (Sigma Chemicals) with 10% fetal bovine serum (Invitrogen Life Technology, Rockville, MD) at 37 ◦ C and 5% carbon dioxide. Cells were routinely subcultured when reaching 80% of confluency. 2.3. EROD activities in intact MCF-7 cells The assay method was performed as previously described (Ciolino and Yeh, 1999). In brief, MCF7 cells in 96-well plates were treated with 1 M DMBA and various concentrations of hydroxychalcone. The medium was then removed and the cells were washed twice by 100 l phosphate-buffered saline (PBS). Ethoxyresorufin-O-deethylase (EROD) activity assays were then carried out. Fifty microlitres of ethoxyresorufin (5 M) and salicyclamide (1.5 mM) dissolved in PBS was added in each well, and incubated at 37 ◦ C for 15 min. The reaction was stopped by 50 l of ice-cold methanol, and the resorufin generated was measured by a FLUOstar Galaxy microplate reader (BMG Labtechnologies, Offenburg, Germany) with an excitation wavelength at 544 and 590 nm as the emission wavelength. The activities were quantified against resorufin standards. 2.4. Enzyme inhibition assays Recombinant CYP1A1 and CYP1B1 proteins expressed in baculovirus-infected insect microsomes (Supersomes® ) were purchased from Gentest Corp. (Woburn, MA, USA). Two picomoles of protein was incubated in 100 l PBS, pH 7.2 with 400 nM ethoxyresorufin and hydroxychalcone in different concentrations. The reaction was initiated by 500 M NADPH, and terminated by 100 l of ice-cold methanol after 20 min of incubation. The fluorescence was measured as described earlier. 2.5. Measurement of Cell viability Cell viability was assessed by 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
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staining as described by Mosmann (1983). Briefly, MCF-7 cells were cultured in 96-well plates at 104 cells per well and treated with 1 M DMBA and various concentrations of hydroxychalcone for 24 h. At the end of the treatment, 50 l MTT (1 mg/ml) was added and incubated at 37 ◦ C for 4 h. Cell viability was determined by the absorbance at 600 nm. 2.6. XRE-luciferase gene reporter assay A fragment with five XRE elements from rat cyp1a1 5 -flanking region was amplified from rat genomic DNA as described by Chan et al. (2003). No other enhancer elements were identified in this fragment. The polymerase chain reaction (PCR) product was digested with SmaI and BamHI and subcloned into a firefly luciferase reporter vector pTA–Luc (Clontech, Palo Alto, CA, USA). MCF-7 cells were seeded at 105 cells/well in 24well plates. After 24 h, the cells were transiently transfected with 4.0 g of the XRE-driven firefly luciferase reporter plasmid and 1.0 g of Renilla luciferase control vector (Promega, Madison, WI, USA) in LipofectAmine (Invitrogen Life Technologies). After 16 h, the medium was removed and the cells were treated with 1 M DMBA and various concentrations of hydroxychalcone for 24 h. The cells were lysed and the luciferase substrates (provided in DualLuciferase Assay Kit, Promega) were mixed with the cell lysate. The luciferase bioluminescence was measured by a FLUOstar Galaxy plate reader as described in the manual of the assay kit. The XRE transactivation activities represented by firefly luciferase light units were then normalized by that of Renilla luciferase. 2.7. Measurement of DMBA–DNA adduct formation This assay was performed as previously described (Chan et al., 2003). MCF-7 cells were plated in 6-well plates at 5 × 105 cells per well and allowed to attach for 24 h. Then, 0.1 g [3 H]-DMBA (Amersham, Arlington Height, IL, USA) per ml was administered with or without hydroxychalcone. After 16 h, cells were washed twice with cold PBS, trypsinized and pelleted. Nuclei were separated by incubating the cells for 10 min on ice in lysis buffer A (10 mM Tris–HCl (pH 7.5),
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320 mM sucrose, 5 mM magnesium chloride and 1% Triton X-100). The nuclei were collected by centrifugation at 5000 rpm after the incubation. The collected nuclei were then lysed by 400 l lysis buffer B (1% sodium dodecyl sulphate (SDS) in 0.5 M Tris. 20 mM EDTA and 10 mM NaCl (pH 9)), followed by the treatment of 20 l Proteinase K (20 mg/ml) for 2 h at 48 ◦ C. The samples were allowed to cool to room temperature and the residual protein was salted out by adding 150 l of saturated NaCl. The samples were then subjected to centrifugation at 13,000 rpm for 30 min at 4 ◦ C. Genomic DNA was isolated from the supernatant fraction by ethanol precipitation, and redissolved in autoclaved water. Ten micrograms of each DNA sample, which attained a 260 nm/280 nm ratio of >1.9, was used for scintillation counting. 2.8. Semi-quantitative RT-PCR Assay A reverse transcription-PCR assay was used to quantify mRNA expression. Total RNA was isolated from cells grown in 6-well Costar plates in triplicates by a method previously described (Chan et al., 2003). 1 g of RNA was used for cDNA synthesis, and the final volume was diluted to 20 l. Primers of CYP1A1, CYP1B1 and -actin, sequences as published formerly (Dohr et al., 1995) and a Perkin-Elmer Thermocycler (GeneAmp PCR System 2400, Norwalk, CT, USA) was utilized to amplify the target cDNAs separately after the first strand reaction. All PCR reactions consisted of 0.2 mmol/l dNTP, 2 l cDNA, 0.2 mol/l of each primer, 1× PCR buffer and 1 U of Taq polymerase. The conditions were 94 ◦ C for 45 s, 65 ◦ C for 45 s, 72 ◦ C for 1 min, and a final extension period of 7 min at 72 ◦ C. The maximum cycles of amplification were 25 for CYP1A1, 23 for CYP1B1, and 19 for  actin. The PCR products were separated on 1.8% agarose gel, stained with ethidium bromide, and photographed. A scanner equipped with Scion Image software (Scion Corporation, Frederick, MD, USA) was used to compare the optical density of the amplified fragments. The linearity of signals was verified in separate experiments.
Fig. 2. Inhibitory action of hydroxychalcones on recombinant CYP1A1 and CYP1B1. Recombinant CYP1A1 (Fig. 2A or CYP1B1 (Fig. 2B expressed in insect microsomes (Supersomes® ) was used to determine the IC50 values. Values are means ± S.D., n = 3. The IC50 values for hydroxychalcones on CYP1A1 are 1.2 M for 2 and 2-hydroxychalcones; 6.2 M for 4-hydroxychalcone; 9.3 M for butein and >10 M for 4,2 ,4 -hydroxychalcone. The values on CYP1B1 are 0.2 M for 2-hydroxychalcones; 0.05 M for 2 hydroxychalcone; 0.5 M for butein and 4,2 ,4 -hydroxychalcone; 1.2 M for 4-hydroxychalcone.
The results were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test if significant differences (p < 0.05) were observed. Another software package SigmaPlot (SPSS Inc., Chicago, IL, USA) was used for graphing the Lineweaver–Burk plots.
2.9. Statistical methods
2.10. CYP1A1 and CYP1B1 activities in recombinant microsomes
A Prism® 3.0 (GraphPad Software Inc., CA, USA) software package was utilized for statistical analysis.
Five hydroxychalcones were screened for their inhibitory effects on CYP1 enzymes. Among the five
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compounds, 2-hydroxychalcone and 2 -hydroxychalcone displayed the greatest inhibition on both CYP1A1 (Fig. 2A) and CYP1B1 (Fig. 2B). The IC50 values of 2-hydroxychalcone were determined to be about 1.2 M for CYP1A1 and 0.2 M for CYP1B1, while those of 2 -hydroxychalcone were 1.2 and 0.05 M, respectively. The five hydroxychalcones,
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in general, appeared to exert greater inhibition on CYP1B1 than CYP1A1. 2.11. XRE-luciferase reporter gene assay In the co-administration of DMBA, 2 -hydroxychalcone, as low as 1 M, could significantly decrease
Fig. 3. Effect of hydroxychalcones on DMBA-induced XRE-driven luciferase activities. MCF-7 cells were transiently transfected with a firefly luciferase reporter gene driven by XREs and a renilla luciferase control plasmid (pRL). The cells were treated (A) with DMBA or (B) without DMBA and various concentrations of hydroxychalcones for 24 h. Control cultures received the carrying solvent DMSO only. Values are means ± S.D., n = 3. In Fig. 3A, means with (a) or (b) are significantly (p < 0.05) higher or lower than the cultures treated with DMBA, respectively. Means marked with (*) are significantly higher than the control (0 M) cultures in Fig. 3B.
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(p < 0.05) XRE-transactivation (Fig. 3A). 4-Hydroxychalcone, 4,2 ,4 -trihydroxychalcone, and butein also suppressed the induced transactivation at 5 M or above. In contrast, 2-hydroxychalcone further potentiated the XRE-driven luciferase activity. In the absence of DMBA, both 2 - and 2-hydroxychalcones induced XRE-transactivation (Fig. 3B). However, the magnitude of the induction seen in 2hydroxychalcone was much stronger than that observed in 2 -hydroxychalcone, i.e. 18-fold versus 2.5-fold, respectively. No significant effects were indicated for the other hydroxychalcones. 2.12. EROD activity in MCF-cells Because XREs are located in the promoter domains of cyp1a1 and cyp1b1, hydroxychalcones can regulate the mRNA expression through the XREdependent transcriptional activities. EROD activity in the treated MCF-7 cells denotes the combined actions of hydroxychalcones on CYP1 enzyme inhibition and gene expression. All except 2-hydroxychalcone decreased the DMBA-induced EROD activities (Fig. 4A) in cells, and 2 -hydroxychalcone was the strongest inhibitor among them. 2-Hydroxychalcone, however, doubled and tripled the EROD activity at, respectively, 2.5 and 5 M. The induction of EROD activity was consistent with the XRE-transactivation data, signifying an increase of CYP1 enzyme expression. To avoid a potential experimental flaw caused by the differences in cell viability, the EROD results shown were already normalized by the corresponding MTT absorbance. Cultures receiving the same treatment were assessed for cell viability, and the result is shown in Fig. 4B. No significant variation in cell survival was observed except for cultures treated with butein. Increased viability ranging from 20 to 30% was seen in cultures treated with 1, 2.5, and 5 M of butein. 2.13. DMBA–DNA covalent binding in MCF-7 cells As EROD activities were modified in hydroxychalcone-treated cultures, we further evaluated the DMBA–DNA binding in cells. 2 -Hydroxychalcone consistently produced the least DMBA–DNA covalent formation in a dose–response manner as shown
Fig. 4. EROD activities in hydroxychalcone-treated MCF-7 cells. MCF-7 cells were seeded in 96-well culture plates and co-treated with 1 M DMBA and various concentrations of hydroxychalcones. After 24 h of treatment, the cells were assayed for EROD activity, and cell viability was determined on a separate set of cultures with equivalent treatment. (A) shows the cellular EROD activities for the treatment of 2-hydroxychalcone, 2 -hydroxychalcone, 4hydroxychalcone, 4,2 ,4 -hydroxychalcone, and butein. The EROD activities presented were normalized with the absorbance obtained from MTT assays. All means are significantly lower than the control except those marked with (θ) and (a). Mean marked with (θ) displays no significant difference and means labeled with (a) are significantly higher than the control. (B) illustrates the MTT results of cells under various kinds of treatment. The values are means ± S.D., n = 4. Means with (*) are significantly (p < 0.05) higher than the control culture.
in Fig. 5. Except for 2–hydroxychalcone, others were also able to reduce the covalent binding to a lesser extent. Although EROD activity was induced by 2hydroxychalcone in cells, the DMBA–DNA covalent binding was not significantly elevated.
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Fig. 5. Effects of hydroxychalcones on DMBA-DNA adducts formation in MCF-7 cells. MCF-7 cells were cultured in 6-well plates and treated with tritiated DMBA in 0.1 g/ml and various concentrations of hydroxychalcones were co-administered. After 16 h of treatment, genomic DNA was isolated and the DMBA-DNA lesions were determined by scintillation counting. Means with (*) are significantly (p < 0.05) lower than the cultures treated with DMBA only.
2.14. Messenger RNA abundance of CYP1A1 and CYP1B1 in cultures treated with 2 -hydroxychalcone and 2-hydroxychalcone Subsequent semi-quantitative RT-PCR data (Fig. 6) indicated that both DMBA-induced cyp1a1 and cyp1b1 expression was hampered by the co-treatment of 2 hydroxychalcone in a dose-dependent manner (Fig. 6, Panel A). The optical density readings underneath illustrated that the induced expressions reverted to their basal levels at about 5 M. 2 -Hyroxychalcone appeared to more strongly inhibit the expression of cyp1a1 than cyp1b1. A significant decrease of cyp1b1 expression was observed at 1 M, whereas the same decrease was seen at 5 M for cyp1a1. 2-Hydroxychalcone at 5 M, on the other hand, potentiated the mRNA expressions of cyp1a1 (Fig. 6, Panel B) and cyp1b1 induced by DMBA treatment.
2.15. CYP1 enzyme kinetic studies Enzyme kinetic assays performed on recombinant hCYP1A1 and hCYP1B1 Supersomes® showed that 2 -hydroxychalcone inhibited both CYP1A1 (Ki ∼ 0.3 M; Fig. 7A) and CYP1B1 (Ki ∼ 5 M; Fig. 7B) in a non-competitive manner.
3. Discussion Hydroxychalcones displayed differential effects on CYP1 enzyme expression and inhibition. Among the compounds tested, 2 -hydroxychalcone was the most effective in suppressing CYP1A1 and CYP1B1 enzyme activities and gene expressions. XRE-luciferase reporter assay indicated that the chemical could reduce the cyp1a1 and cyp1b1 expressions through the disruption of XRE-transactivation. When administered without DMBA, the hydroxychalcone was a weak inducer of XRE-transactivation and appeared to be a weak AHR agonist. The inhibition of 2 -hydroxychalcone on CYP1A1 was competitive and that of CYP1B1 was non-competitive. Because these two enzymes are responsible for the biotransformation of PAH, inhibiting the enzymes would lead to a reduction of genotoxic moieties generated from PAH. As a result, a decrease in DMBA–DNA covalent binding was demonstrated in cultures co-treated with 2 -hydroxychalcone and DMBA. 2-Hydroxychalcone, on the other hand, displayed different characteristics. It inhibited CYP1A1 and 1B1 enzyme activities at the recombinant protein level but potentiated their expressions in MCF-7 cells. Because XRE-transactivation induced by DMBA was further augmented by 2-hydroxychalcone, the potentiation of
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Fig. 6. Effect of 2 -hydroxychalcone and 2-hydroxychalcone on DMBA-induced CYP1A1 and 1B1 mRNA expression. MCF-7 cells were treated with 1 mol/l DMBA and 2 - and 2-hydroxychalcone, and cultured for 24 h. Messenger RNA expression of CYP1A1 and CYP1B1 was semi-quantified by RT-PCR. Gel images for the ethidium bromide-stained PCR fragments are displayed on the upper panel, while the corresponding optical density results are located at the lower panel. Values are means ± S.D., n = 3. Means with (*) are significantly lower and (†) are higher (p < 0.05) than samples treated with DMBA only.
CYP1 expression could be initiated in the AHR activation pathway, such as AHR binding. However, XREdependent transactivation might also be driven by a protein tyrosine kinase-mediated signaling pathway (Backlund et al., 1997), independent of the AHR status. The present study did not address the exact XREdependent pathway by which the hydroxychalcones blocked or potentiated enzyme activities. The present study suggested that the position and number of hydroxyl groups in hydroxychalcone might affect the CYP1 enzyme inhibition and gene expression. Although 2-hydroxychalcone and 2 hydroxychalcone showed comparable enzyme inhibition on recombinant CYP1A1 and CYP1B1 as shown in Fig. 2, their effects on XRE-transactivation and cellular EROD activities were opposite. Machala et al. (2001) have also shown that 2-hydroxychalcone is an inhibitor of CYP1A-dependent EROD activity in hepatic microsomes isolated from TCDD-pretreated mice, but gene regulation was not investigated in this study. Our data
revealed that 2 -hydroxychalcone was a more potent CYP1A1 inhibitor than 2-hydroxychalcone in MCF-7 cells because of the suppression at the gene expression level in addition to enzyme inhibition. Our study also indicated that increased hydroxyl-group substitution in the chalcone structure deterred both gene and enzyme levels of inhibition on CYP1A1/1B1. DNA damage caused by PAHs can be alleviated by some phytochemicals. The flavonols, quercetin and galangin (Ciolino et al., 1999a,b), and the flavone baicalein (Chan et al., 2003) can deter the metabolism of DMBA or B[a]P. These compounds inhibit CYP1A1 at both expression and enzyme levels. Besides, some non-flavonoidal compounds such as resveratrol (Ciolino et al., 1999a,b) and curcumin (Ciolino et al., 1998) can also disrupt PAH metabolism by similar mechanisms. Although the CYP1B1 expressions and enzyme activities have not been specifically addressed in these studies, results comparable to CYP1A1 are expected. In the current study, the 2 -hydroxyl substituted
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chalcones were capable of reducing the DMBA–DNA covalent formation with different potencies. It appeared that the addition of hydroxyl groups in the A- or Bring of the 2 -hydroxychalcone backbone reduced the
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protective effect on DMBA–DNA covalent binding. Although an induced CYP1 activity was seen in cultures treated with 2-hydroxychalcone, the extent of DMBA–DNA binding seemed to be unaltered. The
Fig. 7. Lineweaver–Burk plots of 2 -hydroxychalcone on CYP1A1 and CYP1B1 inhibition. EROD assay was performed on human recombinant hCYP1A1 (Fig. 7A) and hCYP1B1 (Fig. 7B) in the presence of the indicated concentrations of 2 -hydroxychalcone and 100–1600 nM ethoxyresorufin. Lineweaver–Burk plots were generated from the reciprocal data, and the value of Ki was derived from the slopes of the regression lines. Values shown are mean ± S.D., n = 3.
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reason was unknown, but the detoxifying or repairing system in the cell could be activated to offset the increase in DNA damaging species. Alternatively, the inhibition could occur in microsomal epoxide hydrolase. The cellular detoxifying enzyme system might also have metabolized the hydroxychalcones that had a strong inhibitory effect on the CYP1 enzymes, and reduced their potency on the inhibition of DMBA–DNA binding. Because of the hydroxyl group at the ortho-position in a benzene ring, 2 -hydroxychalcone and its derivatives have been subjects of much previous investigation for their pharmacological value. The 2 -hydroxyl group enhances the antioxidative activity of the chalcone structure, and has been shown to be inhibitory to lipid peroxidation and Ehrlich ascites tumor development in Swiss albino mice (Anto et al., 1995). Another study has also indicated that there is a correlation between antioxidant and estrogenic properties in hydroxychalcone (Calliste et al., 2001). Considering that redox reactions are also involved in cytochrome p450 catalysis, the antioxidant activity might also play a role in the enzyme inhibition. Other pharmacological effects of chalcones and their derivatives have also been documented. Zhang et al. (2003) have demonstrated that 2-hydroxychalcone and 2 -hydroxychalcone may sensitize tumor cells to anticancer drugs by inhibiting the conjugation of the drug with GSH. Some newly developed chalcone analogues induce cell cycle arrest and apoptosis, and also modulate p-glycoprotein activity (Vincenzo et al., 2000). Other chalcone derivatives may protect p53 from binding to the oncoprotein MDM2, and the binding inactivating p53’s tumor suppressive property (Stoll et al., 2001). In summary, we demonstrated that the number and position of hydroxyl groups substituted in chalcone might differentially affect XRE-transactivation and downstream gene expression. In addition, 2 -hydroxychalcone displayed an agonist/antagonist property toward AHR binding, whereas 2-hydroxychalcone was a potent inducer of XRE-transactivation.
Acknowledgements The authors would like to thank Prof. Georgia Guldan (Food & Nutritional Science Programme, Biochemistry Dept, the Chinese University of Hong Kong)
for the proofreading of this manuscript. This project was supported by the Chinese University of Hong Kong RGC Research Grant Direct Allocation ID No. 2041031.
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H. Wang et al. / Toxicology 207 (2005) 303–313 breast cancer MCF-7 and MDA-MB 231 cells. Arch. Biochem. Biophys. 321, 405–412. Evans, W.E., Relling, M.V., 1999. Pharmacogenomics: translating functional genomics into rational therapeutics. Science 286, 487–491. Hayashi, K., Nagamatsu, T., Honda, S., Suzuki, Y., 1996. Butein (3,4,2 ,4 -tetrahydroxychalcone) ameliorates experimental antiglomerular basement membrane antibody-associated glomerulonephritis. Eur. J. Pharmacol. 316, 297–306. Haraguchi, H., Ishikawa, H., Mizutani, K., Tamura, Y., Kinoshita, T., 1998. Antioxidative and superoxide scavenging activities of retrochalcones in Glycyrrhiza inflata. Bioorg. Med. Chem. 6, 339–347. Henderson, M.C., Miranda, C.L., Stevens, J.F., Deinzer, M.L., Buhler, D.R., 2000. In vitro inhibition of human p450 enzymes by prenylated flavonoids from hops, Humulus lupulus. Xenobiotica 30, 235–251. Hsieh, H.K., Lee, T.H., Wong, J.P., Ko, F.N., Teng, C.M., 1998. Synthesis and anti-inflammatory effect of chalcones and related compounds. Pharm. Res. 15, 39–46. Kim, Y.P., Ban, H.S., Lim, S.S., Kimura, N., Jung, S.H., Ji, J., Lee, S., Ryu, N., Keum, S.R., Shin, K.H., Ohuchi, K., 2001. Inhibition of prostaglandin E2 production by 2 -hydroxychalcone derivatives and the mechanism of action. J. Pharm. Pharmacol. 53, 1295–1302. Kronenberg, S., Esser, C., Carlberg, C., 2000. An aryl hydrocarbon receptor conformation acts as the functional core of nuclear dioxin signaling. Nucleic Acids Res. 28, 2286–2291. Lin, C.N., Lee, T.H., Hsu, M.F., Wang, J.P., Ko, F.N., Teng, C.M., 1997. 2 ,5 -Dihydroxychalcone as a potent chemical mediator and cyclooxygenase inhibitor. J. Pharm. Pharmacol. 49, 530–536. Machala, M., Kubinova, R., Haravova, P., Suchy, V., 2001. Chemoprotective potentials of homoisoflavonoids and chalcones of dracaena cinnabari: modulations of drug-metabolizing enzymes and antioxidant activity. Phytother. Res. 15, 114–118.
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Hydroxychalcones exhibit differential effects on XRE transactivation Huan Wangb,1 , Yun Wangb,1 , Zhen-yu Chena,b , Franky L. Chanc , Lai K. Leunga,b,∗ b
a Food and Nutritional Sciences Programme, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong Department of Biochemistry, The Chinese University of Hong Kong, Rm. 507C MMW Bldg., Shatin, N.T., Hong Kong c Anatomy Department, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong
Received 19 May 2004; received in revised form 12 October 2004; accepted 12 October 2004
Abstract Chalcones are phenolic compounds that can be isolated from plants. Previous studies have described some pharmacological applications for these compounds. Making use of our established reporter gene system, we determined the effect of five hydroxychalcones—2-hydroxychalcone, 2 -hydroxychalcone, 4-hydroxychalcone, 4,2 ,4 -trihydroxychalcone, and 3,4,2 ,4 tetrahydroxychalcone—on the cellular xenobiotic responsive element (XRE)-transactivation. The interference of chalcones acting against polycyclic aromatic hydrocarbon (PAH)–DNA binding was also examined. Enzyme inhibition assays of cytochrome P450 (CYP) 1A1 and CYP1B1 were initially performed on recombinant protein expressed in insect microsomes. 2 -Hydroxychalcone and 2-hydroxychalcone were the most effective among the tested hydroxychalcones. The two hydroxychalcones had comparable IC50 values for CYP1A1 and CYP1B1, which were determined to be at the micromolar and submicromolar range, respectively. However, reporter gene assays indicated that 2 -hydroxychalcone suppressed XRE-transactivation, whereas 2-hydroxychalcone induced it when 7,12-dimethylbenz[a]anthracene (DMBA) was co-administered. In the absence of DMBA, 10 M 2-hydroxychalcone and 2 -hydroxychalcone increased XRE-transactivation by 18- and 2.5-fold, respectively, while other chalcones did not significantly alter the response. Cultures treated with the two hydroxychalcones also displayed separate trends in ethoxyresorufin-O-deethylase (EROD) activity and DMBA–DNA covalent binding. In summary, the present study illustrated that the inhibition of hydroxychalcone on CYP1 enzymes and XRE-transactivation was affected by the position and number of hydroxyl groups in its structure. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Hydroxychalcones; XRE-transactivation; DMBA–DNA binding; CYP1A1/1B1
Abbreviations: XRE, xenobiotic responsive element; B[a]P, benzo[a]pyrene; PAH, polycyclic aromatic hydrocarbon; AHR, aryl hydrocarbon receptor; DMBA, 7,12-dimethylbenz[a]anthracene; GSH, glutathione; EROD, ethoxyresorufin-O-deethylase; EDTA, ethylenediaminetetraacetic acid; PCR, polymerase chain reaction; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; RT-PCR, reverse transcription-polymerase chain reaction; PBS, phosphate buffer saline ∗ Corresponding author. Tel.: +852 2609 8137; fax: +852 2603 7732. E-mail address: [email protected] (L.K. Leung). 1 Contributed equally to this work. 0300-483X/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2004.10.005
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1. Introduction Cytochrome P450 (CYP) enzymes belong to a multi-gene super family which is made up of constitutive and inducible haem-containing oxidative enzymes in 17 families in the human. Among them the CYP1-3 families are responsible for 70–80% of all phase 1-dependent metabolism of prescription drugs (Bertz and Granneman, 1997; Evans and Relling, 1999) and are also key players in the metabolism of other xenobiotics (Shimada and Fujii-Kuriyama, 2004). Their primary role is to metabolize drugs and chemicals through hydroxylation, but the process may generate some toxic intermediate in some cases. CYP1 family proteins consist of 1A1, 1A2, and 1B1, and they are capable of activating procarcinogens. CYP1A1 and 1B1 are both involved in the biotransformation of polycyclic aromatic hydrocarbons (PAHs), while CYP1A2 metabolizes aryl- and heterocyclic amines. Two animal models have implicated their significance on PAH-induced tumorigenesis. In aryl hydrocarbon receptor (AHR) null mice, the PAH benzo[a]pyrene (B[a]P) cannot upregulate cyp1a1 expression and tumors are not induced (Shimizu et al., 2000). A separate mouse model has also illustrated that 7,12-dimethylbenz[a]anthracene (DMBA) administration cannot induce lymphomas in cyp1b1 knockout mice (Buters et al., 1999). Because of the ubiquitous distribution of PAHs in the environment, the regulation of CYP1A1 and 1B1 has attracted much interest. Xenobiotic responsive elements (XRE) are cisacting enhancer elements located in the promoter regions of xenobiotic responsive genes, which include genes encoded for cytochrome P450 1A1 and 1B1. The expressions of these xenobiotic responsive genes can be regulated through a pathway involving AHR, which is a cytosolic protein that can be activated by PAH. The activated AHR then translocates to the nucleus, dimerizes with AHR nuclear translocator (ARNT), and interacts with XRE (Kronenberg et al., 2000). Cyp1a1 and 1b1 genes that encode for PAH-biotransformation enzymes can be upregulated in this pathway (Safe, 2001; Dertinger et al., 2000). Our laboratory has previously illustrated that XRE transactivation can be an indicator for the expressions of cyp1a1 and cyp1b1 (Chan et al., 2003).
Fig. 1. Basic structure of chalcone.
Chalcones belong to the flavonoid family and can be extracted from plants or synthesized in laboratory with a basic structure shown in Fig. 1. Naturally occurring chalcones are mostly hydroxylated (Vincenzo et al., 2000), and many reports have documented their biologically active properties (Haraguchi et al., 1998; Hsieh et al., 1998). Among them, 2 -hydroxyl substituted chalcones have attracted much attention because they can prevent platelet aggregation (Lin et al., 1997), polymixin B-induced edema (Hsieh et al., 1998), LPSinduced septic shock (Batt et al., 1993), and glomerulonephritis (Hayashi et al., 1996) in animal models. 2 -Hydroxychalcone can also suppress the adhesion of activated neutrophils to HUVEC cell cultures (Madan et al., 2000), and its derivatives are inhibitors of TPAinduced COX-2 expression in rat macrophages (Kim et al., 2001). Previous studies have shown that some prenylated forms (Henderson et al., 2000) and analogues (Monostory et al., 2003) of chalcones are competitive inhibitors of the CYP1A enzyme. Because of the structural resemblance to some CYP1-inhibiting flavonoids, this study evaluated the potential inhibitory effect of hydroxychalcones on two CYP1 enzymes and the pathway involving XRE-transactivation.
2. Materials and methods 2.1. Chemicals 2 -Hydroxychalcone, 2-hydroxychalcone, 4hydroxychalcone, 4,2 ,4 -trihydroxychalcone, butein (3,4,2 ,4 -tetrahydroxychalcone), ethoxyresorufin, and DMBA were purchased from Sigma Chemicals (St.
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Louis, MO, USA). All other chemicals, if not stated, were acquired from Sigma Chemicals. 2.2. Cell culture MCF-7 cells (gift from Dr. V.C. Jordan) were cultured in RPMI—1640 phenol red free media (Sigma Chemicals) with 10% fetal bovine serum (Invitrogen Life Technology, Rockville, MD) at 37 ◦ C and 5% carbon dioxide. Cells were routinely subcultured when reaching 80% of confluency. 2.3. EROD activities in intact MCF-7 cells The assay method was performed as previously described (Ciolino and Yeh, 1999). In brief, MCF7 cells in 96-well plates were treated with 1 M DMBA and various concentrations of hydroxychalcone. The medium was then removed and the cells were washed twice by 100 l phosphate-buffered saline (PBS). Ethoxyresorufin-O-deethylase (EROD) activity assays were then carried out. Fifty microlitres of ethoxyresorufin (5 M) and salicyclamide (1.5 mM) dissolved in PBS was added in each well, and incubated at 37 ◦ C for 15 min. The reaction was stopped by 50 l of ice-cold methanol, and the resorufin generated was measured by a FLUOstar Galaxy microplate reader (BMG Labtechnologies, Offenburg, Germany) with an excitation wavelength at 544 and 590 nm as the emission wavelength. The activities were quantified against resorufin standards. 2.4. Enzyme inhibition assays Recombinant CYP1A1 and CYP1B1 proteins expressed in baculovirus-infected insect microsomes (Supersomes® ) were purchased from Gentest Corp. (Woburn, MA, USA). Two picomoles of protein was incubated in 100 l PBS, pH 7.2 with 400 nM ethoxyresorufin and hydroxychalcone in different concentrations. The reaction was initiated by 500 M NADPH, and terminated by 100 l of ice-cold methanol after 20 min of incubation. The fluorescence was measured as described earlier. 2.5. Measurement of Cell viability Cell viability was assessed by 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
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staining as described by Mosmann (1983). Briefly, MCF-7 cells were cultured in 96-well plates at 104 cells per well and treated with 1 M DMBA and various concentrations of hydroxychalcone for 24 h. At the end of the treatment, 50 l MTT (1 mg/ml) was added and incubated at 37 ◦ C for 4 h. Cell viability was determined by the absorbance at 600 nm. 2.6. XRE-luciferase gene reporter assay A fragment with five XRE elements from rat cyp1a1 5 -flanking region was amplified from rat genomic DNA as described by Chan et al. (2003). No other enhancer elements were identified in this fragment. The polymerase chain reaction (PCR) product was digested with SmaI and BamHI and subcloned into a firefly luciferase reporter vector pTA–Luc (Clontech, Palo Alto, CA, USA). MCF-7 cells were seeded at 105 cells/well in 24well plates. After 24 h, the cells were transiently transfected with 4.0 g of the XRE-driven firefly luciferase reporter plasmid and 1.0 g of Renilla luciferase control vector (Promega, Madison, WI, USA) in LipofectAmine (Invitrogen Life Technologies). After 16 h, the medium was removed and the cells were treated with 1 M DMBA and various concentrations of hydroxychalcone for 24 h. The cells were lysed and the luciferase substrates (provided in DualLuciferase Assay Kit, Promega) were mixed with the cell lysate. The luciferase bioluminescence was measured by a FLUOstar Galaxy plate reader as described in the manual of the assay kit. The XRE transactivation activities represented by firefly luciferase light units were then normalized by that of Renilla luciferase. 2.7. Measurement of DMBA–DNA adduct formation This assay was performed as previously described (Chan et al., 2003). MCF-7 cells were plated in 6-well plates at 5 × 105 cells per well and allowed to attach for 24 h. Then, 0.1 g [3 H]-DMBA (Amersham, Arlington Height, IL, USA) per ml was administered with or without hydroxychalcone. After 16 h, cells were washed twice with cold PBS, trypsinized and pelleted. Nuclei were separated by incubating the cells for 10 min on ice in lysis buffer A (10 mM Tris–HCl (pH 7.5),
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320 mM sucrose, 5 mM magnesium chloride and 1% Triton X-100). The nuclei were collected by centrifugation at 5000 rpm after the incubation. The collected nuclei were then lysed by 400 l lysis buffer B (1% sodium dodecyl sulphate (SDS) in 0.5 M Tris. 20 mM EDTA and 10 mM NaCl (pH 9)), followed by the treatment of 20 l Proteinase K (20 mg/ml) for 2 h at 48 ◦ C. The samples were allowed to cool to room temperature and the residual protein was salted out by adding 150 l of saturated NaCl. The samples were then subjected to centrifugation at 13,000 rpm for 30 min at 4 ◦ C. Genomic DNA was isolated from the supernatant fraction by ethanol precipitation, and redissolved in autoclaved water. Ten micrograms of each DNA sample, which attained a 260 nm/280 nm ratio of >1.9, was used for scintillation counting. 2.8. Semi-quantitative RT-PCR Assay A reverse transcription-PCR assay was used to quantify mRNA expression. Total RNA was isolated from cells grown in 6-well Costar plates in triplicates by a method previously described (Chan et al., 2003). 1 g of RNA was used for cDNA synthesis, and the final volume was diluted to 20 l. Primers of CYP1A1, CYP1B1 and -actin, sequences as published formerly (Dohr et al., 1995) and a Perkin-Elmer Thermocycler (GeneAmp PCR System 2400, Norwalk, CT, USA) was utilized to amplify the target cDNAs separately after the first strand reaction. All PCR reactions consisted of 0.2 mmol/l dNTP, 2 l cDNA, 0.2 mol/l of each primer, 1× PCR buffer and 1 U of Taq polymerase. The conditions were 94 ◦ C for 45 s, 65 ◦ C for 45 s, 72 ◦ C for 1 min, and a final extension period of 7 min at 72 ◦ C. The maximum cycles of amplification were 25 for CYP1A1, 23 for CYP1B1, and 19 for  actin. The PCR products were separated on 1.8% agarose gel, stained with ethidium bromide, and photographed. A scanner equipped with Scion Image software (Scion Corporation, Frederick, MD, USA) was used to compare the optical density of the amplified fragments. The linearity of signals was verified in separate experiments.
Fig. 2. Inhibitory action of hydroxychalcones on recombinant CYP1A1 and CYP1B1. Recombinant CYP1A1 (Fig. 2A or CYP1B1 (Fig. 2B expressed in insect microsomes (Supersomes® ) was used to determine the IC50 values. Values are means ± S.D., n = 3. The IC50 values for hydroxychalcones on CYP1A1 are 1.2 M for 2 and 2-hydroxychalcones; 6.2 M for 4-hydroxychalcone; 9.3 M for butein and >10 M for 4,2 ,4 -hydroxychalcone. The values on CYP1B1 are 0.2 M for 2-hydroxychalcones; 0.05 M for 2 hydroxychalcone; 0.5 M for butein and 4,2 ,4 -hydroxychalcone; 1.2 M for 4-hydroxychalcone.
The results were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test if significant differences (p < 0.05) were observed. Another software package SigmaPlot (SPSS Inc., Chicago, IL, USA) was used for graphing the Lineweaver–Burk plots.
2.9. Statistical methods
2.10. CYP1A1 and CYP1B1 activities in recombinant microsomes
A Prism® 3.0 (GraphPad Software Inc., CA, USA) software package was utilized for statistical analysis.
Five hydroxychalcones were screened for their inhibitory effects on CYP1 enzymes. Among the five
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compounds, 2-hydroxychalcone and 2 -hydroxychalcone displayed the greatest inhibition on both CYP1A1 (Fig. 2A) and CYP1B1 (Fig. 2B). The IC50 values of 2-hydroxychalcone were determined to be about 1.2 M for CYP1A1 and 0.2 M for CYP1B1, while those of 2 -hydroxychalcone were 1.2 and 0.05 M, respectively. The five hydroxychalcones,
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in general, appeared to exert greater inhibition on CYP1B1 than CYP1A1. 2.11. XRE-luciferase reporter gene assay In the co-administration of DMBA, 2 -hydroxychalcone, as low as 1 M, could significantly decrease
Fig. 3. Effect of hydroxychalcones on DMBA-induced XRE-driven luciferase activities. MCF-7 cells were transiently transfected with a firefly luciferase reporter gene driven by XREs and a renilla luciferase control plasmid (pRL). The cells were treated (A) with DMBA or (B) without DMBA and various concentrations of hydroxychalcones for 24 h. Control cultures received the carrying solvent DMSO only. Values are means ± S.D., n = 3. In Fig. 3A, means with (a) or (b) are significantly (p < 0.05) higher or lower than the cultures treated with DMBA, respectively. Means marked with (*) are significantly higher than the control (0 M) cultures in Fig. 3B.
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(p < 0.05) XRE-transactivation (Fig. 3A). 4-Hydroxychalcone, 4,2 ,4 -trihydroxychalcone, and butein also suppressed the induced transactivation at 5 M or above. In contrast, 2-hydroxychalcone further potentiated the XRE-driven luciferase activity. In the absence of DMBA, both 2 - and 2-hydroxychalcones induced XRE-transactivation (Fig. 3B). However, the magnitude of the induction seen in 2hydroxychalcone was much stronger than that observed in 2 -hydroxychalcone, i.e. 18-fold versus 2.5-fold, respectively. No significant effects were indicated for the other hydroxychalcones. 2.12. EROD activity in MCF-cells Because XREs are located in the promoter domains of cyp1a1 and cyp1b1, hydroxychalcones can regulate the mRNA expression through the XREdependent transcriptional activities. EROD activity in the treated MCF-7 cells denotes the combined actions of hydroxychalcones on CYP1 enzyme inhibition and gene expression. All except 2-hydroxychalcone decreased the DMBA-induced EROD activities (Fig. 4A) in cells, and 2 -hydroxychalcone was the strongest inhibitor among them. 2-Hydroxychalcone, however, doubled and tripled the EROD activity at, respectively, 2.5 and 5 M. The induction of EROD activity was consistent with the XRE-transactivation data, signifying an increase of CYP1 enzyme expression. To avoid a potential experimental flaw caused by the differences in cell viability, the EROD results shown were already normalized by the corresponding MTT absorbance. Cultures receiving the same treatment were assessed for cell viability, and the result is shown in Fig. 4B. No significant variation in cell survival was observed except for cultures treated with butein. Increased viability ranging from 20 to 30% was seen in cultures treated with 1, 2.5, and 5 M of butein. 2.13. DMBA–DNA covalent binding in MCF-7 cells As EROD activities were modified in hydroxychalcone-treated cultures, we further evaluated the DMBA–DNA binding in cells. 2 -Hydroxychalcone consistently produced the least DMBA–DNA covalent formation in a dose–response manner as shown
Fig. 4. EROD activities in hydroxychalcone-treated MCF-7 cells. MCF-7 cells were seeded in 96-well culture plates and co-treated with 1 M DMBA and various concentrations of hydroxychalcones. After 24 h of treatment, the cells were assayed for EROD activity, and cell viability was determined on a separate set of cultures with equivalent treatment. (A) shows the cellular EROD activities for the treatment of 2-hydroxychalcone, 2 -hydroxychalcone, 4hydroxychalcone, 4,2 ,4 -hydroxychalcone, and butein. The EROD activities presented were normalized with the absorbance obtained from MTT assays. All means are significantly lower than the control except those marked with (θ) and (a). Mean marked with (θ) displays no significant difference and means labeled with (a) are significantly higher than the control. (B) illustrates the MTT results of cells under various kinds of treatment. The values are means ± S.D., n = 4. Means with (*) are significantly (p < 0.05) higher than the control culture.
in Fig. 5. Except for 2–hydroxychalcone, others were also able to reduce the covalent binding to a lesser extent. Although EROD activity was induced by 2hydroxychalcone in cells, the DMBA–DNA covalent binding was not significantly elevated.
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Fig. 5. Effects of hydroxychalcones on DMBA-DNA adducts formation in MCF-7 cells. MCF-7 cells were cultured in 6-well plates and treated with tritiated DMBA in 0.1 g/ml and various concentrations of hydroxychalcones were co-administered. After 16 h of treatment, genomic DNA was isolated and the DMBA-DNA lesions were determined by scintillation counting. Means with (*) are significantly (p < 0.05) lower than the cultures treated with DMBA only.
2.14. Messenger RNA abundance of CYP1A1 and CYP1B1 in cultures treated with 2 -hydroxychalcone and 2-hydroxychalcone Subsequent semi-quantitative RT-PCR data (Fig. 6) indicated that both DMBA-induced cyp1a1 and cyp1b1 expression was hampered by the co-treatment of 2 hydroxychalcone in a dose-dependent manner (Fig. 6, Panel A). The optical density readings underneath illustrated that the induced expressions reverted to their basal levels at about 5 M. 2 -Hyroxychalcone appeared to more strongly inhibit the expression of cyp1a1 than cyp1b1. A significant decrease of cyp1b1 expression was observed at 1 M, whereas the same decrease was seen at 5 M for cyp1a1. 2-Hydroxychalcone at 5 M, on the other hand, potentiated the mRNA expressions of cyp1a1 (Fig. 6, Panel B) and cyp1b1 induced by DMBA treatment.
2.15. CYP1 enzyme kinetic studies Enzyme kinetic assays performed on recombinant hCYP1A1 and hCYP1B1 Supersomes® showed that 2 -hydroxychalcone inhibited both CYP1A1 (Ki ∼ 0.3 M; Fig. 7A) and CYP1B1 (Ki ∼ 5 M; Fig. 7B) in a non-competitive manner.
3. Discussion Hydroxychalcones displayed differential effects on CYP1 enzyme expression and inhibition. Among the compounds tested, 2 -hydroxychalcone was the most effective in suppressing CYP1A1 and CYP1B1 enzyme activities and gene expressions. XRE-luciferase reporter assay indicated that the chemical could reduce the cyp1a1 and cyp1b1 expressions through the disruption of XRE-transactivation. When administered without DMBA, the hydroxychalcone was a weak inducer of XRE-transactivation and appeared to be a weak AHR agonist. The inhibition of 2 -hydroxychalcone on CYP1A1 was competitive and that of CYP1B1 was non-competitive. Because these two enzymes are responsible for the biotransformation of PAH, inhibiting the enzymes would lead to a reduction of genotoxic moieties generated from PAH. As a result, a decrease in DMBA–DNA covalent binding was demonstrated in cultures co-treated with 2 -hydroxychalcone and DMBA. 2-Hydroxychalcone, on the other hand, displayed different characteristics. It inhibited CYP1A1 and 1B1 enzyme activities at the recombinant protein level but potentiated their expressions in MCF-7 cells. Because XRE-transactivation induced by DMBA was further augmented by 2-hydroxychalcone, the potentiation of
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Fig. 6. Effect of 2 -hydroxychalcone and 2-hydroxychalcone on DMBA-induced CYP1A1 and 1B1 mRNA expression. MCF-7 cells were treated with 1 mol/l DMBA and 2 - and 2-hydroxychalcone, and cultured for 24 h. Messenger RNA expression of CYP1A1 and CYP1B1 was semi-quantified by RT-PCR. Gel images for the ethidium bromide-stained PCR fragments are displayed on the upper panel, while the corresponding optical density results are located at the lower panel. Values are means ± S.D., n = 3. Means with (*) are significantly lower and (†) are higher (p < 0.05) than samples treated with DMBA only.
CYP1 expression could be initiated in the AHR activation pathway, such as AHR binding. However, XREdependent transactivation might also be driven by a protein tyrosine kinase-mediated signaling pathway (Backlund et al., 1997), independent of the AHR status. The present study did not address the exact XREdependent pathway by which the hydroxychalcones blocked or potentiated enzyme activities. The present study suggested that the position and number of hydroxyl groups in hydroxychalcone might affect the CYP1 enzyme inhibition and gene expression. Although 2-hydroxychalcone and 2 hydroxychalcone showed comparable enzyme inhibition on recombinant CYP1A1 and CYP1B1 as shown in Fig. 2, their effects on XRE-transactivation and cellular EROD activities were opposite. Machala et al. (2001) have also shown that 2-hydroxychalcone is an inhibitor of CYP1A-dependent EROD activity in hepatic microsomes isolated from TCDD-pretreated mice, but gene regulation was not investigated in this study. Our data
revealed that 2 -hydroxychalcone was a more potent CYP1A1 inhibitor than 2-hydroxychalcone in MCF-7 cells because of the suppression at the gene expression level in addition to enzyme inhibition. Our study also indicated that increased hydroxyl-group substitution in the chalcone structure deterred both gene and enzyme levels of inhibition on CYP1A1/1B1. DNA damage caused by PAHs can be alleviated by some phytochemicals. The flavonols, quercetin and galangin (Ciolino et al., 1999a,b), and the flavone baicalein (Chan et al., 2003) can deter the metabolism of DMBA or B[a]P. These compounds inhibit CYP1A1 at both expression and enzyme levels. Besides, some non-flavonoidal compounds such as resveratrol (Ciolino et al., 1999a,b) and curcumin (Ciolino et al., 1998) can also disrupt PAH metabolism by similar mechanisms. Although the CYP1B1 expressions and enzyme activities have not been specifically addressed in these studies, results comparable to CYP1A1 are expected. In the current study, the 2 -hydroxyl substituted
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chalcones were capable of reducing the DMBA–DNA covalent formation with different potencies. It appeared that the addition of hydroxyl groups in the A- or Bring of the 2 -hydroxychalcone backbone reduced the
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protective effect on DMBA–DNA covalent binding. Although an induced CYP1 activity was seen in cultures treated with 2-hydroxychalcone, the extent of DMBA–DNA binding seemed to be unaltered. The
Fig. 7. Lineweaver–Burk plots of 2 -hydroxychalcone on CYP1A1 and CYP1B1 inhibition. EROD assay was performed on human recombinant hCYP1A1 (Fig. 7A) and hCYP1B1 (Fig. 7B) in the presence of the indicated concentrations of 2 -hydroxychalcone and 100–1600 nM ethoxyresorufin. Lineweaver–Burk plots were generated from the reciprocal data, and the value of Ki was derived from the slopes of the regression lines. Values shown are mean ± S.D., n = 3.
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reason was unknown, but the detoxifying or repairing system in the cell could be activated to offset the increase in DNA damaging species. Alternatively, the inhibition could occur in microsomal epoxide hydrolase. The cellular detoxifying enzyme system might also have metabolized the hydroxychalcones that had a strong inhibitory effect on the CYP1 enzymes, and reduced their potency on the inhibition of DMBA–DNA binding. Because of the hydroxyl group at the ortho-position in a benzene ring, 2 -hydroxychalcone and its derivatives have been subjects of much previous investigation for their pharmacological value. The 2 -hydroxyl group enhances the antioxidative activity of the chalcone structure, and has been shown to be inhibitory to lipid peroxidation and Ehrlich ascites tumor development in Swiss albino mice (Anto et al., 1995). Another study has also indicated that there is a correlation between antioxidant and estrogenic properties in hydroxychalcone (Calliste et al., 2001). Considering that redox reactions are also involved in cytochrome p450 catalysis, the antioxidant activity might also play a role in the enzyme inhibition. Other pharmacological effects of chalcones and their derivatives have also been documented. Zhang et al. (2003) have demonstrated that 2-hydroxychalcone and 2 -hydroxychalcone may sensitize tumor cells to anticancer drugs by inhibiting the conjugation of the drug with GSH. Some newly developed chalcone analogues induce cell cycle arrest and apoptosis, and also modulate p-glycoprotein activity (Vincenzo et al., 2000). Other chalcone derivatives may protect p53 from binding to the oncoprotein MDM2, and the binding inactivating p53’s tumor suppressive property (Stoll et al., 2001). In summary, we demonstrated that the number and position of hydroxyl groups substituted in chalcone might differentially affect XRE-transactivation and downstream gene expression. In addition, 2 -hydroxychalcone displayed an agonist/antagonist property toward AHR binding, whereas 2-hydroxychalcone was a potent inducer of XRE-transactivation.
Acknowledgements The authors would like to thank Prof. Georgia Guldan (Food & Nutritional Science Programme, Biochemistry Dept, the Chinese University of Hong Kong)
for the proofreading of this manuscript. This project was supported by the Chinese University of Hong Kong RGC Research Grant Direct Allocation ID No. 2041031.
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