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European Journal of Pharmacology
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european journal of pharmacology an international journal ep
Effects of lactone derivatives on aromatase (CYP19) activity in H295R human adrenocortical and (anti)androgenicity in transfected LNCaP human prostate cancer cells
Thomas Sanderson a,*, Martin Renaud ª, Deborah Scholten b, Sandra Nijmeijer b, Martin van den Berg b, Simon Cowell , Emma Guns , Colleen Nelson , Thumnoon Mutarapat ª, Somsak Ruchirawat d
a INRS-Institut Armand-Frappier, 531 blv des Prairies, Laval, QC, Canada H7V 1B7
b Institute for Risk Assessment Sciences, Utrecht University, PO Box 80176, 3508 TD, Utrecht, The Netherlands
” The Prostate Centre, Vancouver General Hospital, Vancouver, BC, Canada V6H 3Z6
d Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, Bangkok 10210, Thailand
ARTICLE INFO
Article history: Received 7 March 2008 Received in revised form 28 May 2008 Accepted 22 June 2008 Available online 27 June 2008
Keywords: Aromatase Anti-androgens Lactones LNCaP
H295R Hormone-dependent cancer
ABSTRACT
Certain lactone-containing secondary plant metabolites display potent biological effects, including anti-tumor activities. This is of particular interest as these compounds appear effective against hormone-dependent cancers, such as those of breast and prostate, of which the incidence is on the rise. The mechanisms of anti-tumor action of these compounds are largely unknown. Thirteen synthetic lactone derivatives were evaluated for effects on aromatase activity and mRNA expression in H295R human adrenocortical carcinoma cells. Aromatase (CYP19) is a key enzyme in the synthesis of estrogens from androgens. Over-expression has been associated with increased risk of developing estrogen-dependent mammary tumors, and aromatase inhibitors are effective in their treatment. The androgen receptor is implicated in mediating hormone-dependent prostate tumor growth, and androgen antagonists are effective in the treatment of these cancers. Thus the (anti)androgenic effects of the lactones were also assessed in LNCaP human prostate cancer cells transfected with human androgen receptor and an androgen receptor-responsive luciferase reporter gene. Cells were exposed to lactones (0.1-100 uM) dissolved in dimethyl sulfoxide (0.1% in medium) for 24 h prior to measurement of aromatase activity using a tritiated water-release assay. Three (competitive) inhibitors of aromatase activity were identified (potencies in decrea- sing order): 3-(3,4-dimethoxy-phenyl)-4-(4-methoxy-phenyl)-5H-furan-2-one (CRI-7; IC50=1 µM; Ki=1.0 }M), 3,4-bis-(3,4-dimethoxy-phenyl)-5H-furan-2-one (CRI-8; IC50=2 1M; Ki= 1.2 uM) and 3-(3,4-dimethoxy-phenyl)- 4-(3,4,5-trimethoxy-phenyl)-5H-furan-2-one (CRI-9; IC50=3 uM; K ;= 6.8 uM). Several concentration-dependent inducers of aromatase (>2fold) were also identified (CRI-1, CRI-4 or Vioxx, CRI-11 and CRI-13). These lactones also induced pII promoter-specific CYP19 transcripts. In transfected LNCaP cells, the three aromatase inhibitors CRI-7, 8 and 9 demonstrated concentration-dependent anti-androgenicity above 0.1 uM in the presence of either 0.1 nM of dihydrotestosterone or the synthetic androgen R1881. The other lactones showed no consistent pro- or anti- androgenic effects in these LNCaP cells. Lactone moiety-containing molecules may form the structural basis for the development of potent drugs effective against hormone-dependent cancers.
@ 2008 Published by Elsevier B.V.
1. Introduction
Increasing efforts are being made to identify and evaluate the pro- perties of compounds of natural origin with potent biological activities, as they may be used or form the basis for the development of novel drugs effective against various diseases. Most research has focused on so-called secondary metabolites produced by terrestrial plants and marine orga- nisms. These compounds encompass a wide variety of chemical structural classes, including terpenoids, flavonoids, coumarins, phytoalexins and many others (Butler, 2004, 2005). These secondary metabolites are deem- ed non-essential for basic metabolic processes of the plant or marine
organism, but are thought to have important functions in defence against bacteria and fungi, and against insects, herbivores and other predators; they are also implicated in the growth impairment of competing plants/ marine organisms in close proximity, and may possess pheromonal pro- perties important for attracting mates. Not surprisingly, these chemicals have been found or purported to have various potent biological effects in humans, including anti-bacterial, anti-fungal, anti-inflammatory, anti- oxidant and anti-cancer activities, and are known to modulate sex hor- mone function.
Numerous biologically active secondary metabolites possess a 5H- furan-2-one- or butenolide moiety, or other variations of a lactone base-structure, hereafter called lactone derivatives (de Nys et al., 2006; Zapf et al., 1995). Examples include ellagitannins (ellagic acid) found in berries (Mullen et al., 2002), various furanones found in seaweeds such as Delisea pulchra (de Nys et al., 2006), acetogenins from the Annonaceae
* Corresponding author. Tel .: +1 450 687 5010x8819; fax: +1 450 686 5309. E-mail address: thomas.sanderson@iaf.inrs.ca (T. Sanderson).
family (Bermejo et al., 2005), cardiac glycosides from Digitalis purpurea and many others (Butler, 2004, 2005). Some of these compounds have been suggested to have anti-cancer properties, although very little is known about their actual mechanism(s) of action. One possibility is through inhibition of various inflammatory processes and oxidative stress; this is illustrated by the fact that the 5H-furan-2-one moiety is found in several highly potent cycloxygenase-2 inhibitors and anti-oxidants, inclu- ding the recently withdrawn rheumatoid arthritis drug rofecoxib (Vioxx) (Jachak, 2006; Scott and Lamb, 1999). Consumption of plant secondary metabolites has been suggested to reduce the risk of hormone-dependent cancers, such as those of prostate and breast; thus, another possibility is that they act as inhibitors of hormone synthesis or as antagonists for steroid hormone receptors. Our working hypothesis is that compounds of natural origin with structural characteristics similar to endogenous andro- gens may act as inhibitors of the enzyme aromatase (CYP19, which uses androgens as substrate) and as possible (ant)agonists for the androgen receptor. The current study examines the ability of a group of synthetic
lactone derivatives to interfere with the expression and catalytic activity of aromatase in H295R human adrenocortical carcinoma cells, and to activate or antagonize the androgen receptor in LNCaP human prostate carcinoma cells transiently transfected with human androgen receptor and an an- drogen-responsive luciferase reporter construct. Our objective is to iden- tify structures that inhibit aromatase activity and/or antagonize the androgen receptor and thus may form the basis for the development of novel drugs effective against hormone-dependent cancers.
2. Materials and methods
2.1. Lactones
The lactone derivatives (CRI-1 to 13) were synthesized in the Labo- ratory of Medicinal Chemistry at the Chulabhorn Research Institute (Bangkok, Thailand) by a modification of previously published methods (Dikshit et al., 1990; Kim et al., 2002; Pirali et al., 2006; Thérien, 2001)
Table 1 Structures and IUPAC names of the thirteen CRI (Chulabhorn Research Institute) lactone derivatives used in this study
3,4-diphenyl-5H-furan-2-one (CRI-1)
3-(3,4-dimethoxy-phenyl)-4-phenyl-5H-furan- 2-one (CRI-6)
3-(3,4-difluoro-phenyl)-4-(4-methylsulfonyl- phenyl)-5H-furan-2-one (CRI-11)
0
0
0
0
0
0
OMe
F
OMe
MeO2S
F
4-(4-methoxy-phenyl)-3-phenyl- 5H-furan-2-one (CRI-2)
3-(3,4-dimethoxy-phenyl)-4-(methoxy- phenyl)-5H-furan-2-one (CRI-7)
3-(2,4-difluoro-phenyl)-4-(4-methylsulfyl- phenyl)-5H-furan-2-one (CRI-12)
0
0
0
0
0
F
OMe
MeO
MeO
OMe
MeS
F
4-(4-methylsulfyl-phenyl)-3-phenyl- 5H-furan-2-one (CRI-3)
3,4-bis-(3,4-dimethoxy-phenyl)-5H-furan-2- one (CRI-8)
3-(2,4-difluoro-phenyl)-4-(4-methylsulfonyl- phenyl)-5H-furan-2-one (CRI-13)
0
0
0
0
0
F
MeO
O Me
Me S
MeO
O Me
MeO2S
F
4-(4-methylsulfonyl-phenyl)-3-phenyl- 5H-furan-2-one (CRI-4, rofecoxib) CAS # 162011-90-7
3-(3,4-dimethoxy-phenyl)-4-(3,4,5- trimethoxy-phenyl)-5H-furan-2-one (CRI-9)
0
0
0
0
MeO
O Me
MeO
O Me
O Me
MeO2S
3,4-bis-(4-methoxy-phenyl)-5H-furan-2-one (CRI-5)
3-(3,4-difluoro-phenyl)-4-(4-methylsulfyl- phenyl)-5H-furan-2-one (CRI-10)
0
0
0
0
F
MeO
OMe
MeS
F
(Table 1). All compounds were greater than 95% pure and dissolved in dimethyl sulfoxide (DMSO; D4540, Sigma-Aldrich, St Louis, MO) as 1000- fold concentrated stock solutions.
2.2. H295R cell culture conditions and treatments
H295R cells were obtained from the American Type Culture Col- lection (ATCC CRL-2128, Manassas, VA) and grown in 1:1 (v/v) Dulbecco’s modified Eagle medium/Ham’s F-12 nutrient mix (DMEM/ F12) containing 365 mg/ml L-glutamine and 15 mM HEPES (31330-38, GibcoBRL, Gaithersburg, MD) under an atmosphere containing 5% CO2 at 37 ℃ and saturating humidity. The medium was supplemented with 10 mg/Linsulin, 6.7 µg/L sodium selenite and 5.5 mg/L transferrin (ITS-G; 41400-037, GibcoBRL), 1.25 mg/L bovine serum albumin (A9647, Sigma- Aldrich), 100 U/L penicilline/100 µg/L streptomycin (P7081, Sigma- Aldrich) and 2% steroid-free replacement serum Ultroser SF (Soprachem, France). For the aromatase experiments cells were treated as described previously (Sanderson et al., 2000). In brief, cells plated at a density of 1- 2×105 cells/ml were exposed to various concentrations of the synthetic lactone derivatives dissolved in DMSO (final solvent concentration of 0.1% v/v). Negative control cells were exposed to 0.1% (v/v) DMSO. As positive control for aromatase inhibition, cells were exposed to 1 µM of the steroidal aromatase inhibitor 4-hydroxyandrostenedione (F2552, Sigma-Aldrich); as positive control for induction, 300 µM of 8-bromo- cyclic adenosine monophosphate (8Br-cAMP; B7880, Sigma-Aldrich) and 20 µM of forskolin (F6886, Sigma-Aldrich) were used. DMSO at 0.1% had no effect on CYP19 expression or catalytic activity relative to unexposed cells. Protein concentrations were determined by the method of Lowry et al. (Lowry et al. (1951), using BSA as standard. All exposures were for 24 h, unless stated otherwise.
2.3. Isolation and amplification of RNA from H295R cells
RNA was isolated using the RNA Insta-Pure System (Eurogentec, Belgium) according to the instructions of the supplier and stored at -80 ℃. The purity of the RNA preparations was verified by denaturing agarose gel electrophoresis. Reverse-transcriptase polymerase chain reactions (RT-PCRs) were performed using the Access RT-PCR System (Promega, Madison, WI). Amplification of pII promoter-derived CYP19 cDNA was accomplished using the primer pairs and experimental con- ditions reported recently (Heneweer et al., 2004; Sanderson et al., 2004), using a Biometra T3000 thermocycler (Montréal Biotech Inc, Montréal, QC). The sequence of pII-specific mRNA was derived from NCBI accession number S52794. RT-PCR of B-actin transcript was used as reference
120
CRI-7
Aromatase activity in H295R cells (% control)
100
-O-CRI-8
CRI-9
80
60
40
20
0
0
1
10
100
1000
Lactone concentration (uM)
1000
10
900
9
Vmax (pmole/h/mg protein)
800
8
700
7
Km (nM)
600
6
500
5
400
4
300
Km
3
200
Vmax
2
100
1
0
0
0
5
10
15
20
25
30
Inhibitor (CRI-7) concentration (uM)
600
6
Vmax (pmole/h/mg protein)
500
5
Km (nM)
400
4
300
3
200
Km
2
100
Vmax
1
0
0
0
5
10
15
20
25
30
Inhibitor (CRI-8) concentration (uM)
300
9
8
Vmax (pmole/h/mg protein)
250
7
Km (nM)
200
6
150
5
4
100
Km
3
50
Vmax
2
1
0
0
0
5
10
15
20
25
30
Inhibitor (CRI-9) concentration (uM)
(Sanderson et al., 2002). Amplification products were detected using agarose gel electrophoresis and ethidium bromide (46067, Fluka-Sigma- Aldrich) staining. Intensities of the ethidium bromide stains were quan- tified using a Fluor-S MultiImager (BioRad, Mississauga, ON).
2.4. Aromatase assay in H295R cells
The catalytic activity of aromatase in H295R cells was determined based on a tritiated water-release assay (Lephart and Simpson, 1991) with minor modifications. Cells were exposed to 54 nM 1B-[3H]andro- stenedione with a specific activity of 25.5 Ci/mmol (NET-926; Perkin Elmer, Boston, MA) dissolved in serum-free culture medium and incu- bated for 1.5 h at 37 ℃ in an atmosphere containing 5% CO2. All further steps were as reported previously (Sanderson et al., 2000). Aromatase activity was expressed in pmoles of androstenedione converted per hour per milligram cellular protein. The specificity of the aromatase assay based on the release of tritiated water was verified by using the selective aromatase inhibitor 4-hydroxyandrostenedione (Brodie et al., 1977). Under our experimental conditions H295R cells had a basal aromatase activity of about 2.4+0.2 pmol/h/mg protein. Aromatase activity remained stable for up to 40 passages. For the enzyme-kinetic experiments, H295R cells were exposed to various concentrations (0.1-
30 µM) of lactone derivatives in the presence of increasing concentra- tions of 1B-[3H]androstenedione (25-1000 nM) and incubated for 90 min prior to analysis of aromatase activity.
2.5. Cyclic AMP measurements in H295R cells
Intracellular cAMP concentrations were determined using a com- mercial enzyme-linked immunoassay kit (KGE002, R&D systems, Minneapolis, MN) according to the instructions provided, and using cell culture and exposure conditions (4 h) optimized previously (Sanderson et al., 2002, 2004). To enhance sensitivity, an acetylation step was included following the instructions of the supplier.
2.6. LNCaP cell culture conditions
LNCaP cells (#CRL-1740, ATCC) were cultured in RPMI 1640 med- ium (R8758, Sigma-Aldrich) supplemented with 1 mM HEPES buffer (H0887, Sigma-Aldrich), 100 mM sodium pyruvate (S8636, Sigma- Aldrich), 100 U/ml penicillin/streptomycin and 10% fetal bovine serum (FBS; F1051, Sigma-Aldrich). A suspension of 1 × 105 cells/ml was added to 12-well plates at 1 ml/well in culture medium containing dextran- coated charcoal-treated FBS to remove steroid hormones (Hyclone, Logan, UT). Plates were incubated overnight under an atmosphere containing 5% CO2 at 37 ℃ and saturating humidity.
2.7. Transfections, exposures to (anti-)androgens and luciferase assays in LNCaP cells
Plasmids containing a human androgen receptor gene expression construct (phAR), an androgen-responsive luciferase reporter con- struct under control of a rat promoter (pARR3tk-luc) and a Renilla luciferase reporter construct (pRLtk; Promega) were simultaneously and transiently transfected into LNCaP cells using LIPOFECTIN™ (Invitrogen, Burlington, ON) as described earlier (Portigal et al., 2002). Each well contained 1.5 µg of the hAR plasmid, 1.0 µg of the pARR3tk-luc reporter and 0.02 µg pRLtk. The combined transfection of phAR and pARR3tk-luc allows the LNCaP cells, which normally express low levels of androgen receptor (Cleutjens et al., 1997), to become highly responsive to wild-type human androgen receptor activation. The Renilla luciferase construct is used as a co-transfection plasmid to normalize for differences in transfection efficiency and cell viability. Cells were incubated overnight and then exposed to the lactones either in the presence or absence of 100 pM dihydrotestosterone (DHT;
400
Aromatase activity in H295R cells
350
+-CRI-1
*- CRI-3
300
CRI-4
-O-CRI-10
+CRI-11
(% control)
250
4-CRI-13
200
150
100
50
0
0
1
10
100
1000
Lactone concentration (uM)
400
400
forskolin
r=0.934
Aromatase activity (% control)
Aromatase activity
pll-CYP19/beta-actin amplification response ratio (% control ratio)
350
o pli-CYP19 mRNA
350
CRI-
300
300
250
CRI-13
250
CRI-3
CRI-11
r=0.980
200
CRI-10
CRI-4
200
150
150
100
100
100
150
200
250
300
350
CAMP levels (% control)
Steraloids, Newport, RI) or methyltrienolone (R1881; Perkin Elmer). Cells were harvested 48 h later in cold phosphate-buffered saline (PBS) containing 1 mM EDTA, pelleted, and resuspended in 0.1 ml Passive Lysis Buffer (Promega). Samples were then mixed thoroughly using a vortex and frozen at -80 ℃ until further analysis. Luciferase assays were performed using the dual-luciferase reporter assay system according to the instructions supplied. Light production was measured using a luminometer (model LB 96V, Berthold Technologies, Germany).
2.8. MTT reduction assay
Mitochondrial function, as an indicator of cytotoxicity, was asses- sed by measuring the capacity of H295R and LNCaP cells to reduce MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan (Denizot and Lang, 1986). Formazan production was moni- tored spectrophotometrically and was linear over a period of 20 min using an absorbance and reference wavelength of 560 and 690 nm, respectively.
2.9. Data analysis
All data are presented as means with standard deviations (n=3 or n=4). Statistically significant differences from control groups were determined by a two-tailed t-test using a correction for multiple com- parisons (Zar, 1999) and a significance level of 0.05. Non-linear regression analyses of enzyme-kinetic data were performed using Prism 5.0 (GraphPad Software, Inc, San Diego, CA) in order to calculate apparent Km (Michaelis-Menten constant, a measure of affinity of substrate for the enzyme) and Vmax (maximum velocity) values. Ki (inhibition constant) values for competitive inhibitors were determined by plotting apparent Km values versus inhibitor concentrations.
3. Results
3.1. Cytotoxicity of lactones in H295R and LNCaP cells
The three sulfide-containing lactone-derivatives CRI-3, 10 and 12 caused a concentration-dependent decrease in MTT reduction capacity of H295R and LNCaP cells. Statistically significant impairment occurred at concentrations of 10 uM and cell viabilities after exposure to 100 µM of CRI-3, 10 and 12 were 61 +4, 74±8 and 60±7%, respec- tively, compared to solvent control (100±6%). None of the other lactones impaired cellular MTT reduction activity in the 0.3-100 µM concentration range.
120
140
140
Relative luciferase expression (% of maximum response)
Relative luciferase expression (% response 0.1 nM DHT)
Relative luciferase expression (% response 0.1 nM R1881)
100
+DHT
120
O-R1881
120
80
100
100
80
80
60
60
60
40
40
40
20
-CRI-7
CRI-7
20
CRI-8
20
CRI-8
-A-CRI-9
-A-CRI-9
0
0.00
0.01
0.10
1.00
10.00
100.00
0
0.0
0.1
1.0
10.0
100.0
1000.0
0
0.0
0.1
1.0
10.0
100.0
1000.0
Concentration (nM)
Lactone concentration (uM)
Lactone concentration (uM)
3.2. Effects of lactones on aromatase activity and CYP19 expression in H295R cells
Three lactone derivatives, CRI-7, 8 and 9 inhibited the catalytic activity of aromatase in H295R cells (Fig. 1), with IC50 values between 1 and 3 uM (Fig. 1). At a concentration of 100 uM CRI-7, 8 and 9 demonstrated inhibition efficacies of 81, 76 and 93%, respectively (Fig. 1); this in comparison to the positive control 4-hydroxyandro- stenedione, which had an IC50 value of 10 nM and caused 90% inhi- bition at 1 µM (not shown). Detailed enzyme-kinetic analysis showed that the three lactone derivatives acted as competitive inhibitors of the aromatase enzyme (Fig. 2), with apparent inhibition constants (Ki) for CRI-7, 8 and 9 of 1.0, 1.2 and 6.8 uM, respectively.
Several lactone derivatives, CRI-1, 4, 11 and 13 caused a concen- tration-dependent induction of the catalytic activity of aromatase (Fig. 3). A certain degree of induction was also observed for CRI-3 and 10, but catalytic activity decreased strongly at concentrations above 10 µM (Fig. 3), largely due to the aforementioned cytotoxicity of the compounds. Of the concentration-dependent inducers of aromatase, CRI-1 appeared to be the most potent and efficacious (Fig. 3). To further investigate the mechanism of induction by these lactones in H295R cells, their effect on promoter-specific expression of CYP19 transcript and intracellular cAMP accumulation was evaluated. CRI-1, 4, 11 and 13, at 100 uM significantly (p<0.05) increased pII promoter-derived CYP19 mRNA levels in H295R cells by 175±14, 132+ 10,148±20, and 157±12%, respectively, compared with solvent control (100±15%). CRI-3 (137± 36%) and CRI-10 (106±7%), at a concentration of 10 µM (before the occurrence of cytotoxicity) did not cause a statistically significant in- crease in CYP19 transcript. Effects of the lactones on intracellular cAMP levels (Fig. 4) correlated well with their ability to induce catalytic activity (r=0.934) and mRNA expression (r=0.980). CRI-2, 5, 6 and 12 had no statistically significant effects on either the catalytic activity of aromatase or its mRNA expression (not shown); effects on cAMP were therefore not investigated.
3.3. (Anti-)androgenic effects of lactones in transiently transfected LNCaP cells
LNCaP cells transiently transfected with human androgen receptor and an androgen-responsive luciferase reporter construct were highly sensitive to the potent androgens DHT and R1881 (Fig. 5). The two agonists did not differ statistically significantly in potency and had EC50 values in the 0.1-0.3 nM range. None of the thirteen lactone derivatives demonstrated pro-androgenic activity in these transiently transfected LNCaP cells (not shown). However, in the presence of an approximately 50% maximal stimulatory concentration of DHT or R1881 (0.1 nM), CRI-7, 8 and 9 caused a concentration-dependent
decrease in androgen-stimulated luciferase activity (Fig. 5). The other lactones had no effect (not shown). The order of anti-androgenic potencies of the three lactones varied depending on whether DHT or R1881 was used as agonist (Fig. 5). In the case of DHT, the order of decreasing potency (IC50) was CRI-8 (=3 µM)>CRI-9 (=40 µM)>CRI-7 (~70 µM); in the case of R1881, it was CRI-7 (=2uM)>CRI-9 (=5 µM)> CRI-8 (=60 µM).
4. Discussion
4.1. Inhibitors of aromatase activity in H295R cells
The present study has shown that CRI-7, 8 and 9 are relatively potent and highly efficacious inhibitors of aromatase activity, with EC50 values in the lower micromolar range. The competitive nature of this inhibition suggests that the compounds interact with the substrate pocket of the aromatase enzyme. H295R human adrenocortical carcinoma cells have been used in several previous studies to study the effects of chemicals on the steroid hormone biosynthesis pathway (Gracia et al., 2007; Hilscherova et al., 2004; Johansson et al., 2002; Ohno et al., 2002; Zhang et al., 2005), and on aromatase activity in particular (Canton et al., 2005; Heneweer et al., 2004, 2005; Letcher et al., 2005; Sanderson et al., 2000, 2001a,b, 2002, 2004). The inhibitory potencies of the lactones in the current study are similar to those found for a number of naturally occurring flavonoids, such as chrysin and apigenin (Sanderson et al., 2004). However, the structure- activity relationship for aromatase inhibition by the lactones is not yet clear. There is some resemblance between the lactones in the current study and the flavonoids mentioned earlier, with the 2-oxo group of the lactone structure likely having a similar role to the 4-oxo group in the flavonoids for interaction with the heme prosthetic group of the aromatase enzyme. In the case of the three inhibitory lactones CRI-7, 8 and 9, they have uniquely in common the presence of two methoxy groups in the 3- and 4-position of the 3-phenyl ring, in combination with at least one methoxy group on the 4-phenyl ring. Considering that replacing a hydroxy group with a methoxy group in the flavonoids reduces their inhibitory potency (Sanderson et al., 2004), it would be important to find out whether various hydroxylated analogues of these lactone derivatives would be more potent inhibitors than the currently tested ones.
4.2. Inducers of aromatase activity in H295R cells
Six lactone derivatives induced aromatase activity in H295R cells, with CRI-1 being the most potent and efficacious. CRI-3 and 10 produced biphasic concentration-response curves, with a post-maximal decline in activity caused by their cytotoxicity at concentrations above 10 µM.
Further examination of the mechanism of induction of aromatase activity revealed that pII promoter-specific CYP19 transcript levels were increased statistically significantly after a 24 h exposure of H295R cells to 100 µM of CRI-1, 4, 11 or 13. The weaker inducers CRI-3 and 10 did not cause statistically significant induction of pII promoter-derived CYP19 transcript levels. Nevertheless, the six lactones that induced aromatase activity significantly increased intracellular cAMP levels, which were highly and statistically significantly (p<0.001) correlated with plI- derived CYP19 mRNA levels and catalytic activity. This is consistent with the fact that the pII promoter, which is the most active promoter of aromatase gene expression in H295R cells (Sanderson et al., 2004; Watanabe and Nakajin, 2004), is strongly stimulated by cAMP analogues and forskolin. Previous studies have shown that various xenobiotics such as the pesticides atrazine and vinclozolin (Sanderson et al., 2002), and the flavonoids genistein and quercetin (Sanderson et al., 2004) induce aromatase activity and CYP19 mRNA levels in H295R cells via stimulation of intracellular cAMP. A likely mechanism common to inducers of aromatase in H295R cells is inhibition of phosphodiesterase (PDE) activity, which would result in decreased breakdown of cAMP, leading to an increased steady-state concentration in the cell. Genistein and quercetin are well known PDE inhibitors (Kuppusamy and Das, 1992; Nichols and Morimoto, 2000), and atrazine too has recently been shown to inhibit isolated bovine heart PDE activity with a potency (IC50=1.8 uM) greater than that of isobutyl methylxanthine (IC50=4.6 [M) (Roberge et al., 2004). It is possible, given the structural similarities between the lactones and naturally occurring flavonoids, that the current lactone derivatives may act as PDE inhibitors in H295R cells.
4.3. Pro- or anti-androgenicity in transiently transfected LNCaP cells
The lactone derivatives did not produce an androgenic response in LNCaP human prostate carcinoma cells transiently transfected with human androgen receptor and an androgen-responsive luciferase con- struct. In contrast, the well known potent androgens DHT and R1881 caused a concentration-dependent increase in androgen receptor-de- pendent luciferase induction with EC50 values in the mid picomolar range (0.1-0.3 nM). Thus, none of the lactones were agonists of the androgen receptor. In the presence of a 50% effective concentration of the agonists DHT or R1881 (0.1 nM), three of the thirteen lactones (CRI-7, 8 and 9) produced an anti-androgenic effect, indicating that they act as androgen receptor antagonists. A possible explanation for the difference in rank of antagonistic potency of the lactones in the presence of either DHT or R1881 may lie in the known differences in the nature of the interaction of these two androgens with the ligand-binding pocket of the androgen receptor (Matias et al., 2000; Pereira de Jesus-Tran et al., 2006). In the case of DHT electrostatic bonds are the predominant factor driving affinity for the androgen receptor, whereas in the case of R1881 and the structurally almost identical anabolic steroid tetrahy- drogestrinone Van der Waals interactions are more important (Pereira de Jesus-Tran et al., 2006). Thus, dependent on the hydrophobicity and electronic properties of the lactones they would have different abilities to compete effectively with DHT versus R1881 as androgen receptor antagonists. Given that DHT and R1881 have similar potencies in our LNCaP system and that the lactones are more effective in antagonizing the androgenic response to R1881, one could suggest that hydrophobic interactions play a predominant role in the affinity of these lactones for the androgen receptor. Interestingly, it is precisely the three lactones CRI-7, 8 and 9 that also inhibited aromatase activity. This is consistent with our initial hypothesis stating that structures with structural resemblance to endogenous androgens would interfere with both the catalytic activity of aromatase and the function of the androgen receptor. However, a better understanding of the detailed nature of these inter- ferences requires further studies, such as androgen receptor binding experiments and an investigation of the ability of these lactones to act as a substrate for the aromatase enzyme. Also important is the exploration of other potential mechanisms of anti-androgenicity, such as increased
androgen receptor degradation or down-regulation, before a thorough evaluation of the (quantitative) structure-activity relationships for this group of compounds and their suitability as prototype anti-hormonal drugs can be made.
Acknowledgments
This work was funded in part by a Natural Science and Engineering Council of Canada (NSERC) Discovery grant (no. 313313) to Thomas Sanderson, and an NSERC undergraduate student bursary and a Fon- dation Armand-Frappier Master student bursary to Martin Renaud.
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