ELSEVIER
Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet
Toxicology Letters
TL
Chemopreventive actions by enterolactone and 13 VIOXX®-related lactone derivatives in H295R human adrenocortical carcinoma cells
Majorie B.M. van Duursena,*, Sandra M. Nijmeijerª, Somsak Ruchirawatb, Martin van den Berga
a Endocrine Toxicology, Institute for Risk Assessment Sciences (IRAS), Utrecht University, Utrecht, The Netherlands
b Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, Bangkok, Thailand
ARTICLE INFO
Article history: Received 29 July 2009 Received in revised form 29 October 2009 Accepted 1 November 2009 Available online 11 November 2009
Keywords:
CYP17 Chemoprevention Lactone derivatives Sex steroidogenesis H295R
ABSTRACT
Cytochrome P450c17 (CYP17) has been linked to various hormone-related diseases, including breast cancer, thus being a potential target for cancer chemoprevention. We studied the naturally occurring phytochemical enterolactone (ENL) and 13 VIOXX®-related lactone derivatives (CRI-1 to CRI-13) for their effects on CYP17 activity and expression and on cell cycle status in the human H295R adrenocor- ticocarcinoma cell line. Of the tested compounds, only CRI-3, -7, -10 and -12 showed to be inhibitors of CYP17 activity in H295R cells. This inhibition was not due to decreased mRNA expression, but was appar- ently caused by post-translational modification of the CYP17 enzyme. The MAPK kinase (MEK) inhibitor PD98059 induced CYP17 activity by 24%, while co-incubation of the CRI-s with PD98059, reduced CYP17 activity even further than the reduction caused by the CRI-s alone. In addition, CRI-3, -7, -10 and -12 arrested the cell cycle in the G(2)/M phase. The structure-activity similarities of the CRI-s with known micro-tubule binding agents strongly suggest that cell cycle arrest is a result of interaction with tubu- lin. We conclude that the proposed cancer chemopreventive actions of ENL are not mediated through interaction with CYP17 or cell cycle status. Of the VIOXX®-related lactone derivatives, CRI-7 could prove useful in the prevention of hormone-dependent cancers, such as breast cancer, since in vitro it shows low cytotoxicity, it is a potent inhibitor of CYP17 activity and strong inducer of cell cycle arrest.
@ 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
During the past years, a shift in cancer research interest is seen from the treatment of cancer toward cancer chemopreven- tion, which is the use of natural, synthetic or biologic chemical agents that reverse, suppress or prevent carcinogenic progression to invasive cancer (Tsao et al., 2004). It is generally accepted that diet plays an important role in cancer incidence and prevention. The American Institute for Cancer Research/World Cancer Research Fund has calculated that as much as 30-40% of all cancers can be prevented by appropriate diets, physical activity and mainte- nance of appropriate body weight (Glade, 1999). Besides nutritional compounds, the daily diet also comprises a large group of phyto- chemicals. Phytochemicals are a diverse group of chemicals which do not possess a nutritional value and that can be found in fruits, vegetables, grains and other plant foods. Phytochemicals have been suggested to exert a variety of health effects including the pro- tection against hormone-dependent cancers, such as breast cancer
(Murillo and Mehta, 2001; Adlercreutz, 2002; Limer and Speirs, 2004). This hypothesis is mainly based on the fact that the incidence and mortality of breast cancer are much lower in Asian countries than in Western countries (Magee and Rowland, 2004). This differ- ence is often attributed to the traditional Asian diet, which is low in fat and high in fiber and phytochemicals (Tham et al., 1998). Uptil now, over 5000 phytochemicals have been identified and classified (Liu, 2004). The term phytoestrogens is also often used to spec- ify phytochemicals that are structurally similar to estrogens or are converted in the gut to compounds that exert estrogen-like actions (usually estrogen receptor, ER-mediated). Phytoestrogens are gen- erally classified as isoflavones, coumestans and lignans (Bingham et al., 1998). Most extensively studied are the isoflavones, with genistein and daidzein being the most important, which are present in large amounts in soybeans and soy products. Coumestans, like coumestrol, can mainly be found in alfalfa and clover sprouts, thus not comprising a substantial part of the human daily diet. Lignans, on the other hand, are widespread in the Western diet and can be found in high fiber foods and whole grain products, but also in berries and garlic (Mazur and Adlercreutz, 1998). The main dietary, plant-derived lignans, matairesinol and seco-isolariciresinol, are converted by intestinal bacteria to the active compounds entero- diol (END) and enterolactone (ENL). END can be further converted into ENL in the mammalian gut (Borriello et al., 1985).
* Corresponding author at: Institute for Risk Assessment Sciences (IRAS), Utrecht University, PO Box 80177, Yalelaan 2, 3584 CM Utrecht, The Netherlands. Tel .: +31 30 253 5398; fax: +31 30 253 5077.
E-mail address: m.vanduursen@uu.nl (M.B.M. van Duursen).
CRI-1
CRI-2
CRI-3
MeO
MeS
CRI-4 (Vioxx®)
CRI-5
CRI-6
OMe
MeO2S
MeO
OMe
OMe
CRI-7
CRI-8
CRI-9
OMe MeO
OMe
MeO
OMe
MeO
OMe
MeO
OMe
MeO
MeO
OMe
CRI-10
CRI-11
CRI-12
F
F
F
MeS
F
MeO2S
F
MeS
F
CRI-13
F
OH
HO
MeO2S
F
Enterolactone
OMe
Combretastatin
Many epidemiological studies have been performed to investi- gate the effects of phytochemicals on hormone-dependent cancers, such as breast cancer. However, the experimental designs (cohort, case-control, prospective studies), research methods (such as food questionnaire, phytochemical urinary or plasma levels) and results (decreased, increased, no effect on cancer risk) are very diverse (Adlercreutz, 2002; Peeters et al., 2003; Dixon, 2004; Magee and Rowland, 2004; van Gils et al., 2005). Still, while only a few studies have investigated the effects of lignans, mainly ENL, on hormone- dependent cancers, most studies describe a modest protective trend between lignan intake and breast cancer risk (Keinan-Boker et al., 2004; Linseisen et al., 2004; Olsen et al., 2004).
The suggested protection against hormone-dependent cancer by phytochemicals is often attributed to their ability to bind to the ER (Kuiper et al., 1997; Pearce and Jordan, 2004). However, mounting evidence suggests that phytochemicals can alter endoge- nous sex steroidogenesis and metabolism, thus altering the natural hormonal homeostasis of a cell, which can influence the initiation, promotion or proliferation of hormone-dependent tumors (Kirk et al., 2001; Sanderson and van den Berg, 2003; Castagnetta et al., 2004). Lignans also appear to have an effect on hormone balance. In
vitro, ENL appears to be a moderate inhibitor of aromatase (Makela et al., 2000) and 5x-reductase (Evans et al., 1995). In vivo, they are positively correlated with plasma sex hormone binding globulin (SHBG) levels and negatively correlated with free plasma estradiol and testosterone (Adlercreutz et al., 1986).
De novo synthesis of sex steroid hormones involves the multiple enzymatic conversions of cholesterol and includes several rate- limiting steps, which makes fine-regulation of sex steroid levels possible. The final step in estrogen synthesis, for example, is the aromatization of androgens to estrogens by cytochrome P450 19 (CYP19 or aromatase) (Adams and Li, 1975). CYP19 has been shown to play an important role in breast tumor growth and suppression of local estrogen synthesis by aromatase inhibitors has been proven to be an effective treatment in post-menopausal breast cancer. Another key enzyme in sex steroidogenesis is cytochrome P450c17 (CYP17). The CYP17 enzyme is linked to various hormone-related diseases, such as polycystic ovarian syndrome (PCOS), congenital adrenal hyperplasia, prostate cancer and breast cancer, underlin- ing the importance of CYP17 in human sex steroid homeostasis. CYP17 displays both 17«-hydroxylase and 17,20-lyase activity. This dual function of CYP17 allows the adrenal glands and gonads to
synthesize cortisol (17a-hydroxylase activity) and sex steroid pre- cursors (17,20-lyase activity) (Chung et al., 1987; Kagimoto et al., 1988). CYP17 mediates sex steroidogenesis via the 45 pathway by the conversion of pregnenolone to 17a-hydroxypregnenolone and then to dehydroepiandrosterone (DHEA). DHEA is then fur- ther converted in two steps, either through androstenedione or androstenediol, to testosterone. Alternatively, androgens are being produced through the 44 pathway via conversion of pro- gesterone to 17a-hydroxyprogesterone to androstenedione and subsequently testosterone (Fluck et al., 2003; Payne and Hales, 2004). Several studies have shown that sex steroidogenesis by human CYP17 favors the 45 pathway (Lee-Robichaud et al., 1995; Fluck et al., 2003).
Considering the clinical success of aromatase (CYP19) inhibitors in breast cancer treatment, a similar approach for targeting CYP17 could prove useful for cancer treatment, but also prevention. Because of the suggested cancer chemopreventive properties of ENL, we investigated its effects on CYP17 activity and expression in the human adrenocorticocarcinoma cell line H295R. The H295R cell line is currently the standard bioassay for the assessment of effects on steroidogenic enzymes in vitro (Sanderson, 2006) (Gracia et al., 2006). Furthermore, we studied the effects of 13 synthetic lac- tone derivatives (Fig. 1) that show structural similarity the in 2004 withdrawn rheumatoid arthritis drug VIOXX® (CRI-4, rofecoxib). Besides their structural resemblance with VIOXX®, the CRI-s also have structural similarities with naturally occurring compounds such as combretastatin, enterolactone and resveratrol. These are all compounds that have been proven safe and have been suggested to possess anti-cancer properties. These VIOXX®-related lactones have been synthesized with the objective to study their potential use as cancer chemopreventive compounds.
2. Materials and methods
2.1. Chemicals
ENL, PD98059, trilostane and SU-10603 (7-chloro-3,4-dihydro-2-(3-pyridyl)- 1-(2H)-naphthalenone) were obtained from Sigma (Zwijndrecht, NL). The VIOXX®-related lactones (CRI-1 to CRI-13, Fig. 1. For IUPAC nomenclature see Sangjun et al., 2009) were synthesized at the Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, Bangkok, Thailand. 2-methoxyestradiol (2-MeOE2) was purchased from Steraloid Inc. (Newport, RI).
2.2. Cell culture
H295R cells (human adrenocorticocarcinoma cell line, ATCC) were cultured in Dulbecco’s modified Eagle’s Medium and Ham’s F12 medium (1:1) contain- ing 365 mg/ml L-glutamine and 15 mM HEPES (GibcoBRL, Breda, The Netherlands). The medium was supplemented with 10 mg/l insulin, 6.7 µg/l sodium selenite and 5.5 mg/l transferrin (ITS-G, GibcoBRL), 1.25 mg/ml bovine serum albumin, 100 U/ml penicillin, 100 µg/ml streptomycin and 2% steroid-free replacement serum Ultroser SF (Biosepra, France).
H295R cells were plated onto 24-well plates (CYP17 activity, cell cycle status) or 12-well plates (mRNA expression) and allowed to attach for 24h. Then cells were exposed to either the test compounds or the solvent vehicle-control (0.1%, v/v DMSO) for 24 h.
2.3. Cell viability
The cell viability was determined by measuring the capacity of the cells to reduce MTT (3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to the blue- colored formazan by the mitochondrial enzyme succinate dehydrogenase (Denizot and Lang, 1986). Cells were incubated with MTT for 30 min, which is in the linear phase of formazan formation. Formazan was extracted by adding 1 ml isopropanol at room temperature. Formazan formation was measured spectrophotometrically at an absorbance wavelength of 595 nm. Cell viability was determined as % of vehicle control-treated cells.
2.4. CYP17 activity
CYP17 activity was determined as described earlier (Canton et al., 2006). In short, cells were exposed to the various lactones for 24 h. After the exposure, CYP17 activ- ity was determined by adding pregnenolone as substrate. To ensure the conversion of pregnenolone into 17@-hydroxypregnenolone and subsequently into DHEA (45
pathway), 3ß-hydroxysteroid dehydrogenase was blocked with trilostane. DHEA synthesis was measured using a commercially available RIA kit (Immunotech, Bech- man Coulter Company) according to the manufacturers instructions. The selective CYP17 inhibitor SU-10603 was used as positive control (Gower, 1974). An MTT assay for cell viability was performed for each plate after measuring CYP17 activity to ensure that any effects were not caused by cytotoxicity.
Duplicate plates were seeded and exposed to perform protein measurements. Cellular protein contents were measured with the method of Lowry et al. (1951) using BSA as protein standard.
2.5. mRNA isolation and expression
Total RNA was isolated by phenol-chloroform extraction using RNA Instapure (Eurogentec, Liege, Belgium). Purity and concentration of the isolated RNA were determined spectrophotometrically at an absorbance wavelength of 260 and 280 nm. RNA samples were stored at -70 ℃ until analysis.
Complementary DNA (cDNA) was synthesized using 1 µg of RNA and the iScript cDNA Synthesis Kit, according to the manufacturer’s instructions (Biorad, Veenen- daal, The Netherlands). The obtained cDNA was diluted 10 times and was stored at 4 ℃ until further analysis.
Quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) was performed in a 96-well plate 0.2 ml thin-wall PCR plates using the iCycler Thermal Cycler (Bio-Rad, Veenendaal, The Netherlands). A PCRmastermix was made con- taining 12.5 ul of IQ SYBR Green Supermix, a forward and reverse primer (each 100 nM) and 0.5 ml of water per sample. For the qPCR, 10 ul of cDNA of each sam- ple was added to 15 ul of the PCRmastermix. The mixture was heated initially at 95 ℃ for 15 min and then followed by 40 cycles with denaturation at 94 ℃ for 30 s, annealing/extension at 60 ℃ for 30 s. In each run a negative control sample (non-RT samples) was included.
CYP17 and beta-actin primers were obtained from the RTPrimer database (Pattyn et al., 2006) and run through National Center for Biotechnology Infor- mation (NCBI) Blast (nucleotide nonredundant database) to confirm specificity. Primer sequences for CYP17 (NM_000102.3) were TGC TTA TTA AGA AGG GCA AGG ACT T (FP) and TGT TGG ACG CGA TGT CTA GAG T (RP) location 426-450 and 473-495, respectively, yielding a 69 bp product size. Primer sequences for beta-actin (NM_001101.3) were TTG TTA CAG GAA GTC CCT TGC C (FP) and ATG CTA TCA CCT CCC CTG TGT G (RP) location 1480-1501 and 1580-1559 in exon 6, respectively, yielding a 101 bp product size. As SYBR Green I is not specific for the PCR product, agarose gel electrophoresis with ethidium bromide staining was performed dur- ing optimization of the RT-qPCR method to confirm that the right amplicon size was obtained. In addition, after each RT-qPCR a melt curve was run to ensure that primer-dimers and other nonspecific products were omitted.
2.6. Cell cycle status
After incubation, cells were trypsinized and washed twice with PBS. Then, the cells were fixed by incubating the cells with 500 pl ice-cold ethanol (70%) for 30 min at room temperature and subsequently treated with RNase A (0.1 mg/ml) for 15 min at 37 ℃. Finally, the cells were washed with PBS and stained with propidium iodide (PI, 0.05 mg/ml) for 10 min at room temperature. PI-elicited fluorescence was mea- sured using a FACScan flow cytometer (Becton-Dickinson, Erembodegen, Belgium). 2-MeOE2 (10 µM) was used as positive control for cell cycle arrest in the G(2)/M phase.
2.7. Data analysis
All experiments were performed at least twice in triplicate. Data were analysed using Prism 3.0 (GraphPad Software, San Diego, CA, USA). Statistical differences of the means were determined by one-way ANOVA analysis followed by a Tukey-Kramer multiple comparisons test or Student’s t-test. Differences were con- sidered statistically significant with P< 0.05.
3. Results
3.1. No effect of lactones on H295R cell viability
MTT reduction, an indicator of mitochondrial function, was used as marker for cytotoxicity. After 24h, none of the tested lactones (CRI-s and ENL) had a significant effect on MTT reduction in H295R cells up to 30 p.M (data not shown). Although for CRI-4 (VIOXX®), a slight (<20%) yet no significant decrease in MTT reducing capacity was observed at the highest concentration tested (30 p.M).
3.2. CRI-3, -7, -10 and -12 inhibit CYP17 activity
Basal CYP17 activity in H295R cells was 75.4±5.5 pmol/h/mg protein. The CYP17 inhibitor SU-10603 (1 p.M) decreased CYP17
140
120
Activity (% of control)
100
80
60
40
-4-CRI-3
+CRI-4
CRI-7
20
+CRI-10
-0-CRI-12
0
SU10603
0,01
0,1
1
10
100
CRI concentration (uM)
activity to <5% of the control activity. All lactone derivatives were tested for their ability to affect CYP17 activity in H295R cells at a concentration of 30 p.M. After 24h, CRI-1, -2, -4, -5, -6, -8, -9, -11 and -13 and the natural lactone ENL showed no effect on CYP17 activity at 30 p.M. CRI-3, -7, -10 and -12 decreased CYP17 activ- ity to 72.2 ±5.2%, 34.2 ±0.5%, 45.0±4.2% and 36.6±1.6% of the vehicle control-treated cells, respectively. For these derivatives full concentration-response curves were obtained (Fig. 2) showing IC50 values for CYP17 inhibition by SU-10603, CRI-3, -7, -10 and -12 of 0.05, 24, 4.3, 9.7 and 6.8 MM respectively. CRI-4 (VIOXX®) was also included in these studies to further study its potential effect, since at 30 µM a slight, yet no significant, increase of CYP17 activity was observed. However, no significant effect of CRI-4 on CYP17 activ- ity was seen at the concentrations tested. The large variation and slight decrease at 30 and 100 p.M was probably caused by moderate cytotoxicity.
3.3. Inhibition of CYP17 activity is not caused by an effect on CYP17 mRNA expression
To determine whether the decrease in CYP17 activity by CRI- 3, -7, -10 and -12 was caused by a decrease in gene expression, mRNA levels of CYP17 were determined by quantitative real-time PCR (RT-qPCR) in H295R cells after a 24-h exposure and normalized using beta-actin expression. Beta-actin expression was not affected by CRI-treatment (data not shown). RT-qPCR analysis showed that CRI-3, -4, -10 and -12 did not affect CYP17 mRNA expression in H295R cells (Fig. 3). CRI-7, on the other hand, increased CYP17 expression significantly by about 137%.
400
mRNA expression (% of control)
350
CYP17
300
250
200
150
100
50
0
control
CRI-3
CRI-4
CRI-7
CRI-10
CRI-12
120
☐ without PD
☐ with PD
**
100
T
CYP17 activity (% of control)
T
80
T
T
$
*
$
T
60
*
*
T
T
$
$
40
20
0
DMSO
CRI-3
CRI-7
CRI-10
CRI-12
3.4. CRI-s interfere with the mitogen-activated protein kinase (MAPK) pathway
Previous studies have shown that CYP17 expression is regulated through the MAPK pathway (Sewer and Waterman, 2003) and that under influence of the MAP kinase kinase (MEK) inhibitor PD98059, CYP17 expression and activity increases in H295R cells (Kempna et al., 2007). To study possible effects of CRI-3, -4, -7, -10 and -12 on CYP17 through the MAPK pathway, CYP17 expression and activ- ity in H295R cells were determined in combination with the MEK inhibitor PD98059. Incubation of H295R cells with 10 p.M PD98059 alone resulted in a significant 24.1 ± 3.3% increase of CYP17 activity compared with vehicle control-treated cells (Fig. 4). When incu- bated alone, CRI-4 (VIOXX®) had no significant effect on CYP17 activity (data not shown) and all other tested CRI-s significantly decreased CYP17 activity with a potency as expected based on the concentration-response curves from Fig. 2. The decrease in CYP17 activity was 12.6%, 49.0%, 23.5% and 37.8% compared with the DMSO-treated cells, for CRI-3, -7, -10 and -12 alone, respectively. Co-incubation of CRI-3 and -12 with PD98059 further diminished the CYP17 activity significantly with 11.7% and 7.1%, respectively. This trend was also seen for CRI-7 and -10 where co-incubation with PD98059 caused a non-significant further decrease of CYP17 activity with 7.1% and 8.9%, respectively.
Other second messenger pathway modulators were also tested for their ability to influence CYP17 activity and modulate the lactone-induced inhibition of CYP17, similarly to the experiment shown in Fig. 4. PMA (PKC inducer, 100 nM), 8Br-cAMP (PKA inducer, 1 µM) and staurosporine (PK inhibitor, 10nM) had no effect on CYP17 activity. Also, co-incubation of the second mes- senger modulators with the various CRI-s showed that none of the second messenger pathway modulators were able to modulate the CRI-induced inhibition of CYP17 activity (data not shown).
3.5. CRI-s cause a cell cycle arrest in the G(2)/M phase
Modulation of the MAPK pathway appears to influence the CYP17 inhibitory properties of the CRI-s. Considering that the MAPK pathway is required for G1- to S-phase progression in the cell cycle (Meloche and Pouyssegur, 2007) we studied the cell cycle status of H295R cells after a 24-h treatment with 30 p.M CRI-3, -4, -7, -10
90,0
DMSO
(A)
80,0
2MeOE2
*
70,0
CRI-3
CRI-7
CRI-10
*
% of total cells
60,0
E CRI-12
50,0
40,0
*
30,0
20,0
10,0
0,0
<G1/GO
G1
S
G(2)/M
90,0
DMSO
(B)
80,0
CRI-7
70,0
% of total cells
zPD98059
60,0
TM7 + PD98059
50,0
*
*
*
*
40,0
30,0
20,0
*
*
10,0
0,0
<G1/GO
G1
S
G(2)/M
or -12. 2-MeOE2 was used as positive control for cell cycle arrest in the G(2)/M phase. Although the MTT assay showed that none of the treatments were cytotoxic to the H295R cells, all treatments caused a 5-10% statistically significant increase in number of dead (apoptotic) cells in the entire cell population compared with the vehicle control-treated cells. In the viable, dividing cell population, ENL and CRI-4 (VIOXX®) had no significant effect on the cell cycle status (data not shown). However, CRI-3, -7, -10 and -12 decreased the number of cells in the G1 phase by about 50% and caused a con- current increase in the number of cells in the G(2)/M phase (Fig. 5A). Incubation of the H295R cells with PD98059 (10 M) only caused a moderate effect on the cell cycle status. No significant changes were observed in the cell cycle phase distribution and, in addition, PD98059 had no significant effect on CRI-7-induced cell cycle arrest (Fig. 5B).
4. Discussion
Cytochrome P450 enzymes have long been left out of the picture in anti-cancer research. However, since the success story of the use of aromatase (CYP19) inhibitors in breast cancer treatment more CYP enzymes have become interesting potential anti-cancer targets (Bruno and Njar, 2007). Furthermore, the fact that many CYPs are expressed extrahepatically, makes them very suitable as potential therapeutic but also chemopreventive targets for synthetic com- pounds (like drugs) and natural compounds. CYP19 expression has been shown in a variety of tissues, including adipose stromal cells in human breast tissue (Agarwal et al., 1996). The finding that CYP17 is also expressed in adipose tissue (Puche et al., 2002), strength- ens the hypothesis that this enzyme is also locally responsible for
steroidogenesis and a potential target for anti-cancer or cancer chemopreventive compounds.
Increased CYP17 gene expression, for example through a sin- gle nucleotide polymorphism in the 5’-untranslated region, has been associated with increased enzyme activity and increased cir- culating estrogen levels (Carey et al., 1994; Feigelson et al., 1998). Increased circulating estrogen levels are directly correlated with increased risk for estrogen-dependent breast cancer. Although an association between a polymorphism in the CYP17 gene and breast cancer susceptibility is generally not supported in the literature, polymorphic CYP17 has been associated with modulation of breast cancer risk factors, such as BRCA status, menstrual cycle length and circulating hormone levels (Miyoshi et al., 2003; Little and Simard, 2005; Verla-Tebit et al., 2005; Chakraborty et al., 2007; Henningson et al., 2007).
In our study we aimed to investigate CYP17 as potential target enzyme in cancer chemoprevention for our proposed chemopre- ventive agents. So far, only two studies have described the effect of dietary lignans on breast cancer risk in relation with CYP17 geno- type. Premenopausal women with a polymorphic CYP17 genotype and highest quartiles of ENL plasma levels had a significant reduced breast cancer risk, showing odds ratios (95% CI) for breast cancer risk of 0.12 (0.03-0.50) in women with at least one polymorphic allele (McCann et al., 2002) and as low as 0.01 (0.00-0.21) in women with two polymorphic alleles (Piller et al., 2006). Because the lig- nan ENL appeared to have a modulating effect on CYP17 and thus breast cancer risk, we studied the effects of the naturally occur- ring lignan ENL and 13 structurally related synthetic derivatives to get an idea of a structure-activity relationship of a potential CYP17 modulator. These synthetic derivatives contain a lactone and two aromatic rings and are structurally related to VIOXX®. We used the human adrenocorticocarcinoma cell line H295R, since this cell line has been shown to be an excellent model for studies on the steroidogenic pathway (Gracia et al., 2006).
We found no effect of ENL on CYP17 activity and expression in H295R cells, suggesting no direct interaction on steroidogenesis through this enzyme. Of the VIOXX®-related lactone derivatives, CRI-3, -7, -10 and -12 decreased CYP17 activity in H295R cells in a concentration-dependent manner. The mechanism of this inhi- bition, however, did not become fully clear. No effects were seen at mRNA expression level, except with CRI-7, but this compound even appeared to increase CYP17 mRNA expression. Although this increase was unexpected, it was significant and observed in three independent experiments performed in triplicate. Enzyme kinetic studies indicated that CRI-7 acts as a non-competitive inhibitor of CYP17 activity, suggesting no direct interaction with the active site of the CYP17 enzyme (data not shown). Another possibility might be that even though the transcription of CYP17 is increased by CRI-7, the post-translational modulation of the CYP17 enzyme by CRI-7 overcomes a potential increase of CYP17 activity. This was strengthened by the fact that the MEK inhibitor PD98059 further reduced the CRI-induced inhibition of CYP17 activity, suggesting that the CRI-s might affect the phosphorylation state of CYP17 through the mitogen-activated protein kinase (MAPK) pathway (Roux and Blenis, 2004), an important cellular signaling machin- ery to transduce extracellular signals into intracellular responses. This concurs with previous findings that phosphorylation of the CYP17 enzyme is necessary for CYP17, more specifically 17,20- lyase activity (Zhang et al., 1995), and that phosphorylation of CYP17 appears to be at least in part cAMP dependent (Biason- Lauber et al., 2000; Kempna et al., 2007). Moreover, the role for the MAPK pathway was previously demonstrated in H295R cells, where silencing of the extracellular signal-regulated kinase (ERK1/2) mimicked cAMP-dependent activation of CYP17 expres- sion (Sewer and Waterman, 2003). This concurs with our finding that PD98059 alone increased CYP17 activity; although, the expo-
sure of the H295R cells to 8Br-cAMP did not affect CYP17 activity in our studies.
The ERK1/2 pathway is the prototypical MAPK pathway, which is activated preferentially by mitogenic factors, differentiation stim- uli and cytokines. In normal cells, sustained activation of ERK1/2 is necessary for G1- to S-phase progression and is associated with induction of positive regulators of the cell cycle and inactivation of antiproliferative genes (Meloche and Pouyssegur, 2007). How- ever, in our study, PD98059 alone had no significant effect on the cell cycle phase distribution in H295R cells nor did it affect CRI-7- induced shift in cycle phase distribution. Probably, this is because PD98059 only inhibits one of the two MEKs (MEK1). Furthermore, we used 10 uM only, while other studies showed that concentra- tions up to 50 p.M of PD98059 even induce merely a moderate effect on cell cycle in various cell types (Sekharam et al., 1998). The CRI- s on the other hand, did show significant effects on the cell cycle in H295R cells. A small increase in apoptotic cells was observed, although the MTT assay did not indicate any cytotoxic effects of the CRI-s. Most likely the MTT assay was not sensitive enough to pick up the moderate effects on cell viability caused by the CRI-s. In addition, CRI-3, -7, -10 and -12 caused a vast cell cycle arrest in the G(2)/M phase at 30 p.M. This cell cycle arrest is most likely not MAPK pathway-mediated, since this would have resulted in an arrest in the G1 phase, as explained above. In addition, two other potent compounds that induce a cell cycle arrest in the G(2)/M phase, 2-MeOE2 and combretastatin, do so by interacting with tubulin. 2-MeOE2, the endogenous phase II metabolite of estradiol, inhibits tubulin polymerization, a process involved in pulling the chromosomes to the opposite poles during mitosis (D’Amato et al., 1994). Combretastatin is also a micro-tubule binding agent, isolated from the South African willow tree (Combretum caffrum), damaging mitotic spindles and causing a cell cycle arrest (Quan et al., 2008). Both compounds are under clinical investigation for their potential cancer chemopreventive actions. Interestingly, combretastatin has the same cis-stilbene backbone structure as the CRI-s (see Fig. 1) and this structure has been shown to be important for its biological activity (Wood et al., 1995; Zhang et al., 2007). In our recently pub- lished study with the VIOXX®-related lactone derivatives in MCF-7 cells, a preliminary structure-activity relationship for these lactone derivatives was extensively discussed (Sangjun et al., 2009). It was concluded that the presence of a methoxy or thiomethyl group on the para position of the left aromatic ring appears to play an essen- tial role in the ability to induce the cells to arrest in the G(2)/M phase. However, this cannot fully explain the difference between the inactivity of CRI-2 and the ability of CRI-3 to arrest the cells in the G(2)/M phase. Especially, since the substitution on the right aromatic ring does not appear to influence the effect on cell cycle. However, for effects on CYP17, the right ring substitution, either with methoxy or fluor, does appears to have an effect. This can be seen by the increased inhibitory activity of CYP17 with increasing MeO (CRI-2 < CRI-5<CRI-7) or F substitution (CRI-3 < CRI-10 <CRI- 12). Apparently, the combination of one group (either MeS or MeO) on the left ring and two groups (either MeO or F) on the right ring are responsible for the biological actions of these compounds, with CRI-7 being the most optimal structure for affecting both cell cycle and CYP17 activity in H295R cells.
The VIOXX®-related lactone derivatives (CRI-1 to CRI-13) we studied here are synthetic agents that have been synthesized with the objective to find a potential cancer chemopreventive com- pound. The functional groups on the core structure have been varied to possibly increase the efficacy of their cancer chemopre- ventive actions. VIOXX® is a selective COX-2 inhibitor prescribed for the treatment of arthritis that has been withdrawn from the market due to safety concerns of increased risk of cardiovascular events in patients on VIOXX® (Graham et al., 2004) However, the chemical structures of the VIOXX®-derivatives are also similar to
naturally occurring chemopreventive compounds that have been proven safe, such as combretastatin, which has been investigated in a number of clinical trials, ENL and the stilbene-derivative resver- atrol, which are part of our daily diet. For cancer chemoprevention, intervention of biological processes involved in carcinogenesis requires modulation of molecular targets. Some classes of phyto- chemicals appear to be suitable for this purpose, since they are biologically active compounds that occur in our daily diet (Dorai and Aggarwal, 2004; Kundu and Surh, 2008; Syed et al., 2008). From our study, it appears that the suggested chemopreventive actions of ENL are not mediated through effects on CYP17 or cell cycle status. Other studies have reported a decrease in aromatase activity in vitro (Brooks and Thompson, 2005) and an increase in plasma SHBG (Adlercreutz et al., 1986) by ENL, which could explain the findings that high plasma ENL is associated with decreased circulating estrogen levels in vivo and its suggested chemopre- ventive actions. Of the VIOXX®-related lactone derivatives, CRI-7 appears to be the best potential chemopreventive agent with mul- tiple modes of action. The inhibition of abnormal cell proliferation via modulation of cell cycle progression as well as inhibition of local hormone production are important strategies for chemopre- vention of hormone-dependent cancers. We have shown that in vitro CRI-7 shows low cytotoxicity, it is a potent inhibitor of CYP17 activity and strong inducer of cell cycle arrest. In addition, previ- ous studies in our lab with H295R cells have shown that CRI-7 is an aromatase inhibitor and anti-androgenic in LNCaP cells (in the pres- ence of 100 nM dihydrotestosterone), transiently transfected with the human androgen receptor (AR) and AR-responsive luciferase (Sanderson et al., 2008). Also, moderate anti-estrogenic effects were observed in MCF-7 cells for some of the VIOXX®-related lac- tone derivatives (Sangjun et al., 2009). These data suggest that CRI-7 could prove useful in the prevention of hormone-dependent can- cers, such as breast cancer and prostate cancer. However, further studies should be performed to clarify the mechanism of action and to confirm these actions in vivo.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
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