Resveratrol activates adenylyl-cyclase in human breast cancer cells: a novel, estrogen receptor-independent cytostatic mechanism

Abdalla M. El-Mowafy1,3 and Moussa Alkhalaf2

1 Department of Applied Therapeutics, Faculty of Pharmacy, Health Sciences Center, Kuwait University, PO Box 24923, Safat 13110, Kuwait
2 Department of Biochemistry, Faculty of Medicine, Health Sciences Center, Kuwait University, PO Box 24923, Safat 13110, Kuwait

3 To whom correspondence should be addressed Email: aelmowafy{at}hsc.kuniv.edu.kw


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resveratrol (RSVL) is a well-established chemopreventive agent in human breast cancer models. The molecular basis of its action is far less characterized. We investigated the effects of RSVL on activity of adenylate- and guanylate-cyclase (AC, GC) enzymes; two known cytostatic cascades in MCF-7 breast cancer cells. RSVL increased cAMP levels in both time- and concentration-dependent manners (t1/2, 6.2 min; EC50 0.8 µM). In contrast, it had no effect on cGMP levels. The stimulatory effects for RSVL on AC were not altered either by the protein synthesis inhibitor (actinomycin-D, 5 µM) or the estrogen-receptor (ER) blockers (tamoxifen and ICI182,780, 1 µM each). Likewise, cAMP formation by RSVL was insensitive to either the broad-spectrum phosphodiesterase (PDE) inhibitor (IBMX, 0.5 mM) or the cAMP-specific PDE inhibitor (rolipram, 10 µM). Instead, these PDE inhibitors significantly augmented maximal cAMP formation by RSVL. Parallel experiments showed that either RSVL or rolipram inhibited the proliferation of these cells in a concentration-responsive manner. Further, concurrent treatment with RSVL and rolipram significantly enhanced their individual cytotoxic responses. The antiproliferative effects were appreciably reversed by the kinase-A inhibitors, Rp-cAMPS (100–300 µM) or KT-5720 (10 µM). Pretreatment with the cPLA2 inhibitor arachidonyl trifluoromethyl ketone (10 µM) markedly antagonized the cytotoxic effects of RSVL, but had no effect on that of rolipram. Altogether, the present study demonstrates, for the first time, that the chemotherapeutic agent RSVL is an agonist for the cAMP/kinase-A system, a documented pro-apoptic and cell-cycle suppressor in breast cancer cells.

Abbreviations: AC, adenylate-cyclase; ATK, arachidonyl trifluoromethyl ketone; ER, estrogen receptor; GC, guanylate-cyclase; IBMX, 3-isobuyl-1-methylxanthine; PDE, phosphodiesterase; RSVL, resveratrol.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Resveratrol (trans-3,4',5-trihydroxystilbene, RSVL) is a naturally occurring, biologically active phytoalexin that is commonly found in grapes, nuts and berries (1,2). RSVL is also abundant in red wine, a liquor whose consumption was established to reduce the incidence of cardiovascular disease and tumorigenesis (2,3). These epidemiological notions prompted extensive research that eventually established RSVL as an anti-mitotic, anti-neoplastic, anti-oxidant, anti-platelet and anti-inflammatory agent (48). Besides, as a phytoestrogen, RSVL appears to mediate some of its actions by modulating the estrogen machinery (9,10). These observations highlighted the potential of RSVL as an anti-breast cancer agent. In vitro studies on human cancer cell lines revealed that RSVL confers anti-proliferative and/or pro-apoptic actions (11). However, the exact cellular mechanisms whereby RSVL elicits such actions remained largely elusive. Likewise, there has been a large need to correlate known signaling mechanisms of RSVL with its chemoprevention. We recently demonstrated the capacity of RSVL to activate guanylate-cyclase (GC) in the vasculature (1). Others showed its agonism on adenylate-cyclase (AC) in polymorphonuclear leucocytes and platelets (12,13). Interestingly, products of these enzymes; namely cAMP and cGMP, can trigger both cytostatic and pro-apoptic signals in breast cancer cells (14,15). Together, these findings prompted us to investigate whether RSVL may activate AC and/or GC in human breast cancer cells.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Chemicals
Cyclic-AMP and cGMP kits were purchased from Biomol (Plymouth Meeting, PA). Trans-RSVL, 3-isobuyl-1-methylxanthine (IBMX) and actinomycin-D were purchased from Sigma (Deisenhofen, Germany). 2-Bis-(O- aminophenoxy) ethane-N,N,N,N-tetraacetic acid tetra (acetoxymethyl) ester (BAPTA-AM), calphostin-C, KT-5720, rolipram, ICI182,870, arachidonyl trifluoromethyl ketone (ATK), tamoxifen and adenosine-3',5'-cyclic monophosphorothioate-Rp-isomer (Rp-cAMPS) were obtained from Calbiochem (San Diego, CA).

Cell culture and treatments
The MCF-7 human breast cancer cell line was kindly provided by Dr Bohdan Wasylyk (IGBMC, Strasbourg, France). The cells were grown in phenol red-free RPMI-1640 medium (Gibco BRL, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS), 4 mM L-glutamine, 100 U/ml of penicillin and 100 µg/ml streptomycin. For the proliferation assays, cells were plated at ~5 x 104 cells/25-mm diameter multi-wells and maintained in a 5% CO2 humidified atmosphere at 37°C for 24 h. Then, the cells were treated with increasing concentration of RSVL or rolipram in a medium containing 5% of FBS. After 3 days the cells were harvested by treatment with trypsin and EDTA and counted with a Coulter counter (Type Z2). RSVL and rolipram were prepared as stock solutions in ethanol, and dilutions were made such that final concentration of solvent(s) was kept below 0.1%. Control wells were treated with equivalent volume of solvent.

Determination of cyclic nucleotides
cAMP/cGMP were measured by enzymeimmunoassay according to our reported procedures (16,17). For this purpose, MCF-7 cells were plated at 3 x 106 cells/25 cm2 flasks and maintained in a 5% CO2 humidified atmosphere at 37°C for 48 h. The effect of 5 µM of RSVL was tested in a medium containing 5% FBS, and either of the phosphodiesterase inhibitors, IBMX (0.5 mM) or rolipram (10 µM). Reactions were terminated by ice cooling, cells were rinsed with PBS twice and then cyclic nucleotides were extracted in 0.1 M HCl. Cyclic nucleotides were quantified by enzyme immunoassay using a kit from Biomol, and their levels were normalized to basal cellular concentrations of these nucleotides, and to cell number.

Statistical analyses
Statistical significance between two groups was evaluated by Student's t-test for unpaired data. Comparison among multiple groups was made through the one-way analysis of variance (ANOVA) test, followed by Tukey's post hoc test to determine significant differences among the means of the data groups. A probability of P < 0.05 was accepted as a significant difference.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Figure 1 indicates that RSVL inhibits the growth of human breast cancer (MCF-7) cells, which conforms to previous studies with this stilbene in various cancer models (11). Because of the antiproliferative effects of cAMP and cGMP in MCF-7 cells and the reported capacity of RSVL to increase these nucleotides in various biological systems (1,12,13), we investigated these possibilities in MCF-7 cells. Short-term treatment with RSVL appreciably increased cAMP levels (~2.5-fold) in these cells (Figure 2A and B). This response occurred in a time- and concentration-dependent manner, yielding a t1/2 value of 6.2 min and EC50 value of 0.8 µM (Figures 1 and 2). We did not observe any difference in cAMP formation by RSVL in low (0.5–1%) serum- or phenol red-free medium. Conversely, up to 100 µM, RSVL had no effect on cGMP levels (Figure 2), thus indicating the specificity of RSVL actions on the cAMP pathway. Generally, cAMP accumulation results from either stimulation of AC activity or, alternatively, from inhibition of cAMP-phosphodiesterases (PDE). Therefore, the impact of RSVL on cAMP level was first determined in presence of a maximal concentration of the cAMP-specific, PDE-IV, inhibitor (rolipram, 10 µM). These pre-treatments did not abolish the RSVL-evoked cAMP formation. Instead, rolipram markedly enhanced cAMP formation by RSVL (Figure 3A). These findings imply that the actions of RSVL are not due to inhibition of PDE, but stimulation of AC, in this system. On the other hand, the broad-spectrum PDE inhibitor (IBMX, 0.5 mM) elicited similar effects to rolipram on RSVL-evoked cAMP formation. However, at their maximally effective concentrations, IBMX was a more potent cAMP producer than rolipram (Figures 2 and 3A). This disparity is most probably entailed to their differential efficacy against various PDE isoforms. It is well established that 11 different genes encoding PDEs can give rise to several PDE isoforms, of which at least 10 isoforms can hydrolyze cAMP (18). All the latter enzymes can be inhibited by IBMX (18). In contrast, rolipram is specific only for the PDE-IV isoform, thus allowing partial hydrolysis of cAMP by the rest of the PDE isoforms. This could explain the reduced cytotoxicity of rolipram as compared with IBMX. Figure 3B indicates that rolipram, per se, significantly attenuated cellular proliferation and that concurrent treatments with RSVL/rolipram had a significantly higher cytotoxicity than either of their individual effects.



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Fig. 1. Effect of RSVL on proliferation rate of MCF-7 cells. Values represent the mean viable cell no. ± SE of five determinations, normalized to control (untreated) values. Control cell number was 31 4349 ± 9211. *Significantly lower than untreated cells, P < 0.05.

 


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Fig. 2. (A) Time course study for RSVL (10 µM)-induced cAMP formation in MCF-7 cells. Cells were pre-treated with IBMX (0.5 mM) for 15 min prior to the addition of RSVL. Values represent the mean ± SEM of six determinations, normalized to control concentrations. (B) Concentration–response curve for the effects of RSVL on cAMP and cGMP levels in MCF-7 cells. Pre-treatment with IBMX (0.5 mM) occurred for 15 min prior to addition of RSVL, which lasted for an additional 15 min. Values represent the mean ± SEM of five to seven determinations, normalized to control concentrations.

 


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Fig. 3. (A) Effect of rolipram (10 µM) and/or RSVL (1–10 µM) on cAMP formation in MCF-7 cells. Rolipram was added 15 min prior to RSVL and incubations continued for an additional 15 min before termination of the reaction. Values represent the mean ± SEM of five determinations. $, Significant from control; *, from rolipram only-treated groups at P < 0.05. (B) Individual and combined effects of rolipram (10 µM) and RSVL (1 µM) on cellular proliferation. Rolipram was added 15 min prior to RSVL. Values represent the mean ± SEM of six determinations. $, Significant from control; *, from rolipram only-treated group (P < 0.05).

 
RSVL has been known as a phytoestrogen that can modulate estrogen receptor (ER)-mediated effects in numerous biological systems (9,10). Accordingly, it was of interest to verify whether binding to ER is required for the stimulation of AC by RSVL. To this end, responses to RSVL were challenged by two ER antagonists, namely tamoxifen and ICI182,780 (1 µM each). Neither of these inhibitors significantly altered RSVL-evoked cAMP formation. Likewise, RSVL response was insensitive to the protein synthesis inhibitor, actinomycin-D (5 µM) (Figure 4), a finding that is consistent with the short-term stimulation of AC activity by RSVL (minutes).



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Fig. 4. Differential effects of the protein synthesis inhibitor (actinomycin-D, 5 µM) and the ER blockers; tamoxifen and ICI182,780 (1 µM each) on RSVL (1–10 µM)-induced cAMP formation in MCF-7 cells. Pre-treatments with blockers occurred for 20 min. RSVL was then added for 15 min before reactions were terminated. Values represent the mean ± SEM of five determinations.

 
Other than Gs-protein-linked receptors, effector signaling mechanisms that converge into activation of AC commonly involve PKC, calcium/calmodulin and the cPLA2 cascades (19). Accordingly, inhibitors for these signals have been employed to verify how RSVL might stimulate AC activity in MCF-7 cells. As evident from Figure 5, the RSVL-induced AC activation was resistant to calphostin-C (1 µM), a broad-spectrum PKC inhibitor and to the intracellular calcium chelator, BAPTA-acetoxymethyl ester (BAPTA-AM, 20 µM). In contrast, this response was abrogated by the specific cPLA2 inhibitor (ATK, 10 µM) implying that these short-term effects of RSVL are mediated by cPLA2. To check for the specificity of ATK actions in our system, control experiments were conducted on ISO (1 µM)-induced cAMP formation, a response known to emanate from direct coupling of ß-adrenoceptors to AC. The ISO-evoked activation of AC (nearly 3-fold) remained unchanged in presence of ATK (Figure 5), thus confirming the specificity of actions achieved by this inhibitor, and the involvement of cPLA2 in RSVL-triggered cAMP formation. To further link this RSVL-stimulated signaling pathway with its chemopreventive effects in MCF-7 cells, the cytotoxicity of RSVL was assessed in presence of inhibitors for kinase-A (Rp-cAMPS, 300 µM) and for cPLA2 (ATK). Figure 6 demonstrates that both inhibitors significantly antagonized the cytotoxic effects of RSVL. Similar responses were due to another kinase-A inhibitor (KT-5720) (data not shown). Thus, collectively, these observations conclude that RSVL specifically stimulates AC in human breast cancer cells through a cPLA2-dependant pathway.



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Fig. 5. Effects of the PKC inhibitor (calphostin-C, 1 µM), the calcium chelator (BAPTA-AM, 20 µM), and the cPLA2 inhibitor (ATK, 10 µM) on RSVL (1–10 µM, 15 min)- or isoproterenol (1 µM, 5 min)-induced cAMP formation in MCF-7 cells. Pre-treatments with these inhibitors occurred in IBMX (0.5 mM)-containing medium for 15 min. Values represent the mean ± SEM of five determinations. *Significant from control values (P < 0.05).

 


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Fig. 6. Effects of the kinase-A inhibitor (Rp-cAMPS, 300 µM), and the cPLA2 inhibitor (ATK, 10 µM) on RSVL-induced suppression of MCF-7 cell proliferation. *Significant from RSVL-treated group (P < 0.05).

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Evidence from epidemiological and experimental studies that natural edible compounds can protect against diseases is currently best exemplified by RSVL, a phytoalexin (3,4',5-trihydroxystilbene) that is commonly found in grapes and berries (4). RSVL is also a principal component of red wine, a liquor whose consumption proved to inversely associate with the incidence of cardiovascular disease (3,17). This phenomenon, commonly designated as the French paradox of red wine, prompted an explosion of research on the health benefits of RSVL; of which the chemopreventive and chemotherapeutic properties of RSVL have been highly appreciated (11,20). At the molecular level, RSVL was shown to block tumor cell proliferation, to promote their apoptosis and to interfere with phases of carcinogenesis (11,21). A variety of cellular effectors have been recognized as targets for RSVL action in tumor cells; however, mostly pertained to disruption of DNA synthesis and the cell-cycle machinery (2225). Recently, the therapeutic significance of AC and GC has been revisited, and their potentials to ameliorate cardiovascular and malignant diseases have been reappraised (12,13,26). RSVL activated GC and/or AC in cardiovascular tissues and some blood cells (1,1214). Conversely, similar effects for RSVL have never been investigated in tumor-based systems. The activity of the AC/kinase-A cascade has been positively correlated with inhibition of cellular proliferation, and induction of apoptosis in tumor cells including the human breast cancer, MCF-7 cells (27). We presently demonstrated the capacity of RSVL to stimulate AC in the latter cells and subsequently assessed the cytotoxic impact of this signal; aiming to unravel the underpinnings of RSVL's anti-breast cancer actions. Current data show the capacity of RSVL to upregulate the AC-kinase-A pathway, an enzyme known to transduce vital cytosolic and nuclear signals in tumor cells (11). We also observed that RSVL stimulated AC in T47-D and MDA-MB-231 breast cancer cells, thus indicating that recruitment of AC by RSVL is not confined to MCF-7 cells, and is also independent of the ERs. In all these cells, elevated cAMP levels were maintained all over the time course of experiments for cell growth (3 days) (unpublished observations). However, unlike its capacity to stimulate AC, RSVL failed to influence GC activity in these cells. Interestingly, in the vasculature, we have demonstrated opposite stimulatory actions for RSVL on these enzymes (1), thus attesting to the diversity of RSVL actions among biological systems.

Currently, positive correlation has been observed between RSVL-induced cAMP formation and its cytotoxicity. Moreover, inhibition of kinase-A activity appreciably attenuated the cytotoxic effects of RSVL. Additional evidence for correspondence between these effects came from the use of the cAMP-specific, PDE-IV inhibitor ‘rolipram’, which mimicked the chemopreventive effects of RSVL. Indeed, these observations for RSVL can be substantiated by actions of other drugs in breast cancer models. For instance, treatment of breast cancer cells with exogenous 8-Br-cAMP, or with drugs such as vincristine, paclitaxel and suramin, both stimulated AC activity and substantially suppressed tumor cell growth (15,28,29). On the other hand, the present effects of RSVL on cAMP were not mediated by inhibition of PDE, as RSVL effects on both cAMP and cell growth were virtually additive to those by maximal concentration of rolipram. Likewise, RSVL stimulatory effects on AC were not mediated by calcium or PKC, two established positive modulators of AC isoforms. In contrast, effects for RSVL were ablated by the cPLA2 inhibitor, ATK, suggesting that cPLA2 is an upstream effector mechanism in the activation of AC by RSVL. This scenario for AC activation was also evident in coronary arteries (16). Accordingly, the present study reinforces the impact of cPLA2 as an effector mechanism in regulating breast cancer cell proliferation. Other than RSVL, stimulation of this phospholipase occurred also in breast cancer cells in response to vitamin D (30). It appears, therefore, that cPLA2 and AC are principal cellular triggers for the chemopreventive actions of natural edible compounds, such as RSVL and vitamin D.

Another aspect of RSVL biological profiles is its ability to bind to the ER and, hence, to modulate estradiol effects in numerous target tissues (3133). For instance, the cardiovascular benefits of RSVL were partly ascribed to estrogen-like effects, whilst its anti-breast cancer effects were mediated by down-regulation of the estrogen machinery (3,4,11). Thus, it was of our interest to verify whether RSVL effects on AC involve the estrogen machinery. Our data indicated that the RSVL-evoked cAMP formation was insensitive to ER blockers, thus clearly dissociating these cellular events. In agreement, Nakagawa et al. demonstrated that RSVL induces apoptosis of human breast cancer cells in both ER-negative and ER-positive cells (34). However, this latter study along with our present observations cannot preclude ER-mediated cytotoxic responses for RSVL in ER-positive cells. For instance, we found that treatment with ER-blockers partly (~20–30%) reduced RSVL cytotoxic effects in MCF-7 cells (data not presented).

In conclusion, the present study highlights a new cellular mechanism for the anti-breast cancer effects of RSVL: that is activation of the AC/PKA cascade. This pathway appears to disrupt necessary signaling mechanisms in breast cancer cells, thereby augmenting the cytotoxicity of RSVL. Such effects for RSVL were not mediated by interaction with ER, thus also indicating the versatility of the mechanisms entailing chemopreventive effects of RSVL. These studies may well substantiate the clinical utility of RSVL against breast cancer.


    Acknowledgments
 
We wish to thank Professor J.N.Sharma (Applied Therapeutics Department) for critical reading and fruitful comments on the manuscript. This study was supported, in part, by Kuwait university research grants PT01/00 awarded to A.El-Mowafy.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 24, 2002; revised January 24, 2003; accepted January 28, 2003.