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Clinical Cancer Research Vol. 12, 3478-3484, June 1, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Sulindac Sulfide and Exisulind Inhibit Expression of the Estrogen and Progesterone Receptors in Human Breast Cancer Cells

Jin T.E. Lim1, Andrew K. Joe1, Masumi Suzui2, Masahito Shimizu1, Muneyuki Masuda3 and I. Bernard Weinstein1

Authors' Affiliations: 1 Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, New York, New York; 2 Department of Pathology, University of the Ryukyus, Okinawa, Japan; and 3 Department of Otolaryngology, Kyushu University, Maidashi, Fukuoka, Higashi, Japan

Requests for reprints: I. Bernard Weinstein, Herbert Irving Comprehensive Cancer Center, Columbia University College of Physicians and Surgeons, 701 West 168th Street, New York, NY 10032. Phone: 212-305-6921; Fax: 212-305-6889; E-mail: Weinstein{at}cuccfa.ccc.columbia.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results and Discussion
 References
 
In previous studies, we found that sulindac sulfide and exisulind (sulindac sulfone, Aptosyn) cause growth inhibition, arrest cells in the G1 phase of the cell cycle, and induce apoptosis in human breast cancer cell lines. These effects were associated with decreased expression of cyclin D1. The present study focuses on the effects of sulindac sulfide and exisulind on hormone signaling components in breast cancer cells. We found that estrogen receptor (ER)–positive and progesterone receptor (PR)–positive T47D breast cancer cells were somewhat more sensitive to growth inhibition by sulindac sulfide or exisulind than ER-negative PR-negative MB-MDA-468 breast cancer cells. Further studies indicated that sulindac sulfide and exisulind caused marked down-regulation of expression of the ER and PR-A and PR-B in T47D cells. However, neither compound caused a major change in expression of the retinoic acid receptor {alpha} (RAR{alpha}), RARß, or RAR{alpha} in T47D cells. Sulindac sulfide and exisulind also caused a decrease in expression of the ER in estrogen-responsive MCF-7 breast cancer cells. Both compounds also markedly inhibited estrogen-stimulated activation of an estrogen-responsive promoter in transient transfection reporter assays. Treatment of T47D cells with specific protein kinase G (PKG) activators did not cause a decrease in ER or PR expression. Therefore, although sulindac sulfide and exisulind can cause activation of PKG, the inhibitory effects of these two compounds on ER and PR expression does not seem to be mediated by PKG. Our findings suggest that the growth inhibition by sulindac sulfide and exisulind in ER-positive and PR-positive human breast cancer cells may be mediated, in part, by inhibition of ER and PR signaling. Thus, these and related compounds may provide a novel approach to the prevention and treatment of human breast cancers, especially those that are ER positive.

It is estimated that in the United States, in the year 2006, there will be 214,640 new cases of breast cancer, and 40,970 women will die from this disease (1). Indeed, breast cancer is the most common cancer among both Caucasian and African American women. African-American women with breast cancer have a poorer prognosis than Caucasian women (1). The precise basis for this ethnic difference is not known, although some studies have suggested that this relates to the fact that the estrogen receptor (ER) is less frequently expressed in African-American cases of breast cancer (2, 3). Many breast cancers are hormone dependent, and estrogen can stimulate breast tumor growth. Hence, there is concern about an increase in breast cancer risk in menopausal women receiving hormone replacement therapy (4, 5). Current antihormonal regimens for breast cancer therapy target the ER signaling pathway in three different ways: (a) blocking the ligand binding domain of the ER (tamoxifen and faslodex), (b) inhibiting endogenous estrogen production (oophorectomy, anastrozole, and letrozole), and (c) disrupting stability of the ER protein (fulvestrant; for review, see refs. 6, 7).

Sulindac is a nonsteroidal anti-inflammatory drug that has been used to treat patients with chronic inflammatory diseases and for its analgesic, antipyretic, and platelet inhibitory activities. The drug has also been used for the treatment of patients with familial adenomatous polyposis of the colon (8). Exisulind (sulindac sulfone, Aptosyn), an oxidative metabolite of sulindac that lacks cyclooxygenase inhibitory activity, has also been shown to induce polyp regression and prevent polyp recurrence in patients with familial adenomateous polyposis (9). In previous studies, we found that sulindac sulfide, another metabolite of sulindac, and exisulind induced inhibition of growth and apoptosis in several human breast cancer cell lines (10). Similar effects were seen in human prostate cancer cell lines (11), and in a more recent study, we found that these compounds inhibited expression of the androgen receptor (AR) and AR signaling in the AR-positive LNCaP prostate cancer cell line (12). Exisulind inhibited the growth of LNCaP xenografts tumors in mice (13), and in a clinical study, it reduced rising serum prostate-specific antigen levels in high-risk post-radical prostectomy patients with prostate cancer (14). In view of these results, it was of interest to determine whether exisulind and sulindac sulfide affect the ER signaling pathway in human breast cancer cells. Indeed, the present study shows that treatment with sulindac sulfide or exisulind markedly decreases expression of both the ER and the progesterone receptor (PR) in ER-positive human breast cancer cells. These novel effects may provide a further rationale for the use of these and related compounds in the chemoprevention and treatment of breast cancer.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results and Discussion
 References
 
Compounds and plasmids. Sulindac sulfide and exisulind (Aptosyn) were obtained from OSI Pharmaceuticals, Inc. (Long Island, NY). The compounds were dissolved in 100% DMSO and added to the cell culture medium at a final concentration of 0.1% DMSO. YC-1, 8-bromo-cyclic guanosine 3',5'-monophosphate (8-Br-cGMP), and 8-bromo-3',5' cyclic AMP (8-Br-cAMP) were purchased from Alexis Corp. (San Diego, CA).

Cell culture procedures. The MCF-10F, MCF-7, T47D, and MB-MDA-468 human breast cancer cell lines were purchased from the American Type Culture Collection (Rockville, MD). The cells were grown in DMEM medium supplemented with 10% fetal bovine serum, 100 units/mL of penicillin G, and 100 mg/mL of streptomycin (Life Technologies, Inc., Frederick, MD) and maintained at 37°C in a humidified atmosphere containing 5% CO2. For cell growth assays, the cells were plated, in triplicate, at a density of 1 x 105 per well in six-well plates. Twenty-four hours later, various concentrations of sulindac sulfide, exisulind, or DMSO (0.1% final) were added to the culture media. The number of attached viable cells were counted at 24, 48, and 72 hours after adding the compounds, using a Coulter Counter, model ZF, (Coulter Electronics, Inc., Krefeld, Germany). The percentage of growth, using the DMSO control-treated cells as the 100% value, and their respective SDs (based on triplicate assays) were determined using Microsoft Excel 6.0 software. DMSO at 0.1% had no significant effect on the growth of these cell lines. IC50values were calculated as previously described (11, 15).

Immunoblotting. The methods for preparation of cell extracts and immunoblotting were described previously (11, 15). The primary antibodies used were ER{alpha} (1:1,000; Santa Cruz, Inc., Santa Cruz, CA), PR-A and PR-B (1:500; Lab Vision, Inc., Freemont, CA), retinoic acid receptor {alpha} (RAR{alpha}; 1:1,000; Santa Cruz), RARß (1:1,000; Santa Cruz), retinoid X receptor ß (RXRß; 1:1,000; Santa Cruz), and ß-actin (1:500; Sigma, Inc., St. Louis, MO).

Transient transfection reporter assays. MCF-7 cells were plated at a density of 1 x 105 per plate in 60-mm diameter plates. Twenty-four hours later, the cells were transiently transfected with 2 µg of an ERE-luciferase reporter plasmid and 1 µg of a pCMV-ß-galactosidase reporter plasmid (internal control), using the manufacturer's protocol (Life Technologies). After 20 hours, the cells were treated with 0.1% DMSO, 0.2 mmol/L sulindac sulfide, or 0.5 mmol/L exisulind in phenol red–free DMEM medium (Life Technologies) containing 10% charcoal-stripped fetal bovine serum, plus or minus the addition of 5 mmol/L ß-estradiol. Twenty hours later, whole-cell extracts were prepared, and luciferase assays were done according to the manufacturer's instructions (Promega, Madison, WI). ß-Galactosidase activity (Promega) was also assayed using the same whole cell extracts, and this value was used to normalize the luciferase assays for transfection efficiency. Each assay was done in triplicate, and the mean relative luciferase activity was calculated, as previously described (12).

Reverse transcription-PCR. Total RNA was isolated from cells using the Trizol reagent and methods described previously (12). The primers used for amplification were as follows: ER, forward 5'-TACTACCTGGAGAACGAGCC-3' and reverse 5'-TGGTGGCTGGACACATATAG-3'; PR, forward 5'-TGCTCAAGGAGGGCCTGCCGCAGGT-3' and reverse 5'-CTACTGAAAGAAGTTGCCTCTCGCC-3'; glyceraldehyde-3-phosphate dehydrogenase, forward 5'-GCCACATCGCTCAGACACCA-3' and reverse 5'-GATGACCCTTTTGGCTCCCC-3'. Amplification was carried out for 35 cycles of denaturation at 94°C, annealing at 52°C, and extension at 72°C, for 30 seconds each. PCR products were separated using a 2% agarose gel and identified by ethidium bromide staining.

Statistical analysis. Statistical analysis of the data was done using the Student's t test (Ps < 0.001) with the computer software SigmaPlot version 8.0 (SigmaPlot, SPSS, Chicago, IL).


   Results and Discussion
Top
 Abstract
 Materials and Methods
 Results and Discussion
References
 
Sulindac sulfide and exisulind inhibit growth of estrogen-dependent and estrogen-independent breast cancer cell lines. In previous studies, we found that the estrogen-dependent MCF-7 breast cancer cell line was more sensitive to growth inhibition by sulindac sulfide than the MCF-10F normal mammary epithelial cell line (10). To examine possible differences in the growth inhibitory effects of sulindac sulfide and exisulind on estrogen-dependent and estrogen-independent breast cancer cells, we used the ER-positive and PR-positive T47D and the ER-negative and PR-negative MDA-MB-468 breast cancer cells. We also included for comparison the normal human mammary epithelial cell line MCF-10F cells, which does not express ER{alpha} or PR (16). All three cell lines were grown in increasing concentrations of sulindac sulfide or exisulind for 24, 48, or 72 hours. (Fig. 1 ). The T47D cells were somewhat more sensitive to growth inhibition by sulindac sulfide than MDA-MB-468 cells during the first 48 hours (Fig. 1A). Thus, at 24 hours, the respective IC50 values were 0.25 and 0.4 mmol/L. However, at 72 hours the IC50 values for both T47D and MDA-MB-468 cells decreased to a value of 0.15 mmol/L. The MCF-10F cells were the least sensitive to growth inhibition by sulindac sulfide because even at 72 hours, the IC50 value exceeded 0.4 mmol/L. In the absence of sulindac sulfide, MCF-10F, MDA-MB-468, and T47D cells had similar exponential doubling times of ~24 hours (data not shown). The T47D cells were considerably more sensitive to growth inhibition by exisulind than MDA-MB-468 cells during the first 48 hours. This was readily apparent at 24 hours, and at 48 hours, the respective IC50values were 0.3 and >0.6 mmol/L, respectively. However, at 72 hours, the two cell types had similar growth inhibition with IC50 values of 0.2 and 0.3 mmol/L (Fig. 1B). The MCF-10F cells were less sensitive to growth inhibition by exisulind than either the T47D cells and the MDA-MB-468 cells because at 72 hours, the IC50 value for the former cells was >0.6 mmol/L (Fig. 1B).


Figure 1
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  		Fig. 1. T47D breast cancer cells are more sensitive to growth inhibition than MDA/MB/468 or MCF-10F cells. A, the three cell lines were treated with sulindac sulfide (SS) at the indicated concentrations for 24, 48, or 72 hours, and their growth was plotted as % growth with respect to the DMSO (control)–treated cells. B, the three cell lines were similarly treated with exisulind. Points, mean of triplicate assays; bars, SD. Similar results were obtained in a repeat study.

 
These results, together with our previous studies with MCF-7 cells (10), suggest that sulindac sulfide and exisulind preferentially inhibit the growth of breast cancer cells when compared with normal mammary epithelial cells. Furthermore, because at early time points, the T47D cells were more sensitive to the growth inhibitory effects of sulindac sulfide and exisulind than the MDA-MB-468 cells, both compounds may exert their inhibitory effects, at least in part, through an ER-dependent pathway. However, because MDA-MB-468 cells were also inhibited by both sulindac sulfide and exisulind, these compounds can apparently also inhibit growth via ER-independent mechanisms.

Sulindac sulfide and exisulind down-regulate expression of the ER and PR proteins. In view of the above results, and our previous study indicating that both sulindac sulfide and exisulind markedly inhibit expression of the androgen receptor in prostate cancer cells (12), we examined possible effects of these compounds on hormone receptors in breast cancer cells. Estrogen activity in breast tissue is mediated by two specific nuclear steroid receptors that function as transcription factors (ER{alpha} and ERß). The structure of human ERß is homologous to that of ER{alpha} within the DNA and ligand binding domains, but the two receptors are encoded on different chromosomes and can exert different functions (17). Although ER{alpha} and ERß can form biologically functional receptor heterodimers, ER{alpha} and ERß have distinct cellular distributions, regulate separate sets of genes, and can oppose each other's effects on the expression of certain genes (18). To assess possible effects on expression of the ER{alpha} protein, T47D cells were treated with 0.2 mmol/L sulindac sulfide or 0.5 mmol/L exisulind for increasing periods of time, and cell extracts were examined by immunoblotting (Fig. 2A ). We found that within 20 hours after treatment with either compound, there was a marked and persistent decrease in the intracellular level of ER{alpha}, when compared with the level in DMSO-treated control cells (Fig. 2A). In contrast, the expression of ß-actin (loading control) was not altered by treatment with either sulindac sulfide or exisulind (Fig. 2A, bottom).


Figure 2
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  		Fig. 2. Sulindac sulfide (SS) and exisulind inhibit the expression of ER{alpha}, PR-A, and PR-B in T47D breast cancer cells. A, sulindac sulfide and exisulind decrease cellular levels of the ER and PR proteins in T47D cells. Exponentially growing cells were treated with either DMSO, 0.2 mmol/L sulindac sulfide, or 0.5 mmol/L exisulind for the indicated number of hours. Extracts were immunoblotted for the expression of ER-{alpha}, PR-A, PR-B, and ß-actin. B, sulindac sulfide or exisulind can down-regulate ER and PR mRNA in T47D breast cancer cells. Cells were treated with DMSO, 0.2 mmol/L sulindac sulfide, or 0.5 mmol/L exisulind for the indicated number of hours. RNA extracts were then analyzed by reverse transcription-PCR for levels of ER{alpha}, PR, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs, as described in Materials and Methods. C, T47D cells were treated with the indicated concentrations of sulindac sulfide or exisulind for 20 hours. Extracts were then analyzed by immunoblotting for levels of the RAR{alpha}, RARß, and RXR{alpha} proteins using the respective antibodies.

 
The clinical significance of PR positivity in human breast cancer is less well understood than that of ER{alpha} positivity. Human PR proteins exist as two isoforms (PR-A and PR-B) that are transcribed from a single gene but regulated by separate promoters (19). PR-A lacks the first 164 NH2-terminal amino acids contained in PR-B, a sequence that is required for the transactivation function of PR-B (20). In general, breast cancers that are ER positive are also PR positive and tend to have a better prognosis. However, breast cancers that are PR positive and ER negative may have a worse prognosis (21). T47D breast cancer cells express high levels of PR-A and PR-B, but unlike other breast cancer cell lines, PR expression in T47D cells is independent of estrogen control (22). Therefore, it was of interest to examine possible effects of sulindac sulfide and exisulind on PR expression in T47D cells. We found that treatment of these cells with 0.2 mmol/L sulindac sulfide caused a marked decrease in cellular levels of both PR-A and PR-B at 48 hours, and treatment with 0.5 mmol/L exisulind caused a decrease in both proteins within 20 hours (Fig. 2A). Thus, sulindac sulfide and exisulind cause a decrease in cellular levels of three nuclear steroids (ER{alpha}, PR-A, and PR-B) in T47D cells (Fig. 2A). In preliminary studies, we found that ERß is expressed in T47D cells, and treatment with exisulind also inhibited its expression, but this inhibition was not as strong as with ERß (data not shown).

Sulindac sulfide and exisulind decrease the levels of ER{alpha} and PR mRNAs. We next investigated the effects of sulindac sulfide and exisulind on cellular levels of ER{alpha} and PR mRNAs in T47D cells using reverse transcription-PCR assays. We found a marked decrease in ER{alpha} mRNA expression within 20 hours after the cells were treated with 0.2 mmol/L sulindac sulfide or 0.5 mmol/L exisulind (Fig. 2B, top). Because our immunoblot results showed equivalent effects on both isoforms of the PR protein (Fig. 2A), we used oligonucleotides that amplify the 3' terminal region of the mRNAs for both PR-A and RP-B (nucleotides 1761-2363). Treatment of T47D cells with 0.2 mmol/L sulindac sulfide caused a decrease in the expected 602-bp PR band (19) after 48 hours, and treatment with 0.5 mmol/L exisulind caused a decrease in this band within 20 hours (Fig. 2B, middle). Treatment with either compound did not affect expression of the control glyceraldehyde-3-phosphate dehydrogenase mRNA (Fig. 2B, bottom), and there was no significant decrease in ER{alpha} or PR mRNA expression in T47D cells treated with the vehicle (0.1% DMSO) during the 48 hours time course. These results are consistent with the above-described protein expression studies (Fig. 2A) and suggest that sulindac sulfide and exisulind cause a decrease in cellular levels of the ER{alpha} and the PR proteins by inhibiting expression of the mRNAs of the corresponding genes.

Sulindac sulfide and exisulind do not have a marked effect on expression of RAR{alpha}, RARß, or RXR{alpha}. To determine whether treatment with either sulindac sulfide or exisulind inhibits the expression of other members of the family of steroid hormone nuclear receptors, we examined the effects of these compounds on three members of the RAR/RXR receptor subfamily. RARs include RAR{alpha}, RARß, and RAR{gamma}, each of which has high affinity for all trans-retinoic acid. RXRs include RXR{alpha}, RXRß, and RXR{gamma}. The RXRs are activated by 9-cis-retinoic acid, a stereoisomer and photoisomer of all trans-retinoic acid that is expressed in vivo in both liver and kidney cells (23). T47D cells were treated with 0.1% DMSO, 0.2 mmol/L sulindac sulfide, or 0.5 mmol/L exisulind for 24 hours. Extracts were then immunoblotted using primary antibodies against RAR{alpha}, RARß, and RXR{alpha} (Fig. 2C). Treatment with 0.2 mmol/L sulindac sulfide had no effect on RAR{alpha} expression and caused a partial inhibition of RARß expression, and treatment with exisulind caused partial inhibition of RAR{alpha} expression (Fig. 2C). Treatment with either compound had no effect on RXR{alpha}. These results contrast with the marked reduction in ER{alpha}, PR-B, or PR-A when T47D cells were treated with sulindac sulfide or exisulind for 20 hours (Fig. 2A).

Sulindac sulfide and exisulind inhibit the transcriptional activity of an estrogen-responsive promoter. To examine whether the inhibitory effects of sulindac sulfide and exisulind on ER{alpha} expression in T47D cells extend to other ER-positive breast cancer cells, we did similar studies with the ER-positive MCF-7 human breast cancer cell line. We found that, as in T47D cells (Fig. 2A), there was a marked and persistent decrease in the intracellular level of ER{alpha} within 20 hours after treating MCF-7 cells with 0.2 mmol/L sulindac sulfide or 0.5 mmol/L exisulind (Fig. 3A ). The expression of ß-actin (loading control) was not altered by treating MCF-7 cells with either sulindac sulfide or exisulind (Fig. 3A, bottom). The reduction in cellular levels of ER{alpha} in both T47D (Fig. 2A) and MCF-7 cells (Fig. 3A) suggested that these drugs might inhibit the expression of genes whose transcription is controlled by an estrogen-responsive element (ERE). Therefore, we did transient transfection reporter assays by transfecting an ERE-luciferase reporter plasmid into MCF-7 breast cancer cells. A pCMV-ß-galactosidase reporter was cotransfected with the latter plasmid to serve as a control for transfection efficiency. The cells were incubated in phenol red–free DMEM medium with 10% charcoal-stripped fetal bovine serum to deplete them of estrogenic activity. As expected, the addition of ß-estradiol stimulated luciferase activity (Fig. 3B). The latter activity was markedly inhibited by 0.2 mmol/L sulindac sulfide or 0.5 mmol/L exisulind (Fig. 3B). Similar results were obtained with T47D breast cancer cells, although activation of the ERE-luciferase reporter obtained with ß-estradiol was weaker than in MCF-7 cells (data not shown). These findings suggest that the decrease in ER{alpha} expression caused by sulindac sulfide and exisulind could exert biological effects by decreasing the expression of ER-responsive genes in ER-positive breast cancer cells.


Figure 3
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  		Fig. 3. Sulindac sulfide (SS) and exisulind inhibit ER{alpha} expression and activation of an estrogen-responsive reporter construct in MCF-7 breast cancer cells. A, MCF-7 cells were treated with either DMSO, 0.2 mmol/L sulindac sulfide, or 0.5 mmol/L exisulind for the indicated number of hours, and extracts were immunoblotted for expression of the ER{alpha} and ß-actin proteins. B, MCF-7 cells were cotransfected with plasmids that encode an estrogen-responsive luciferase reporter and a CMV-ß-galactosidase reporter, and the cells were grown in estrogen-depleted medium. They were treated with or without 5 nmol/L ß-estradiol (E2), 0.2 mmol/L sulindac sulfide, or 0.5 mmol/L exisulind, as indicated, for 20 hours. Extracts were then prepared and analyzed for relative luciferase activity (see Materials and Methods). Columns, mean of triplicate assays; bars, SD.

 
Activation of protein kinase G does not affect expression of ER{alpha} or the PR. Previous studies indicate that sulindac sulfide and exisulind can inhibit the cGMP-specific phoshodiesterases 2 and 5, thus causing an increase in intracellular levels of cGMP and activation of the enzyme protein kinase G (PKG). Activation of PKG can then induce apoptosis by a cascade of events involving activation of the mitogen-activated protein kinase kinase kinase 1/stress-activated protein kinase kinase 1/c-Jun NH2-terminal kinase 1 pathway and inhibition of the expression of ß-catenin and cyclin D1 (24–26). To determine whether sulindac sulfide or exisulind mediates down-regulation of the ER{alpha} and PR via activation of PKG, we assayed expression of these two receptors after treating T47D cells with various compounds that cause activation of PKG (24–26). These included YC-1, a guanylate cyclase activator; 8-Br-cGMP, a cell-permeable form of cGMP; and sulindac sulfide or exisulind, both of which inhibit cellular cGMP-phosphodiesterases (24–26). As a control, we also treated the cells with 8-Br-cAMP, a cell-permeable form of cAMP. We found that neither YC-1, 8-Br-cGMP, nor 8-Br-cAMP inhibited expression of ER{alpha}, PR-A, or PR-B (Fig. 4 ). However, as in our previous study (Fig. 2A), sulindac sulfide and exisulind inhibited the expression of all three receptors (Fig. 4). These results suggest that inhibition of expression of ER{alpha}, PR-A, and PR-B by sulindac sulfide and exisulind is not mediated via activation of PKG. Nor does it seem that cAMP plays an important role in this process.


Figure 4
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  		Fig. 4. PKG activators do not inhibit expression of ER{alpha}, PR-A, or PR-B in T47D breast cancer cells. The cells were treated with the indicated concentrations of DMSO, YC-1, 8-Br-cGMP (cGMP), 8-Br-cAMP (cAMP), sulindac sulfide (SS), or exisulind for 20 hours. Extracts were then analyzed by immunoblotting for the ER{alpha}, PR-A, or PR-B and ß-actin proteins, with the respective antibodies. Similar results were obtained in a repeat experiment.

 
Hormone receptor status has prognostic value and is useful in the clinical management of patients with breast cancer (27). About 50% of primary breast tumors are positive for both ER{alpha} and PR, and <5% are ER{alpha} negative but still positive for PR (27). Several studies have shown that ER{alpha} status is the most important variable in predicting whether metastatic breast cancers will respond to hormonal therapy (28). PR status is also believed to be an independent predictor of response to hormonal therapy (29). Well-differentiated tumors are more likely to be ER{alpha} positive and PR positive than poorly differentiated tumors. Total PR levels correlate with an increased likelihood of response to tamoxifen, longer time to treatment failure, and greater overall survival (30, 31). Therefore, the present studies may be of clinical interest because they indicate that sulindac sulfide and exisulind inhibit expression of both the ER{alpha} and PR genes in ER-positive and PR-positive human breast cancer cells. This provides a unique approach to inhibiting the action of these receptors because current hormonal approaches to the treatment of breast cancer either inhibit estrogen production (aromatase inhibitors) or modify the function of the ER{alpha} (selective ER modifiers) but do not directly inhibit expression of the ER{alpha}. Nor are we aware of any compounds that inhibit expression of the PR.

Nuclear steroid hormone receptors are divided into three categories: type I (AR, glucocorticoid receptor, mineralcorticoid receptor, and PR), type II (T3R, RAR, RXR, and vitamin D receptor), and type III (ER and orphan receptors; refs. 6, 23). In previous studies, we found that treatment of the AR-positive prostate cancer cell line LNCaP with sulindac sulfide or exisulind at concentrations that induce growth inhibition and apoptosis caused a marked decrease in cellular levels of the AR protein and mRNA (12). In the present study, sulindac sulfide and exisulind had no effect or only partially inhibited the expression of RAR{alpha}, RARß, or RXR{alpha} (Fig. 2C). Thus, our findings with prostate and breast cancer cells suggest that these two compounds preferentially inhibit expression of types I and III steroid hormone receptors. Treatment of cells with sulindac sulfide, exisulind, or related cGMP-phoshodiesterase inhibitors leads to activation of PKG (24–26). The results obtained in the present study (Fig. 4) and our previous study on prostate cancer cells (12) suggest that PKG activation is not in itself sufficient to inhibit expression of the ER{alpha}, PR, and AR proteins. The precise mechanism remains to be determined. Finally, we should emphasize that exisulind and related compounds can induce growth inhibition in estrogen-independent breast cancer cell lines (ref. 10; Fig. 1B). Therefore, the clinical use of these compounds may not be limited to patients with ER-positive breast cancers. Nevertheless, the present results suggest that inhibition of expression of ER{alpha} and the PR might augment the antiproliferative effects of these compounds in estrogen-dependent tumors and possibly precursor lesions. This may be of particular importance in the chemoprevention of breast cancer.

We should, however, emphasize that further studies are required to determine whether exisulind and related compounds can also inhibit the expression of ER{alpha} and PR in tumors in vivo.


   Footnotes
 
Grant support: OSI Pharmaceuticals, Inc.; T.J. Martell Foundation (I.B. Weinstein); and National Foundation for Cancer Research (I.B. Weinstein).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 9/20/05; revised 1/31/06; accepted 3/23/06.


   References
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 Abstract
 Materials and Methods
 Results and Discussion
 References
 

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