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Reduced expression of endothelial and inducible nitric oxide synthase in a human breast cancer cell line which has acquired estrogen independence
Cancer Letters 144 (1999) 65±74
Reduced expression of endothelial and inducible nitric oxide synthase in a human breast cancer cell line which has acquired estrogen independence Jan H.J. Martin a,*, Osama Alalami a, Hendrik W. van den Berg b a
Division of Biomedical Sciences, University of Wolverhampton, 62±68 Lich®eld Street, Wolverhampton WV1 1SB, UK b Department of Oncology, The Whitla Medical Building, The Queen's University of Belfast, Belfast, UK Received 19 February 1999; received in revised form 10 May 1999; accepted 10 May 1999
Abstract We have recently reported the presence of inducible nitric oxide synthase (iNOS) in the human breast cancer cell line ZR-75-1. The purpose of the present study was to examine differences in expression of endothelial (eNOS) and inducible nitric oxide synthase in normal human mammary epithelial cells (HMEC) compared with two variants of the ZR-75-1 cell line. One variant has acquired estrogen independence, the other has acquired resistance to tamoxifen. Immunohistochemical investigations demonstrated that 100% of HMEC cells staining positive for both eNOS and iNOS. ZR-75-1 cells showed 100% staining for eNOS and 52% positive staining for iNOS. There was no difference in staining between the parent cell line and cells which had acquired resistance to tamoxifen (ZR-75-9a1). However, in the breast cancer cell line which had acquired estrogen independence (ZR-PR-LT), less than 5% of cells exhibited positive staining for eNOS and staining for iNOS was undetectable. l-Arginine increased NO production in both ZR-75-9a1 and ZR-PR-LT cells. Progesterone was able to down regulate NO production in both ZR-75-1 and ZR-75-9a1 cells and this effect was reversible by RU486. These results support the suggestion that loss of NOS expression may be associated with the progression of breast cancers. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Breast cancer; Nitric oxide; Nitric oxide synthase; Estrogen independence; Tamoxifen resistance; Human mammary epithelial cells
1. Introduction The exact role of nitric oxide (NO) in tumor biology has not yet been fully elucidated. This is due to the fact that NO has been implicated in many different aspects of cancer biology including both proand anti- tumor functions. NO may have a role in carcinogenesis by inducing DNA strand breaks [1] and by impairing the tumor suppressor function of p53 [2]. It has also been impli* Corresponding author. Tel.: 144-1902-321-154; fax: 1441902-321-161.
cated as part of a signalling cascade for neovascularization [3] and it can increase tumor blood ¯ow [4]. The presence of inducible NOS (iNOS) has been correlated with metastatic disease [5] and a NOS inhibitor signi®cantly reduces bone metastasis [6]. With regard to its anti-tumor role, NO has a cytostatic/cytotoxic role towards tumor cells [7,8]. Cytotoxic effects are mediated via interference with DNA replication and several enzymes, including aconitase and ubiquinone oxireductase [9]. NO has also been shown to induce apoptosis in tumor cells [10,11]. The balance between these opposing roles may depend upon the local concentration of NO, with
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00198-6
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high concentrations of NO exerting anti-proliferative effects and low concentrations facilitating tumor growth [3]. Although tamoxifen is still the treatment of choice for breast cancer patients with estrogen receptor (ER)positive tumors [12] nearly all patients will eventually become resistant to this treatment [13]. Treatment failure may be associated with a variety of changes in tumor characteristics leading to a more malignant phenotype, including the development of anti-estrogen resistance and progression to estrogen independence. We have previously characterized two such variants of the ZR-75-1 human breast cancer cell line. In ZR-75-9a1 cells [14] which have acquired tamoxifen resistance, there is loss of detectable ERs and progesterone receptors (PGR), and an increase in epidermal growth factor receptor (EGFR) expression [15]. The ZR-PR-LT cell line [16], which is an estrogen-independent variant, expresses high numbers of PGR and has reduced EGFR expression. We have recently reported the presence of iNOS in the ZR-75-1 human breast cancer cell line [17] and the aim of the current study was to investigate expression of both endothelial NOS (eNOS) and iNOS, in two more malignant variant cell lines. We report that the tamoxifen resistant ZR-PR-9a1 cells have similar eNOS and iNOS expression to the parent cell line whereas ZR-PR-LT cells line have much reduced levels of both eNOS and iNOS. 2. Materials and methods 2.1. Materials Anti-eNOS, bovine endothelial cell (599-613) (rabbit) and anti-iNOS mouse macrophage (11311144) (rabbit) were obtained from Calbiochem (Nottingham, UK). The StreptABComplex/HRP Duet (mouse/rabbit) was from Dako Ltd (Buckinghamshire, UK). Sulphanilimide and naphthylethylenediamine dihydrochloride were from BDH (Lutterworth, Leicestershire, UK). RU486 was a gift from Excelgen, France. All other chemicals were from Sigma Chemical Company (Poole, UK). 2.2. Cell culture Human
mammary
epithelial
cells
(HMEC)
(Biowhittaker, Berkshire, England) were routinely maintained in Mammary Epithelial Basal Medium (modi®ed MCDB 170) supplemented with bovine pituitary extract (52 mg/ml), human recombinant epidermal growth factor (hEGF) (10 ng/ml), hydrocortisone (0.5 mg/ml), gentamycin (50 mg/ml), amphotericin-B (50 ng/ml) and insulin (5 mg/ml). The ZR-75-1 human breast cancer cell line [18] (ECACC, Porton Down, Salisbury, UK) was routinely maintained in RPMI 1640 medium supplemented with HEPES (20 mM), fetal calf serum (10%), penicillin (50 units/ml), streptomycin (50 mg/ml) and glutamine (300 mg/ml). ZR-75-9a1 cells [14] were cultured in the same medium supplemented with 8 mM tamoxifen. ZR-PR-LT cells [16] were maintained in phenol red-free RPMI 1640 medium supplemented with 5% heat-treated, charcoal-stripped serum. Cells were grown in 5% CO2 at 378C. 2.3. Determination of NO production Cells (5 £ 104 ) were plated into 24-well plates and allowed to attach for 24 h. Medium was then replaced with fresh medium with or without the drug and the nitrite assay was performed 2 days later. Nitric oxide was measured as the amount of nitrite (NO22) which is a stable end-product of NO metabolism. Cell free supernatants were collected and stored at 2208C until analysis. Nitrite concentration in the medium was quantitated by a colourimetric assay based on the Greiss reaction [19]. Brie¯y, samples (100 ml) were mixed with an equal volume of Greiss reagent (1% sulphanilimide, 0.1% naphthylethylenediamine dihydrochloride and 2.5% H3PO4) and incubated at room temperature for 10 min. The absorbance was measured at 550 nm in a microplate reader. Sodium nitrite was used as a standard. 2.4. Immunocytochemistry Cells (4 £ 105 ) were plated into 24-well plates and allowed to attach for 24 h. Following washing with PBS, cells were permeabilized with 2% saponin for 10 min at room temperature and then ®xed in 5% formalin±saline. Incubation with normal rabbit serum (1:20) blocked non-speci®c binding. Cells were incubated with the primary antibody to eNOS or iNOS (1:200) for 30 min. IgG (1:100) was used as
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the secondary antibody as part of the StreptABComplex/HRP Duet kit with the ABC/fast red technique being used to demonstrate presence of eNOS or iNOS in breast cancer cells. Negative controls were cells not treated with the primary antibody.
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2.5. Statistical analysis Statistical signi®cance was determined using the Student's t-test and the differences were regarded as signi®cant for values of P , 0:05.
Fig. 1. Immunohistochemical staining (red stain) for eNOS in (A1) human mammary epithelial cells and (A2) negative control; (B1) ZR-75-1 cells and (B2) negative control; (C1) ZR-75-9a1 cells and (C2) negative control; (D1) ZR-PR-LT cells and (D2) negative control; using bovine endothelial anti-nitric oxide synthase, (599-613) (rabbit) antibody (£400).
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3. Results 3.1. Immunohistochemical staining for eNOS The presence of cells staining positive for eNOS monoclonal antibody was demonstrated by the appearance of red-stained cells (Fig. 1). One-hundred percent of HMEC (Fig. 1A) and 100% of ZR-75-1
cells (Fig. 1B) stained positive for eNOS. Only 50% of ZR-75-9a1 cells stained positive for eNOS (Fig. 1C) and less than 5% of ZR-PR-LT cells stained positive for eNOS (Fig. 1D). 3.2. Immunohistochemical staining for iNOS The presence of cells staining positive for iNOS
Fig. 2. Immunohistochemical staining (red stain) for iNOS in (A1) human mammary epithelial cells and (A2) negative control; (B1) ZR-75-1 cells and (B2) negative control; (C1) ZR-75-9a1 cells and (C2) negative control; (D1) ZR-PR-LT cells and (D2) negative control; using mouse macrophage inducible anti-nitric oxide synthase (1131-1144) antibody (£400).
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monoclonal antibody was demonstrated by the appearance of red-stained cells (Fig. 2). One-hundred percent of HMECs stained positive for iNOS (Fig. 2A). The percentage of ZR-75-1 cells staining positive for iNOS was 52% (Fig. 2B, previously reported in Ref. [17]). Only 40% of ZR-75-9a1 cells stained positive for iNOS (Fig. 2C). No ZR-PR-LT cells were positively stained for iNOS (Fig. 2D). 3.3. Effect of l-arginine, l-nitro-l-arginine methyl ester (l-NAME) and tamoxifen on nitrite secretion by ZR-75-9a1 and ZR-PR-LT cells During a 2-day culture period, untreated ZR-75-9a1 cells secreted 3 nmol NO22 (5 £ 104 cells) into the
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culture medium (Fig. 3) and ZR-PR-LT cells secreted 1 nmol NO22 (5 £ 104 cells) into the culture medium (Fig. 4). A 2-day treatment of ZR-75-9a1 cells with larginine (10 mM) resulted in an increase to 64 nmol NO22/5 £ 104 cells (Fig. 3) and a 2-day treatment of ZR-PR-LT cells with l-arginine (10 mM) resulted in an increase to 42 nmol NO22/5 £ 104 cells (Fig. 4). These increases were signi®cant at the P , 0:001 level. Treatment of ZR-75-9a1 (Fig. 3) and ZR-PRLT (Fig. 4) cells with concentrations of l-arginine ranging from 10 mM±5mM also produced signi®cant increases in the amount of nitrite secreted into the conditioned medium. For both control ZR-75-9a1 cells (Fig. 3) and those treated with l-arginine, treatment with l-NAME
Fig. 3. Effect of l-arginine, l-NAME and tamoxifen on nitrite secretion by ZR-75-9a1 cells. Cells (5 £ 104 ) were seeded into wells of a culture plate in 1 ml of RPMI 1640 medium and allowed to attach for 24 h. Medium was then replaced with medium with (open bar) or without l-arginine (10±10000 mM), l-NAME (2 mM) (dotted) or tamoxifen (10 28 M) (striped) and cultured for 2 days. After 2 days the supernatant was removed and aliquots (100 ml) were analyzed for nitrite concentration. This was determined by reacting with an equal volume of Greiss reagent and measuring the absorbance at 540 nm in a Bio-Rad plate reader. The results of a typical experiment which was repeated seven times are presented. Values presented are means of experimental replicates (n 3) with SE.
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Fig. 4. Effect of l-arginine, l-NAME and tamoxifen on nitrite secretion by ZR-PR-LT cells. Cells (5 £ 104 ) were seeded into wells of a culture plate in 1 ml of RPMI 1640 medium and allowed to attach for 24 h. Medium was then replaced with medium with (open bar) or without l-arginine (10±10000 mM), l-NAME (2 mM) (dotted) or tamoxifen (10 28 M) (striped) and cultured for 2 days. After 2 days the supernatant was removed and aliquots (100 ml) were analyzed for nitrite concentration. This was determined by reacting with an equal volume of Greiss reagent and measuring the absorbance at 540 nm in a Bio-Rad plate reader. The results of a typical experiment which was repeated seven times are presented. Values presented are means of experimental replicates (n 3) with SE.
(2 mM) caused a signi®cant (P , 0:05) reduction in NO22 secreted into the medium. Similar results were obtained for ZR-PR-LT cells (Fig. 4). Treatment with tamoxifen (10 28M) did not cause a signi®cant change in nitric oxide production for l-arginine-treated ZR75-9a1 (Fig. 3) or ZR-PR-LT (Fig. 4) cells. Similar results have been obtained in seven separate experiments. 3.4. Effect of progesterone and RU486 on nitrite secretion by ZR-75-1, ZR-75-9a1 and ZR-PR-LT cells During a 2-day culture period, untreated ZR-75-1 cells secreted 3.7 nmol NO22 (5 £ 104 cells) into the
medium (Fig. 5). Treatment of cells with concentrations of 0.1±1000nM progesterone caused signi®cant (P , 0:05) decrease in NO22 secreted into the culture medium, which could be reversed by RU486 (10 24 M). Untreated ZR-75-9a1 cells secreted 3.5 nmol NO22 (5 £ 104 cells) into the medium (Fig. 5). Treatment of these cells with concentrations of 0.1±1000nM progesterone also caused signi®cant (P , 0:001) decreases in NO22 secreted into the culture medium. This could be reversed by RU486 (10 24 M). During a 2-day culture period, untreated ZR-PR-LT cells secreted 0.5 nmol NO22 (5 £ 104 cells) into the medium (Fig. 5). Although treatment of ZR-PR-LT cells with concentrations of 0.1±1000nM progesterone reduced nitrite
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Fig. 5. Effect of progesterone and RU486 on nitrite secretion by ZR-75-1, ZR-75-9a1 and ZR-PR-LT cells. Cells (5 £ 104 ) were seeded into wells of a culture plate in 1 ml of RPMI 1640 medium and allowed to attach for 24 h. Medium was then replaced with medium with or without progesterone (0.1±1000 nM) and RU486 (10 24 M), and cultured for 2 days. After 2 days the supernatant was removed and aliquots (100 ml) were analyzed for nitrite concentration. This was determined by reacting with an equal volume of Greiss reagent and measuring the absorbance at 540 nm in a Bio-Rad plate reader. ZR-75-1 without RU486 (open bar) and with RU486 (vertical stripe). ZR-75-9a1 without RU486 (dotted) and with RU486 (horizontal stripe). ZR-PR-LT without RU486 (checked bar) and with RU486 (angled stripe). The results of a typical experiment which was repeated ®ve times are presented. Values presented are means of experimental replicates (n 3) with SE.
secretion, this was not statistically signi®cant. Similar results have been obtained in ®ve separate experiments 3.5. Effect of phorbol 12-myristate 13-acetate (PMA) on nitrite secretion by ZR-75-9a1 and ZR-PR-LT cells During a 2-day culture period, untreated ZR-75-9a1 cells secreted 3.5 nmol NO22 (5 £ 104 cells) into the culture medium (Fig. 6). A 2-day treatment of ZR-759a1 cells with PMA (1000 nM) resulted in an increase to 23 nmol NO22/5 £ 104 cells. This increase was signi®cant at the P , 0:001 level. Treatment of ZR75-9a1 cells with concentrations of PMA ranging from 200±800nM also produced signi®cant increases in the amount of nitrite secreted into the conditioned medium (Fig. 6). During a 2-day culture period, untreated ZR-PR-LT
cells secreted 1 nmol NO22 (5 £ 104 cells) into the culture medium (Fig. 6). A 2-day treatment of ZRPR-LT cells with PMA (1000 nM) resulted in a slight increase to 1.5 nmol NO22/5 £ 104 cells but this was not statistically signi®cant (Fig. 6). Treatment of ZRPR-LT cells with concentrations of PMA ranging from 200±800nM did not produce signi®cant changes in the amount of nitrite secreted into the conditioned medium (Fig. 6). Similar results have been obtained in ®ve separate experiments. 4. Discussion The role of nitric oxide in cancer biology is at present poorly understood as it has both pro- and anti- tumor functions. We have previously demon-
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Fig. 6. Effect of phorbol 12-myristate 13-acetate (PMA) on nitrite secretion by ZR-75-9a1 and ZR-PR-LT cells. ZR-75-9a1 cells (X) or ZR-PR-LT cells (O) (5 £ 104 ) were seeded into wells of a culture plate in 1 ml of RPMI 1640 medium and allowed to attach for 24 h. Medium was then replaced with medium with or without PMA (200±1000 nM), and cultured for 2 days. After 2 days the supernatant was removed and aliquots (100 ml) were analyzed for nitrite concentration. This was determined by reacting with an equal volume of Greiss reagent and measuring the absorbance at 540 nm in a Bio-Rad plate reader. The results of a typical experiment which was repeated ®ve times are presented. Values presented are means of experimental replicates (n 3) with SE.
strated the presence of the l-arginine/nitric oxide pathway in ZR-75-1 human breast cancer cells [17]. The aim of the current study was to investigate expression of both eNOS and iNOS in normal human mammary epithelial cells and in two more malignant variant breast cancer cell lines, namely the estrogen-independent ZR-PR-LT cell line [16] and the tamoxifen-resistant ZR-75-9a1 cell line [14]. Our results demonstrate that normal breast cells show 100% staining for iNOS. This is in marked contrast to all three tumor cell lines in which expression of iNOS was reduced, with only 52% positive staining for ZR-75-1 cells and 40% positive staining for ZR-75-9a1 and ZR-PR-LT cells, which failed to express any positivity for iNOS. Similarly, although both the normal breast cells and ZR-75-1 cells expressed 100% staining for eNOS, there was considerably less positivity for eNOS in the more malignant ZR-75-9a1 and ZR-PR-LT cells. We have also shown that the tamoxifen-resistant cell line exhibited similar expression of iNOS to the
parent cell line. Similar basal levels of NO production were observed which could be increased by l-arginine and induced by PMA. These similarities to the parent cell line [17] may be due to the instability of the ZR75-9a1 cell line. Although the tamoxifen-resistant cells exhibit loss of detectable ERs and PGRs, the ZR-75-9a1 cell line is unstable and in the absence of tamoxifen these cells will revert back to an ER- and PGR- positive phenotype [14]. The ZR-PR-LT cell line expresses high numbers of PGR [16] and has reduced EGFR expression [15]. We have shown that this estrogen-independent cell line expresses signi®cantly lower levels of both eNOS and iNOS compared with the parent cell line, with correspondingly lower NO production. In contrast to ZR-75-1 cells, in ZR-PR-LT cells, NO production could not be stimulated by PMA. Previous studies have investigated the role of estrogen in the nitric oxide pathway. Estrogen has been reported to upregulate eNOS gene expression [20] and several potential signalling mechanisms have been proposed [21]. A correlation between eNOS expression and ER expression has been reported in several human breast cancer cell lines [22]. Our results for the estrogen-independent breast cancer cells supports these previous ®ndings. Our explanation for a lack of the two NOS isoforms in more malignant breast cells is two-fold. Firstly, in ER-positive cells it is thought that estradiol may enhance production of NO which then acts as a free radical to induce mutations leading to a more malignant phenotype [22]. NO itself has been shown to decrease transcription of the iNOS gene [23] consequently downregulating iNOS expression in the more malignant cells. Secondly, as the cells adapt to estrogen-independent growth and fail to express detectable ER, the upregulation of eNOS by estradiol has been lost, again leading to decreased expression of eNOS in the more malignant cells. Treatment of ZR-75-1 and ZR-75-9a1 cells with progesterone resulted in downregulation of NO production. Our results are consistent with previous studies demonstrating that progesterone inhibits both iNOS gene expression and NO production [24]. Progesterone is thought to inhibit NOS by binding to the glucocorticoid receptor. It has been suggested that the activated glucocorticoid receptor prevents the binding of transcription factor NF-kB to the NOS
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promoter thereby inhibiting the induction of NOS transcription [25]. In our experiments, RU486 which has a high af®nity for both the progesterone and glucocorticoid receptors was able to reverse the inhibitory effects of progesterone. Interestingly, PMA increased NO production in both ZR-75-1 [17] and ZR-75-9a1 breast cancer cells but was unable to do so in ZR-PR-LT cells. This may suggest a defect in protein kinase C intracellular signalling pathways in the estrogen-independent cell line. However, further investigation is required to clarify this hypothesis. In conclusion, we have demonstrated reduced expression of eNOS and iNOS in a human breast cancer cell line adapted to growth in estrogen-free conditions. These results suggest a role for nitric oxide synthase in human breast cancer and also suggest an interaction of estrogen/estrogen receptor with nitric oxide function.
[9]
[10]
[11]
[12] [13]
[14]
References [1] Y. Yoshie, H. Ohshima, Synergistic induction of DNA strand breakage by catechol-estrogen and nitric oxide: implications for hormonal carcinogenesis, Free Radical Bio. Med. 24 (1998) 341±348. [2] S. Calmels, P. Hainaut, H. Ohshima, Nitric oxide induces conformational and functional modi®cations of wild-type p53 tumor suppressor protein, Cancer Res. 57 (1997) 3365±3369. [3] D.C. Jenkins, I.G. Charles, L.L. Thomsen, D.W. Moss, L.S. Holmes, S.A. Baylis, P. Rhodes, K. Westmore, P.C. Emson, S. Moncada, Roles of nitric oxide in tumor growth, Proc. Natl. Acad. Sci. USA 92 (1995) 4392±4396. [4] S.P. Andrade, H.R. Hart, P.J. Piper, Inhibitors of nitric oxide synthase selectively reduce ¯ow in tumor-associated neovasculature, Br. J. Pharmacol. 107 (1992) 1092±1097. [5] A. Due-nas-Gonzalez, C.M. Isales, del Mar Abad-Hernandez M, Gonzalez-Sarmiento R., Sangueza O., Rodriguez-Commes J., Expression of inducible nitric oxide synthase in breast cancer correlates with metastatic disease, Modern Pathol. 10 (1997) 645±649. [6] T. Iwasaki, M. Higashiyama, K. Kuriyama, A. Sasaki, M. Mukai, K. Shinkai, T. Horai, H. Matsuda, H. Akedo, NGnitro-l-arginine methyl ester inhibits bone metastasis after modi®ed intracardiac injection of human breast cancer cells in a nude mouse model, Jpn. J. Cancer Res. 88 (1997) 861±866. [7] D.J. Stuehr, C.F. Nathan, Nitric oxide, a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells, J. Exp. Med. 169 (1989) 1543±1555. [8] J.H.J. Martin, S.W. Edwards, Changes in mechanisms of monocyte/macrophage mediated cytotoxicity during culture, Reactive oxygen intermediates are involved in monocyte-
[15]
[16]
[17]
73
mediated cytotoxicity whereas reactive nitrogen intermediates are employed by macrophages in tumor cell killing, J. Immunol. 150 (1993) 3478±3486. J. Drapier, Hibbs Jr J.B., Murine cytotoxic activated macrophages inhibit aconitase in tumor cells: inhibition involves the iron-sulfur prosthetic group and is reversible, J. Clin. Invest. 78 (1986) 790±795. M. Sandoval, X. Liu, P.D. Oliver, X.J. Zhang, D.A. Clark, M.J.S. Miller, Nitric oxide induces apoptosis in a human colonic epithelial cell line, T84, Mediat. In¯amm. 4 (1995) 248±250. S. Cui, J.S. Reichner, R.B. Mateo, J.E. Albina, Activated murine macrophages induce apoptosis in tumor cells through nitric oxide-dependent or independent mechanisms, Cancer Res. 54 (1994) 2462±2467. V.C. Jordan, Long-term Tamoxifen Treatment for Breast Cancer, University of Wisconsin Press, Madison, 1994, pp. 1± 289. F. Leonessa, V. Boulay, A. Wright, E.W. Thompson, N. Brunner, R. Clarke, The biology of breast tumor progression: acquisition of hormone-independence and resistance to cytotoxic drugs, Acta Oncol. 31 (1992) 115±123. H.W. van den Berg, M. Lynch, J. Martin, J. Nelson, G.R. Dickson, A.D. Crockard, Characterisation of a tamoxifenresistant variant of the ZR-75-1 human breast cancer cell line (ZR-75-9a1) and stability of the resistant phenotype, Br. J. Cancer 59 (1989) 522±526. B. Long, Mc Kibben B.M., Lynch M., van den Berg H.W., Changes in epidermal growth factor receptor expression and response to ligand associated with acquired tamoxifen resistance or oestrogen independence in the ZR-75-1 human breast cancer cell line, Br. J. Cancer 65 (1992) 865±869. H.W. van den Berg, J. Martin, M. Lynch, High progesterone receptor concentration in a variant of the ZR-75-1 human breast cancer cell line adapted to growth in oestrogen-free conditions, Br. J. Cancer 61 (1990) 504±507. O. Alalami, J.H.J. Martin, ZR-75-1 Human breast cancer cells: expression of inducible nitric oxide synthase and effect of tamoxifen and phorbol ester on nitric oxide production, Cancer Lett. 123 (1998) 99±105.
[18] L.W. Engel, N.A. Young, T.S. Tralka, M.E. Lippman, S.J. O'Brien, M.J. Joyce, Establishment and characterisation of three new continuous cell lines derived from human breast carcinomas, Cancer Res. 38 (1978) 3352±3364. [19] L.C. Green, D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok, S.R. Tannenbaum, Analysis of nitrate, nitrite, and [ 15N]nitrate in biological ¯uids, Anal. Biochem. 126 (1982) 131±138. [20] A.N. MacRitchie, S.S. Jun, Z. Chen, Z. German, I.S. Yuhanna, T.S. Sherman, P.W. Shaul, Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium, Circ. Res. 81 (1997) 355±362. [21] K. Kauser, G.M. Rubanyi, Potential cellular signalling mechanisms mediating upregulation of endothelial nitric oxide production by estrogen, J. Vasc. Res. 34 (1997) 229±236. [22] R. Zeillinger, E. Tantscher, C. Schneeberger, W. Tschugguel, S. Eder, G. Sliutz, J.C. Huber, Simultaneous expression of
74
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nitric oxide synthase and estrogen receptor in human breast cancer cell lines, Breast Cancer Res. Tr. 40 (1996) 205±207. [23] S.K. Park, D. Grzybicki, H.L. Lin, S. Murphy, Modulation of inducible nitric oxide synthase expression in astroglial cells, Neuropharmacology 33 (1994) 1419±1423. [24] L. Miller, E.W. Alley, W.J. Murphy, S.W. Russell, J.S. Hunt, Progesterone inhibits inducible nitric oxide synthase gene
expression and nitric oxide production in murine macrophages, J. Leukocyte Biol. 59 (1996) 442±450. [25] H. Kleiner, C. Euchenhofer, I. Ihrig-Biedert, U. Forstermann, Glucocorticoids inhibit the induction of nitric oxide synthase II by down-regulating cytokine-induced activity of transcription factor-kB, Mol. Pharmacol. 49 (1996) 15±21.
Reduced expression of endothelial and inducible nitric oxide synthase in a human breast cancer cell line which has acquired estrogen independence Jan H.J. Martin a,*, Osama Alalami a, Hendrik W. van den Berg b a
Division of Biomedical Sciences, University of Wolverhampton, 62±68 Lich®eld Street, Wolverhampton WV1 1SB, UK b Department of Oncology, The Whitla Medical Building, The Queen's University of Belfast, Belfast, UK Received 19 February 1999; received in revised form 10 May 1999; accepted 10 May 1999
Abstract We have recently reported the presence of inducible nitric oxide synthase (iNOS) in the human breast cancer cell line ZR-75-1. The purpose of the present study was to examine differences in expression of endothelial (eNOS) and inducible nitric oxide synthase in normal human mammary epithelial cells (HMEC) compared with two variants of the ZR-75-1 cell line. One variant has acquired estrogen independence, the other has acquired resistance to tamoxifen. Immunohistochemical investigations demonstrated that 100% of HMEC cells staining positive for both eNOS and iNOS. ZR-75-1 cells showed 100% staining for eNOS and 52% positive staining for iNOS. There was no difference in staining between the parent cell line and cells which had acquired resistance to tamoxifen (ZR-75-9a1). However, in the breast cancer cell line which had acquired estrogen independence (ZR-PR-LT), less than 5% of cells exhibited positive staining for eNOS and staining for iNOS was undetectable. l-Arginine increased NO production in both ZR-75-9a1 and ZR-PR-LT cells. Progesterone was able to down regulate NO production in both ZR-75-1 and ZR-75-9a1 cells and this effect was reversible by RU486. These results support the suggestion that loss of NOS expression may be associated with the progression of breast cancers. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Breast cancer; Nitric oxide; Nitric oxide synthase; Estrogen independence; Tamoxifen resistance; Human mammary epithelial cells
1. Introduction The exact role of nitric oxide (NO) in tumor biology has not yet been fully elucidated. This is due to the fact that NO has been implicated in many different aspects of cancer biology including both proand anti- tumor functions. NO may have a role in carcinogenesis by inducing DNA strand breaks [1] and by impairing the tumor suppressor function of p53 [2]. It has also been impli* Corresponding author. Tel.: 144-1902-321-154; fax: 1441902-321-161.
cated as part of a signalling cascade for neovascularization [3] and it can increase tumor blood ¯ow [4]. The presence of inducible NOS (iNOS) has been correlated with metastatic disease [5] and a NOS inhibitor signi®cantly reduces bone metastasis [6]. With regard to its anti-tumor role, NO has a cytostatic/cytotoxic role towards tumor cells [7,8]. Cytotoxic effects are mediated via interference with DNA replication and several enzymes, including aconitase and ubiquinone oxireductase [9]. NO has also been shown to induce apoptosis in tumor cells [10,11]. The balance between these opposing roles may depend upon the local concentration of NO, with
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00198-6
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high concentrations of NO exerting anti-proliferative effects and low concentrations facilitating tumor growth [3]. Although tamoxifen is still the treatment of choice for breast cancer patients with estrogen receptor (ER)positive tumors [12] nearly all patients will eventually become resistant to this treatment [13]. Treatment failure may be associated with a variety of changes in tumor characteristics leading to a more malignant phenotype, including the development of anti-estrogen resistance and progression to estrogen independence. We have previously characterized two such variants of the ZR-75-1 human breast cancer cell line. In ZR-75-9a1 cells [14] which have acquired tamoxifen resistance, there is loss of detectable ERs and progesterone receptors (PGR), and an increase in epidermal growth factor receptor (EGFR) expression [15]. The ZR-PR-LT cell line [16], which is an estrogen-independent variant, expresses high numbers of PGR and has reduced EGFR expression. We have recently reported the presence of iNOS in the ZR-75-1 human breast cancer cell line [17] and the aim of the current study was to investigate expression of both endothelial NOS (eNOS) and iNOS, in two more malignant variant cell lines. We report that the tamoxifen resistant ZR-PR-9a1 cells have similar eNOS and iNOS expression to the parent cell line whereas ZR-PR-LT cells line have much reduced levels of both eNOS and iNOS. 2. Materials and methods 2.1. Materials Anti-eNOS, bovine endothelial cell (599-613) (rabbit) and anti-iNOS mouse macrophage (11311144) (rabbit) were obtained from Calbiochem (Nottingham, UK). The StreptABComplex/HRP Duet (mouse/rabbit) was from Dako Ltd (Buckinghamshire, UK). Sulphanilimide and naphthylethylenediamine dihydrochloride were from BDH (Lutterworth, Leicestershire, UK). RU486 was a gift from Excelgen, France. All other chemicals were from Sigma Chemical Company (Poole, UK). 2.2. Cell culture Human
mammary
epithelial
cells
(HMEC)
(Biowhittaker, Berkshire, England) were routinely maintained in Mammary Epithelial Basal Medium (modi®ed MCDB 170) supplemented with bovine pituitary extract (52 mg/ml), human recombinant epidermal growth factor (hEGF) (10 ng/ml), hydrocortisone (0.5 mg/ml), gentamycin (50 mg/ml), amphotericin-B (50 ng/ml) and insulin (5 mg/ml). The ZR-75-1 human breast cancer cell line [18] (ECACC, Porton Down, Salisbury, UK) was routinely maintained in RPMI 1640 medium supplemented with HEPES (20 mM), fetal calf serum (10%), penicillin (50 units/ml), streptomycin (50 mg/ml) and glutamine (300 mg/ml). ZR-75-9a1 cells [14] were cultured in the same medium supplemented with 8 mM tamoxifen. ZR-PR-LT cells [16] were maintained in phenol red-free RPMI 1640 medium supplemented with 5% heat-treated, charcoal-stripped serum. Cells were grown in 5% CO2 at 378C. 2.3. Determination of NO production Cells (5 £ 104 ) were plated into 24-well plates and allowed to attach for 24 h. Medium was then replaced with fresh medium with or without the drug and the nitrite assay was performed 2 days later. Nitric oxide was measured as the amount of nitrite (NO22) which is a stable end-product of NO metabolism. Cell free supernatants were collected and stored at 2208C until analysis. Nitrite concentration in the medium was quantitated by a colourimetric assay based on the Greiss reaction [19]. Brie¯y, samples (100 ml) were mixed with an equal volume of Greiss reagent (1% sulphanilimide, 0.1% naphthylethylenediamine dihydrochloride and 2.5% H3PO4) and incubated at room temperature for 10 min. The absorbance was measured at 550 nm in a microplate reader. Sodium nitrite was used as a standard. 2.4. Immunocytochemistry Cells (4 £ 105 ) were plated into 24-well plates and allowed to attach for 24 h. Following washing with PBS, cells were permeabilized with 2% saponin for 10 min at room temperature and then ®xed in 5% formalin±saline. Incubation with normal rabbit serum (1:20) blocked non-speci®c binding. Cells were incubated with the primary antibody to eNOS or iNOS (1:200) for 30 min. IgG (1:100) was used as
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the secondary antibody as part of the StreptABComplex/HRP Duet kit with the ABC/fast red technique being used to demonstrate presence of eNOS or iNOS in breast cancer cells. Negative controls were cells not treated with the primary antibody.
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2.5. Statistical analysis Statistical signi®cance was determined using the Student's t-test and the differences were regarded as signi®cant for values of P , 0:05.
Fig. 1. Immunohistochemical staining (red stain) for eNOS in (A1) human mammary epithelial cells and (A2) negative control; (B1) ZR-75-1 cells and (B2) negative control; (C1) ZR-75-9a1 cells and (C2) negative control; (D1) ZR-PR-LT cells and (D2) negative control; using bovine endothelial anti-nitric oxide synthase, (599-613) (rabbit) antibody (£400).
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3. Results 3.1. Immunohistochemical staining for eNOS The presence of cells staining positive for eNOS monoclonal antibody was demonstrated by the appearance of red-stained cells (Fig. 1). One-hundred percent of HMEC (Fig. 1A) and 100% of ZR-75-1
cells (Fig. 1B) stained positive for eNOS. Only 50% of ZR-75-9a1 cells stained positive for eNOS (Fig. 1C) and less than 5% of ZR-PR-LT cells stained positive for eNOS (Fig. 1D). 3.2. Immunohistochemical staining for iNOS The presence of cells staining positive for iNOS
Fig. 2. Immunohistochemical staining (red stain) for iNOS in (A1) human mammary epithelial cells and (A2) negative control; (B1) ZR-75-1 cells and (B2) negative control; (C1) ZR-75-9a1 cells and (C2) negative control; (D1) ZR-PR-LT cells and (D2) negative control; using mouse macrophage inducible anti-nitric oxide synthase (1131-1144) antibody (£400).
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monoclonal antibody was demonstrated by the appearance of red-stained cells (Fig. 2). One-hundred percent of HMECs stained positive for iNOS (Fig. 2A). The percentage of ZR-75-1 cells staining positive for iNOS was 52% (Fig. 2B, previously reported in Ref. [17]). Only 40% of ZR-75-9a1 cells stained positive for iNOS (Fig. 2C). No ZR-PR-LT cells were positively stained for iNOS (Fig. 2D). 3.3. Effect of l-arginine, l-nitro-l-arginine methyl ester (l-NAME) and tamoxifen on nitrite secretion by ZR-75-9a1 and ZR-PR-LT cells During a 2-day culture period, untreated ZR-75-9a1 cells secreted 3 nmol NO22 (5 £ 104 cells) into the
69
culture medium (Fig. 3) and ZR-PR-LT cells secreted 1 nmol NO22 (5 £ 104 cells) into the culture medium (Fig. 4). A 2-day treatment of ZR-75-9a1 cells with larginine (10 mM) resulted in an increase to 64 nmol NO22/5 £ 104 cells (Fig. 3) and a 2-day treatment of ZR-PR-LT cells with l-arginine (10 mM) resulted in an increase to 42 nmol NO22/5 £ 104 cells (Fig. 4). These increases were signi®cant at the P , 0:001 level. Treatment of ZR-75-9a1 (Fig. 3) and ZR-PRLT (Fig. 4) cells with concentrations of l-arginine ranging from 10 mM±5mM also produced signi®cant increases in the amount of nitrite secreted into the conditioned medium. For both control ZR-75-9a1 cells (Fig. 3) and those treated with l-arginine, treatment with l-NAME
Fig. 3. Effect of l-arginine, l-NAME and tamoxifen on nitrite secretion by ZR-75-9a1 cells. Cells (5 £ 104 ) were seeded into wells of a culture plate in 1 ml of RPMI 1640 medium and allowed to attach for 24 h. Medium was then replaced with medium with (open bar) or without l-arginine (10±10000 mM), l-NAME (2 mM) (dotted) or tamoxifen (10 28 M) (striped) and cultured for 2 days. After 2 days the supernatant was removed and aliquots (100 ml) were analyzed for nitrite concentration. This was determined by reacting with an equal volume of Greiss reagent and measuring the absorbance at 540 nm in a Bio-Rad plate reader. The results of a typical experiment which was repeated seven times are presented. Values presented are means of experimental replicates (n 3) with SE.
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Fig. 4. Effect of l-arginine, l-NAME and tamoxifen on nitrite secretion by ZR-PR-LT cells. Cells (5 £ 104 ) were seeded into wells of a culture plate in 1 ml of RPMI 1640 medium and allowed to attach for 24 h. Medium was then replaced with medium with (open bar) or without l-arginine (10±10000 mM), l-NAME (2 mM) (dotted) or tamoxifen (10 28 M) (striped) and cultured for 2 days. After 2 days the supernatant was removed and aliquots (100 ml) were analyzed for nitrite concentration. This was determined by reacting with an equal volume of Greiss reagent and measuring the absorbance at 540 nm in a Bio-Rad plate reader. The results of a typical experiment which was repeated seven times are presented. Values presented are means of experimental replicates (n 3) with SE.
(2 mM) caused a signi®cant (P , 0:05) reduction in NO22 secreted into the medium. Similar results were obtained for ZR-PR-LT cells (Fig. 4). Treatment with tamoxifen (10 28M) did not cause a signi®cant change in nitric oxide production for l-arginine-treated ZR75-9a1 (Fig. 3) or ZR-PR-LT (Fig. 4) cells. Similar results have been obtained in seven separate experiments. 3.4. Effect of progesterone and RU486 on nitrite secretion by ZR-75-1, ZR-75-9a1 and ZR-PR-LT cells During a 2-day culture period, untreated ZR-75-1 cells secreted 3.7 nmol NO22 (5 £ 104 cells) into the
medium (Fig. 5). Treatment of cells with concentrations of 0.1±1000nM progesterone caused signi®cant (P , 0:05) decrease in NO22 secreted into the culture medium, which could be reversed by RU486 (10 24 M). Untreated ZR-75-9a1 cells secreted 3.5 nmol NO22 (5 £ 104 cells) into the medium (Fig. 5). Treatment of these cells with concentrations of 0.1±1000nM progesterone also caused signi®cant (P , 0:001) decreases in NO22 secreted into the culture medium. This could be reversed by RU486 (10 24 M). During a 2-day culture period, untreated ZR-PR-LT cells secreted 0.5 nmol NO22 (5 £ 104 cells) into the medium (Fig. 5). Although treatment of ZR-PR-LT cells with concentrations of 0.1±1000nM progesterone reduced nitrite
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Fig. 5. Effect of progesterone and RU486 on nitrite secretion by ZR-75-1, ZR-75-9a1 and ZR-PR-LT cells. Cells (5 £ 104 ) were seeded into wells of a culture plate in 1 ml of RPMI 1640 medium and allowed to attach for 24 h. Medium was then replaced with medium with or without progesterone (0.1±1000 nM) and RU486 (10 24 M), and cultured for 2 days. After 2 days the supernatant was removed and aliquots (100 ml) were analyzed for nitrite concentration. This was determined by reacting with an equal volume of Greiss reagent and measuring the absorbance at 540 nm in a Bio-Rad plate reader. ZR-75-1 without RU486 (open bar) and with RU486 (vertical stripe). ZR-75-9a1 without RU486 (dotted) and with RU486 (horizontal stripe). ZR-PR-LT without RU486 (checked bar) and with RU486 (angled stripe). The results of a typical experiment which was repeated ®ve times are presented. Values presented are means of experimental replicates (n 3) with SE.
secretion, this was not statistically signi®cant. Similar results have been obtained in ®ve separate experiments 3.5. Effect of phorbol 12-myristate 13-acetate (PMA) on nitrite secretion by ZR-75-9a1 and ZR-PR-LT cells During a 2-day culture period, untreated ZR-75-9a1 cells secreted 3.5 nmol NO22 (5 £ 104 cells) into the culture medium (Fig. 6). A 2-day treatment of ZR-759a1 cells with PMA (1000 nM) resulted in an increase to 23 nmol NO22/5 £ 104 cells. This increase was signi®cant at the P , 0:001 level. Treatment of ZR75-9a1 cells with concentrations of PMA ranging from 200±800nM also produced signi®cant increases in the amount of nitrite secreted into the conditioned medium (Fig. 6). During a 2-day culture period, untreated ZR-PR-LT
cells secreted 1 nmol NO22 (5 £ 104 cells) into the culture medium (Fig. 6). A 2-day treatment of ZRPR-LT cells with PMA (1000 nM) resulted in a slight increase to 1.5 nmol NO22/5 £ 104 cells but this was not statistically signi®cant (Fig. 6). Treatment of ZRPR-LT cells with concentrations of PMA ranging from 200±800nM did not produce signi®cant changes in the amount of nitrite secreted into the conditioned medium (Fig. 6). Similar results have been obtained in ®ve separate experiments. 4. Discussion The role of nitric oxide in cancer biology is at present poorly understood as it has both pro- and anti- tumor functions. We have previously demon-
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Fig. 6. Effect of phorbol 12-myristate 13-acetate (PMA) on nitrite secretion by ZR-75-9a1 and ZR-PR-LT cells. ZR-75-9a1 cells (X) or ZR-PR-LT cells (O) (5 £ 104 ) were seeded into wells of a culture plate in 1 ml of RPMI 1640 medium and allowed to attach for 24 h. Medium was then replaced with medium with or without PMA (200±1000 nM), and cultured for 2 days. After 2 days the supernatant was removed and aliquots (100 ml) were analyzed for nitrite concentration. This was determined by reacting with an equal volume of Greiss reagent and measuring the absorbance at 540 nm in a Bio-Rad plate reader. The results of a typical experiment which was repeated ®ve times are presented. Values presented are means of experimental replicates (n 3) with SE.
strated the presence of the l-arginine/nitric oxide pathway in ZR-75-1 human breast cancer cells [17]. The aim of the current study was to investigate expression of both eNOS and iNOS in normal human mammary epithelial cells and in two more malignant variant breast cancer cell lines, namely the estrogen-independent ZR-PR-LT cell line [16] and the tamoxifen-resistant ZR-75-9a1 cell line [14]. Our results demonstrate that normal breast cells show 100% staining for iNOS. This is in marked contrast to all three tumor cell lines in which expression of iNOS was reduced, with only 52% positive staining for ZR-75-1 cells and 40% positive staining for ZR-75-9a1 and ZR-PR-LT cells, which failed to express any positivity for iNOS. Similarly, although both the normal breast cells and ZR-75-1 cells expressed 100% staining for eNOS, there was considerably less positivity for eNOS in the more malignant ZR-75-9a1 and ZR-PR-LT cells. We have also shown that the tamoxifen-resistant cell line exhibited similar expression of iNOS to the
parent cell line. Similar basal levels of NO production were observed which could be increased by l-arginine and induced by PMA. These similarities to the parent cell line [17] may be due to the instability of the ZR75-9a1 cell line. Although the tamoxifen-resistant cells exhibit loss of detectable ERs and PGRs, the ZR-75-9a1 cell line is unstable and in the absence of tamoxifen these cells will revert back to an ER- and PGR- positive phenotype [14]. The ZR-PR-LT cell line expresses high numbers of PGR [16] and has reduced EGFR expression [15]. We have shown that this estrogen-independent cell line expresses signi®cantly lower levels of both eNOS and iNOS compared with the parent cell line, with correspondingly lower NO production. In contrast to ZR-75-1 cells, in ZR-PR-LT cells, NO production could not be stimulated by PMA. Previous studies have investigated the role of estrogen in the nitric oxide pathway. Estrogen has been reported to upregulate eNOS gene expression [20] and several potential signalling mechanisms have been proposed [21]. A correlation between eNOS expression and ER expression has been reported in several human breast cancer cell lines [22]. Our results for the estrogen-independent breast cancer cells supports these previous ®ndings. Our explanation for a lack of the two NOS isoforms in more malignant breast cells is two-fold. Firstly, in ER-positive cells it is thought that estradiol may enhance production of NO which then acts as a free radical to induce mutations leading to a more malignant phenotype [22]. NO itself has been shown to decrease transcription of the iNOS gene [23] consequently downregulating iNOS expression in the more malignant cells. Secondly, as the cells adapt to estrogen-independent growth and fail to express detectable ER, the upregulation of eNOS by estradiol has been lost, again leading to decreased expression of eNOS in the more malignant cells. Treatment of ZR-75-1 and ZR-75-9a1 cells with progesterone resulted in downregulation of NO production. Our results are consistent with previous studies demonstrating that progesterone inhibits both iNOS gene expression and NO production [24]. Progesterone is thought to inhibit NOS by binding to the glucocorticoid receptor. It has been suggested that the activated glucocorticoid receptor prevents the binding of transcription factor NF-kB to the NOS
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promoter thereby inhibiting the induction of NOS transcription [25]. In our experiments, RU486 which has a high af®nity for both the progesterone and glucocorticoid receptors was able to reverse the inhibitory effects of progesterone. Interestingly, PMA increased NO production in both ZR-75-1 [17] and ZR-75-9a1 breast cancer cells but was unable to do so in ZR-PR-LT cells. This may suggest a defect in protein kinase C intracellular signalling pathways in the estrogen-independent cell line. However, further investigation is required to clarify this hypothesis. In conclusion, we have demonstrated reduced expression of eNOS and iNOS in a human breast cancer cell line adapted to growth in estrogen-free conditions. These results suggest a role for nitric oxide synthase in human breast cancer and also suggest an interaction of estrogen/estrogen receptor with nitric oxide function.
[9]
[10]
[11]
[12] [13]
[14]
References [1] Y. Yoshie, H. Ohshima, Synergistic induction of DNA strand breakage by catechol-estrogen and nitric oxide: implications for hormonal carcinogenesis, Free Radical Bio. Med. 24 (1998) 341±348. [2] S. Calmels, P. Hainaut, H. Ohshima, Nitric oxide induces conformational and functional modi®cations of wild-type p53 tumor suppressor protein, Cancer Res. 57 (1997) 3365±3369. [3] D.C. Jenkins, I.G. Charles, L.L. Thomsen, D.W. Moss, L.S. Holmes, S.A. Baylis, P. Rhodes, K. Westmore, P.C. Emson, S. Moncada, Roles of nitric oxide in tumor growth, Proc. Natl. Acad. Sci. USA 92 (1995) 4392±4396. [4] S.P. Andrade, H.R. Hart, P.J. Piper, Inhibitors of nitric oxide synthase selectively reduce ¯ow in tumor-associated neovasculature, Br. J. Pharmacol. 107 (1992) 1092±1097. [5] A. Due-nas-Gonzalez, C.M. Isales, del Mar Abad-Hernandez M, Gonzalez-Sarmiento R., Sangueza O., Rodriguez-Commes J., Expression of inducible nitric oxide synthase in breast cancer correlates with metastatic disease, Modern Pathol. 10 (1997) 645±649. [6] T. Iwasaki, M. Higashiyama, K. Kuriyama, A. Sasaki, M. Mukai, K. Shinkai, T. Horai, H. Matsuda, H. Akedo, NGnitro-l-arginine methyl ester inhibits bone metastasis after modi®ed intracardiac injection of human breast cancer cells in a nude mouse model, Jpn. J. Cancer Res. 88 (1997) 861±866. [7] D.J. Stuehr, C.F. Nathan, Nitric oxide, a macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells, J. Exp. Med. 169 (1989) 1543±1555. [8] J.H.J. Martin, S.W. Edwards, Changes in mechanisms of monocyte/macrophage mediated cytotoxicity during culture, Reactive oxygen intermediates are involved in monocyte-
[15]
[16]
[17]
73
mediated cytotoxicity whereas reactive nitrogen intermediates are employed by macrophages in tumor cell killing, J. Immunol. 150 (1993) 3478±3486. J. Drapier, Hibbs Jr J.B., Murine cytotoxic activated macrophages inhibit aconitase in tumor cells: inhibition involves the iron-sulfur prosthetic group and is reversible, J. Clin. Invest. 78 (1986) 790±795. M. Sandoval, X. Liu, P.D. Oliver, X.J. Zhang, D.A. Clark, M.J.S. Miller, Nitric oxide induces apoptosis in a human colonic epithelial cell line, T84, Mediat. In¯amm. 4 (1995) 248±250. S. Cui, J.S. Reichner, R.B. Mateo, J.E. Albina, Activated murine macrophages induce apoptosis in tumor cells through nitric oxide-dependent or independent mechanisms, Cancer Res. 54 (1994) 2462±2467. V.C. Jordan, Long-term Tamoxifen Treatment for Breast Cancer, University of Wisconsin Press, Madison, 1994, pp. 1± 289. F. Leonessa, V. Boulay, A. Wright, E.W. Thompson, N. Brunner, R. Clarke, The biology of breast tumor progression: acquisition of hormone-independence and resistance to cytotoxic drugs, Acta Oncol. 31 (1992) 115±123. H.W. van den Berg, M. Lynch, J. Martin, J. Nelson, G.R. Dickson, A.D. Crockard, Characterisation of a tamoxifenresistant variant of the ZR-75-1 human breast cancer cell line (ZR-75-9a1) and stability of the resistant phenotype, Br. J. Cancer 59 (1989) 522±526. B. Long, Mc Kibben B.M., Lynch M., van den Berg H.W., Changes in epidermal growth factor receptor expression and response to ligand associated with acquired tamoxifen resistance or oestrogen independence in the ZR-75-1 human breast cancer cell line, Br. J. Cancer 65 (1992) 865±869. H.W. van den Berg, J. Martin, M. Lynch, High progesterone receptor concentration in a variant of the ZR-75-1 human breast cancer cell line adapted to growth in oestrogen-free conditions, Br. J. Cancer 61 (1990) 504±507. O. Alalami, J.H.J. Martin, ZR-75-1 Human breast cancer cells: expression of inducible nitric oxide synthase and effect of tamoxifen and phorbol ester on nitric oxide production, Cancer Lett. 123 (1998) 99±105.
[18] L.W. Engel, N.A. Young, T.S. Tralka, M.E. Lippman, S.J. O'Brien, M.J. Joyce, Establishment and characterisation of three new continuous cell lines derived from human breast carcinomas, Cancer Res. 38 (1978) 3352±3364. [19] L.C. Green, D.A. Wagner, J. Glogowski, P.L. Skipper, J.S. Wishnok, S.R. Tannenbaum, Analysis of nitrate, nitrite, and [ 15N]nitrate in biological ¯uids, Anal. Biochem. 126 (1982) 131±138. [20] A.N. MacRitchie, S.S. Jun, Z. Chen, Z. German, I.S. Yuhanna, T.S. Sherman, P.W. Shaul, Estrogen upregulates endothelial nitric oxide synthase gene expression in fetal pulmonary artery endothelium, Circ. Res. 81 (1997) 355±362. [21] K. Kauser, G.M. Rubanyi, Potential cellular signalling mechanisms mediating upregulation of endothelial nitric oxide production by estrogen, J. Vasc. Res. 34 (1997) 229±236. [22] R. Zeillinger, E. Tantscher, C. Schneeberger, W. Tschugguel, S. Eder, G. Sliutz, J.C. Huber, Simultaneous expression of
74
J.H.J. Martin et al. / Cancer Letters 144 (1999) 65±74
nitric oxide synthase and estrogen receptor in human breast cancer cell lines, Breast Cancer Res. Tr. 40 (1996) 205±207. [23] S.K. Park, D. Grzybicki, H.L. Lin, S. Murphy, Modulation of inducible nitric oxide synthase expression in astroglial cells, Neuropharmacology 33 (1994) 1419±1423. [24] L. Miller, E.W. Alley, W.J. Murphy, S.W. Russell, J.S. Hunt, Progesterone inhibits inducible nitric oxide synthase gene
expression and nitric oxide production in murine macrophages, J. Leukocyte Biol. 59 (1996) 442±450. [25] H. Kleiner, C. Euchenhofer, I. Ihrig-Biedert, U. Forstermann, Glucocorticoids inhibit the induction of nitric oxide synthase II by down-regulating cytokine-induced activity of transcription factor-kB, Mol. Pharmacol. 49 (1996) 15±21.