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AT1 receptor and ACE mRNA are increased in chemically induced carcinoma of rat mammary gland
Molecular and Cellular Endocrinology 244 (2005) 42–46
AT1 receptor and ACE mRNA are increased in chemically induced carcinoma of rat mammary gland Katarina Tybitanclova a,∗ , Dana Macejova a , Jan Liska b , Julius Brtko a , Stefan Zorad a a
Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlarska 3, 833 06 Bratislava, Slovakia b Institute of Histology and Embryology, Medical Faculty of Comenius University, Bratislava, Slovakia Received 26 November 2004; accepted 30 January 2005
Abstract Angiotesin II has except of strong vasoconstrict effect also ability to potentiate protein synthesis and cellular growth. The aim of this study was to investigate the gene expression of components of the renin-angiotensin system, angiotensinogen, renin, angiotensin-converting enzyme and AT1 receptor, in rat mammary gland and in chemically induced carcinoma of this gland. Retinoids are known to inhibit cell proliferation and induce cell differentiation so they are considered as a promising chemopreventive agents. We studied the effect of 13-cis-retinoic acid on the gene expression of mentioned elements of the renin-angiotensin system in tumour tissue. The expressions in control and carcinoma tissue were investigated using RT-PCR and activity of angiotensin-converting enzyme was measured. The amount of angiotensin-converting enzyme and AT1 receptor mRNA and angiotensin-converting enzyme activity always showed a significant increase in the carcinoma tissue in comparison with the control. Administration of 13-cis-retinoic acid to rats with induced mammary gland carcinoma was without significant effect on either tumour numbers or tumour burden and volume. Similarly, 13-cis-retinoic acid did not change the angiotensin-converting enzyme expression and activity. The AT1 receptor gene expression displayed a clear tendency to decrease in tumour tissue after retinoic acid treatment. Our results demonstrate the presence of angiotensin-converting enzyme and AT1 receptor in control and carcinoma tissue of mammary gland. We assume that both proteins might play a role in development of tumour cells and vasculature. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: AT1 receptor; Angiotensin-converting enzyme; Mammary gland; 13-cis-Retinoic acid; Rat
1. Introduction The one of widely used experimental systems for the study of mammary adenocarcinomas is the model in which tumours are induced in Sprague–Dawley rats by 1-methyl-1nitrosourea (MNU) (Wang et al., 2001). This MNU-induced tumour rat model was originally described by Gullino et al. (1975) and exhibits similarities with the human ER + breast tumours (Gould, 1995). Tumour histology and incidence, number of malignant tumours/rat and latency all showed carcinogen dose–response (McCormick et al., 1981). The susceptibility of the mammary gland to MNU-induced carcinogenesis is strongly age-dependent and is maximal when the carcinogen is administered to rats between the ages of 45 and 60 days, that is the age of sexual maturity. Rat mammary tumours are also generally strongly hormone-dependent for both induction and growth (Russo et al., 1990). ∗
Corresponding author. Tel.: +421 2 54772800; fax: +421 2 54774247. E-mail address: k [email protected] (K. Tybitanclova).
0303-7207/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2005.01.015
Angiotensin II (Ang II), the major active peptide of the reninangiotensin system (RAS), is generated from angiotensinogen. Important enzymes involved in Ang II formation are renin (EC 3.4.23.15) and angiotensin-converting enzyme (ACE)(EC 3.4.15.1). Most of the known physiological effects of Ang II are mediated through angiotensin receptor of AT1 subtype. On the one hand, the RAS is an endocrine system regulating the level of arterial pressure; on the other hand, it is an autocrineparacrine system participating in tissue remodelling (Ardaillou and Michel, 1999). In addition to its strong contractile action in smooth muscle cells, Ang II stimulates growth and/or proliferative responses in its target cells. The growth factor-like effects of Ang II include increases in tyrosine phosphorylation of numerous intracellular proteins, activation of MAPK and related pathways, and increased expression of several early response genes including c-fos, c-jun and c-myc (De Gasparo et al., 2000). Thus, Ang II regulates and enhances the activity of growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-, plasminogen activator-inhibitor-1 and
K. Tybitanclova et al. / Molecular and Cellular Endocrinology 244 (2005) 42–46
increases extracellular matrix proteins synthesis (Ardaillou and Michel, 1999). It has been suggested that transformation of cells from normal to malignant may indirectly or directly result from increased production of growth-stimulatory factors (Dickson and Lippman, 1995). ACE inhibitors and AT1 receptor antagonists have cytostatic properties on in vitro cultures of many normal and neoplastic cells (Fujimoto et al., 2001; Muscella et al., 2002; Greco et al., 2003; Molteni et al., 2003). The inhibition of Ang II synthesis and/or the blockade of Ang II receptors is likely to be an important mechanism for this cytostatic action. For example, RAS inhibitors are effective, in particular, in reducing the growth of human breast adenocarcinomas and in controlling excessive angiogenesis in MA-16 viral-induced mammary carcinoma of the mouse (Molteni et al., 2003). It is well accepted that Vitamin A and its biologically active derivatives regulate the expression of many different genes within the body. Retinoids interact with their nuclear receptors of two distinct classes: the retinoic acid receptors (RAR␣,-,-) and the retinoid X receptors (RXR␣,-,-) (Kurlandsky et al., 1995). Although 13-cis-retinoic acid (isotretinoin)(13cRA) is a naturally occurring form of retinoic acid, it is thought that many of its transcriptional activities are mediated by all-trans-retinoic acid or possibly 9-cis retinoic acid, after isomerization of 13cRA (Blaner, 2001). In vitro it has been shown that retinoids inhibit anchoragedependent growth of tumour cells, including carcinomas of the breast, and they also exert non-specific cytotoxic effect and induce apoptosis in various types of cancer cells. So, the role of retinoids as a cancer chemopreventive as well as cancer chemotherapeutic agents has been examined in a variety of in vivo model systems and in clinical trials (Sun and Lotan, 1998). In our study we investigated the presence of components of the RAS in rat mammary gland and in MNU-induced mammary gland carcinoma. In addition, we studied the effect of 13-cisretinoic acid on tumour progress and the gene expression of the RAS.
43
Table 1 The effect of retinoic acid on MNU-induced mammary gland carcinoma in rat
MNU 13cRA
Number of tumours/animal
Tumour burden (g)/animal
Tumour volume (cm3 )/animal
4.00 ± 0.63 3.75 ± 0.96
22.80 ± 5.26 12.13 ± 6.82
45.60 ± 10.90 22.00 ± 12.50
MNU: MNU treated rats, 13cRA: MNU rats receiving 13-cis-retinoic acid.
and Sacchi (1987). Two micrograms of total RNA was reverse transcribed using ready-to-go-you-prime First-Strand Beads Kit and pd(N)6 random hexamer primers (Amersham Pharmacia Biotech). PCR amplification was performed in a total volume of 25 l containing 2.5 l 10× PCR buffer, 0.5 l 12.5 mM dNTP, 1 U DyNAzymeTM II DNA polymerase (Finnzymes, Biotech), 4 l of first strand cDNA and 25 pmol of each primers for AT1 receptor gene 5 -GCA CAA TCG CCA TAA TTA TCC-3 (sense) and 5 -CAC CTA TGT AAG ATC GCT TC-3 (antisense), for ACE gene 5 -CCT GAT CAA CAA GGA GTT TGC AGA G-3 (sense) and 5 -GCC AGC CTT CCC AGG CAA ACA GCA C-3 (antisense), for angiotensinogen gene 5 -TTG TTG AGA GCT TGG GTC CCT TCA-3 (sense) and 5 -CAG ACA CTG AGG TGC TGT TGT CCA-3 (antisense) and for renin gene 5 -TCT CAG CAA CAT GGA CTA TGT GC-3 (sense) and 5 -TTA GCG GGC CAA GGC GAA CC-3 (antisense). Reactions were normalized using housekeeping gene glyceraldehyd3-phosphate dehydrogenase (GAPDH): 5 -AGA TCC ACA ACG GAT ACA TT-3 (sense) and 5 -TCC CTC AAG ATT GTC AGC AA-3 (antisense). PCR reactions were performed under conditions we described previously (Pinterova et al., 2000). The negative control consists of omission of the RT for each sample and resulted in no bands after RT-PCR. Specific PCR products were separated by electrophoresis in 2% agarose gels in the presence of ethidium bromide stain and subsequently quantified using camera ULTRA LUM KS 4000 and 1D Image Analysis Software (Eastman Kodak).
2.3. Angiotensin-converting enzyme activity Angiotensin-converting activity was assayed in homogenates of mammary gland tissue and tumour tissue with hippuryl-l-histidyl-l-leucine(glycine-114 C) (HHL) as the substrate for the enzyme as previously described (Heemskerk et al., 1999).
2.4. Statistical analysis
2. Materials and methods
The results are expressed as the means ± S.E.M. Statistical comparisons were made using Rank Sum Test. A value of p < 0.05 was considered statistically significant.
2.1. Animals
3. Results
Female Sprague–Dawley rats (obtained from Charles-River Laboratories, Germany) were housed at 23 ± 2 ◦ C and 12-h light/12-h dark cycle. To induce mammary gland tumours we used a model in which rats were injected intraperitoneally with MNU in dose 50 mg kg−1 on the 53rd, 82nd and 102nd day of age. From 55th day of age, one group of MNU treated rats was receiving 13cRA (1 mg kg−1 ) intragastrically three times per week until the end of the experiment (Macejova et al., 2001). The animals were decapitated (on the 164th day of age) and mammary gland and mammary gland tumour tissue were excised, rapidly frozen in liquid nitrogen and stored until assayed. Principles of laboratory animal care and all procedures were approved by the Animal Care Committee of the IEE SAS Bratislava, Slovak Republic. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.
2.2. Total RNA isolation and semi-quantitative RT-PCR Total RNA was isolated from frozen tissue by using guanidiniumthiocyanate/phenol chloroform extraction method according to Chomczynski
In our experiment, all mammary gland carcinomas were classified as malignant and various combinations of pappilary, cribriform or comedo patterns of ductal adenocarcinomas occurred in tumours. The average values of tumour incidence, tumour burden and tumour volume are shown in Table 1. We examined the gene expression of angiotensinogen, renin, ACE and AT1 receptor in six normal rat mammary glands against six MNU-induced mammary carcinomas. We did not detect the angiotensinogen and renin mRNA in any group of experimental animals. The quantification of the specific band densities expressed as ratio ACE/GAPDH revealed the presence of ACE mRNA in mammary gland of control and MNU-treated rats (Fig. 1). MNU-group showed significant increase in comparison with control animals. As demonstrated in Fig. 2, we obtained similar results for AT1 receptor, which was expressed
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K. Tybitanclova et al. / Molecular and Cellular Endocrinology 244 (2005) 42–46 Table 2 The enzymatic activity of angiotensin-converting enzyme in rat mammary gland of control and carcinoma tissue ACE activity (nmol of product/100 g proteins/h) Control MNU 13cRA
9.7 ± 1.4 (n = 6) 21.8 ± 3.7a (n = 6) 18.4 ± 5.7 (n = 6)
MNU: MNU treated rats, 13cRA: MNU rats receiving 13-cis-retinoic acid. a Significant difference from control (p < 0.05).
Fig. 1. The gene expression of angiotensin-converting enzyme (ACE) in mammary gland of control rats (C), MNU-treated rats (MNU) and MNU-treated rats receiving 13-cis-retinoic acid (13CRA). Significant differences: ** p < 0.01 vs. control.
nomas. In MNU rats, which were received 13cRA we noticed a tendency to decreased carcinoma growth in terms of tumour weight and volume compared to untreated rats (Table 1). Fig. 1 and Table 2 document that the treatment of MNU rats with 13cRA was without statistically significant effect on either level of ACE mRNA or ACE enzymatic activity. The gene expression of AT1 receptor in tumour tissue after 13cRA administration displayed a clear tendency to decrease but there were also found no statistical significant differences. 4. Discussion
Fig. 2. The gene expression of AT1 receptor in mammary gland of control rats (C), MNU-treated rats (MNU) and MNU-rats treated with 13-cis-retinoic acid (13CRA). Significant differences: + p < 0.05 vs. control.
in mammary gland of control and MNU-treated rats with significant increase in carcinoma tissue. Fig. 3A and B represents a typical electrophoresis images of PCR amplified fragments of ACE and AT1 receptor in control and tumour tissues. The measurement of enzymatic activity of ACE in the homogenates of control and carcinoma tissue of mammary gland (Table 2) is in accordance with determination of ACE gene expression. In addition, we investigated the influence of 13cRA application on development of MNU-induced mammary gland carci-
The results obtained in the present study demonstrate that ACE and AT1 receptor are expressed in both normal mammary gland and transplants of ductal MNU-induced mammary gland carcinomas in female Sprague–Dawley rats. ACE and also AT1 receptor are much more expressed in tumour tissues. The present findings of a high level of ACE and AT1 receptor mRNA in adenocarcinomas is in agreement with observations of Guerra et al. (1993) but these authors, using a model of medroxyprogesterone acetate-induced mammary tumours in BALB/c mice, determined that the presence of AT1 receptor mRNA is associated only to neoplastic epithelial cells. On the other hand, Greco et al. (2002) showed that AT1 mRNA is expressed in normal and cancerous human breast cells in primary culture. Development of the mammary epithelium is unique in that mammary ducts must grow into a pad of adipose tissue (Neville et al., 1998) and adipocytes were found also in MNU-induced tumours (Agarwal et al., 2000). Based on this and on the basis that rat white adipose tissue expresses all the RAS components (Pinterova et al., 2000), we assumed their possible presence in mammary gland. Since we did not detect the gene expression of angiotensinogen and renin, we may not suggest the presence of the complete local RAS in mentioned gland. On the other hand,
Fig. 3. The typical images of specific PCR product of (A) angiotensin-converting enzyme (ACE), (B) AT1 receptor (AT1 R) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 309 bp) genes in mammary gland of control rats, C, MNU-treated rats (MNU) and MNU-rats treated with 13-cis-retinoic acid (13CRA).
K. Tybitanclova et al. / Molecular and Cellular Endocrinology 244 (2005) 42–46
AT1 receptor as well as ACE expression might also originate from vascular tissue of the mammary gland. It is well known that normal and malignant mammary tissues are able to synthesise locally acting hormone-like substances including polypeptide growth factors (Dickson and Lippman, 1995). The synthesis of Ang II in tissues depends on the local level of ACE, whereas renin and angiotensinogen may be taken up from the circulation by the tissues (Ardaillou and Michel, 1999). We found simultaneous progress in the activity and the gene expression of ACE in mammary gland adenocarcinomas compared to normal gland. It was in correspondence with increased AT1 receptor expression in tumours and it might be supposed that peptide Ang II is locally produced within the carcinoma tissue. The local Ang II formation implies its distinct, paracrine or autocrine role. Furthermore, the effect of Ang II in the tissues is modulated by the specificities of the target cells and the density of Ang II receptors at the cell surface (Ardaillou and Michel, 1999). Because of its direct vasoconstrict effect, Ang II may increase blood flow through tumour tissue. Multiple growth factors, including FGFs (fibroblast growth factors), EGF-related factors, IGFs, TGFs, PDGF and vascular endothelial growth factor (VEGF) were found in the normal and malignant mammary epithelium. They are though to be involved in the process of blood vessel invasion into the tumour area (angiogenesis) and also in tumour growth and metastasize (Dickson and Lippman, 1995). It has been shown by many authors that Ang II contributes to proliferation via increased expression of growth factors and their receptors, for example, at vascular neointima formation after arterial injury (Powell et al., 1989), in human primary cultured breast cancer cells (Greco et al., 2003), in human pancreatic cancer cells (Fujimoto et al., 2001), in breast cancer epithelial cells MCF-7 (Muscella et al., 2002) and human endothelial progenitor cells (Imanishi et al., 2004). In addition, Ang II receptor blockers and ACE inhibitors have shown antineoplastic activity as well as angiogenesis inhibition in tumoural experimental models (Escobar et al., 2004; Uemura et al., 2003; Molteni et al., 2003; Greco et al., 2003). In the present investigation, the Ang II receptor increase in adenocarcinomas clearly indicates that Ang II plays a role in the regulation of mammary gland tumour cells growth and angiogenesis. The numerous studies showed that retinoids have anticarcinogenic activities due to their ability to modulate the cell growth and differentiation (Sun and Lotan, 1998; Ponthan et al., 2001; Agarwal et al., 2000). Although the 13cRA treatment of rats bearing MNU-induced mammary gland tumours resulted in marked tumour size reduction, it did not reach statistical significance under our experimental conditions. A clear tendency to AT1 expression decline and a little drop of ACE expression and activity in cancer tissues after 13cRA administration compared to untreated animals are another indications that point out a possible involvement of both proteins in carcinogenesis. In conclusion, the present study provides evidence of the presence of mRNA for ACE and AT1 receptor in both normal and cancerous mammary gland tissue in Sprague–Dawley rats. The physiological relevance for these proteins in mammary gland
45
tissue is currently not clear, however, there is a obvious assumption that Ang II through AT1 receptors may be effective as a growth factor, and thus play a role in the proliferation of tumour cells and vasculature development within tumour tissue. In the model of MNU-induced mammary gland cancer, the response to 13cRA treatment, a promising chemopreventive agent, was without statistical significant effect. Acknowledgements This work was supported grant of VEGA 2/3190/23 and Centre of Excellence ICA1-CT-2000-70008. References Agarwal, V.R., Bischoff, E.D., Hermann, T., Lamph, W.W., 2000. Induction of adipocyte-specific gene expression is correlated with mammary tumor regression by the retinoid X receptor-ligand LGD1069 (targretin). Cancer Res. 60, 6033–6038. Ardaillou, R., Michel, J.B., 1999. The relative roles of circulating and tissue renin-angiotensin systems. Nephrol. Dial. Transplant. 14, 283–286. Blaner, W.S., 2001. Cellular metabolism and actions of 13-cis-retinoic acid. J. Am. Acad. Dermatol. 45, S129–S135. Chomczynski, P., Sacchi, N., 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162, 156–159. De Gasparo, M., Catt, K.J., Inagami, T., Wright, J.W., Unger, Th., 2000. International union of pharmacology. XXIII. The angiotensin II receptors. Pharmacol. Rev. 52, 415–472. Dickson, R.B., Lippman, M.E., 1995. Growth factors in breast cancer. Endocr. Rev. 16, 559–589. Escobar, E., Rodriguez-Reyna, T.S., Arrieta, O., Sotelo, J., 2004. Angiotensin II, cell proliferation and angiogenesis regulator: biologic and therapeutic implications in cancer. Curr. Vasc. Pharmacol. 2, 385–399. Fujimoto, Y., Sasaki, T., Tsuchida, A., Chayama, K., 2001. Angiotensin II type 1 receptor expression in human pancreatic cancer and growth inhibition by angiotensin II type 1 receptor antagonist. FEBS Lett. 495, 197–200. Gould, M.N., 1995. Rodent models for the study of etiology, prevention and treatment of breast cancer. Semin. Cancer Biol. 6, 147–152. Greco, S., Elia, M.G., Muscella, A., Storelli, C., Marsigliante, S., 2002. AT1 angiotensin II receptor mediates intracellular calcium mobilization in normal and cancerous breast cells in primary culture. Cell Calcium 32, 1–10. Greco, S., Muscella, A., Elia, M.G., Salvatore, P., Storelli, C., Mazzotta, A., Manca, C., Marsigliante, S., 2003. Angiotensin II activates extracellular signal regulated kinases via protein kinase C and epidermal growth factor receptor in breast cancer cells. J. Cell. Physiol. 196, 370–377. Guerra, F.K., Ciuffo, G.M., Elizalde, P.V., Charreau, E.H., Saavedra, J.M., 1993. Enhanced expression of angiotensin II receptor subtypes and angiotensin converting enzyme in medroxyprogesterone-induced mouse mammary adenocarcinomas. Biochem. Biophys. Res. Commun. 193, 93–99. Gullino, P.M., Pettigrew, H.M., Grantham, F.H., 1975. N-nitrosomethylurea as mammary gland carcinogen in rats. J. Natl. Cancer Inst. 54, 401–414. Heemskerk, F.M., Zorad, S., Xu, N., Gutkind, S.J., Saavedra, J.M., 1999. Characterization of AT2 receptor expression in NIH 3T3 fibroblasts. Cell. Mol. Neurobiol. 19, 277–288. Imanishi, T., Hano, T., Nishio, I., 2004. Angiotensin II potentiates vascular endothelial growth factor-induced proliferation and network formation of endothelial progenitor cells. Hypertens. Res. 27, 101–108. Kurlandsky, S.B., Gamble, M.V., Ramakrishnan, R., Blaner, W.S., 1995. Plasma delivery of retinoic acid to tissues in the rat. J. Biol. Chem. 270, 17850–17857.
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Macejova, D., Liˇska, J., Brtko, J., 2001. Mammary gland carcinoma-related increase of type I iodothyronine 5 -deiodinase activity in Sprague–Dawley rats. Gen. Physiol. Biophys. 20, 293–302. McCormick, D.L., Adamowski, C.B., Fiks, A., Moon, R.C., 1981. Lifetime dose–response relationships for mammary tumor induction by a single administration of N-methyl-N-nitrosourea. Cancer Res. 41, 1690–1694. Molteni, A., Ward, W.F., Ts’ao, C.H., Taylor, J., Small W.Jr., Brizio-Molteni, L., Veno, P.A., 2003. Cytostatic properties of some angiotensin I converting enzyme inhibitors and of angiotensin II type I receptor antagonists. Curr. Pharm. Des. 9, 751–761. Muscella, A., Greco, S., Elia, M.G., Storelli, C., Marsigliante, S., 2002. Angiotensin II stimulation of Na+/K+ATPase activity and cell growth by calcium-independent pathway in MCF-7 breast cancer cells. J. Endocrinol. 173, 315–323. Neville, M.C., Medina, D., Monks, J., Hovey, R.C., 1998. The mammary fat pad. J. Mammary Gland Biol. Neoplasia 3, 109–116. Pinterova, L., Krizanova, O., Zorad, S., 2000. Rat epididymal fat tissue express all components of the renin-angiotensin system. Gen. Physiol. Biophys. 19, 329–334. Ponthan, F., Borgstrom, P., Hassan, M., Wassberg, E., Redfern, C.P., Kogner, P., 2001. The Vitamin A analogues: 13-cis retinoic acid, 9-cis retinoic
acid, and Ro 13-6307 inhibit neuroblastoma tumour growth in vivo. Med. Pediatr. Oncol. 36, 127–131. Powell, J.S., Clozel, J.P., Muller, R.K., Kuhn, H., Hefti, F., Hosang, M., Baumgartner, H.R., 1989. Inhibitors of angiotensin-converting enzyme prevent myointimal proliferation after vascular injury. Science 245, 186–188. Russo, J., Gusterson, B.A., Rogers, A.E., Russo, I.H., Wellings, S.R., Van Zwieten, M.J., 1990. Biology of disease: comparative study of human and rat mammary tumorigenesis. Lab. Invest. 62, 244–278. Sun, S.Y., Lotan, R., 1998. Retinoids as chemopreventive and therapeutic agents. Drugs Future 23, 621–634. Uemura, H., Ishiguro, H., Nakaigawa, N., Nagashima, Y., Miyoshi, Y., Fujinami, K., Sakaguchi, A., Kubota, Y., 2003. Angiotensin II receptor blocker shows antiproliferative activity in prostate cancer cells: a possibility of tyrosine kinase inhibitor of growth factor. Mol. Cancer Ther. 2, 1139–1147. Wang, Y., Hu, L., Yao, R., Wang, M., Crist, K.A., Grubbs, C.J., Johanning, G.L., Lubet, R.A., You, M., 2001. Altered gene expression profile in chemically induced rat mammary adenocarcinomas and its modulation by an aromatase inhibitor. Oncogene 20, 7710– 7721.
AT1 receptor and ACE mRNA are increased in chemically induced carcinoma of rat mammary gland Katarina Tybitanclova a,∗ , Dana Macejova a , Jan Liska b , Julius Brtko a , Stefan Zorad a a
Institute of Experimental Endocrinology, Slovak Academy of Sciences, Vlarska 3, 833 06 Bratislava, Slovakia b Institute of Histology and Embryology, Medical Faculty of Comenius University, Bratislava, Slovakia Received 26 November 2004; accepted 30 January 2005
Abstract Angiotesin II has except of strong vasoconstrict effect also ability to potentiate protein synthesis and cellular growth. The aim of this study was to investigate the gene expression of components of the renin-angiotensin system, angiotensinogen, renin, angiotensin-converting enzyme and AT1 receptor, in rat mammary gland and in chemically induced carcinoma of this gland. Retinoids are known to inhibit cell proliferation and induce cell differentiation so they are considered as a promising chemopreventive agents. We studied the effect of 13-cis-retinoic acid on the gene expression of mentioned elements of the renin-angiotensin system in tumour tissue. The expressions in control and carcinoma tissue were investigated using RT-PCR and activity of angiotensin-converting enzyme was measured. The amount of angiotensin-converting enzyme and AT1 receptor mRNA and angiotensin-converting enzyme activity always showed a significant increase in the carcinoma tissue in comparison with the control. Administration of 13-cis-retinoic acid to rats with induced mammary gland carcinoma was without significant effect on either tumour numbers or tumour burden and volume. Similarly, 13-cis-retinoic acid did not change the angiotensin-converting enzyme expression and activity. The AT1 receptor gene expression displayed a clear tendency to decrease in tumour tissue after retinoic acid treatment. Our results demonstrate the presence of angiotensin-converting enzyme and AT1 receptor in control and carcinoma tissue of mammary gland. We assume that both proteins might play a role in development of tumour cells and vasculature. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: AT1 receptor; Angiotensin-converting enzyme; Mammary gland; 13-cis-Retinoic acid; Rat
1. Introduction The one of widely used experimental systems for the study of mammary adenocarcinomas is the model in which tumours are induced in Sprague–Dawley rats by 1-methyl-1nitrosourea (MNU) (Wang et al., 2001). This MNU-induced tumour rat model was originally described by Gullino et al. (1975) and exhibits similarities with the human ER + breast tumours (Gould, 1995). Tumour histology and incidence, number of malignant tumours/rat and latency all showed carcinogen dose–response (McCormick et al., 1981). The susceptibility of the mammary gland to MNU-induced carcinogenesis is strongly age-dependent and is maximal when the carcinogen is administered to rats between the ages of 45 and 60 days, that is the age of sexual maturity. Rat mammary tumours are also generally strongly hormone-dependent for both induction and growth (Russo et al., 1990). ∗
Corresponding author. Tel.: +421 2 54772800; fax: +421 2 54774247. E-mail address: k [email protected] (K. Tybitanclova).
0303-7207/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2005.01.015
Angiotensin II (Ang II), the major active peptide of the reninangiotensin system (RAS), is generated from angiotensinogen. Important enzymes involved in Ang II formation are renin (EC 3.4.23.15) and angiotensin-converting enzyme (ACE)(EC 3.4.15.1). Most of the known physiological effects of Ang II are mediated through angiotensin receptor of AT1 subtype. On the one hand, the RAS is an endocrine system regulating the level of arterial pressure; on the other hand, it is an autocrineparacrine system participating in tissue remodelling (Ardaillou and Michel, 1999). In addition to its strong contractile action in smooth muscle cells, Ang II stimulates growth and/or proliferative responses in its target cells. The growth factor-like effects of Ang II include increases in tyrosine phosphorylation of numerous intracellular proteins, activation of MAPK and related pathways, and increased expression of several early response genes including c-fos, c-jun and c-myc (De Gasparo et al., 2000). Thus, Ang II regulates and enhances the activity of growth factors, such as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-, plasminogen activator-inhibitor-1 and
K. Tybitanclova et al. / Molecular and Cellular Endocrinology 244 (2005) 42–46
increases extracellular matrix proteins synthesis (Ardaillou and Michel, 1999). It has been suggested that transformation of cells from normal to malignant may indirectly or directly result from increased production of growth-stimulatory factors (Dickson and Lippman, 1995). ACE inhibitors and AT1 receptor antagonists have cytostatic properties on in vitro cultures of many normal and neoplastic cells (Fujimoto et al., 2001; Muscella et al., 2002; Greco et al., 2003; Molteni et al., 2003). The inhibition of Ang II synthesis and/or the blockade of Ang II receptors is likely to be an important mechanism for this cytostatic action. For example, RAS inhibitors are effective, in particular, in reducing the growth of human breast adenocarcinomas and in controlling excessive angiogenesis in MA-16 viral-induced mammary carcinoma of the mouse (Molteni et al., 2003). It is well accepted that Vitamin A and its biologically active derivatives regulate the expression of many different genes within the body. Retinoids interact with their nuclear receptors of two distinct classes: the retinoic acid receptors (RAR␣,-,-) and the retinoid X receptors (RXR␣,-,-) (Kurlandsky et al., 1995). Although 13-cis-retinoic acid (isotretinoin)(13cRA) is a naturally occurring form of retinoic acid, it is thought that many of its transcriptional activities are mediated by all-trans-retinoic acid or possibly 9-cis retinoic acid, after isomerization of 13cRA (Blaner, 2001). In vitro it has been shown that retinoids inhibit anchoragedependent growth of tumour cells, including carcinomas of the breast, and they also exert non-specific cytotoxic effect and induce apoptosis in various types of cancer cells. So, the role of retinoids as a cancer chemopreventive as well as cancer chemotherapeutic agents has been examined in a variety of in vivo model systems and in clinical trials (Sun and Lotan, 1998). In our study we investigated the presence of components of the RAS in rat mammary gland and in MNU-induced mammary gland carcinoma. In addition, we studied the effect of 13-cisretinoic acid on tumour progress and the gene expression of the RAS.
43
Table 1 The effect of retinoic acid on MNU-induced mammary gland carcinoma in rat
MNU 13cRA
Number of tumours/animal
Tumour burden (g)/animal
Tumour volume (cm3 )/animal
4.00 ± 0.63 3.75 ± 0.96
22.80 ± 5.26 12.13 ± 6.82
45.60 ± 10.90 22.00 ± 12.50
MNU: MNU treated rats, 13cRA: MNU rats receiving 13-cis-retinoic acid.
and Sacchi (1987). Two micrograms of total RNA was reverse transcribed using ready-to-go-you-prime First-Strand Beads Kit and pd(N)6 random hexamer primers (Amersham Pharmacia Biotech). PCR amplification was performed in a total volume of 25 l containing 2.5 l 10× PCR buffer, 0.5 l 12.5 mM dNTP, 1 U DyNAzymeTM II DNA polymerase (Finnzymes, Biotech), 4 l of first strand cDNA and 25 pmol of each primers for AT1 receptor gene 5 -GCA CAA TCG CCA TAA TTA TCC-3 (sense) and 5 -CAC CTA TGT AAG ATC GCT TC-3 (antisense), for ACE gene 5 -CCT GAT CAA CAA GGA GTT TGC AGA G-3 (sense) and 5 -GCC AGC CTT CCC AGG CAA ACA GCA C-3 (antisense), for angiotensinogen gene 5 -TTG TTG AGA GCT TGG GTC CCT TCA-3 (sense) and 5 -CAG ACA CTG AGG TGC TGT TGT CCA-3 (antisense) and for renin gene 5 -TCT CAG CAA CAT GGA CTA TGT GC-3 (sense) and 5 -TTA GCG GGC CAA GGC GAA CC-3 (antisense). Reactions were normalized using housekeeping gene glyceraldehyd3-phosphate dehydrogenase (GAPDH): 5 -AGA TCC ACA ACG GAT ACA TT-3 (sense) and 5 -TCC CTC AAG ATT GTC AGC AA-3 (antisense). PCR reactions were performed under conditions we described previously (Pinterova et al., 2000). The negative control consists of omission of the RT for each sample and resulted in no bands after RT-PCR. Specific PCR products were separated by electrophoresis in 2% agarose gels in the presence of ethidium bromide stain and subsequently quantified using camera ULTRA LUM KS 4000 and 1D Image Analysis Software (Eastman Kodak).
2.3. Angiotensin-converting enzyme activity Angiotensin-converting activity was assayed in homogenates of mammary gland tissue and tumour tissue with hippuryl-l-histidyl-l-leucine(glycine-114 C) (HHL) as the substrate for the enzyme as previously described (Heemskerk et al., 1999).
2.4. Statistical analysis
2. Materials and methods
The results are expressed as the means ± S.E.M. Statistical comparisons were made using Rank Sum Test. A value of p < 0.05 was considered statistically significant.
2.1. Animals
3. Results
Female Sprague–Dawley rats (obtained from Charles-River Laboratories, Germany) were housed at 23 ± 2 ◦ C and 12-h light/12-h dark cycle. To induce mammary gland tumours we used a model in which rats were injected intraperitoneally with MNU in dose 50 mg kg−1 on the 53rd, 82nd and 102nd day of age. From 55th day of age, one group of MNU treated rats was receiving 13cRA (1 mg kg−1 ) intragastrically three times per week until the end of the experiment (Macejova et al., 2001). The animals were decapitated (on the 164th day of age) and mammary gland and mammary gland tumour tissue were excised, rapidly frozen in liquid nitrogen and stored until assayed. Principles of laboratory animal care and all procedures were approved by the Animal Care Committee of the IEE SAS Bratislava, Slovak Republic. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.
2.2. Total RNA isolation and semi-quantitative RT-PCR Total RNA was isolated from frozen tissue by using guanidiniumthiocyanate/phenol chloroform extraction method according to Chomczynski
In our experiment, all mammary gland carcinomas were classified as malignant and various combinations of pappilary, cribriform or comedo patterns of ductal adenocarcinomas occurred in tumours. The average values of tumour incidence, tumour burden and tumour volume are shown in Table 1. We examined the gene expression of angiotensinogen, renin, ACE and AT1 receptor in six normal rat mammary glands against six MNU-induced mammary carcinomas. We did not detect the angiotensinogen and renin mRNA in any group of experimental animals. The quantification of the specific band densities expressed as ratio ACE/GAPDH revealed the presence of ACE mRNA in mammary gland of control and MNU-treated rats (Fig. 1). MNU-group showed significant increase in comparison with control animals. As demonstrated in Fig. 2, we obtained similar results for AT1 receptor, which was expressed
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K. Tybitanclova et al. / Molecular and Cellular Endocrinology 244 (2005) 42–46 Table 2 The enzymatic activity of angiotensin-converting enzyme in rat mammary gland of control and carcinoma tissue ACE activity (nmol of product/100 g proteins/h) Control MNU 13cRA
9.7 ± 1.4 (n = 6) 21.8 ± 3.7a (n = 6) 18.4 ± 5.7 (n = 6)
MNU: MNU treated rats, 13cRA: MNU rats receiving 13-cis-retinoic acid. a Significant difference from control (p < 0.05).
Fig. 1. The gene expression of angiotensin-converting enzyme (ACE) in mammary gland of control rats (C), MNU-treated rats (MNU) and MNU-treated rats receiving 13-cis-retinoic acid (13CRA). Significant differences: ** p < 0.01 vs. control.
nomas. In MNU rats, which were received 13cRA we noticed a tendency to decreased carcinoma growth in terms of tumour weight and volume compared to untreated rats (Table 1). Fig. 1 and Table 2 document that the treatment of MNU rats with 13cRA was without statistically significant effect on either level of ACE mRNA or ACE enzymatic activity. The gene expression of AT1 receptor in tumour tissue after 13cRA administration displayed a clear tendency to decrease but there were also found no statistical significant differences. 4. Discussion
Fig. 2. The gene expression of AT1 receptor in mammary gland of control rats (C), MNU-treated rats (MNU) and MNU-rats treated with 13-cis-retinoic acid (13CRA). Significant differences: + p < 0.05 vs. control.
in mammary gland of control and MNU-treated rats with significant increase in carcinoma tissue. Fig. 3A and B represents a typical electrophoresis images of PCR amplified fragments of ACE and AT1 receptor in control and tumour tissues. The measurement of enzymatic activity of ACE in the homogenates of control and carcinoma tissue of mammary gland (Table 2) is in accordance with determination of ACE gene expression. In addition, we investigated the influence of 13cRA application on development of MNU-induced mammary gland carci-
The results obtained in the present study demonstrate that ACE and AT1 receptor are expressed in both normal mammary gland and transplants of ductal MNU-induced mammary gland carcinomas in female Sprague–Dawley rats. ACE and also AT1 receptor are much more expressed in tumour tissues. The present findings of a high level of ACE and AT1 receptor mRNA in adenocarcinomas is in agreement with observations of Guerra et al. (1993) but these authors, using a model of medroxyprogesterone acetate-induced mammary tumours in BALB/c mice, determined that the presence of AT1 receptor mRNA is associated only to neoplastic epithelial cells. On the other hand, Greco et al. (2002) showed that AT1 mRNA is expressed in normal and cancerous human breast cells in primary culture. Development of the mammary epithelium is unique in that mammary ducts must grow into a pad of adipose tissue (Neville et al., 1998) and adipocytes were found also in MNU-induced tumours (Agarwal et al., 2000). Based on this and on the basis that rat white adipose tissue expresses all the RAS components (Pinterova et al., 2000), we assumed their possible presence in mammary gland. Since we did not detect the gene expression of angiotensinogen and renin, we may not suggest the presence of the complete local RAS in mentioned gland. On the other hand,
Fig. 3. The typical images of specific PCR product of (A) angiotensin-converting enzyme (ACE), (B) AT1 receptor (AT1 R) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 309 bp) genes in mammary gland of control rats, C, MNU-treated rats (MNU) and MNU-rats treated with 13-cis-retinoic acid (13CRA).
K. Tybitanclova et al. / Molecular and Cellular Endocrinology 244 (2005) 42–46
AT1 receptor as well as ACE expression might also originate from vascular tissue of the mammary gland. It is well known that normal and malignant mammary tissues are able to synthesise locally acting hormone-like substances including polypeptide growth factors (Dickson and Lippman, 1995). The synthesis of Ang II in tissues depends on the local level of ACE, whereas renin and angiotensinogen may be taken up from the circulation by the tissues (Ardaillou and Michel, 1999). We found simultaneous progress in the activity and the gene expression of ACE in mammary gland adenocarcinomas compared to normal gland. It was in correspondence with increased AT1 receptor expression in tumours and it might be supposed that peptide Ang II is locally produced within the carcinoma tissue. The local Ang II formation implies its distinct, paracrine or autocrine role. Furthermore, the effect of Ang II in the tissues is modulated by the specificities of the target cells and the density of Ang II receptors at the cell surface (Ardaillou and Michel, 1999). Because of its direct vasoconstrict effect, Ang II may increase blood flow through tumour tissue. Multiple growth factors, including FGFs (fibroblast growth factors), EGF-related factors, IGFs, TGFs, PDGF and vascular endothelial growth factor (VEGF) were found in the normal and malignant mammary epithelium. They are though to be involved in the process of blood vessel invasion into the tumour area (angiogenesis) and also in tumour growth and metastasize (Dickson and Lippman, 1995). It has been shown by many authors that Ang II contributes to proliferation via increased expression of growth factors and their receptors, for example, at vascular neointima formation after arterial injury (Powell et al., 1989), in human primary cultured breast cancer cells (Greco et al., 2003), in human pancreatic cancer cells (Fujimoto et al., 2001), in breast cancer epithelial cells MCF-7 (Muscella et al., 2002) and human endothelial progenitor cells (Imanishi et al., 2004). In addition, Ang II receptor blockers and ACE inhibitors have shown antineoplastic activity as well as angiogenesis inhibition in tumoural experimental models (Escobar et al., 2004; Uemura et al., 2003; Molteni et al., 2003; Greco et al., 2003). In the present investigation, the Ang II receptor increase in adenocarcinomas clearly indicates that Ang II plays a role in the regulation of mammary gland tumour cells growth and angiogenesis. The numerous studies showed that retinoids have anticarcinogenic activities due to their ability to modulate the cell growth and differentiation (Sun and Lotan, 1998; Ponthan et al., 2001; Agarwal et al., 2000). Although the 13cRA treatment of rats bearing MNU-induced mammary gland tumours resulted in marked tumour size reduction, it did not reach statistical significance under our experimental conditions. A clear tendency to AT1 expression decline and a little drop of ACE expression and activity in cancer tissues after 13cRA administration compared to untreated animals are another indications that point out a possible involvement of both proteins in carcinogenesis. In conclusion, the present study provides evidence of the presence of mRNA for ACE and AT1 receptor in both normal and cancerous mammary gland tissue in Sprague–Dawley rats. The physiological relevance for these proteins in mammary gland
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