Stewart Barker

Mechanism for Aldosterone Potentiation of Angiotensin II–Stimulated Rat Arterial Smooth Muscle Cell Proliferation

After earlier studies in which secretion of aldosterone was demonstrated to be important in rat arterial smooth muscle cell (RASMC) proliferation in vitro, the presence of both 11&bgr;-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) gene transcription were shown in these cells by real-time reverse transcription–polymerase chain reaction (RT-PCR). In proliferation studies, tritiated thymidine incorporation into RASMC and RASMC cell number were both significantly increased by angiotensin II (Ang II) (10−7 mol/L) compared with controls (P<0.01), but this effect was inhibited by the 3&bgr;-hydroxysteroid-dehydrogenase inhibitor trilostane (10−6 mol/L and 10−5 mol/L, P<0.05). Aldosterone alone added to RASMC did not significantly change tritiated thymidine incorporation when compared with controls, but the Ang II–induced increase was significantly enhanced by aldosterone at 10−10 mol/L and 10−8 mol/L (P<0.05). Neither corticosterone nor 18-hydroxydeoxycorticosterone had any such potentiating effect. RT-PCR analysis and real-time quantitative RT-PCR revealed an increase of Ang II type-1 (AT1) receptor mRNA in RASMC treated by aldosterone (10−8 mol/L) compared with untreated controls, and this was correlated with a small but significant increase in AT1 receptor protein (P<0.05), as assessed by immunoblotting analysis. These data confirm that steroid production by RASMC is critical in the response to Ang II, and the data support the view that aldosterone specifically is required for the full proliferative response to Ang II in RASMC. One way it may act is by modulating the expression and functions of the AT1 receptor.

Localisation of renin-angiotensin system (RAS) components in breast

Angiotensin II has mitogenic and angiogenic effects and its receptors are widespread, particularly in epithelial tissue. Tissue renin angiotensin systems (tRASs) may be a local source of angiotensin II that has specific paracrine functions. To investigate the presence of a tRAS in normal human breast and tumours. Immunocytochemistry, and quantitative RT–PCR was used to establish: (i) the presence and localisation of RAS components, (ii) the possibility of their involvement in cancer. (1) mRNA coding for angiotensinogen, prorenin, angiotensin converting enzyme (ACE), and both AT1 and AT2 receptors was demonstrated in normal and diseased breast tissues. (2) (pro)renin was identified in epithelial cells in both normal and diseased tissue, but in invasive carcinoma, its distribution was mostly confined to fibroblasts or could not be detected at all. (3) Angiotensin converting enzyme was shown in epithelial cells in both normal and malignant tissue. The results are consistent with the hypothesis that a tRAS is present in the breast, and is disrupted in invasive cancer.

Non-competitive steroid inhibition of oestrogen receptor functions

Currently available antioestrogens, such as tamoxifen, are competitive inhibitors that bind to the ligand binding sites of oestrogen receptors, ERα and ERβ. The search for alternative anti‐hormone therapies is prompted by the need for drugs that are effective when tumours become tamoxifen resistant. The existence of different receptor isoforms also raise the possibility of improving selectivity. Earlier use of the 3β‐hydroxysteroid dehydrogenase inhibitor, trilostane (4α,5‐ epoxy‐17β‐hydroxy‐3‐oxo‐5α‐androstane‐2α‐carbonitrile), suggested that it had beneficial actions in breast cancer that were only partially attributable to inhibition of steroidogenesis. The present studies on the interactions of trilostane with oestrogen receptors show that it (i) inhibits oestrogen‐stimulated proliferation in MCF‐7 breast cancer cells, (ii) enhances the affinity of oestradiol binding to ER in rat uteri and specifically increases oestradiol binding to an ERβ‐like isoform, (iii) inhibits ERα and ERβ binding to the classical vitellogenin gene oestrogen response element (ERE) and (iv) inhibits oestrogen‐stimulated gene transcription in ERE‐linked reporter systems in MCF‐7 cells. The results demonstrate a novel, presumably allosteric, mode of antioestrogen action. The beneficial actions of trilostane in breast cancer may be attributed to the combination of this antioestrogen effect with its well documented suppression of steroidogenesis.

The renin–angiotensin system in the breast and breast cancer

Much evidence now suggests that angiotensin II has roles in normal functions of the breast that may be altered or attenuated in cancer. Both angiotensin type 1 (AT1) and type 2 (AT2) receptors are present particularly in the secretory epithelium. Additionally, all the elements of a tissue renin-angiotensin system, angiotensinogen, prorenin and angiotensin-converting enzyme (ACE), are also present and distributed in different cell types in a manner suggesting a close relationship with sites of angiotensin II activity. These findings are consistent with the concept that stromal elements and myoepithelium are instrumental in maintaining normal epithelial structure and function. In disease, this system becomes disrupted, particularly in invasive carcinoma. Both AT1 and AT2 receptors are present in tumours and may be up-regulated in some. Experimentally, angiotensin II, acting via the AT1 receptor, increases tumour cell proliferation and angiogenesis, both these are inhibited by blocking its production or function. Epidemiological evidence on the effect of expression levels of ACE or the distribution of ACE or AT1 receptor variants in many types of cancer gives indirect support to these concepts. It is possible that there is a case for the therapeutic use of high doses of ACE inhibitors and AT1 receptor blockers in breast cancer, as there may be for AT2 receptor agonists, though this awaits full investigation. Attention is drawn to the possibility of blocking specific AT1-mediated intracellular signalling pathways, for example by AT1-directed antibodies, which exploit the possibility that the extracellular N-terminus of the AT1 receptor may have previously unsuspected signalling roles.

Enzyme-linked immunosorbent assay of HER-2/neu gene product (p185) in breast cancer: its correlation with sex steroid receptors, cathepsin D and histologic grades

One hundred and nine primary breast cancers were analyzed to assess the presence of the HER-2/neu gene product (p185), the oestrogen (ER) and the progesterone (PR) receptors, and the total cathepsin D status. An enzyme-linked immunosorbent assay (ELISA kit, Oncogene Science Inc.) was used for the evaluation of p185 in pellets obtained after a 100,000 x g centrifugation, ER and PR were measured by enzyme immunoassay (EIA kit, Abbott Laboratories), and the total cathepsin D content was evaluated by immunoradiometric assay (IRMA kit, CIS Biointernational). We showed that the ELISA kit is feasible to quantify the p185 present in breast cancer cell membranes, and that the detector antibody recognises a protein of apprroximatly M(r) 185,000. The detected antigen was inversely related to both ER and PR, but it did not correlate to total cathepsin D. No significant differences were found in the expression of p185, ER, PR, cathepsin D between infiltrating ductal carcinomas without special features (NOS) and non-ductal (non-NOS) carcinomas. Nevertheless, in NOS carcinomas, a trend was observed in the p185 levels expressed by the tumours with different histological grades, in that p185 concentration was higher in the poorly differentiated grade 3 with respect to grade 2 and grade 1.

Anti-cancer actions of a recombinant antibody (R6313/G2) against the angiotensin II AT1 receptor

Although several tumour types express both AT1 and AT2 angiotensin II receptors, and angiotensin II stimulates cell proliferation, angiotensin-converting enzyme inhibitors and angiotensin receptor blockers are not effective anti-cancer agents. Development of a biologically active monoclonal antibody (6313/G2) against the AT1 receptor prompted the testing of a recombinant short-chain variable fragment form (R6313/G2) against breast cancer cells in vitro and in vivo. Cell lines MCF-7, MDA-MB-231 and T47D all expressed both receptor subtypes. In vitro, R6313/G2 suppressed cell proliferation in the presence of 100 nM angiotensin II, with IC50s of 30 nM, 153 nM and 2.8 microM for the three cell types respectively; in contrast, the AT1 receptor blocker losartan was effective only in T47D cells, at 25 microM. Studies on MCF-7 and T47D cells showed R6313/G2 also opposed the angiotensin II-induced inhibition of caspase-3/7 activity. In vivo, hollow fibres containing the cell lines were implanted in nu/nu balb-c mice at two sites, s.c. and i.p. Treatments of R6313/G2 at 2.5 nmol/kg and 25 nmol/kg twice per day for 7 days dose dependently reduced cell numbers for all three cell lines, but here MCF-7 cells responded most sensitively and MDA-MB-231 cells least. Although T47D cells were refractory at the s.c. site, growth was inhibited at the i.p. location, and otherwise results were similar at the two sites. In xenografts, MCF-7 cell tumours were dose dependently reduced by R6313/G2, and 13 and 27 nmol/kg R6313/G2 twice/day gave means of 74 and 76% tumour regression after 7 days. The data suggest that the anti-cancer action of R6313/G2 is considerably more effective than AT1 antagonists.

The expression of endothelin-1 and endothelin-converting enzyme-1 (ECE-1) are independently regulated in bovine aortic endothelial cells

The endopeptidase called endothelin-converting enzyme-1 (ECE-1) is thought to play a physiological role in endothelin-1 (ET-1) biosynthesis. For human ECE-1, differential splicing of messenger RNA (mRNA) results in the synthesis of three isoforms, termed ECE-1a, ECE-1b, and ECE-1c. The isoform(s) responsible for the hydrolysis of the biosynthetic intermediate big ET-1 in endothelial cells have yet to be assigned. To investigate whether the expression of mRNAs for preproET-1 and ECE-1 are regulated in parallel, a variety of conditions were used to compare levels of ET-1 synthesis by bovine aortic endothelial cells (BAECs) with levels of mRNA for preproET-1, ECE-1a, ECE-1c, and the combined ECE-1 isoforms (ECE-1a/b/c). Stimulation of BAECs with tumor necrosis factor-alpha or transforming growth factor-beta increased ET-1 synthesis, and treatment of BAECs with 2-chloroadenosine or staurosporine caused concentration-dependent reductions in ET-1 synthesis. Estimates of mRNA levels by reverse transcription-polymerase chain reaction (RT-PCR) with linear cycling conditions showed changes in preproET-1 expression to correlate well with ET-1 secretion. In contrast, RT-PCR analysis of ECE-1 expression by using primer pairs to measure ECE-1a, ECE-1c, or all the ECE-1 isoforms simultaneously showed no correlation between their mRNA levels and those of preproET-1. This indicates that under the conditions investigated, expression of ECE-1 is not coordinated with ET-1 synthesis in BAECs.

Glucocorticoid receptors in the euryhaline teleost Anguilla anguilla.

To determine the importance of glucocorticoids in the salt water adaptation of European yellow eel we have evaluated the concentration, affinity and physical properties of glucocorticoid receptors (GR) in gill from both sea water- (SW) and freshwater-adapted (FW) animals. Using ligand binding techniques we demonstrated that high affinity GR were present in both cytosolic and nuclear fractions obtained from whole gill. Isoelectric focusing (IEF) of branchial GR indicated the presence of two distinct species, with pI values of 6.1 and 6.7. The form at pI 6.7 sedimented with a Svedberg constant of 4S on glycerol density gradients while the pI 6. 1 sedimented in fractions corresponding to 9S. Treatment of the pI 6. 1 form with urea (4 M) resulted in generation of the form with pI 6. 7. The evidence thus suggested that the oligomeric urea-sensitive form (pI 6.1) contained a form of GR which, as a monomer, focused at pI 6.7. IEF revealed the same concentrations of the pI 6.7 form in both SW and FW. However, there was significantly more (3-fold) pI 6. 1 isoform in FW than in SW, and this form decreased gradually during the course of seawater transfer. A transient increase of the nuclear-bound GR was also observed during SW adaptation. The balance between these forms could represent a dynamic parameter with important implications regarding GR function and gill responses to cortisol in salt water adaptation in teleosts.

Adrenomedullin acts as a local mediator of vascular homeostasis through interactions which lead to reduced endothelin-1 synthesis and secretion

Adrenomedullin acts as a local mediator of vascular homeostasis through interactions which lead to reduced endothelin-1 synthesis and secretion

Inhibitory effect of adrenomedullin on basal and tumour necrosis factor alpha-stimulated endothelin-1 synthesis in bovine aortic endothelial cells is independent of cyclic AMP.

Adrenomedullin (ADM) is a potent vasodilator and reverses the vasoconstrictor action of endothelin-1 (ET-1). These studies aimed to determine the effect of ADM on ET-1 synthesis in bovine aortic endothelial cells (BAEC) and to identify the possible mechanisms involved. In this cell model, ADM increased cyclic AMP production by BAEC with threshold concentrations of 100 pM and an EC(50) of 1 nM. This effect was not blocked by co-treatment with the CGRP type 1 receptor antagonist CGRP(8--37). ADM caused a potent concentration-dependent inhibition of ET-1 release that was correlated with reduced preproET-1 mRNA levels. This reached a maximal reduction of 70% compared to basal levels after 2 and 6 hr exposure of BAEC to 1 nM ADM, with significant decreases at concentrations as low as 10 pM. However, a 100-fold discrepancy between the threshold ADM concentration for cyclic AMP production and inhibition of ET-1 release was observed. Treatment of BAEC with tumour necrosis factor alpha (TNFalpha; 10 ng/mL) caused a 2-fold increase over basal ET-1 release. ADM caused a more marked reduction in stimulated ET-1 synthesis with a threshold of 1 pM, and suppression of ET-1 release to basal levels at 100 nM. 8-Bromo cyclic AMP, showed no concentration-dependent inhibition of ET-1 release, yet caused a 50% reduction in TNFalpha-stimulated intercellular adhesion molecule-1 (ICAM-1) mRNA levels. Thus, physiological ADM concentrations inhibit ET-1 synthesis independently of cyclic AMP in BAEC at the level of preproET-1 mRNA expression. The high sensitivity of this inhibition implicates ADM as an important physiological regulator of endothelial ET-1 production.