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Henry Wellcome Labs for Integrative Neuroscience and Endocrinology (L.A.N., A.L.), University of Bristol, Bristol BS1 3NY, United Kingdom; Novartis Institutes for BioMedical Research (H.A.S.), Oncology Research, CH-4057 Basel, Switzerland
Address all correspondence and requests for reprints to: Dr. L. Nolan, Henry Wellcome Labs for Integrative Neuroscience and Endocrinology, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, United Kingdom. E-mail: lesley.a.nolan{at}bris.ac.uk.
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Somatotroph adenomas predominantly express SSTR2 and SSTR5 somatostatin receptors. Prolactinomas tend to express SSTR1 and SSTR5, and corticotroph adenomas predominantly express SSTR5 in approximately 50% with low levels of SSTR2 in about one third (8). Most recent reports suggest an even higher percentage of SSTR5 expression in patients with primary Cushing’s disease (9). The presence of SSTR5 receptors on corticotroph adenomas and the numerous pathways of somatostatin action, which include phosphotyrosine phosphatases, potassium and calcium channel modulation, phospholipase C, and adenylyl cyclase second messenger pathways, suggests that somatostatin analogs with modified receptor subtype affinities have the potential to be developed as new treatment modalities for corticotroph adenomas (10).
With few exceptions (4), SSTR1, 2, 3, and 5 have been found to be antiproliferative in endocrinologically inactive pituitary adenoma cells in vitro and in nonendocrine tumors (11, 12). Octreotide, which acts predominantly on SSTR2 receptors, has not proven effective in inhibiting ACTH secretion in patients with Cushing’s disease (9, 10, 13, 14). Nevertheless, the multiligand somatostatin analog pasireotide is potentially of interest in the therapeutics of Cushing’s disease as it has 30–40 times the binding affinity of octreotide at the type 1 and type 5 receptors and occupies the receptors for 11 h compared with 90 min with octreotide (15, 16, 17). In a small study of six human corticotroph adenoma cell samples studied in vitro, pasireotide reduced total cell proliferation measured by quantification of fluorescent vital stain uptake, by between 10–70% after 48–96 h (5), suggesting that a useful therapeutic effect might be anticipated in a subgroup of patients with this condition. However, studies of this nature are difficult to perform and interpret because the amount of tissue available is usually very small and may be contaminated with fragments of normal pituitary. In addition, tumor phenotypes even within the same principal secretory subgroup are highly variable, and almost without exception, human pituitary adenomas do not grow in vitro so, therefore, cannot be used easily as an experimental model of trophic activity (18). Pituitary cell lines such as mouse corticotroph AtT20 cells are equally unhelpful in this context as they are already abnormal from a trophic point of view and isolated from the effects of surrounding cells and normal physiological influences. Use of well-validated in vivo animal models in which mitotic activity in normal anterior pituitary cells can be manipulated reproducibly over a reasonably short time frame is a useful alternative strategy to begin to examine the general antiproliferative effects of somatostatin analogs that might be relevant to their clinical use.
In the present study, we have examined the effects of pasireotide and octreotide on mitotic and apoptotic activity and changes in ACTH labeling index in the male rat pituitary under basal conditions and after surgical bilateral adrenalectomy as a model of nonneoplastic, enhanced trophic activity. This pathophysiological stimulus induces a highly reproducible wave of increased mitotic activity in normal rat pituitary cells that reaches significance within 48 h of surgery before returning to baseline after between 7–14 d (19).
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Materials and Methods |
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Groups of adrenalectomy and sham-operated rats were given sc injections of 30 µg pasireotide (Novartis Pharma AG, Basel, Switzerland) in 0.25 ml saline—a dose toward the higher end of that known to be tolerated without risk of gastrointestinal side effects—at the time of surgery followed by a single daily sc injection of 60 µg pasireotide. Control groups received equal volumes of saline vehicle and were killed with treatment groups 2, 4, and 6 d after surgery. To further define the somatostatin receptor subtype implicated in the mitotic and apoptotic effects of pasireotide, an additional experiment was carried out, in which groups of adrenalectomized and sham-operated rats were treated with either a single injection of pasireotide as above or twice daily injections of 30 µg octreotide (Novartis Pharma AG), an analog with high affinity for SSTR2 but low affinity for SSTR1, 4, and 5. Groups of rats were weighed 5 d postoperatively, and pituitary glands were harvested 6 d after adrenalectomy. Trunk blood was collected in heparinized tubes and centrifuged at 3000 rpm for 10 min. Plasma was stored at –20 C and used to assay plasma ACTH levels with an ACTH immunoradiometric assay kit according to the manufacturer’s instructions (DiaSorin ACTH IRMA; DiaSorin Ltd., Wokingham, UK). As an additional indicator of mitotic rate, all animals were given a single 200 mg/kg of body weight ip injection of bromodeoxyuridine (BrdU; 10 mg/ml in 0.007 M NaOH/0.9% NaCl; Roche, Welwyn Garden City, UK) 24 h before being killed. Rats were not injected on the day they were killed, which occurred between 0800 and 0900 h. All procedures were carried out in accordance with UK Home Office animal welfare regulations.
BrdU and ACTH immunohistochemistry
Standard pituitary tissue sections for trophic analysis and immunohistochemistry were prepared exactly as described (20). Pituitary sections were processed for BrdU immunohistochemistry according to a previously published protocol (21) with minor modifications (22). Briefly, dewaxed and rehydrated sections were transferred to a hot antigen unmasking solution (0.01 M citric acid in water; pH 6.0) and incubated for 10 min in a microwave oven on a power setting that maintained the solution just below its boiling point. Sections were then cooled in water to room temperature before permeabilization for 10 min in 0.001% trypsin (Roche) diluted in 0.1% CaCl2/20 mM Tris buffer (pH 7.5). After three washes in PBS, the sections were denatured in 2 N HCl in PBS for 30 min, washed again in PBS, gently agitated for 30 min in blocking serum (3% normal horse serum, 0.5% Triton X-100 in PBS) and incubated overnight at 4 C with monoclonal anti-BrdU antibody (Becton Dickinson no. 347580; 1/100 diluted in blocking serum; Becton Dickinson, Oxford, UK). Sections were washed in three changes of PBS, incubated for 1 h at room temperature with biotinylated antimouse IgG (Vector Labs, Peterborough, UK; 1/200 diluted in blocking serum), and washed again in fresh PBS before blocking endogenous peroxidases for 30 min with 0.6% (vol/vol) hydrogen peroxide in PBS. After a further three washes in PBS, sections were incubated with R.T.U. Vectastain Elite ABC reagent (PK-7100; Vector Labs) for 1 h at room temperature, rinsed in PBS, and developed for approximately 8 min in diaminobenzidine substrate according to the manufacturer’s instructions (SK-4100; Vector Labs). The resulting brown color reaction was stopped in water, and sections were rinsed three times in PBS, gently agitated for 30 min in blocking serum (3% normal goat serum, 0.5% Triton X-100 in PBS), and incubated overnight at 4 C with either rabbit polyclonal anti-ACTH (National Institute of Diabetes and Digestive and Kidney Diseases; 1/2000 diluted in blocking serum). Sections were washed in three changes of PBS, incubated for 1 h at room temperature with biotinylated antirabbit IgG (Vector Labs; 1/200 diluted in blocking serum), washed in a further three changes of PBS, incubated with R.T.U. Vectastain Elite ABC reagent for 1 h at room temperature, rinsed in PBS, and developed for approximately 2 min in VIP substrate according to the manufacturer’s instructions (SK-4600; Vector Labs). The resulting purple color reaction was stopped in water, and the sections were lightly counterstained with hematoxylin.
Image analysis for trophic activity
Apoptotic and mitotic event prevalence was analyzed on 2-µm-thick hematoxylin-and-eosin-stained pituitary sections at x1000 magnification (19) using a real-time system [AxioHOME Zeiss (23)] that projects an image of a computer screen fractionally above the histological section. Identifier tags placed over manually identified cells and trophic events remain in registration with the targets irrespective of subsequent stage movements and magnification changes. For each animal, three random areas of approximately 47,000 µm2 were scored for the presence of mitotic and apoptotic figures. By defining counting boundaries at low power and counting events at high power, selection bias and double scoring were eliminated, allowing the error in quantifying the number of normal cells surrounding these events to be limited to 2% or less and the overall error in estimating the prevalence of trophic events to be reduced to approximately 0.001%.
The histological characteristics used to define apoptotic cells were clusters of two or more extremely dense round or oval structures varying in diameter from approximately 0.7–4 µm and surrounded by normal cells (24). Earlier stages of apoptosis cannot be visualized using hematoxylin and eosin staining and light microscopy. Results were expressed as a percentage of the total cell numbers counted for each animal.
Immunopositive cell counts were performed at x1000 magnification. For each animal, three areas totaling 0.15 mm2, containing an average of 2000 cells were scored as positive or negative for the presence of immunoreactive BrdU and/or ACTH. In this study, no specific mathematical corrections were made for anticipated increases in corticotroph cell size after adrenalectomy (25). Therefore, changes in corticotroph number are likely to be overestimated by 20–35% (19). All slides were coded and counted by one blinded observer (L.A.N.), and the results expressed as the mean ± SE with differences between groups evaluated using one-way ANOVA followed by Tukey-Kramer multiple comparison post tests. P < 0.05 was considered statistically significant.
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Results |
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). Concurrent administration of either pasireotide or octreotide to adrenalectomized animals caused a further small reduction in body weight over the same time period, as did octreotide alone in sham-operated animals (Fig. 1).
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; n = 8 in all groups). As expected, a numerically small but nevertheless highly significant increase in mitotic activity was observed in adrenalectomized animals treated with vehicle at 2, 4, and 6 d after surgery (0.316 ± 0.027% at d 6; Fig. 2, bottom panel). Pasireotide completely blocked this wave of adrenalectomy-induced increase in mitotic activity, although baseline mitotic activity was not suppressed at any time point. Pasireotide administered to adrenal-intact rats had no effect on mitotic index when compared with intact rats treated with vehicle alone (Fig. 2, bottom panel). There was no measurable effect of pasireotide and/or surgery on the apoptotic index at any time point (Fig. 2, top panel). The effects of octreotide on anterior pituitary trophic activity were indistinguishable from those of pasireotide at the dose, route, and time point studied (Fig. 3).
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, top panel). As pasireotide completely blocked the wave of increased anterior pituitary mitotic activity that normally follows adrenalectomy, these data indicate that differentiation of preexisting ACTH-immunonegative cells rather than cell division is largely responsible for the observed increase in ACTH-immunopositive cells after adrenalectomy. Six days after surgery, the proportion of ACTH-positive cells, although increased compared with values after 4 d, was not significantly different in adrenalectomized rats treated with vehicle, pasireotide, or octreotide (13.55 ± 0.45% vs. 12.96 ± 0.55% vs. 12.79 ± 0.39%, respectively; n = 6). The percentages of ACTH-positive cells in sham-operated animals treated with vehicle or octreotide were not significantly different either from each other (7.33 ± 0.14% vs. 8 ± 0.37%, respectively) or from sham-operated animals treated with vehicle or pasireotide for 4 d.
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, bottom panel). In sham-operated rats treated with vehicle for 4 d and BrdU for 24 h before being killed, the anterior pituitary BrdU-labeling index was 2.07 ± 0.17%. In adrenalectomized animals, the BrdU-labeling index was significantly increased (3.68 ± 0.22%; n = 5–6; P < 0.001; Fig. 4, bottom panel). Concurrent treatment of adrenalectomized rats with pasireotide prevented this increase, but as expected, given data derived by direct quantification of mitotic figures (Fig. 2, bottom panel), pasireotide had no effect on baseline BrdU-labeling index in sham-operated rats (Fig. 4, bottom panel).
The proportion of BrdU-labeled cells that was also immunopositive for ACTH was extremely low in all groups (Fig. 4, bottom panel), again supporting the observation that the observed increase in ACTH labeling index after adrenalectomy does not result from division of differentiated, hormone-producing cells.
The effects of pasireotide and octreotide on plasma ACTH
Six days after surgery, adrenalectomy resulted in a more than 9-fold increase in plasma ACTH compared with controls (P < 0.001) (Fig. 5). Concurrent treatment of adrenalectomized animals with either pasireotide or octreotide suppressed plasma ACTH levels by 41% (P < 0.01) and 34% (P < 0.05) of vehicle-treated controls, respectively (Fig. 5). No suppression of baseline ACTH secretion was seen in sham-operated animals treated with octreotide.
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Discussion |
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The antiproliferative effects of somatostatin analogs may be mediated by direct effects such as cell cycle arrest and induction of apoptosis or as a result of indirect effects on growth factor production or angiogenesis (2). Induction of apoptosis via somatostatin receptors is considered by some to be limited to signaling via SSTR3 as is the case in transfected CHO-K1 (Chinese hamster ovary) cells (29), although in one study using AtT20 cells, octreotide was able to induce apoptosis during the cell cycle (30). No effect of octreotide pretreatment on apoptotic index was found in somatotroph adenoma cells (3), but a reduction in Ki-67 labeling index was found in sections from the same tumor biopsies, suggesting a primarily antiproliferative effect of octreotide on these cells.
Although human corticotroph adenomas express multiple somatostatin receptor subtypes (5, 9) and SSTR2 and SSTR5 agonists have been shown to suppress basal and stimulated ACTH secretion in human corticotroph adenoma cells in vitro (9, 10), octreotide (principally an SSTR2 agonist) has not been shown to inhibit ACTH secretion in Cushing’s disease in vivo (13, 14). Neither has an in vitro effect of octreotide on cell proliferation been identified in either normal or adenomatous human corticotrophs. Pasireotide binds to multiple somatostatin receptors with high affinity (SSTR5 > 2 > 3 > 1) and has a more pronounced inhibitory effect on ACTH secretion from AtT20 mouse corticotroph tumor cells than octreotide (9, 27). It has not been shown to have any effects on AtT20 cell proliferation or rates of apoptosis (9), but a reduction in cell proliferation of from 10–70% in six of six human corticotroph adenoma cell samples maintained in vitro has been observed within the 1–100 nm concentration range (5). As might be expected, the effects of pasireotide on ACTH secretion and cell proliferation appear to be relatively independent (5) in the same way that in human somatotroph adenomas, reduction in GH secretion and tumor shrinkage in response to somatostatin analogs are poorly correlated (3, 6).
After adrenalectomy in the male rat there is a modest increase in the anterior pituitary mitotic index that peaks within 2–6 d of surgery and returns to baseline levels over the next 14 d or so, despite the continued absence of circulating glucocorticoids (19). The transient nature of the trophic response which results principally from glucocorticoid withdrawal at the level of the pituitary (31) contrasts with the sustained ACTH secretory response that is induced by increased CRH stimulation (31). In the current study, we have shown that concurrent treatment with pasireotide or octreotide blocks the adrenalectomy-induced increase in male rat anterior pituitary mitosis but had no measurable effects on basal cell turnover throughout the 6-d time period after surgery.
Previous studies have shown that, after adrenalectomy, most newly formed ACTH cells are derived from differentiation of preexisting hormonally undifferentiated cells and that the same progenitor-like population responds mitotically to both adrenalectomy and gonadectomy (32). In the present study, double immunochemistry with antibodies against BrdU and ACTH was used to quantify the proportion of dividing cells with hormonal identity. Four days after adrenalectomy, the proportion of double-labeled cells remained extremely low irrespective of whether the animals were adrenalectomized or whether they received pasireotide treatment. The very small increase in the proportion of dividing cells that also labeled for ACTH presumably represents rare division in recently differentiated corticotrophs reaching the end of their proliferative potential. The data presented in the current study indicate that, whereas pasireotide suppresses division in a subpopulation of anterior pituitary cells, these cells are not preexisting corticotrophs, neither are they destined to become differentiated corticotrophs in response to adrenalectomy in this rat model.
In light of previously published effects of pasireotide and octreotide on the inhibition of secretion of GH and cell proliferation in human somatotroph adenomas, it has been suggested that their inhibitory effect on cell proliferation in the rat anterior pituitary could be directly mediated through differentiated somatotrophs (2, 6). However, our own unpublished data quantifying the proportion of GH-positive cells in the anterior pituitary at either 2, 4, or 7 d after adrenalectomy show no significant change compared with that found in adrenal-intact controls (26.4 ± 1.34% vs. 31.8 ± 1.66%, respectively, at 4 d postoperatively). These data indicate that the mitotic wave induced by adrenalectomy alone does not occur within the differentiated somatotroph population.
The findings in the present study suggest that basal cell turnover in the rat anterior pituitary is mediated by a small subpopulation of progenitor cells, the activity of which is not influenced by exposure to either pasireotide or octreotide. Daughter cells generated by progenitor cell division that do not undergo early apoptosis, remain hormonally null in the short-term. These nascent cells respond to multiple stimuli including acute reduction in circulating corticosterone levels by further limited division and concurrent somatostatin receptor expression. Cells derived from this pool may undergo apoptosis or differentiate further into new hormone-positive cells, which presumably include corticotrophs. From the present study and others, it seems likely that the differentiation pathway is unaffected by octreotide or pasireotide. The very small increase in BrdU/ACTH double-labeled cells after adrenalectomy occurs in recently differentiated corticotrophs that are approaching the end of their capacity to divide. They represent an extremely small fraction of the BrdU-only-labeled cells and the ACTH-only-labeled cells. In this model, somatostatin analogs only have a small window of opportunity to influence cell proliferation and can only do so during limited and specific periods of cell cycling. In summary, surgical adrenalectomy in the male rat stimulates cell division in a specific subpopulation of hormonally null cells that are equally sensitive to the antiproliferative effects of pasireotide and octreotide, suggesting that this effect is primarily mediated by the SSTR2 receptor subtype. In line with these data is the clinical observation that patients with Nelson’s disease but not primary pituitary-dependent Cushing’s show responsiveness to octreotide (33). Basal cell turnover, apoptotic index, and division and differentiation of ACTH-positive cells in the anterior pituitary are unaffected by treatment with either of these somatostatin receptor agonists used in the present study.
In conclusion, the presence and amplitude of ACTH antisecretory and antiproliferative responses to the somatostatin analogs pasireotide and octreotide depends on current and recent changes in anterior pituitary progenitor cell hormonal microenvironment. Base line anterior pituitary cell turnover is unaffected by pasireotide and octreotide, but progenitor cell mitotic activity induced by corticosterone withdrawal is blocked by these somatostatin analogs. Basal ACTH secretion is unaffected by pasireotide and octreotide, but the secretory response to bilateral adrenalectomy is greatly reduced.
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Acknowledgments |
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Footnotes |
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Abbreviation: BrdU, Bromodeoxyuridine.
We would like to acknowledge financial support from Novartis and The Wellcome Trust.
Disclosure Statement: L.A.N and A.L. received grant support (January 2006 to February 2007) from Novartis Pharma. H.A.S. is employed by Novartis Pharma.
Received December 20, 2006.
Accepted for publication February 27, 2007.
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-subunit and chromogranin A secretion and on cell viability in human non-functioning pituitary adenomas in vitro. J Clin Endocrinol Metab 89:5181–5188This article has been cited by other articles:
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  A. Ben-Shlomo, H. Schmid, K. Wawrowsky, O. Pichurin, E. Hubina, V. Chesnokova, N.-A. Liu, M. Culler, and S. Melmed Differential Ligand-Mediated Pituitary Somatostatin Receptor Subtype Signaling: Implications for Corticotroph Tumor Therapy J. Clin. Endocrinol. Metab., November 1, 2009; 94(11): 4342 - 4350. [Abstract] [Full Text] [PDF]
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