1-Adrenergic Receptor Antagonists: Novel Therapy for Pituitary Adenomas
Manory A. Fernando and
Anthony P. Heaney
Division of Endocrinology, Cedars-Sinai Research Institute-Geffen School of Medicine at University of California at Los Angeles, Los Angeles, California 90048
Address all correspondence and requests for reprints to: Anthony Heaney, Division of Endocrinology, B-127, Cedars-Sinai Medical Center, 8700 Beverly Boulevard, Los Angeles, California 90048. E-mail: HeaneyA{at}cshs.org.
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ABSTRACT
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Pituitary tumors are common and cause considerable morbidity due to local invasion and altered hormone secretion. Doxazosin (dox), a selective
1-adrenergic receptor antagonist, used to treat hypertension, also inhibits prostate cancer cell proliferation. We examined the effects of dox on murine and human pituitary tumor cell proliferation in vitro and in vivo. dox treatment inhibited proliferation of murine pituitary tumor cells, induced G0-G1 cell cycle arrest, and reduced phosphorylated retinoblastoma levels. In addition, increased annexin-fluorescein isothiocyanate immunoreactivity and cleaved caspase-3 levels, in keeping with dox-mediated apoptosis, were observed in the human and murine pituitary tumor cells, and dox administration to mice, harboring corticotroph tumors, decreased tumor growth and reduced plasma ACTH levels. dox-mediated antiproliferative and proapoptotic actions were not confined to
-adrenergic receptor-expressing pituitary tumor cells and were unaffected by cotreatment with the
-adrenergic receptor blocker, phenoxybenzamine. dox treatment led to reduced phosphorylated inhibitory
B (I
B)-
expression, and nuclear factor-
B transcription and decreased basal and TNF
-induced proopiomelanocortin transcriptional activation. These results demonstrate that the selective
1-adrenergic receptor antagonist dox inhibits pituitary tumor cell growth in vitro and in vivo by mechanisms that are in part independent of its
-adrenergic receptor-blocking actions and involve down-regulation of nuclear factor-
B signaling. dox is proposed as a possible novel medical therapy for pituitary tumors.
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INTRODUCTION
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PITUITARY TUMORS ACCOUNT for 1020% of intracranial neoplasms, are generally slow growing, and rarely metastasize (1). However they cause considerable morbidity due to hormonal hypersecretion and destruction of local structures leading to visual defects and hypopituitarism (2). The goals of pituitary tumor treatment are ablation or reduction of tumor size, normalization of excess pituitary hormones, and prevention of tumor recurrence with preservation of normal pituitary function (3). Medical treatment options for prolactin (PRL)- and GH-secreting tumors such as dopamine agonists and somatostatin analogs control PRL and GH secretion effectively in 75% and 5075% of patients, respectively (4, 5). However, currently there is no suitable medical therapy for ACTH-secreting and nonfunctioning pituitary tumors (2, 6), and the main treatment option is surgery (7, 8). Pituitary tumor surgery leads to remission in approximately 8090% of microadenomas (tumors
1 cm diameter) but depends heavily on the experience of the surgeon (7). Initial cure rates in macroadenomas (tumors
1 cm diameter) are only about 50%, and multiple surgeries or pituitary irradiation may then be necessary but cause optic nerve damage and /or pituitary failure in approximately 50% of patients (9, 10, 11).
Adrenergic receptors belong to the G protein-coupled receptor superfamily, include the
1,
2, ß1, ß2, and ß3 receptor families, and are important in sympathetic nervous system activity (12).
1-Adrenergic receptors are further subdivided into
1A,
1B, and
1D subtypes (13), and antagonists include quinazoline-based prazosin, doxazosin (dox), and terazosin and the sulfonamide derivative tamsulosin. They are used to treat systemic hypertension (14) but have also been reported to inhibit growth and induce apoptosis in prostate cancer and reduce prostate-specific antigen levels (15, 16).
We demonstrate that the selective
1-adrenergic receptor antagonist, dox, potently inhibits human and murine somatolactotroph, corticotroph, and gonadotroph pituitary tumor cell proliferation and induces pituitary tumor cell apoptosis in vitro. In vivo, dox reduces murine corticotroph AtT20 pituitary tumor cell growth and suppresses plasma ACTH levels. dox-mediated antiproliferative and proapoptotic effects are not dependent on
1-adrenergic receptor expression, but involve down-regulation of nuclear factor-
B (NF
B) signaling. These results support the potential use of dox as a possible novel therapy for patients with pituitary adenomas.
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RESULTS
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The
1-Adrenergic Receptor Antagonist, dox, Inhibits Pituitary Tumor Proliferation
The effects of the
1-adrenergic receptor antagonist dox on murine pituitary tumor cell proliferation were examined in vitro using a [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) cell proliferation assay in which a lower absorbance value indicates reduced cell proliferation. After dox treatment, murine LH-secreting, LßT2 pituitary tumor cell proliferation was inhibited, after 24 h treatment (vehicle, 0.34 ± 0.005 vs. 10 µM dox, 0.32 ± 0.004; 20 µM dox, 0.27 ± 0.003; 30 µM dox, 0.20 ± 0.001; mean ± SEM; P < 0.05). After single-dose treatment the dox-mediated proliferation inhibition was also evident at 48 h (vehicle, 0.41 ± 0.005 vs. 10 µM dox, 0.39 ± 0.005; 20 µM dox, 0.22 ± 0.001; 30 µM dox, 0.21 ± 0.003; mean ± SEM; P < 0.001), and became most pronounced after 72 h treatment (vehicle, 0.48 ± 0.007 vs. 10 µM dox, 0.45 ± 0.005; 20 µM dox, 0.27 ± 0.003; 30 µM dox, 0.24 ± 0.002; mean ± SEM; P < 0.01) (Fig. 1A
). Similar findings were observed after dox treatment of murine ACTH-secreting corticotroph AtT20 pituitary tumor cells, after 24 h treatment (vehicle, 0.26 ± 0.003 vs. 10 µM dox , 0.19 ± 0.001; 20 µM dox, 0.06 ± 0.002; 30 µM dox, 0.01 ± 0.001; mean ± SEM; P < 0.001), which again persisted after 48 h (vehicle, 0.42 ± 0.01 vs. 10 µM dox, 0.31 ± 0.01; 20 µM dox, 0.02 ± 0.002; 30 µM dox, 0.02 ± 0.002; mean ± SEM; P < 0.001), and after 72 h dox treatment (vehicle, 0.92 ± 0.029 vs. 10 µM dox, 0.55 ± 0.016; 20 µM dox, 0.02 ± 0.003; 30 µM dox, 0.02 ± 0.002; mean ± SEM; P < 0.001) (Fig. 1B
).

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Fig. 1. dox Inhibits Pituitary Tumor Cell Proliferation
After dox treatment (1030 µM) of gonadotroph LßT2 cells (A) and corticotroph AtT20 pituitary tumor cells (B) for 24, 48, and 72 h, cell proliferation was measured using MTS cell proliferation assay. dox treatment decreased proliferation of gonadotroph LßT2 and corticotroph AtT20 cells. (*, P < 0.05; **, P < 0.001). V, Vehicle.
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dox Treatment Leads to G0-G1 Cell Cycle Arrest
To determine whether the observed reduced pituitary tumor cell proliferation rates were due to cell cycle arrest and /or increased apoptosis, flow cytometric analysis was next performed on the dox-treated cells. dox treatment of murine corticotroph AtT20 cells for 24 h led to G0-G1 cell cycle arrest (vehicle G1, 68 ± 0.24 vs. 10 µM dox G1, 72 ± 1.4; 15 µM dox G1, 79 ± 3.8; 20 µM dox G1, 85 ± 1.5; 25 µM dox G1, 96 ± 3.8; mean ± SEM; P < 0.05) and decreased the number of cells in the synthesis phase (S phase) (% S phase: vehicle, 25 ± 0.45 vs. 10 µM dox, 22 ± 0.94; 15 µM dox, 16 ± 3.4; 20 µM dox, 10 ± 1.5; 25 µM dox, 2 ± 1.9; mean ± SEM; P < 0.01) (Fig. 2A
). Similarly, dox treatment of rat somatolactotroph GH3 cells for 48 h led to a G0-G1 cell cycle arrest (vehicle G1, 57 ± 0.96 vs. 20 µM dox G1, 63 ± 1.8; 30 µM dox G1, 72 ± 2.6; mean ± SEM; P < 0.01) and also decreased the population of cells in S phase (% S phase: vehicle, 36 ± 0.8 vs. 20 µM dox, 31 ± 0.7; 30 µM dox, 18 ± 1.0; mean ± SEM; P < 0.001) (Fig. 2B
), confirming that dox treatment induced cell cycle arrest.

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Fig. 2. dox Induces Pituitary Tumor Cell Cycle Arrest
Representative fluorescent activated cell sorting analysis of murine corticotroph AtT20 cells (A) (1025 µM, 24 h), and rat somatolactotroph GH3 cells (B), after dox treatment (2030 µM, 48 h) showed dose-dependent increase in G0-G1 phase and decreased S phase population of cells. (*, P < 0.01; **, P < 0.001). V, Vehicle.
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Western blot analysis of protein extracts derived from dox-treated GH3 cells revealed decreased phosphorylated retinoblastoma (pRb) (vehicle, 1.0 vs. 5 µM dox, 0.81 ± 0.03; 20 µM dox, 0.36 ± 0.36; 30 µM dox, 0.07 ± 0.002; mean ± SEM; P < 0.05) and proliferating cell nuclear antigen (PCNA) expression (Fig. 3A
). This effect was most evident at 30 µM dox, but was also seen after lower dose (5 µM) dox treatment. dox treatment also led to decreased pRb expression in murine corticotroph AtT20 pituitary tumor cells (vehicle, 1.0 vs. 5 µM dox, 0.82 ± 0.16; 10 µM dox, 0.45 ± 0.12; 15 µM dox, 0.25 ± 0.14; 20 µM dox, 0.16 ± 0.16; 25 µM dox, 0.0056 ± 0.006; 30 µM dox, 0.0004 ± 0.000; mean ± SEM; P < 0.01) and in gonadotroph LßT2 pituitary tumor cells (vehicle, 1.0 vs. 15 µM dox, 0.87 ± 0.01; 20 µM dox, 0.63 ± 0.2; 25 µM dox, 0.73 ± 0.1; 30 µM dox, 0.04 ± 0.039; mean ± SEM; P < 0.01) (Fig. 3
, B and C). Furthermore, in murine gonadotroph LßT2 cells, dox treatment led to increased expression of the cyclin-dependent kinase inhibitor p27 (Fig. 3D
), providing a potential mechanism by which dox regulates gonadotroph tumor pRb expression. In support of our findings in the murine pituitary tumor cells, dox treatment (1020 µM, 48 h) of individual human nonfunctioning (Fig. 4A
) and corticotroph (Fig. 4B
) pituitary tumor cultures led to a significant decrease in pRb and PCNA expression. The observed decrease in pRb and PCNA in human and murine pituitary tumor cells confirms the antiproliferative effect of dox and provides a mechanism for the observed G0/G1 cell cycle arrest. Similar inhibitory effects on pRb expression were seen with prazosin, another quinazoline based
1-adrenergic receptor antagonist (data not shown).

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Fig. 3. dox Decreases Rb Protein Phosphorylation
Western blot analysis of dox-treated (530 µM) GH3 cells (A) showed decreased pRb and decreased PCNA. dox-treated (530 µM) murine corticotroph AtT20 (B) and gonadotroph LßT2 (C) cells also revealed decreased pRb and increased expression of p27 (D), in keeping with dox-mediated inhibition of pituitary tumor cell proliferation. ß-Actin confirmed equal protein loading. (*, P < 0.05; **, P < 0.01; ***, P < 0.001). V, Vehicle.
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Fig. 4. dox Inhibits Human Pituitary Tumor Cell Proliferation
Western blot analysis of dox-treated human nonfunctioning (A), and ACTH secreting pituitary tumors (B), revealed decreased pRb and decreased PCNA levels. Ponceau S staining confirmed equal protein loading. V, Vehicle.
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dox Induces Pituitary Tumor Apoptosis in Vitro
We next used annexin fluorescence to examine dox-mediated effects on pituitary tumor cell survival. dox treatment (1030 µM, 48 h) of rat somatolactotroph GH3 cells (Fig. 5A
) led to a dose-dependent increase in annexin-positive cells (% apoptotic cells: GH3 cells, vehicle, 17 ± 1.5 vs. 10 µM dox, 18 ± 0.8; 20 µM dox, 28 ± 4.0; dox 30 µM, 67 ± 4.0; mean ± SEM, P < 0.001). A dose-dependent increase in apoptosis was also observed after dox treatment of murine gonadotroph LßT2 cells (Fig. 5B
), (% apoptotic cells: vehicle, 9 ± 0.3, vs. 10 µM dox, 13 ± 0.8; 15 µM dox, 15 ± 0.4; 20 µM dox, 28 ± 1.0; 25 µM dox, 39 ± 2.7; 30 µM dox, 63 ± 4.6; mean ± SEM; P < 0.001). Similar results were observed with prazosin treatment of murine pituitary tumor cells (data not shown). dox treatment of six individual human pituitary tumor primary cultures (one GH-, one ACTH-, one PRL-secreting, and three nonfunctioning tumors) (Fig. 5C
) also led to increased apoptosis (apoptosis fold increase: vehicle, 1.0 vs. 10 µM dox, 1.6 ± 0.4; 20 µM dox, 2.5 ± 0.8; mean ± SEM; P < 0.01) in comparison with vehicle-treated cells, demonstrating that dox induced programmed cell death in addition to inhibiting pituitary tumor cell proliferation.

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Fig. 5. dox Induces Pituitary Tumor Cell Apoptosis
dox Treatment (1030 µM) for 48 h showed increased annexin staining in a dose-dependent manner in rat somatolactotroph GH3 cells (A), murine gonadotroph LßT2 cells (B), and human pituitary tumor cells (C), (n = 6). (*, P < 0.01; **, P < 0.001). V, Vehicle.
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To examine the mechanism of pituitary tumor apoptosis, Western blot analysis was performed on protein extracts derived from the dox-treated cells. In GH3 cells an approximately 40-fold increase in cleaved caspase-3 expression was observed (cleaved caspase-3 fold increase: vehicle, 1.0 vs. 10 µM dox, 1.1 ± 0.1; 20 µM dox, 2.2 ± 1.1; 25 µM dox, 6.5 ± 1.2; 30 µM dox, 39.7 ± 7.8; mean ± SEM; P < 0.001) (Fig. 6A
), and an approximately 13-fold induction of cleaved caspase-3 was seen in dox-treated AtT20 cells (Fig. 6B
) (cleaved caspase-3 fold increase: vehicle, 1.0 vs. 5 µM dox, 0.6 ± 1.4; 10 µM dox, 1.6 ± 0.3; 20 µM dox, 5 ± 0.2; 25 µM dox, 12.5 ± 0.4; 30 µM dox, 13.4 ± 1.1; mean ± SEM, P < 0.001) (Fig. 6B
). Similar findings were observed in dox-treated
T3 cells where an approximately 4-fold increased apoptosis was observed even after low-dose (5 µM) dox treatment (cleaved caspase-3 fold increase: vehicle, 1.0 vs. 5 µM dox, 4.1 ± 0.2; 20 µM dox, 6.9 ± 0.4; mean ± SEM; P < 0.01) (Fig. 6C
), and LßT2 pituitary tumor cells (cleaved caspase-3 fold increase: vehicle, 1.0 vs. 15 µM dox, 7 ± 2.1; 20 µM dox, 16 ± 4.1; 25 µM dox, 16 ± 3.5; mean ± SEM; P = 0.054) (Fig. 6D
), confirming caspase-3 involvement in dox-mediated pituitary tumor apoptosis.

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Fig. 6. dox Induces Apoptosis of Pituitary Tumor Cells by Activation of Caspase-3
Western blot analysis of dox-treated pituitary tumor cell extracts demonstrated an increase in active cleaved caspase-3 and a concordant decrease in uncleaved total caspase-3 in rat somatolactotroph GH3 cells (A), murine corticotroph AtT20 (B), gonadotroph T3 (C) and LßT2 cells (D) (*, P < 0.05; **, P < 0.01). V, Vehicle; Casp-3, caspase-3.
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The
1-Adrenergic Receptor Antagonist, dox, Inhibits in Vivo Pituitary Tumor Growth and Reduces Plasma ACTH Levels
As dox potently inhibited pituitary tumor cell proliferation and induced apoptosis in vitro, we next examined the effects of dox treatment on murine corticotroph pituitary tumor growth in vivo. Murine corticotroph AtT20 pituitary tumor cells (
200,000) were inoculated sc in 4-wk-old female athymic nude mice (n = 10). Animals were then randomized to receive either dox treatment (5 mg/kg·d) (n = 5) or vehicle alone (n = 5). This in vivo dose is approximately equivalent to 9 µM in vitro. dox was administered, dissolved in sterile water (100 µl) by daily oral gavage feeding using a 24-gauge gavage needle. One animal in the dox-treated group died at 2 wk after gavage complications. By 2 wk, three of the remaining four dox-treated animals and all five vehicle-treated animals developed visible tumors and became debilitated, necessitating their euthanization at 4 wk. Tumor weights were lower in the dox-treated animals compared with vehicle-treated mice (vehicle, 0.28 ± 0.07 g vs. dox, 0.09 ± 0.04 g; P = 0.03) (Fig. 7B
). In addition, plasma ACTH levels were lower in the dox-treated tumor bearing mice compared with vehicle-treated animals (vehicle, 2195 ± 445 pg/ml vs. dox, 902 ± 180 pg/ml; P = 0.03) (Fig. 7C
). These results demonstrate the potent antiproliferative and antihormonal effects of dox on murine corticotroph pituitary tumor cell growth in vivo.

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Fig. 7. dox Treatment Inhibits Pituitary Corticotroph Tumor Growth in Vivo
Athymic nude mice were inoculated sc with murine corticotroph AtT20 pituitary tumor cells ( 200,000). Animals were randomized to receive dox (5 mg/kg·d) (n = 4) or vehicle (n = 5) treatment. A representative photograph of vehicle and dox-treated mice is shown in panel A. Tumor weights (B) and serum ACTH levels (C) were lower in dox-treated mice (*, P < 0.03).
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dox-Mediated Antiproliferative and Proapoptotic Actions Are Independent of the
1-Adrenergic Receptor
To elucidate the potential mechanism(s) of dox-mediated antiproliferative and proapoptotic actions, we first examined
1-adrenergic receptor mRNA expression in murine pituitary tumor cell lines and in six human pituitary tumors by RT-PCR.
1A-Adrenergic receptor mRNA expression was demonstrated in murine gonadotroph
T3 pituitary tumor cells but was not detectable in murine gonadotroph LßT2, corticotroph AtT20, or somatolactotroph GH3 cells.
1B-Adrenergic receptor mRNA was demonstrated in murine gonadotroph LßT2 and
T3 pituitary tumor cells but was undetectable in GH3 and AtT20 pituitary tumor cells (Fig. 8A
). In all six human pituitary tumor samples (one GH, ACTH, and PRL and three NF) examined,
1A and
1B adrenergic receptor mRNA was demonstrated (Fig. 8B
). The endothelial cells and stromal components in the human pituitary tumor specimens may account for the observed
1-adrenergic receptor expression. In addition, due to the limited sensitivity of RT-PCR, some
1-adrenergic receptor expression in GH3 and AtT20 pituitary tumor cell lines cannot be totally excluded. Nonetheless, these results suggested that dox-induced antiproliferative and proapoptotic effects were not restricted to pituitary tumor cells that expressed the
1-adrenergic receptor. Cotreatment of gonadotroph pituitary tumor LßT2 cells and
T3 cells, which expressed the
1A- and/or
1B-adrenergic receptor with dox and the irreversible
-adrenergic receptor antagonist phenoxybenzamine, did not abrogate the antiproliferative (Fig. 8C
) and proapoptotic actions of dox (Fig. 8
, D and E), providing further evidence that dox actions are not
-adrenergic receptor mediated.
The transcription factor, NF
B, induces cell cycle progression, inhibits apoptosis, and is inactivated by hypophosphorylated inhibitory
B (I
B)-
(17). In murine gonadotroph LßT2 cells, dox treatment (2030 µM, 48 h) led to decreased levels of phosphorylated I
B-
expression. dox-mediated reduction in phosphorylated I
B-
was not abrogated in these
1-adrenergic receptor-expressing LßT2 cells by cotreatment with dox and the irreversible
-adrenergic receptor antagonist phenoxybenzamine (1 µM, 48 h) (Fig. 9A
). Similarly, dox treatment decreased phosphorylated I
B-
expression in corticotroph AtT20 pituitary tumor cells (vehicle, 1.0 vs. 5 µM, 0.6 ± 0.1; 25 µM dox, 0.55 ± 0.03; 30 µM dox, 0.1 ± 0.04; mean ± SEM, P < 0.05), (Fig. 9B
). These observations suggested that dox, in part, mediates its antiproliferative and proapoptotic pituitary tumor effects independently of the
-adrenergic receptor and, at least in part, via down-regulation of NF
B signaling pathways.
To investigate this further, AtT20 cells were transiently transfected with an NF
B-luciferase reporter plasmid and treated with dox (1530 µM, overnight). dox treatment led to an approximately 4060% decrease in NF
B luciferase activity in comparison with vehicle-treated cells (NF
B-luc fold change: vehicle, 1.0 vs. 15 µM dox, 0.61 ± 0.04; 20 µM dox, 0.57 ± 0.13; 25 µM dox, 0.32 ± 0.09; 30 µM dox, 0.37 ± 0.12; mean ± SEM, P < 0.05) (Fig. 9C
). As expected, TNF
treatment (50 ng/ml, 2 h) induced NF
B reporter activity approximately 10-fold (18). Pretreatment of the corticotroph NF
B transfectants with dox (1530 µM, overnight) abrogated TNF
(50 ng/ml, 2 h)-induced corticotroph NF
B transactivation in a dose-dependent manner (NF
B-luc fold change: vehicle, 1.0 vs. TNF
, 10.0 ± 2.6; TNF
+ 15 µM dox, 7.7 ± 2.0; TNF
+ dox 20 µM, 3.2 ± 1.6; TNF
+ dox 25 µM, 2.2 ± 0.8; TNF
+ dox 30 µM, 1.2 ± 0.2; mean ± SEM; P < 0.05) (Fig. 9D
). The rat proopiomelanocortin (POMC) promoter contains an NF
B-responsive region between 141 and 151 upstream of the transcription initiation site. To determine whether our observed dox-mediated actions on NF
B might also impact POMC transcription, a rat POMC promoter luciferase reporter (480 to + 63) was transiently transfected into the AtT20 cells, and POMC-luc transfectants were treated with dox. As depicted, dox treatment (1530 µM overnight) decreased POMC-luciferase activity by approximately 60% (POMC-luc fold change: vehicle, 1.0 vs. 15 µM dox, 0.39 ± 0.10; 20 µM dox, 0.23 ± 0.05; 25 µM dox, 0.28 ± 0.06; 30 µM dox, 0.16 ± 0.04; mean ± SEM, P < 0.001) (Fig. 9E
). Furthermore, TNF
(50 ng/ml, 2 h)-induced POMC-luciferase activity was abrogated by pretreatment of the POMC-luciferase transfectants with dox (1530 µM, overnight) (POMC-luc fold change: vehicle, 1.0 vs. TNF
, 1.5 ± 0.1; TNF
+ 15 µM dox, 0.64 ± 0.04; TNF
+ 20 µM dox, 0.28 ± 0.02; TNF
+ 25 µM dox, 0.17 ± 0.01; TNF
+ 30 µM dox, 0.05 ± 0.001; mean ± SEM; P < 0.001) (Fig. 9F
), indicating that dox-mediated POMC transcriptional inhibition is due, in part, to reduced NF
B-mediated POMC regulation.
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DISCUSSION
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Pituitary tumors are common, mostly benign, and slow growing (1, 2, 3). Although surgery in specialized centers offers cure rates in excess of 80% for most microadenomas, patients with macroadenomas often require repeated pituitary surgeries, or pituitary irradiation that may not be effective for years (7, 8, 10, 11). The latter commonly causes hypopituitarism, occasional optic tract dysfunction, and, rarely, secondary brain tumor (9, 19). The United States prevalence of ACTH-secreting and nonfunctioning pituitary tumors is approximately 10,000 and 25,000, respectively [Central Brain Tumor Registry of U.S. (CB Trust); http://www.cbtrust.org]. No safe and efficacious medical therapy that inhibits tumor growth and hormone production currently exists for these patients (2, 21, 22), and such a treatment would be extremely valuable. We show here that the
1-adrenergic receptor antagonist dox decreased human and murine pituitary tumor cell proliferation and increased pituitary tumor cell apoptosis in vitro. dox treatment also reduced murine corticotroph pituitary tumor growth in vivo, supporting a potential role for dox in inducing regression and inhibiting regrowth of pituitary tumors.
Cell proliferation and G0/G1 to S phase transition is a highly regulated process involving many cell cycle-specific proteins (23), and retinoblastoma (Rb) protein plays a key role as a suppressor of cell growth (24, 25). Although, Rb loss or mutation is an infrequent finding in human pituitary tumors (26), some studies have reported reduced pituitary tumor pRb expression (27), and heterozygous Rb +/ mice develop
-MSH-secreting intermediate lobe pituitary tumors (28, 29). dox treatment of human and murine pituitary tumor cells decreased Rb phosphorylation in association with tumor cell cycle arrest. In addition, increased p27 was observed in gonadotroph LßT2 cells after dox treatment, underpinning the role of Rb in dox-mediated antiproliferative actions.
An optimal medical therapy for pituitary tumors would not only inhibit pituitary tumor cell proliferation, but also induce regression of existing tumor mass (3). dox treatment increased human and murine pituitary tumor cell apoptosis, and increased expression of the apoptotic enzyme cleaved caspase-3 in murine pituitary tumor cells. However, although in vitro induction of cleaved caspase-3 was most marked after higher dox doses (2530 µM), lower dox doses (515 µM) led to cleaved caspase-3 induction in gonadotroph LßT2 and
T3 tumor cells, suggesting differential pituitary tumor cell subtype sensitivity to some of dox-mediated antitumor actions. Notably, the gonadotroph tumor cells exhibit
1A and/or
1B adrenergic receptor expression, and it is interesting to speculate that dox-mediated
1-adrenergic receptor-dependent and -independent actions may converge to augment some of the antiproliferative actions of this drug.
Although the exact mechanism(s) of dox-mediated pituitary antiproliferative and proapoptotic effects are unclear, several lines of evidence suggest they are, in part, not mediated via the
1-adrenergic receptor. First, we observed dox-mediated antiproliferative and proapoptotic effects in GH3 and AtT20 pituitary tumor cells that did not exhibit
1-adrenergic receptor expression. In addition, cotreatment of
1-adrenergic receptor-expressing pituitary tumor cells with the irreversible
-adrenergic receptor inhibitor, phenoxybenzamine, did not abrogate dox-mediated antiproliferative and proapoptotic effects.
Several alternate pathways have been proposed for dox-mediated antiproliferative and apoptotic effects, including induction of TGFß signaling (30, 31) and inhibition of NF
B signaling by induction of its inhibitor I
B-
(32), and we have demonstrated decreased phosphorylated I
B-
expression in murine gonadotroph LßT2 and corticotroph AtT20 pituitary tumor cells after dox treatment. NF
B is a key transcription factor that induces expression of multiple target genes involved in cell proliferation, tumor invasion, and metastasis including c-Myc, cyclin D1, and vascular endothelial growth factor (17). We have also demonstrated that dox decreases basal and TNF
-induced NF
B transcription in a dose-dependent manner, indicating that dox down-regulates NF
B signaling. Furthermore, dox treatment of corticotroph AtT20 cells led to lower basal and TNF
-induced POMC transcription levels, and we speculate that dox inhibits NF
B-mediated POMC transcription to mediate its antihormonal effects. Murine corticotroph AtT20 cells appeared more sensitive to dox-mediated antiproliferative and cell cycle-inhibitory effects (Figs. 1
and 2
) than other pituitary tumor subtypes. Although the reasons for this are unclear, we speculate this may be partly explained by dox-mediated actions to inhibit NF
B-regulated corticotroph tumor cell growth.
Furthermore, in vivo, dox treatment reduced murine corticotroph AtT20 pituitary tumor cell growth in mice and led to lower plasma ACTH levels in tumor-bearing animals compared with vehicle-treated animals, indicating its potential utility to inhibit pituitary tumor hormonal excess as well as inhibit tumor growth.
dox Is used to treat hypertension and benign prostate hyperplasia and is well tolerated in most patients (13, 33), with minimal side effects of dizziness, headache, and fatigue. Furthermore, dox administration to normotensive patients does not significantly decrease blood pressure (34). The established safety and efficacy of dox, along with its potent antiproliferative and proapoptotic effects, make it an attractive potential therapy for pituitary adenomas, particularly because we observed inhibition of pituitary tumor cell proliferation with similar or lower dox concentrations than those previously shown to inhibit prostate cancer growth. Furthermore, dox treatment with approved doses reduced growth and induced apoptosis in patients with prostatic hyperplasia (14, 15, 33). The results presented here indicate the potential utility of dox treatment in pituitary tumors not only to inhibit tumor growth and induce tumor regression but also to inhibit cytokine-mediated corticotroph tumor hormone secretion.
In conclusion, because dox is a potent inhibitor of pituitary tumor proliferation and induces apoptosis in pituitary tumors, it is proposed as a possible novel, safe, and efficacious medical therapy for treating pituitary adenomas.
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MATERIALS AND METHODS
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Pituitary Tumor Tissues
Fresh consecutive surgically resected pituitary tumors were obtained in accordance with Cedars-Sinai Institutional Review Board guidelines. For RT-PCR, total RNA was extracted from tissue aliquots using TRIZOL reagent (Invitrogen Corp., Carlsbad, CA). For pituitary primary cultures, pituitary tumors were mechanically dispersed and enzymatically digested for 30 min at 37 C with 10 mg collagenase and 1 mg hyaluronidase (Sigma, St. Louis, MO) in 10 ml DMEM serum-free medium. Pituitary tumor cells were then cultured for 24 h in DMEM with 10% serum before treatment with vehicle or dox (1020 µM) for 4872 h. Lyophilized dox was provided by Pfizer, Inc. (New York, NY) and solubilized in dimethylsulfoxide at 10%; final concentrations were
0.03%.
PCR for
1-Adrenergic Receptors
Total RNA (3 µg) from human pituitary tumor tissue and pituitary tumor cell lines were treated with DNAse I, at 37 C for 30 min, and reverse transcribed using SuperScript First-Strand Synthesis system for RT-PCR (Invitrogen Corp.). Rat and human
1-adrenergic receptor-specific oligonucleotide primers were used to amplify
1-adrenoreceptor sequences using PCR as previously described (35, 36). PCR for 18S ribosomal RNA was performed as an internal control on both reverse transcriptase-positive and -negative samples to confirm cDNA product integrity. Aliquots of the PCR products were electrophoresed on 1% agarose gels and stained with ethidium bromide to visualize PCR products.
Cell Culture
Subconfluent rat somatolactotroph GH3 cells, murine corticotroph AtT20 cells (American Type Culture Collection, Manassas, VA) and murine gonadotroph LßT2 cells and
T3 cells (gifts from P. Mellon, University of California San Diego, San Diego, CA) were cultured in DMEM/F12 or DMEM medium (Invitrogen Corp.) supplemented with 15% horse serum and 2.5% fetal bovine serum (GH3) or 10% fetal bovine serum (AtT20, LßT2,
T3) and antibiotics at 37 C in 5% CO2 for 24 h before treatment with dox (530 µM).
Cell Proliferation Assay
Murine gonadotroph LßT2 cells and corticotroph AtT20 cells (
3000) were plated onto 96-well plates and treated with dox (1030 µM) for 2472 h or dox (2030 µM) plus the irreversible
-adrenergic receptor antagonist phenoxybenzamine (15 µM) for 48 h (Sigma). Phenoxybenzamine was added 30 min before dox in dox plus phenoxybenzamine cotreatment experiments. After treatment, cells were incubated with One Solution (containing tetrazolium compound MTS and phenazine ethosulfate reagent) at 37 C for 2 h. Absorbance (490 nm), which correlates closely with cell proliferation, was then measured using CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS), according to manufacturers instructions (Promega Corp., Madison, WI).
Cell Cycle Distribution
After dox treatment, rat GH3 cells were trypsinized, centrifuged (1500 x g for 5 min), washed in PBS, and treated with RNase A (10 U/sample). DNA was stained with 50 µg/ml propidium iodide for 1 h at room temperature and protected from light before analysis with a FACScan (Becton Dickinson, Franklin Lakes, NJ). DNA histogram analysis was performed using ModFit LT software (Verity Software House Inc., Topsham, ME).
Apoptosis Assay
Pituitary tumor cells were either treated with 1030 µM dox alone (48 h), 1 µM phenoxybenzamine alone (48 h) or 2030 µM dox plus 1 µM phenoxybenzamine (Sigma) for 48 h, following a 30-min preincubation in 1 µM phenoxybenzamine alone. Cells were then washed in PBS, trypsinized, and centrifuged, and cell suspensions were incubated with an fluorescein isothiocyanate-labeled monoclonal antibody to annexin and propidium iodide for 30 min at room temperature (Pharmingen, San Diego, CA) and then analyzed by flow cytometry. Propidium iodide-labeled nuclei were gated on light scatter to remove debris and necrotic cells, and the percentage of annexin-fluorescein isothiocyanate-positive (apoptotic) cells were determined as described previously (20, 37).
Western Blot Analysis
Protein lysates (50 µg) prepared in radioimmunoprecipitation buffer (RIPA) from dox-treated human, rat somatolactotroph GH3, murine corticotroph AtT20, gonadotroph LßT2, and
T3 pituitary tumor cells were electrophoresed on 12% SDS-PAGE gels and transferred to polyvinylidine difluoride membranes. Membranes were incubated with antibodies to phospho-specific Rb (Ser807/811) (1:1000); cleaved and total caspase-3 (1:500); phosphorylated I
B-
(1:1000) (Cell Signaling Technology, Beverly, MA); PCNA (1:1000) (DAKO Corp., Carpentaria, CA); and p27 (1:1000) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in Tris-buffered saline solution with 5% nonfat milk and 0.05% Tween solution overnight at 4 C. After washing, membranes were incubated with appropriate IgG horseradish peroxidase conjugates and immunoreactive protein bands visualized with enhanced chemiluminescence (Amersham International, Buckinghamshire, UK). Ponceau S staining was used to confirm equivalent total protein loading.
Animals
In accordance with Institutional Animal Care and Use Committee of Cedars-Sinai Medical Center guidelines, 4-wk-old female Nu/Nu mice were inoculated sc with murine ACTH-secreting AtT20 pituitary tumor cells (
200,000). Animals were then randomized to receive dox, 5 mg/kg·d, or vehicle. dox dissolved in distilled water (100 µl) was gavage fed daily using a 24-gauge gavage needle (Harvard Bioscience, Holliston, MA). At the end of the study, 4 wk after tumor cell injection, mice were euthanized by CO2 inhalation, tumors were measured and weighed, and aliquots were frozen and stored for subsequent analysis.
Transfections and Luciferase Assay
AtT20 cells were transiently transfected with a pGL3-NF
B-luciferase construct (generous gift from Dr. Moshe Arditi, Cedars-Sinai Medical Center) containing four copies of NF
B binding sites or a rat POMC promoter-luciferase construct (480 to +63, generous gift from Dr. Jacques Drouin, Institut de Recherches Cliniques, Montreal, Canada) using Lipofectamine 2000 according to manufacturers instructions (Invitrogen Corp.). AtT20 transfectants were pretreated in serum-free medium with dox alone (1530 µM, overnight), or dox before the addition of TNF
(50 ng/ml, 2 h) (Sigma, St. Louis, MO) or TNF
in combination with dox, after which luciferase activity was measured (Promega Corp.). A ß-galactosidase reporter plasmid was cotransfected with the NF
B and POMC promoter constructs to normalize transfection efficiency.
Statistical Analysis
Statistical analysis was performed by ANOVA (Kruskal Wallis) with Dunns multiple comparison tests or nonparametric t test (one tailed/unpaired). P values < 0.05 were considered significant.
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ACKNOWLEDGMENTS
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We thank Dr. William Yong and Dr. Shlomo Melmed for help in obtaining pituitary tumor samples.
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FOOTNOTES
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This work was supported by Cedars-Sinai Research Institute and Pfizer Pharmaceuticals.
First Published Online July 14, 2005
Abbreviations: dox, Doxazosin; I
B, inhibitory factor
B, MTS, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; NF
B, nuclear factor-
B; PCNA, proliferating cell nuclear antigen; POMC, proopiomelanocortin; PRL, prolactin; pRb, phosphorylated retinoblastoma protein; Rb, retinoblastoma protein.
Received for publication November 22, 2004.
Accepted for publication July 6, 2005.
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