PPAR-gamma receptor ligands: novel therapy for pituitary adenomas

Pituitary tumors cause considerable morbidity due to local invasion, hypopituitarism, or hormone hypersecretion. In many cases, no suitable drug therapies are available, and surgical excision is currently the only effective treatment. We show here abundant expression of nuclear hormone receptor PPAR-gamma in all of 39 human pituitary tumors. PPAR-gamma activating thiazolidinediones (TZDs) rosiglitazone and troglitazone induced G(0)-G(1) cell-cycle arrest and apoptosis in human, rat somatolactotroph, and murine gonadotroph pituitary tumor cells, and suppressed in vitro hormone secretion. In vivo development and growth of murine somatolactotroph and gonadotroph tumors, generated by subcutaneous injection of prolactin-secreting (PRL-secreting) and growth hormone-secreting (GH-secreting) GH3 cells, luteinizing hormone-secreting (LH-secreting) LbetaT2 cells, and alpha-T3 cells, was markedly suppressed in rosiglitazone-treated mice, and serum GH, PRL, and LH levels were attenuated in all treated animals (P < 0.009). These results demonstrate that PPAR-gamma is an important molecular target in pituitary adenoma cells and PPAR-gamma ligands inhibit tumor cell growth and GH, PRL, and LH secretion in vitro and in vivo. TZDs are proposed as novel oral medications for managing pituitary tumors.     

The emerging role of pituitary tumor transforming gene in tumorigenesis

Pituitary tumor transforming gene (PTTG) is a newly discovered oncogene, and serves as a marker of malignancy grades in several forms of cancer, particularly endocrine malignancies such as pituitary adenomas. PTTG appears also to have a role in the genesis of some types of cancer. Also known as a human form of securin, PTTG is an anaphase inhibitor that prevents premature chromosome separation through inhibition of separase activity; hence, its degradation is required to start anaphase. Through this important function, PTTG participates in several key cellular events such as mitosis, cell cycle progression, DNA repair and apoptosis. The physiological importance of PTTG is indicated by the study of PTTG-null mice that have cell growth abnormalities in testis and pancreatic beta cells. Overexpression of PTTG has been observed in thyroid and colon cancers. In addition, 90% of pituitary adenomas overexpress PTTG, qualifying it as the best available marker for this disease. Although the exact mechanism is unknown, PTTG participates in the pathogenesis of various tumors, including pituitary tumors, by inducing aneuploidy and upregulating FGF-2, a potent mitogenic and angiogenic factor. Various growth factors, nuclear factors and hormones regulate PTTG expression in different tumor cells, which could be important to understand in order to obtain insight into the tumorigenic and tumor progression process. Here, we review the current knowledge of the biological and pathophysiological roles of PTTG.     

垂体腺瘤MRI侵袭特征与VEGF、bFGF mRNA表达的关系

目的探讨垂体腺瘤MRI侵袭特征与VEGF、bFGFmRNA表达的关系。方法对30例垂体腺瘤患者术前行MRI检查,术后收集新鲜标本应用逆转录PCR(RT-PCR)方法检测垂体腺瘤组织标本中VEGF和bFGFmRNA的表达,分析MRI侵袭性表现与VEGF和bFGFmRNA表达之间的关系。结果在垂体腺瘤相关MRI侵袭表现中,向海绵窦、鞍底侵袭不同程度间的VEGF值有显著性差异(P<0.05),bFGF值无显著性差异(P>0.05);向鞍上生长的不同体积肿瘤的VEGF、bFGF值无显著性差异(P>0.05)。结论垂体腺瘤MRI侵袭特征与VEGFmRNA表达的上调有关。     

PTTG和C-myc在垂体腺瘤中的表达及其与肿瘤复发的关系

目的:通过检测PTTG和C-myc在垂体腺瘤中的表达来探讨它们与垂体腺瘤侵袭和复发的关系。方法:采用免疫组化方法检测PTTG和C-myc在侵袭性、非侵袭性及复发性垂体腺瘤中的表达。结果:PTTG在侵袭性和复发性垂体腺瘤中的表达水平显著高于非侵袭性垂体腺瘤,均P<0.05。C-myc的表达在侵袭组和非侵袭组、复发组与侵袭组、复发组与非侵袭组之间均无统计学意义,均P>0.05。结论:PTTG与垂体腺瘤侵袭性和术后复发可能密切相关。而C-myc可能不能作为评价垂体腺瘤侵袭性的指标。     

(-)-Gossypol suppresses the growth of human prostate cancer xenografts via modulating VEGF signaling-mediated angiogenesis

(-)-Gossypol, a natural BH3-mimetic and small-molecule Bcl-2 inhibitor, shows promise in ongoing phase II clinical trials for human cancers. However, whether (-)-gossypol plays functional roles in tumor angiogenesis has not been directly elucidated yet. In this study, we showed that (-)-gossypol dose dependently inhibited the expression of VEGF, Bcl-2, and Bcl-xL in human prostate cancer cells (PC-3 and DU 145) and primary cultured human umbilical vascular endothelial cells (HUVEC) in vitro. Notably, the growth of human prostate tumor PC-3 xenografts in mice was significantly suppressed by (-)-gossypol at a dosage of 15 mg/kg/d. This inhibitory action of (-)-gossypol in vivo was largely dependent on suppression of angiogenesis in the solid tumors, where VEGF expression and microvessel density were remarkably decreased. Furthermore, (-)-gossypol inhibited VEGF-induced chemotactic motility and tubulogenesis in HUVECs and human microvascular endothelial cells and suppressed microvessel sprouting from rat aortic rings ex vivo. When examined for the mechanism, we found that (-)-gossypol blocked the activation of VEGF receptor 2 kinase with the half maximal inhibitory concentration of 2.38 μmol/L in endothelial cells. Consequently, the phosphorylation of key intracellular proangiogenic kinases induced by VEGF was all suppressed by the treatment, such as Src family kinase, focal adhesion kinase, extracellular signal-related kinase, and AKT kinase. Taken together, the present study shows that (-)-gossypol potently inhibits human prostate tumor growth through modulating VEGF signaling pathway, which further validates its great potential in clinical practice.     

Cancer growth and spread are saltatory and phase-locked to the reproductive cycle through mediators of angiogenesis

The frequency of breast cancer metastatic spread is affected by the menstrual cycle phase of its resection. Breast cancer growth, post-resection spread, and cure frequency are each modulated by the estrous cycle in C(3)HeB/FeJ mice. Tumor metastases are 2- to 3-fold more frequent when the resection is done during diestrus as compared with estrus. Tumor angiogenesis is essential for both cancer growth and lethal metastatic cancer spread. The balance between vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) modulates new blood vessel formation and blood vessel permeability. Sex hormones modulate the expression of these key angiogenesis regulators in the endometrium and uterus. We, therefore, asked whether the estrous cycle modulates the density of CD31-positive vessels within the tumor, the permeability of tumor blood vessels, levels of VEGF and bFGF immunoreactive protein in normal breast and breast cancer, and whether expression of these genes are modulated by the estrous cycle stage in C(3)HeB/FeJ mice. We find that tumor blood vessel density and blood volume do not vary throughout the cycle; however, tumor capillary permeability is regulated by the estrous cycle being highest in diestrus, the cycle stage associated with the highest cancer growth rate and the highest frequency of post-resection cancer metastasis. VEGF protein levels in breast cancer are >100-fold higher than in normal breast. VEGF protein in this mammary tumor varies with the estrus cycle with highest levels in proestrus. In a non-breast tumor, methylcholantrenene A sarcoma, from CD(2)F(1) mice, tumor VEGF protein also varies with the estrus cycle with highest levels in proestrus and diestrus. VEGF gene expression in the mammary tumor does not change significantly across the cycle, but is modulated by the cycle in normal breast tissue. bFGF protein concentration is 6-fold higher in normal breast than in breast cancer. bFGF protein pattern in both tumor and breast are similar, opposite to VEGF, and affected by oophorectomy. bFGF message is modulated by the cycle in both breast cancer and normal breast. The changes in breast cancer capillary permeability, VEGF, and bFGF that occur during each fertility cycle, in breast tissue and breast cancer, putatively in response to cyclical changes in sex hormones, might contribute, at least in part, to both the modulation of cancer growth and post-resection breast cancer spread by the fertility cycle. These fertility cycle-induced effects on tumor biology also seem to extend to non-breast cancer biology.     

侵袭性垂体腺瘤nm23-H2和PTTG蛋白表达及其临床意义

目的　探讨nm2 3 H2和PTTG蛋白表达与垂体腺瘤发生、发展、生物学行为及预后的关系。方法　采用免疫组化S P法检测上述两基因蛋白在 6 2例垂体腺瘤中的表达。结果　nm2 3 H2在非侵袭性垂体腺瘤组的阳性表达率显著高于侵袭性垂体腺瘤组 (P <0 0 5 ) ,腺瘤侵袭海绵窦、包绕颈内动脉组、蝶窦斜坡骨质破坏组、腺瘤囊变卒中组、复发组的nm2 3 H2阳性表达率均明显低于对照组 (P <0 0 5或P <0 0 1)。PTTG在侵袭性垂体腺瘤组与非侵袭性垂体腺瘤组强阳性表达率差异有显著性 (P <0 0 5 )。功能性腺瘤组、海绵窦侵袭组、蝶窦斜坡骨质破坏组的强阳性表达率均与对照组有显著差异 (P <0 0 1或P <0 0 5 )。nm2 3 H2基因蛋白阴性表达和PTTG基因蛋白阳性表达代表了两基因突变 ,两者之间相关性无统计学意义 ,说明它们在垂体腺瘤发生及生物学行为改变过程中可能不是同时或伴随发生。结论　nm2 3 H2和PTTG基因蛋白的异常表达可作为客观评价垂体腺瘤的生物学行为、预后复发的判断、指导术后个体化治疗的良好指标。     

Fischer 344大鼠泌乳素腺瘤动物模型的建立

目的建立Fischer344（F344）大鼠泌乳素腺瘤动物模型。方法采用F344雌性大鼠,行腹腔注射苯甲酸雌二醇或无菌生理盐水,用药50d后行MRI扫描,观察大鼠生存状态,检测垂体重量、血清泌乳素（PRL）水平、病理与PRL免疫组织化学检测变化。结果应用苯甲酸雌二醇50d后,大鼠体重增长受抑,垂体重量与PRL水平显著升高,病理检查可见垂体肿瘤形成且PRL染色阳性,MRI可见肿瘤,成瘤率达100%。结论利用F344大鼠建立的垂体瘤动物模型,成瘤周期短,简单易行,稳定可靠,符合泌乳素腺瘤的生物学特性。     

Direct effects of catecholamines, thyrotropin-releasing hormone, and somatostatin on growth hormone and prolactin secretion from adenomatous and nonadenomatous human pituitary cells in culture.

To determine the mechanism and the site of action of catecholamines as well as hormones including thyrotropin-releasing hormone (TRH)1 and somatostatin on pituitary hormone release in patients with acromegaly and in normal subjects, the effects of these substances on growth hormone (GH) and prolactin (PRL) secretion from adenomatous and nonadenomatous human pituitary cells in culture were examined. When dopamine (0.01-0.1 microM) or bromocriptine (0.01-0.1 microM) was added to the culture media, a significant inhibition of GH and PRL secretion from adenoma cells from acromegalic patients was observed. This inhibition was blocked by D2 receptor blockade with metoclopramide or sulpiride, but not by D1 receptor blockade. Similarly, dopamine suppressed GH and PRL release by nonadenomatous pituitary cells in a dose-dependent manner, which was again blocked by D2 receptor blockade. The minimum effective concentration of dopamine required for a significant inhibition of PRL secretion (0.01 microM) was lower than that for GH release (0.1 microM). Norepinephrine, likewise, caused a suppression of PRL secretion from adenomatous and nonadenomatous pituitary cells. This effect was blocked by sulpiride, phentolamine, however, was ineffective. When TRH was added to the media, both GH and PRL secretion were enhanced in adenoma cells, while only the stimulation of PRL release was observed in nonadenomatous pituitary cells. Coincubation of TRH and dopamine resulted in variable effects on GH and PRL secretion. Somatostatin consistently lowered GH and PRL secretion in both adenomatous and nonadenomatous pituitary cells and completely blocked the TRH-induced stimulation of GH and PRL secretion from adenoma cells. Opioid peptides (1 microM) failed to affect hormone release. These results suggest that no qualitative difference in GH and PRL responses to dopaminergic agonists or to somatostatin exists between adenoma cells of acromegalic patients and normal pituitary cells, and that the direct effect of catecholamines on GH and PRL secretion from human pituitary cells is mediated mainly through dopamine receptor activation.     

PTTG和b-FGF在前列腺癌中的表达与临床病理学特征的相关性研究

目的：探讨垂体肿瘤转化基因和碱性成纤维细胞生长因子在前列腺癌中的表达及其与临床病理学特征之间的关系，并探讨两者表达的相关性。方法：采用免疫组织化学链霉亲合素-生物素-过氧化物酶复合物法，检测51例前列腺癌和51例前列腺增生组织中垂体肿瘤转化基因和碱性成纤维细胞生长因子的表达情况。结果：垂体肿瘤转化基因和碱性成纤维细胞生长因子在前列腺癌和前列腺增生组织中的阳性表达率分别为72．55％和47．06％（P〈0．01），58．82％和35．29％（P〈0．05）。垂体肿瘤转化基因表达程度与前列腺癌的临床分期密切相关（P〈0．05），而与其病理分级无关（P〉0．05）。碱性成纤维细胞生长因子表达程度与前列腺癌的临床分期密切相关（P〈0．05），与其病理分级无关（P〉0．05）。垂体肿瘤转化基因和碱性成纤维细胞生长因子在前列腺癌中的表达程度呈正相关（P〈0．01）。结论：垂体肿瘤转化基因、碱性成纤雏细胞生长因子的高表达可能参与了前列腺癌的发生，在前列腺癌的侵袭和转移中发挥重要作用，且二者具有协同作用。     

Insulin suppresses growth hormone secretion by rat pituitary cells

The effects of insulin on basal and hydrocortisone-induced growth hormone (GH) secretion were studied in rat pituitary tumor cells (GH3). Cells were grown in monolayer culture and exposed to exogenously added insulin for up to 8 d. Basal GH secretion was inhibited by insulin (0.7 nM) after a 48-h lag period by approximately 50% (P less than 0.01, vs. untreated control cells). The suppression of GH secretion was reversible, as removal of added insulin resulted in return of GH secretion to normal levels after 24 h. Maximal suppression of basal GH secretion was achieved by 0.7 nM insulin, and these effects were prevented by simultaneous exposure of the cells to guinea pig anti-insulin serum (1:2,000). No effects of insulin on cell replication were evident, and glucose concentration in the medium did not differ in control or insulin-treated wells. Insulin (7 nM) significantly suppressed the fivefold hydrocortisone-induced GH stimulation during 5 d of incubation with up to 1,000 nM of the steroid (P less than 0.001). These inhibitory effects were similarly observed in glucose- and pyruvate-free medium, and in the presence of 2-deoxyglucose. Insulin also reversed the suppression of prolactin (PRL) secretion induced by hydrocortisone (1 uM), and actually stimulated basal PRL secretion by over 50%. Insulin did not alter the inhibitory effect of hydrocortisone on GH3 cell proliferation. Although higher doses (13 nM) of insulin-like growth factor (IGF-I) also suppressed basal GH secretion, IGF-I did not alter the GH and PRL secretory changes induced by hydrocortisone. The results show that insulin exerts a direct, specific inhibitory effect on basal and hydrocortisone-induced GH secretion by GH3 cells unrelated to glucose utilization by the cells.     

Characterization of the bystander effect of somatostatin receptor sst2 after in vivo gene transfer into human pancreatic cancer cells

Pancreatic cancer is one of the most aggressive and devastating human malignancies. The present study was conducted to determine whether in vivo sst2 gene transfer into human pancreatic tumors would impair tumor progression, and to characterize sst2 antitumoral bystander mechanisms. sst2 administration, using the synthetic vector PEI, strongly inhibited tumor progression of human pancreatic adenocarcinoma, in vivo. sst2 gene transfer induced intratumoral production of its ligand somatostatin. Disruption of this autocrine loop by RNA interference completely reversed sst2 antitumoral activity. Mice depleted of natural killer (NK) cells did not hamper sst2 tumor growth inhibition. However, microvessel density and vascular endothelial growth factor (VEGF) expression were markedly reduced in sst2-transfected tumors, whereas sst3 somatostatin receptor was upregulated. Depleting somatostatin by RNA interference completely abolished the sst2 inhibitory effect on VEGF expression and tumor angiogenesis, and sst2-induced sst3 expression in peripheral tumor vessels. We conclude that in vivo sst2 gene transfer elicited intratumoral somatostatin production and strongly impaired human pancreatic tumor growth. NK cells were not involved in this antitumoral bystander effect. VEGF and tumor vascularization were identified as novel targets for sst2-mediated antitumoral bystander effect. sst3 somatostatin receptor was upregulated in sst2-transfected tumors. Therefore, in vivo gene delivery of sst2 receptor to target the angiogenic process in pancreatic ductal adenocarcinoma might be a new therapeutic approach for treatment of pancreatic cancer in patients with unresectable disease.     

Survivin在大肠癌中的表达及其与血管生长因子的相关性研究

目的　检测大肠癌中凋亡抑制基因Survivin的表达,研究其与大肠癌临床特征和血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)表达的相关性,探讨Survivin在大肠癌发生、发展及转移中的作用及机制。方法　收集6 2例大肠癌标本及其病例资料,应用免疫组织化学方法检测Survivin、VEGF、bFGF在大肠癌标本中的表达情况。结果　Survivin、VEGF、bFGF基因在癌旁正常大肠组织中均不表达,在6 2例大肠癌组织中,30例Survivin表达阳性,阳性率为4 8. 39% ,Survivin与大肠癌的Ducks分期密切相关(P <0 .0 0 1)但与病理分化无关(P >0 .0 5 )。2 9例VEGF表达阳性,阳性率4 6 . 18% ,4 5例bFGF表达阳性,阳性率75 . 5 8%。肿瘤局限于黏膜层及黏膜下层和肌层、浆膜层时,VEGF、bFGF表达无明显差异(P >0 . 0 5 ) ,但当肿瘤侵犯到浆膜层和浆膜外时,VEGF、bFGF蛋白表达阳性率明显增高(P<0 . 0 0 1)。淋巴转移组的VEGF、bFGF阳性表达显著高于未发生淋巴转移组(P <0. 0 0 0 1)。结论　Survivin基因、VEGF、bFGF与大肠癌的Ducks分期有密切关系但与病理分化无关,检测Survivin更有意义。     

Somatostatin receptor (SSTR) subtype-selective analogues differentially suppress in vitro growth hormone and prolactin in human pituitary adenomas. Novel potential therapy for functional pituitary tumors.

Previously, we have shown somatostatin receptor (SSTR) subtype-specific regulation of growth hormone (GH), thyroid-stimulating hormone, and prolactin (PRL) secretion in human fetal pituitary cultures, where GH and thyroid-stimulating hormone are mediated by both SSTR2 and SSTR5, whereas SSTR2 preferentially mediates PRL secretion. We now tested SSTR subtype-selective analogues in primary human GH- and PRL-secreting pituitary adenoma cultures. Analogue affinities determined by membrane radioligand binding in cells stably expressing human SSTR forms were either SSTR2 or SSTR5-selective. Analogues preferential either for SSTR2, including octreotide, lanreotide, and novel compounds with improved affinity for SSTR2, or new SSTR5-selective compounds suppressed GH in tumor cell cultures (up to 44% of control; P < 0.0005). However, novel analogues from both groups were 30-40% more potent than octreotide and lanreotide in suppressing GH (P < 0.05). Heterologous analogue combinations containing both SSTR2- and SSTR5-selective compounds were more potent in decreasing GH than analogues used alone (P < 0.05), or than combinations of compounds specific for the same receptor subtype (P < 0.005). In contrast, SSTR2-selective analogues did not suppress PRL release from six cultured prolactinomas studied. However, new SSTR5-selective analogues suppressed in vitro PRL secretion (30-40%; P < 0.05) in four of six prolactinomas. These results suggest that both SSTR2 and SSTR5 are involved in GH regulation in somatotroph adenoma cells, whereas SSTR5 exclusively regulates PRL secretion from prolactinoma cells. Thus, somatostatin analogues with improved selective binding affinity for these receptor subtypes may be effective in the treatment of either GH- or PRL-secreting adenomas.     

垂体肿瘤转化基因及碱性成纤维细胞生长因子在乳腺癌组织中的表达及意义

目的研究垂体肿瘤转化基因（PTTG）及碱性成纤维细胞生长因子（bFGF）在乳腺癌中的表达及意义。方法收集乳腺癌患者手术标本72例,癌旁正常乳腺组织20例。通过免疫组化方法检测PTTG和bFGF的表达,随访观察发生转移情况及5年生存率。结果 PTTG及bFGF在正常乳腺组织中呈阴性表达,在乳腺癌组织中呈阳性表达,高表达者其发生远处转移的几率及死亡率均明显高于低表达者,且PTTG及bFGF间存在相关关系。结论 PTTG及bFGF参与乳腺癌的形成及发展,其表达高低与乳腺癌的恶性程度及预后相关。在标本中对其行半定量分析可对乳腺癌患者的治疗及预后提供重要的参考。     

EGFR as a therapeutic target for human, canine, and mouse ACTH-secreting pituitary adenomas

Cushing disease is a condition in which the pituitary gland releases excessive adrenocorticotropic hormone (ACTH) as a result of an adenoma arising from the ACTH-secreting cells in the anterior pituitary. ACTH-secreting pituitary adenomas lead to hypercortisolemia and cause significant morbidity and mortality. Pituitary-directed medications are mostly ineffective, and new treatment options are needed. As these tumors express EGFR, we tested whether EGFR might provide a therapeutic target for Cushing disease. Here, we show that in surgically resected human and canine corticotroph cultured tumors, blocking EGFR suppressed expression of proopiomelanocortin (POMC), the ACTH precursor. In mouse corticotroph EGFR transfectants, ACTH secretion was enhanced, and EGF increased Pomc promoter activity, an effect that was dependent on MAPK. Blocking EGFR activity with gefitinib, an EGFR tyrosine kinase inhibitor, attenuated Pomc expression, inhibited corticotroph tumor cell proliferation, and induced apoptosis. As predominantly nuclear EGFR expression was observed in canine and human corticotroph tumors, we preferentially targeted EGFR to mouse corticotroph cell nuclei, which resulted in higher Pomc expression and ACTH secretion, both of which were inhibited by gefitinib. In athymic nude mice, EGFR overexpression enhanced the growth of explanted ACTH-secreting tumors and further elevated serum corticosterone levels. Gefitinib treatment decreased both tumor size and corticosterone levels; it also reversed signs of hypercortisolemia, including elevated glucose levels and excess omental fat. These results indicate that inhibiting EGFR signaling may be a novel strategy for treating Cushing disease.