Maintenance of S-nitrosothiol homeostasis plays an important role in growth suppression of estrogen receptor-positive breast tumors.

INTRODUCTION: Protein denitrosylation by thioredoxin reductase (TrxR) is key for maintaining S-nitrosothiol (SNO) homeostasis, although its role in tumor progression is unknown. Therefore, the present study aimed to assess the role of altered SNO homeostasis in breast cancer cells. METHODS: The impairment of SNO homeostasis in breast cancer cells was achieved with the highly specific TrxR inhibitor auranofin and/or exposure to S-nitroso-L-cysteine. S-nitrosylated proteins were detected using the biotin switch assay. Estrogen receptor (ER) alpha knockdown was achieved using RNA silencing technologies and subcellular localization of ERα was analyzed by confocal microscopy. The Oncomine database was explored for TrxR1 (TXNRD1) expression in breast tumors and TrxR1, ER and p53 expression was analyzed by immunohistochemistry in a panel of breast tumors. RESULTS: The impairment of SNO homeostasis enhanced cell proliferation and survival of ER+ MCF-7 cells, but not of MDA-MB-231 (ER-, mut p53) or BT-474 (ER+, mut p53) cells. This enhanced cell growth and survival was associated with Akt, Erk1/2 phosphorylation, and augmented cyclin D1 expression and was abolished by the ER antagonist fulvestrant or the p53 specific inhibitor pifithrin-α. The specific silencing of ERα expression in MCF-7 cells also abrogated the growth effect of TrxR inhibition. Estrogenic deprivation in MCF-7 cells potentiated the pro-proliferative effect of impaired SNO homeostasis. Moreover, the subcellular distribution of ERα was altered, with a predominant nuclear localization associated with phosphorylation at Thr311 in those cells with impaired SNO homeostasis. The impairment of SNO homeostasis also expanded a cancer stem cell-like subpopulation in MCF-7 cells, as indicated by the increase of percentage of CD44+ cells and the augmented capability to form mammospheres in vitro. Notably, ER+ status in breast tumors was significantly associated with lower TXNDR1 mRNA expression and immunohistochemical studies confirmed this association, particularly when p53 abnormalities were absent. CONCLUSION: The ER status in breast cancer may dictate tumor response to different nitrosative environments. Impairment of SNO homeostasis confers survival advantages to ER+ breast tumors, and these molecular mechanisms may also participate in the development of resistance against hormonal therapies that arise in this type of mammary tumors.

To cardiovascular disease and beyond: new therapeutic perspectives of statins in autoimmune diseases and cancer.

Statins have been successfully used in patients with hypercholesterolemia and cardiovascular diseases, but there is increasing evidence that they exert effects by much exceeding the lowering of cholesterol levels. Statins have antiatherosclerotic, antiinflammatory, antioxidant, immunomodulatory and antithrombotic effects. These "pleiotropic" effects stem from their inhibition of prenylation of the small GTP-binding proteins Ras and Rho, and to the disruption, or depletion, of cholesterol rich membrane micro-domains (membrane rafts). Through these pathways statins modulate immune responses by altering cytokine levels and by affecting the function of cells involved in both innate and adaptive responses. Anti-inflammatory and immunosuppressory properties of statins provide the rationale for their potential application in conditions in which the inflammation and immune response represent key pathogenic mechanisms, such as antiphospholipid syndrome, rheumatoid arthritis and systemic lupus erythematosus. Reduction of atherosclerosis progression in autoimmunity is also a very important effect. Statins pathways of action in systemic autoimmune diseases, and their potential therapeutic use are discussed in this review. The inhibition of mevalonate pathway by statins impairs modification of Ras and Rho GTPases, which play key roles in signaling pathways related to tumor formation, metastasis and cell death. There is experimental and clinical evidence that statins may improve the therapeutic outcome of anticancer drugs. Thus, this review will also discuss recent insights into the molecular mechanisms underlying the anticancer effects of statins and their assessment as promising candidates for inclusion into current therapeutic regimens for the treatment of malignant diseases.

AEE788 is a vascular endothelial growth factor receptor tyrosine kinase inhibitor with antiproliferative and proapoptotic effects in acute myeloid leukemia.

OBJECTIVE: Aberrant activation of tyrosine kinase receptors is frequently observed in acute myelogenous leukemia (AML). Moreover, activating mutations of the fms-like tyrosine kinase 3 (FLT3) receptor can be found in approximately 30% of patients, thereby representing one of the most frequent single genetic alterations in AML. AEE788, a novel dual receptor tyrosine kinase inhibitor of endothelial growth factor and vascular endothelial growth factor (VEGF), is being studied in several solid tumors with remarkable success. It is not known, however, about the efficacy of this inhibitor in the treatment of AML. Therefore, we investigated the effect of AEE788 in the treatment of three human AML cell lines and seven AML patient samples. MATERIALS AND METHODS: Cell survival in THP-1, MOLM-13, and MV4-11 cell lines (the two last harboring the FLT3/internal tandem duplication mutation) and AML blasts incubated with 0.5 to 15 microM AEE788 were quantified. We also studied the activation of VEGF/VEGF receptors loop, FLT3, and their downstream effectors (Akt, extracellular signal-regulated kinase, signal transducers and activators of transcription 5, and nuclear factor-kappaB). RESULTS: Our data showed that AEE788 was a tyrosine kinase inhibitor of FLT3 activity and had antiproliferative and proapoptotic activity in AML-derived cell lines and AML blasts that presented phosphorylation of the FLT3 receptor. Consistently, in these cells AEE788 abrogated VEGF/VEGF receptors activation and the survival signaling pathways studied. CONCLUSION: Taken together, the activity of AEE788 might represent a promising new option of targeting FLT3 for the treatment of AML.

Additive effect of PTK787/ZK 222584, a potent inhibitor of VEGFR phosphorylation, with Idarubicin in the treatment of acute myeloid leukemia.

Acute myeloid leukemia (AML) is a disease with a poor prognosis. It has been demonstrated that AML cells express vascular endothelial growth factor (VEGF) as well as Flt-1 and KDR, resulting in an autocrine pathway for cell survival. PTK787/ZK 222584 is a new oral antiangiogenic molecule that inhibits tyrosine kinase activity of all known VEGF receptors. The present study aimed to investigate the therapeutic efficacy of combining PTK787/ZK 222584 with a chemotherapeutic agent, such as Idarubicin, for treatment of AML. We have analyzed in four AML cell lines and seven AML patient samples, cell proliferation, apoptosis, angiogenesis. and activation of several related intracellular pathways after treatment with PTK787/ZK 222584 alone or combined with Idarubicin. PTK787/ZK 222584 decreased VEGF levels and VEGF receptor phosphorylation in the AML cells showing Fms-like tyrosine kinase 3/internal tandem duplication mutation (Flt3/ITD). Both drugs, given separately, inhibited cell proliferation and promoted apoptosis. Moreover, combined treatment promoted more apoptosis and inhibition of cell proliferation than each compound administered separately in all AML cells. In conclusion, PTK787/ZK 222584 combined with Idarubicin achieved a better therapeutic efficacy than chemotherapy alone in AML cells, especially in those with Flt3/ITD, in which the combination further prevented activation of the angiogenic process.

Utility of alpha subunit determination after thyrotropin-releasing hormone stimulation as an indicator of gonadotropinoma persistence and/or recurrence.

OBJECTIVE: Gonadotropinomas are adenomas of the gonadotropic cells of the anterior pituitary. These cells produce and secrete gonadotropins (follicle-stimulating hormone and luteinizing hormone). Most of these tumors show altered production of gonadotropins and their subunits (the p-FSH, a and, less frequently, p-LH subunits). The thyrotropin-releasing hormone (TRH) stimulation test could differentiate these tumors from nonfunctioning tumors. Equally, this test could be able to distinguish between postsurgical changes and tumoral remnants after surgery. SUBJECTS AND METHOD: We studied 24 patients with pituitary macroadenoma, 14 of who had a histological diagnosis of gonadotroph adenoma. The TRH stimulation test was performed before and after surgery. RESULTS: Both before and after surgery, a positive result to the TRH test was obtained in 50% of gonadotropinomas. Magnetic resonance imaging (MRI) performed after surgery revealed that 83% of the patients with gonadotropinoma had signs of tumoral persistence or recurrence and/or postsurgical changes. Of these patients, 83% (41.6% of the total) showed positive a subunit stimulation after the TRH test. In the group of non-gonadotropinoma macroadenomas, only 33% had a positive result before surgery and another 33% had a positive result after surgery. In the MRI performed after surgery, all showed tumoral persistence/recurrence or postsurgical inflammatory changes. CONCLUSIONS: This test could be useful in the differential diagnosis of gonadotropinomas as well as in the follow-upand postsurgical evaluation of these tumors.

Distribution and determinants of adiponectin, resistin and ghrelin in a randomly selected healthy population.

OBJECTIVE: Adiponectin, resistin, ghrelin and the IGF-I system seem to play an important role in the regulation of body composition throughout life, but the mechanisms are not well understood. The aim of our study was to analyse the distribution among sexes and all decades of the adult life of adiponectin, resistin and ghrelin and their relationship with anthropometric, body composition parameters and the IGF-I system. SUBJECTS: One hundred and thirty-four men and 127 healthy women were included in the study. MEASUREMENTS: Plasma concentration of adiponectin, resistin, ghrelin, total IGF-I, free IGF-I and IGFBP-3 were determined in all subjects. Body composition was evaluated by bioelectrical impedance. RESULTS: Resistin and ghrelin were not affected by age. Plasma adiponectin correlated negatively with age, body mass index (BMI), waist-to-hip ratio (WHR), waist circumference (WC), fat mass (FM) and body fat (BF) in men. Adiponectin correlated negatively with WHR and positively with free IGF-I in women. Resistin correlated positively with BMI and WC only in men, and ghrelin correlated positively with WC, BMI and FM and negatively with free IGF-I in men. In multiple regression analysis adiponectin remained associated with WHR (beta=-0.19, P=0.01) in women. Resistin was positively associated with BMI (beta=0.30, P=0.003) in women and ghrelin was negatively related to free IGF-I (beta=-0.158, P=0.019) in men. CONCLUSIONS: Plasma adiponectin declines with age and is negatively associated with FM in men. Our data suggest the existence of a positive correlation of adiponectin and the IGF-I axis in women and of an inverse relationship between ghrelin and the IGF-I system in men.