Thyroid status regulates the tumor microenvironment delineating breast cancer fate.

The patient's hormonal context plays a crucial role in the outcome of cancer. However, the association between thyroid disease and breast cancer risk remains unclear. We evaluated the effect of thyroid status on breast cancer growth and dissemination in an immunocompetent mouse model. For this, hyperthyroid and hypothyroid Balb/c mice were orthotopically inoculated with triple-negative breast cancer 4T1 cells. Tumors from hyperthyroid mice showed an increased growth rate and an immunosuppressive tumor microenvironment, characterized by increased IL-10 levels and decreased percentage of activated cytotoxic T cells. On the other hand, delayed tumor growth in hypothyroid animals was associated with increased tumor infiltration of activated CD8+ cells and a high IFNγ/IL-10 ratio. Paradoxically, hypothyroid mice developed a higher number of lung metastasis than hyperthyroid animals. This was related to an increased secretion of tumor CCL2 and an immunosuppressive systemic environment, with increased proportion of regulatory T cells and IL-10 levels in spleens. A lower number of lung metastasis in hyperthyroid mice was related to the reduced presence of mesenchymal stem cells in tumors and metastatic sites. These animals also exhibited decreased percentages of regulatory T lymphocytes and myeloid-derived suppressor cells in spleens but increased activated CD8+ cells and the IFNγ/IL-10 ratio. Therefore, thyroid hormones modulate the cellular and cytokine content of the breast tumor microenvironment. A better understanding of the mechanisms involved in these effects could be a starting point for the discovery of new therapeutic targets for breast cancer.

Oxidative Stress Produced by Hyperthyroidism Status Induces the Antioxidant Enzyme Transcription through the Activation of the Nrf-2 Factor in Lymphoid Tissues of Balb/c Mice.

Hyperthyroidism is an endocrine disorder characterized by excessive secretion of thyroid hormones T3 and T4. Thyroid hormones exert pleiotropic actions on numerous tissues and induce an overall increase in metabolism, with an increase in energy demand and oxygen consumption. Therefore, the purpose of this study was to investigate the effects of hyperthyroidism on the production of reactive oxygen species (ROS) in lymph node and spleen cells of euthyroid and hyperthyroid mice, analyzing antioxidant mechanisms involved in the restitution of the cellular redox state. For this, thirty female Balb/c (H-2d) mice were randomly divided into two groups: euthyroid (by treatment with placebo) and hyperthyroid (by treatment with 12 mg/l of T4 in drinking water for 30 days). We found a significant increase in ROS and an increase in the genomic and protein expression of the antioxidant enzymes catalase (CAT) and glutathione peroxidase-1 (GPx-1) in lymph node and spleen cells of hyperthyroid mice. In vitro treatment with H2O2 (250 µM) of the lymphoid cells of euthyroid mice increased the expression levels of CAT and GPx-1. The hyperthyroidism increased the phosphorylation levels of Nrf2 (nuclear factor erythroid 2-related factor) and the kinase activity of protein kinase C (PKC) and extracellular signal-regulated kinase (ERK). Additionally, we found an increase in the expression of the classic isoenzymes of PKCα, ß and γ. In conclusion, these results indicated that the increase in ROS found in the hyperthyroid state induces the antioxidant enzyme transcription through the activation of the Nrf-2 factor in lymphoid tissues. This shows the influence of hyperthyroidism on the regulation of the cellular antioxidant system.

Thyroid hormones induce doxorubicin chemosensitivity through enzymes involved in chemotherapy metabolism in lymphoma T cells.

Thyroid hormones (THs) - 3,3',5-triiodo-L-thyronine (T3) and L-thyroxine (T4) - are important regulators of the metabolism and physiology of most normal tissues. Cytochrome P450 family 3A members are drug metabolizing enzymes involved in the activation and detoxification of several drugs. CYP3A4 is the major enzyme involved in the metabolism of chemotherapeutic drugs. In this work, we demonstrate that THs induce a significant increase in CYP3A4 mRNA levels, protein expression and metabolic activity through the membrane receptor integrin αvß3 and the activation of signalling pathways through Stat1 and NF-κB. We reasoned that TH-induced CYP3A4 modulation may act as an important regulator in the metabolism of doxorubicin (Doxo). Experiments in vitro demonstrated that in CYP3A4-knocked down cells, no TH-mediated chemosensitivity to Doxo was observed. We also found that THs modulate these functions by activating the membrane receptor integrin αvß3. In addition, we showed that the thyroid status can modulate CYP450 mRNA levels in tumor and liver tissues, and the tumor volume in response to chemotherapy in vivo. In fact, Doxo treatment in hypothyroid mice was associated with lower tumors, displaying lower levels of CYP enzymes, than euthyroid mice. However, higher mRNA levels of CYP enzymes were found in livers from Doxo treated hypothyroid mice respect to control. These results present a new mechanism by which TH could modulate chemotherapy response. These findings highlight the importance of evaluating thyroid status in patients during application of T-cell lymphoma therapeutic regimens.

Hypothyroidism-related zinc deficiency leads to suppression of T lymphocyte activity.

PURPOSE: Hypothyroidism has been shown to induce immunosuppression and both the thyroid status and immunity are affected by zinc deficiency. However, the impact of hypothyroidism on zinc metabolism and its possible relationship with the immune status has not yet been deeply explored. Here, our aim was to study whether hypothyroidism may alter zinc metabolism and thus lead to the impairment of T lymphocyte activity. METHODS: Variations in the distribution of zinc in the body were evaluated in PTU-treated hypothyroid mice. The effects of hypothyroidism and zinc deficiency were studied on T lymphocyte proliferation after stimulation both in vitro and in vivo. For in vitro assays, thyroid hormone-free or zinc chelator (TPEN or DTPA)-supplemented media were used. For in vivo assays, lymphocyte activity was evaluated in cells from hypothyroid, T3-treated, and zinc-supplemented mice. RESULTS: Hypothyroid mice showed lower levels of zinc in femur and lymph nodes than controls. T3 and zinc supplementation reversed these effects. In vitro, both thyroid hormone and zinc deficiency led to a decreased response to mitogen stimulation. However, only zinc deficiency was able to induce lymphocyte apoptosis. Mitogen-stimulated T cells from hypothyroid mice showed impaired proliferation, accompanied by decreased activation of PKC and lower levels of p-ERK, effects that were reversed by T3 replacement or zinc supplementation. CONCLUSIONS: Our results show an important role of zinc deficiency in hypothyroid-mediated T-cell suppression and suggest the importance of evaluating zinc levels and restoring them when necessary to maintain an efficient immune response in hypothyroid patients.

Beneficial Effect of Fluoxetine and Sertraline on Chronic Stress-Induced Tumor Growth and Cell Dissemination in a Mouse Model of Lymphoma: Crucial Role of Antitumor Immunity.

Clinical data and experimental studies have suggested a relationship between psychosocial factors and cancer prognosis. Both, stress effects on the immune system and on tumor biology were analyzed independently. However, there are few studies regarding the stress influence on the interplay between the immune system and tumor biology. Moreover, antidepressants have been used in patients with cancer to alleviate mood disorders. Nevertheless, there is contradictory evidence about their action on cancer prognosis. In this context, we investigated the effect of chronic stress on tumor progression taking into account both its influence on the immune system and on tumor biology. Furthermore, we analyzed the action of selective serotonin reuptake inhibitors, fluoxetine and sertraline, in these effects. For this purpose, C57BL/6J mice submitted or not to a chronic stress model and treated or not with fluoxetine or sertraline were subcutaneously inoculated with EL4 cells to develop solid tumors. Our results indicated that chronic stress leads to an increase in both tumor growth and tumor cell dissemination. The analysis of cell cycle regulatory proteins showed that stress induced an increase in the mRNA levels of cyclins A2, D1, and D3 and a decrease in mRNA levels of cell cycle inhibitors p15, p16, p21, p27, stimulating cell cycle progression. Moreover, an augment of mRNA levels of metalloproteases (MMP-2 and MMP-9), a decrease of inhibitors of metalloproteases mRNA levels (TIMP 1, 2, and 3), and an increase in migration ability were found in tumors from stressed animals. In addition, a significant decrease of antitumor immune response in animals under stress was found. Adoptive lymphoid cell transfer experiments indicated that the reduced immune response in stressed animals influenced both the tumor growth and the metastatic capacity of tumor cells. Finally, we found an important beneficious effect of fluoxetine or sertraline treatment on cancer progression. Our results emphasize the crucial role of the immune system in tumor progression under stress situations. Although a direct effect of stress and drug treatment on tumor biology could not be ruled out, the beneficial effect of fluoxetine and sertraline appears to be mainly due to a restoration of antitumor immune response.

Integrin αvß3 acting as membrane receptor for thyroid hormones mediates angiogenesis in malignant T cells.

The interaction of lymphoid tumor cells with components of the extracellular matrix via integrin αvß3 allows tumor survival and growth. This integrin was demonstrated to be the membrane receptor for thyroid hormones (THs) in several tissues. We found that THs, acting as soluble integrin αvß3 ligands, activated growth-related signaling pathways in T-cell lymphomas (TCLs). Specifically, TH-activated αvß3 integrin signaling promoted TCL proliferation and angiogenesis, in part, via the upregulation of vascular endothelial growth factor (VEGF). Consequently, genetic or pharmacologic inhibition of integrin αvß3 decreased VEGF production and induced TCL cell death in vitro and in human xenograft models. In sum, we show that integrin αvß3 transduces prosurvival signals into TCL nuclei, suggesting a novel mechanism for the endocrine modulation of TCL pathophysiology. Targeting this mechanism could constitute an effective and potentially low-toxicity chemotherapy-free treatment of TCL patients.

Niacin Modulates Pro-inflammatory Cytokine Secretion. A Potential Mechanism Involved in its Anti-atherosclerotic Effect.

The pathogenesis of atherosclerosis includes the assignment of a critical role to cells of the monocyte/macrophage lineage and to pro-inflammatory cytokines. Niacin is known to improve lipid metabolism and to produce beneficial modification of cardiovascular risk factors. The aim of this work was to investigate if Niacin is able to modulate pro-inflammatory cytokine production in macrophages in a murine model of atherosclerosis. For this purpose C57Bl/6J mice fed with atherogenic diet (AGD) or with conventional chow diet were used. The AGD group showed an increase in body weight and in total plasma cholesterol, with no differences in triglyceride or HDL levels. Lesions in arterial walls were observed. The characterization of Niacin receptor showed an increase in the receptor number of macrophages from the AGD group. Macrophages from control and AGD animals treated in vitro with an inflammatory stimulus showed elevated levels of IL-6, IL-1 and TNF-α, that were even higher in macrophages from AGD mice. Niacin was able to decrease the production of pro-inflammatory cytokines in stimulated macrophages. Similar effect of Niacin was observed in an in vivo model of inflammation. These results show an attenuating inflammatory mechanism for this therapeutic agent and would point out its potential action in plaque stabilization and in the prevention of atherosclerosis progression. Furthermore, the present results provide the basis for future studies on the potential contribution of Niacin to anti-inflammatory therapies.

Oral administration of fluoxetine alters the proliferation/apoptosis balance of lymphoma cells and up-regulates T cell immunity in tumor-bearing mice.

Antidepressants have a controversial role with regard to their influence on cancer and immunity. Recently, we showed that fluoxetine administration induces an enhancement of the T-cell mediated immunity in naïve mice, resulting in the inhibition of tumor growth. Here we studied the effects of fluoxetine on lymphoma proliferation/apoptosis and immunity in tumor bearing-mice. We found an increase of apoptotic cells (active Caspase-3(+)) and a decrease of proliferative cells (PCNA(+)) in tumors growing in fluoxetine-treated animals. In addition, differential gene expressions of cell cycle and death markers were observed. Cyclins D3, E and B were reduced in tumors from animals treated with fluoxetine, whereas the tumor suppressor p53 and the cell cycle inhibitors p15/INK4B, p16/INK4A and p27/Kip1 were increased. Besides, the expression of the antiapoptotic factor Bcl-2 and the proapoptotic factor Bad were lower and higher respectively in these animals. These changes were accompanied by increased IFN-γ and TNF-α levels as well as augmented circulating CD8(+) T lymphocytes in tumor-bearing mice treated with the antidepressant. Therefore, we propose that the up-regulation of T-cell mediated antitumor immunity may be contributing to the alterations of tumor cell proliferation and apoptosis thus resulting in the inhibition of tumor progression.

Involvement of thyroid hormones in the alterations of T-cell immunity and tumor progression induced by chronic stress.

BACKGROUND: Stress alters the neuroendocrine system, immunity, and cancer. Although the classic stress hormones are glucocorticoids and catecholamines, thyroid hormones have also been related to stress. We recently reported that chronic restraint stress impairs T-cell mediated immunity and enhances tumor growth in mice. METHODS: To study the participation of these hormones on the stress-induced alterations of the immune function and lymphoma growth, mice were subjected to acute or chronic stress, with or without thyroxin supplementation. Hormone levels, immune status, and cancer progression were evaluated. RESULTS: Differential endocrine alterations were observed in response to acute and chronic stress. Although corticosterone and noradrenaline levels were increased by acute stress, they were restored after prolonged exposure to the stressor. Instead, thyroid hormone levels were only reduced in chronically stressed animals in comparison with control subjects. Correlating, chronic but not acute stress impaired T-cell reactivity. Thyroxin replacement treatment of chronic restraint stress-exposed mice, which restored the euthyroid status, reversed the observed reduction of T-cell lymphoproliferative responses. Moreover, therapeutic thyroid replacement also reversed the alterations of lymphoma growth induced by chronic stress in syngeneic mice bearing tumors as well as Interleukin-2 production and specific cytotoxic response against tumor cells. Finally, we found that the isoforms theta and alpha of the protein kinase C are involved in these events. CONCLUSIONS: These results show for the first time that thyroid hormones are important neuroendocrine regulators of tumor evolution, most probably acting through the modulation of T-cell mediated immunity affected by chronic stress.

Fluoxetine directly counteracts the adverse effects of chronic stress on T cell immunity by compensatory and specific mechanisms.

Chronic stress and depression are widely known to down-regulate the immune system, and several antidepressants can reverse this impairment, with or without effects in normal subjects. Although the central nervous system is undoubtedly involved in these events, some psychotropic drugs can also exert direct effects on lymphoid cells. We have recently shown that the antidepressant fluoxetine enhances T cell proliferation and T(H)1 cytokine production in vivo, without changes on CD4/CD8 subsets. In vitro, a direct action of fluoxetine upon T lymphocyte reactivity by complex mechanisms was also described. In another work, we also found that chronic stress reduces T cell mediated immunity, namely a decrease of T cell response to mitogens, T(H)1 cytokine production and CD4+-but not CD8+--T lymphocytes. Here we investigated the effects of fluoxetine on chronic stress-driven immune system depression. We found that fluoxetine restored T cell proliferation and interleukin-2, interferon-gamma and tumor necrosis factor-alpha production by compensatory mechanisms. In addition, CD4/CD8 ratio was also normalized by antidepressant administration, but this seems to be a non-compensatory effect associated specifically to stress. No changes were observed in other lymphoid cells, i.e. natural killer cells and B lymphocytes. Finally, we observed that fluoxetine is able to reverse T cell reactivity impairment in vitro by a direct action at clinically relevant doses. These results highlight the relevance of pharmacological treatment of stress and depression, and may help to begin elucidating the complex events triggered--directly and/or indirectly--by antidepressants in non-neuronal cell types.

Different effect of chronic stress on learning and memory in BALB/c and C57BL/6 inbred mice: Involvement of hippocampal NO production and PKC activity.

Nitric oxide (NO) has been involved in many pathophysiological brain processes. Recently, we showed that neuronal nitric oxide synthase (nNOS)-mediated decrease in NO production is involved in memory impairment induced by chronic mild stress (CMS) in BALB/c mice. Two genetically different inbred murine strains, C57BL/6 and BALB/c, show distinct behavioral responses, neurodevelopmental and neurochemical parameters. Here, we perform a comparative study on CMS effects upon learning and memory in both strains, analyzing the role of NO production and its regulation by protein kinase C (PKC). Stressed BALB/c, but not C57Bl/6 mice, showed a poor learning performance in both the open field and passive avoidance inhibitory tasks. Also, CMS induced a diminished NO production by nNOS, associated with an increment in gamma and zeta PKC isoenzymes in BALB/c mice. In C57BL/6 mice, CMS had no effect on NO production, but increased delta and decreased betaI PKC isoforms. In vivo administration of a NOS inhibitor induced behavioral alterations in both strains. These results suggest a differential effect of stress, with BALB/c being more vulnerable to stress than C57BL/6 mice. This effect could be related to a differential regulation of NOS and PKC isoenzymes, pointing to an important role of NO in learning and memory.

Inhibitory effect of fluoxetine on lymphoma growth through the modulation of antitumor T-cell response by serotonin-dependent and independent mechanisms.

Fluoxetine, a selective serotonin reuptake inhibitor, is widely used for the treatment of depressive symptoms of cancer patients. However, there are contradictory evidences about its effects on immunity and cancer. Thus, we studied the effects of fluoxetine on tumor growth and on antitumoral T-cell-mediated immunity. In vivo chronic fluoxetine treatment inhibited tumor growth, and increased latency of appearance of solid tumors and survival of mice. Fluoxetine administration also increased mitogen-induced T-cell proliferation and Tumor Necrosis Factor-alpha (TNF-alpha) and Interferon-gamma (IFN-gamma) expression, without altering CD4(+)/CD8(+) ratio. In vitro, fluoxetine did not affect tumor cells proliferation, but it exerted a direct effect on T lymphocytes. Both fluoxetine and serotonin stimulated proliferation induced by a suboptimal mitogen concentration but inhibited proliferation at the optimal one. When both drugs were combined the results indicated that the effects of fluoxetine are in part independent of its ability to elevate serotonin extracellular levels. Finally, continue fluoxetine administration in nude mice - devoid of T lymphocytes - did not modify tumor progression, thus supporting the hypothesis of an immuno-modulatory effect of this drug on T cells that drives tumor growth control. These findings indicate, for the first time, that fluoxetine inhibits tumor growth through modulation of T-cell-mediated immunity by the already known serotonin-dependent pathway and by a novel independent mechanism.

Loss of hippocampal neuronal nitric oxide synthase contributes to the stress-related deficit in learning and memory.

Nitric oxide (NO) has been involved in many pathophysiological brain processes. However, the exact role of NO in the cognitive deficit associated to chronic stress exposure has not been elucidated. In this study, we investigated the participation of hippocampal NO production and their regulation by protein kinase C (PKC) in the memory impairment induced in mice subjected to chronic mild stress model (CMS). CMS mice showed a poor learning performance in both open field and passive avoidance inhibitory task respect to control mice. Histological studies showed a morphological alteration in the hippocampus of CMS mice. On the other hand, chronic stress induced a diminished NO production by neuronal nitric oxide synthase (nNOS) correlated with an increment in gamma and zeta PKC isoenzymes. Partial restoration of nNOS activity was obtained after PKC activity blockade. NO production by inducible nitric oxide synthase isoform was not detected. The magnitude of oxidative stress, evaluated by reactive oxygen species production, after excitotoxic levels of NMDA was increased in hippocampus of CMS mice. Moreover, ROS formation was higher in the presence of nNOS inhibitor in both control and CMS mice. Finally, treatment of mice with nNOS inhibitors results in behavioural alterations similar to those observed in CMS animals. These findings suggest a novel role for nNOS showing protective activity against insults that trigger tissue toxicity leading to memory impairments.

Different mitogen-mediated Beta-adrenergic receptor modulation in murine T lymphocytes depending on the thyroid status.

OBJECTIVE: The aim of this work was to analyze beta-adrenergic receptor (betaAR) regulation of T-lymphocyte proliferation in mice according to different thyroid hormone statuses. METHODS: T cells from eu-, hypo- (by propylthiouracil treatment) and hyperthyroid (by thyroxine, T4 administration) mice were purified and specific radioligand binding assays were performed. The effects of the beta-agonist isoproterenol (ISO) on intracellular levels of cyclic AMP (cAMP) were determined. Mitogen-induced T-cell proliferation was measured by [(3)H]-thymidine incorporation. Finally, protein kinase C (PKC) activity in cytosol and membrane fractions were determined using radiolabelled enzymatic substrates. RESULTS: Adecrease or a non-significant increase in betaAR number was found on T lymphocytes from hypo- and hyperthyroid mice compared to euthyroid controls. ISO stimulation of cAMP levels was lower in hypothyroid and higher in hyperthyroid T lymphocytes compared to controls. T-selective mitogen-induced proliferation was increased in T4-treated animals, but decreased in hypothyroid mice. During the peak of proliferation, downregulation of betaAR was observed in all animals. However, a higher or a lower decrease was observed in hyper- and hypothyroid T cells, respectively. In parallel, a higher translocation of PKC activity was observed in hyperthyroid cells, and a lower one was found in hypothyroid lymphocytes with respect to controls. CONCLUSIONS: These results indicate that intracellular signals triggered by mitogen activation, namely PKC, would be related to differential betaAR downregulation in T lymphocytes depending on the thyroid hormone status, contributing to the distinct proliferative responses found in hypo- or hyperthyroidism compared to the euthyroid state.

Importancia del empleo de un patrón de 129I para la medición de 125I / Usefulness of a 129I for 125I measurement

En el presente trabajo se muestran las ventajas de la utilización de un patrón de 129I para comprobar diariamente que la respuesta del equipo de detección es constante para el 125I y poder determinar además la eficiencia para dicho nucleido. Una alternativa para conocer ese valor consiste en la aplicación de un método de coincidencia. La comparación de los resultados logrados con uno y otro método, demuestra que ambos valores de eficiencia son iguales. Por otra parte se analizan algunas cuestiones relacionadas con las ecuaciones teóricas utilizadas

Importancia del empleo de un patrón de 129I para la medición de 125I / Usefulness of a 129I for 125I measurement

En el presente trabajo se muestran las ventajas de la utilización de un patrón de 129I para comprobar diariamente que la respuesta del equipo de detección es constante para el 125I y poder determinar además la eficiencia para dicho nucleido. Una alternativa para conocer ese valor consiste en la aplicación de un método de coincidencia. La comparación de los resultados logrados con uno y otro método, demuestra que ambos valores de eficiencia son iguales. Por otra parte se analizan algunas cuestiones relacionadas con las ecuaciones teóricas utilizadas (AU)