Valproic acid radiosensitizes anaplastic thyroid cells through a decrease of the DNA damage repair capacity.

BACKGROUND: Anaplastic thyroid cancer (ATC) represents a rare lethal human malignancy with poor prognosis. Multimodality treatment, including radiotherapy, is recommended to improve local control and survival. Valproic acid (VA) is a clinically available histone deacetylase inhibitor with a well-documented side effect profile. In this study, we aim to investigate the combined effect of VA with photon irradiation in vitro. METHODS: Anaplastic thyroid cancer cells (8505c) were used to investigate the radiosensitizing effect of VA. RESULTS: VA sensitized cells to photon irradiation. VA increased radiation-induced apoptosis and radiation-induced DNA damage measured by γH2AX foci induction. Furthermore, VA prolonged γH2AX foci disappearance over time in irradiated cells and decreased the radiation-induced levels of mRNA of key DNA damage repair proteins of the homologous recombination (HR) and the nonhomologous end joining (NHEJ) pathways. CONCLUSIONS: VA at a clinically safe dose enhance the radiosensitivity of 8505c cells through an increase in radiation-induced apoptosis and a disruption in the molecular mechanism of HR and NHEJ DNA damage repair pathways.

The thyroid status reprograms T cell lymphoma growth and modulates immune cell frequencies.

UNLABELLED: In spite of considerable evidence on the regulation of immunity by thyroid hormones, the impact of the thyroid status in tumor immunity is poorly understood. Here, we evaluated the antitumor immune responses evoked in mice with different thyroid status (euthyroid, hyperthyroid, and hypothyroid) that developed solid tumors or metastases after inoculation of syngeneic T lymphoma cells. Hyperthyroid mice showed increased tumor growth along with increased expression of cell cycle regulators compared to hypothyroid and control tumor-bearing mice. However, hypothyroid mice showed a higher frequency of metastases than the other groups. Hyperthyroid mice bearing tumors displayed a lower number of tumor-infiltrating T lymphocytes, lower percentage of functional IFN-γ-producing CD8(+) T cells, and higher percentage of CD19(+) B cells than euthyroid tumor-bearing mice. However, no differences were found in the distribution of lymphocyte subpopulations in tumor-draining lymph nodes (TDLNs) or spleens among different experimental groups. Interestingly, hypothyroid TDLN showed an increased percentage of regulatory T (Treg) cells, while hyperthyroid mice displayed increased number and activity of splenic NK cells, which frequency declined in spleens from hypothyroid mice. Moreover, a decreased number of splenic myeloid-derived suppressor cells (MDSCs) were found in tumor-bearing hyperthyroid mice as compared to hypothyroid or euthyroid mice. Additionally, hyperthyroid mice showed increased cytotoxic activity, which declined in hypothyroid mice. Thus, low levels of intratumoral cytotoxic activity would favor tumor local growth in hyperthyroid mice, while regional and systemic antitumor response may contribute to tumor dissemination in hypothyroid animals. Our results highlight the importance of monitoring the thyroid status in patients with T cell lymphomas. KEY MESSAGES: T cell lymphoma phenotype is paradoxically influenced by thyroid status. Hyperthyroidism favors tumor growth and hypothyroidism rises tumor dissemination. Thyroid status affects the distribution of immune cell types in the tumor milieu. Thyroid status also modifies the nature of local and systemic immune responses.

Selective cytoprotective effect of histamine on doxorubicin-induced hepatic and cardiac toxicity in animal models.

The aim of the present work was to evaluate the potential protective effect of histamine on Doxorubicin (Dox)-induced hepatic and cardiac toxicity in different rodent species and in a triple-negative breast tumor-bearing mice model. Male Sprague Dawley rats and Balb/c mice were divided into four groups: control (received saline), histamine (5 mg/kg for rats and 1 mg/kg for mice, daily subcutaneous injection starting 24 h before treatment with Dox), Dox (2 mg/kg, intraperitoneally injected three times a week for 2 weeks) and Dox+histamine (received both treatments). Tissue toxicity was evaluated by histopathological studies and oxidative stress and biochemical parameters. The combined effect of histamine and Dox was also investigated in vitro and in vivo in human MDA-MB-231 triple-negative breast cancer model. Heart and liver of Dox-treated animals displayed severe histological damage, loss of tissue weight, increased TBARS levels and DNA damage along with an augment in serum creatine kinase-myocardial band. Pretreatment with histamine prevented Dox-induced tissue events producing a significant preservation of the integrity of both rat and mouse myocardium and liver, through the reduction of Dox-induced oxidative stress and apoptosis. Histamine treatment preserved anti-tumor activity of Dox, exhibiting differential cytotoxicity and increasing the Dox-induced inhibition of breast tumor growth. Findings provide preclinical evidence indicating that histamine could be a promising candidate as a selective cytoprotective agent for the treatment of Dox-induced cardiac and hepatic toxicity, and encourage the translation to clinical practice.

Thyroid status modulates T lymphoma growth via cell cycle regulatory proteins and angiogenesis.

We have shown in vitro that thyroid hormones (THs) regulate the balance between proliferation and apoptosis of T lymphoma cells. The effects of THs on tumor development have been studied, but the results are still controversial. Herein, we show the modulatory action of thyroid status on the in vivo growth of T lymphoma cells. For this purpose, euthyroid, hypothyroid, and hyperthyroid mice received inoculations of EL4 cells to allow the development of solid tumors. Tumors in the hyperthyroid animals exhibited a higher growth rate, as evidenced by the early appearance of palpable solid tumors and the increased tumor volume. These results are consistent with the rate of cell division determined by staining tumor cells with carboxyfluorescein succinimidyl ester. Additionally, hyperthyroid mice exhibited reduced survival. Hypothyroid mice did not differ significantly from the euthyroid controls with respect to these parameters. Additionally, only tumors from hyperthyroid animals had increased expression levels of proliferating cell nuclear antigen and active caspase 3. Differential expression of cell cycle regulatory proteins was also observed. The levels of cyclins D1 and D3 were augmented in the tumors of the hyperthyroid animals, whereas the cell cycle inhibitors p16/INK4A (CDKN2A) and p27/Kip1 (CDKN1B) and the tumor suppressor p53 (TRP53) were increased in hypothyroid mice. Intratumoral and peritumoral vasculogenesis was increased only in hyperthyroid mice. Therefore, we propose that the thyroid status modulates the in vivo growth of EL4 T lymphoma through the regulation of cyclin, cyclin-dependent kinase inhibitor, and tumor suppressor gene expression, as well as the stimulation of angiogenesis.

Induction of apoptosis in T lymphoma cells by long-term treatment with thyroxine involves PKCζ nitration by nitric oxide synthase.

Thyroid hormones are important regulators of cell physiology, inducing cell proliferation, differentiation or apoptosis, depending on the cell type. Thyroid hormones induce proliferation in short-term T lymphocyte cultures. In this study, we assessed the effect of long-term thyroxine (T4) treatment on the balance of proliferation and apoptosis and the intermediate participants in T lymphoma cells. Treatment with T4 affected this balance from the fifth day of culture, inhibiting proliferation in a time-dependent manner. This effect was associated with apoptosis induction, as characterized through nuclear morphological changes, DNA fragmentation, and Annexin V-FITC/Propidium Iodide co-staining. In addition, increased iNOS gene and protein levels, and enzyme activity were observed. The generation of reactive oxygen species, depolarization of the mitochondrial membrane, and a reduction in glutathione levels were also observed. The imbalance between oxidants and antioxidants species is typically associated with the nitration of proteins, including PKCζ, an isoenzyme essential for lymphoma cell division and survival. Consistently, evidence of PKCζ nitration via proteasome degradation was also observed in this study. Taken together, these results suggest that the long-term culture of T lymphoma cells with T4 induces apoptosis through the increased production of oxidative species resulting from both augmented iNOS activity and the loss of mitochondrial function. These species induce the nitration of proteins involved in cell viability, promoting proteasome degradation. Furthermore, we discuss the impact of these results on the modulation of T lymphoma growth and the thyroid status in vivo.

Possible involvement of stress hormones and hyperglycaemia in chronic mild stress-induced impairment of immune functions in diabetic mice.

Stress, an important aspect of modern life, has long been associated with an altered homeostatic state. Little is known about the effect of the life stress on the outcome of diabetes mellitus, especially related to the higher risk of infections. Here, we evaluate the effects of chronic mild stress (CMS) exposure on the evolution of type I diabetes induced by streptozotocin administration in BALB/c mice. Exposure of diabetic mice to CMS resulted in a significant reduction of survival and a sustained increase in blood glucose values. Concerning the immune response, chronic stress had a differential effect in mice with diabetes with respect to controls, showing a marked decrease in both T- and B-cell proliferation. No correlation was found between splenic catecholamine or circulating corticosterone levels and the proliferative response. However, a significant negative correlation was found between glucose levels and concanavalin A- and lipopolysaccharide-stimulated proliferative responses of T and B cells. A positive correlation between blood glucose and splenic catecholamine concentrations was found in diabetic mice but not in controls subjected to CMS. Hence, the present report shows that diabetic mice show a worse performance in immune function after stress exposure, pointing to the importance of considering life stress as a risk factor for patients with diabetes.

Helicobacter spp. from gastric biopsies of stranded South American fur seals (Arctocephalus australis).

Gastrointestinal lesions with uncertain etiology have been widely described among pinnipeds. The aim of our study was to investigate the presence of Helicobacter spp. in the gastric mucosa of South American fur seals (Arctocephalusaustralis). Gastric biopsies from thirteen seals, stranded on the shores of the Southwestern Atlantic Ocean in Argentina, were evaluated for the presence of Helicobacter spp. by PCR and DNA sequence analysis. Six gastric biopsies were positive for Helicobacter spp. Pairwise sequence comparisons showed less than 95% identity to novel Helicobacter spp. described from pinnipeds from North America and Australia. However, phylogenetic analysis revealed that the South American fur seal sequences clustered with 99-100% homology with H. cetorum, a species isolated from dolphins and whales. The presence of H. cetorum in pinnipeds, if confirmed by its isolation from the gastric mucosa of these mammals, demonstrates the wide host range of this bacterium in the marine environment.

Detection of Helicobacter and Campylobacter spp. from the aquatic environment of marine mammals.

The mechanism by which Helicobacter species are transmitted remains unclear. To examine the possible role of environmental transmission in marine mammals, we sought the presence of Helicobacter spp. and non-Helicobacter bacteria within the order Campylobacterales in water from the aquatic environment of marine mammals, and in fish otoliths regurgitated by dolphins. Water was collected from six pools, two inhabited by dolphins and four inhabited by seals. Regurgitated otoliths were collected from the bottom of dolphins' pools. Samples were evaluated by culture, PCR and DNA sequence analysis. Sequences from dolphins' water and from regurgitated otoliths clustered with 99.8-100% homology with sequences from gastric fluids, dental plaque and saliva from dolphins living in those pools, and with 99.5% homology with H. cetorum. Sequences from seals' water clustered with 99.5% homology with a sequence amplified from a Northern sea lion (AY203900). Control PCR on source water for the pools and from otoliths dissected from feeder fish were negative. The findings of Helicobacter spp. DNA in the aquatic environment suggests that contaminated water from regurgitated fish otoliths and perhaps other tissues may play a role in Helicobacter transmission among marine mammals.

Chronic restraint stress impairs T-cell immunity and promotes tumor progression in mice.

Long-term exposure to stressful situations can affect the immune system. The T-cell response is an important component of anti-tumoral immunity. Hence, impairment of the immune function induced by a chronic stressor has been postulated to alter the immunosurveillance of tumors, thus leading to a worse neoplastic prognosis. Here, we show that chronic restraint stress affects T-cell mediated immunity in mice. This was evidenced by a decrease of mitogen-induced T-cell proliferation, a reduction in CD4(+)T lymphocyte number and a decrease of tumor necrosis factor-alpha (TNF-alpha) and Interferon-gamma (IFN-gamma) production in stressed mice. Additionally, mice subjected to chronic restraint stress displayed an enhancement of tumor growth in a syngeneic lymphoma model, i.e. an increase of tumor proliferation and a reduction of animal survival. Finally, stressed mice had a reduced specific cytotoxic response against these tumor cells. These results suggest that chronic exposure to stress promotes cancer establishment and subsequent progression, probably by depressing T-cell mediated immunity. The T-cell immunity impairment as well as the tumor progression enhancement emphasize the importance of the therapeutic management of stress to improve the prognosis of cancer patients.

Impaired immune responses in streptozotocin-induced type I diabetes in mice. Involvement of high glucose.

Diabetes is widely believed to predispose to serious infections. However, the mechanisms linking diabetes and immunosuppression are not well defined. One potential mediator of the altered defence mechanisms is hyperglycaemia. It has been identified as the main factor contributing to the development of diseases associated with diabetes mellitus. In this study we analyse the immune response in diabetes and the direct effect of hyperglycaemia on T and B lymphocyte reactivity. Diabetes induced an early decrease in IgG levels in the secondary response. However, both primary responses against a T-cell-dependent or independent antigen were affected after 6 months of diabetes induction. T- and B- cell proliferation was only decreased at this time. To gain insight into the potential mechanisms involved, we evaluated the influence of hyperglycaemia over the immune response. Pre-incubation of lymph node and spleen cells in a high glucose (HG) containing medium led to a significant time- and dose-dependent decrease in T- and B-cell proliferation. This effect was associated with the presence of HG-derived supernatants. Still viable cells after HG exposition were able to improve their proliferative response when cultured with the mitogen in a fresh standard medium. HG diminished cell viability, increased apoptosis and induced oxidative stress in lymphocytes. These results indicate that HG concentrations can directly affect lymphoid cell growth. An increase in oxidative stress would be implicated in this deleterious effect. The possibility that prolonged exposure to pathologically HG concentrations would result in the immunosuppressive state observed in diabetes is also discussed.

Protein kinase C-dependent NF-kappaB activation is altered in T cells by chronic stress.

Chronic stress has been associated with impaired immune function. In this work we studied the effect of chronic mild stress (CMS) exposure on the early intracellular pathways involved in T cells after stimulation with mitogen. We found that mitogen stimulation of T lymphocytes from CMS-exposed mice resulted in a reduction of the intracellular [Ca2+] rise, an impairment of growth-promoting protein kinase C (PKC) activation, a lower NF-kappaB activation and an increase in the inhibitory cAMP-protein kinase A (PKA) pathway activity with respect to those found in control lymphocytes. However, T cell activation with the direct PKC activator phorbol 12-myristate 13-acetate plus calcium ionophore led to a similar proliferative response in both CMS and control lymphocytes, indicating that signals downstream of PKC would not be affected by stress. In summary, our results show that chronic stress induced an alteration in T cell early transduction signals that result in an impairment of the proliferative response.

In vivo iron and zinc deficiency diminished T- and B-selective mitogen stimulation of murine lymphoid cells through protein kinase C-mediated mechanism.

Zinc and iron are crucial mineral components of human diet, because their deficiency leads to several disorders, including alterations of the immune function. It has been demonstrated, in both humans and rodents, that a diminished number of lymphoid cells and a loss of lymphocyte activity accompany deprivation of these essential minerals. The aim of this work was to analyze if iron and/or zinc imbalances regulate lymphocyte activity and the intracellular signals involved in the effect. Mice from the BALB/c strain were fed with iron- and/or zinc-deficient or mineral-supplemented diets, according to the American Institute of Nutrition Rodent Diets. Levels of iron and zinc were assessed in blood, liver, or bone samples. Selective mitogen stimulation of T- and B-lymphocytes were performed. We found a diminished proliferative response in T- and B-lymphocytes from zinc- and/or iron-deficient animals with respect to controls. These effects were related to decreased mitogen-induced translocation of protein kinase C (PKC) activity to cell membranes on both cell types from all animals fed with deficient diets. Our results demonstrate that iron and zinc deficiencies affect both T- and B-lymphocyte function by PKC-dependent mechanisms.

Downregulation of beta adrenergic receptor expression on B cells by activation of early signals in alloantigen-induced immune response.

Previously we described a decrease in beta-adrenergic receptor expression in B lymphocytes as a consequence of in vivo alloimmunization. This decrease correlates with the highest response of alloantibody production by B cells. In the present report we examined the participation of intracellular signals elicited after alloimmune stimulation. We showed that in vitro stimulation of B cells with mitomycin C-treated allogenic cells induced a reduction in the number of beta-adrenoceptors. This downregulation correlated to changes in basal and in isoproterenol-stimulated intracellular cAMP levels. We found that calcium mobilization and protein kinase C activation triggered after direct allogenic stimulation and/or by the action of T cell-soluble factors induced the reduction in beta-adrenoceptor sites. These findings could be of interest to understand the neuroendocrine mechanisms involved in the regulation of B cell activation.

Altered beta-adrenoceptor function associated to protein kinase C activation in hyperproliferative T lymphocytes.

beta-Adrenoceptor (betaAR) expression and function as well as its modulation via intracellular transduction signals, were analyzed on the T cell lymphoma BW5147. Independently to the kinetic of proliferation and relative to the number of receptors displayed in normal T lymphocytes, BW5147 cells displayed a decreased number of betaAR, uncoupled to adenylate cyclase, but coupled to protein kinase C stimulation. This last effect was impaired by a beta-antagonist and by blockers of the enzymatic pathways involved in T lymphocyte proliferation, inducing a recovery of betaAR sites. Down-regulation of betaAR would implicate the loss of a negative neuroimmune control mechanism for lymphocyte proliferation. The coupling of the remaining sites to a positive signal for cellular activation, would contribute to establish an hyperproliferative state.

Zinc status and immune system relationship: a review.

The essentiality of zinc for humans was first documented by Prasad in the 1960s. The main clinical manifestations associated with zinc deficiency are growth retardation, hypogonadism, diarrhea, and increased susceptibility to infectious diseases. Thus, in the past 25 yr, there was an increased interest of researchers in studying the role of zinc in human immunity. Although mechanistic research has been carried out using animal models, there are several studies in humans with similar results. This work is an attempt to review the information available in this field to understand the important role that zinc plays in the normal development and function of the immune system.

Intracellular signals coupled to muscarinic acetylcholine receptor activation in cerebral frontal cortex from hypoxic mice.

1. The aim of the present work was to determine hypoxia-induced modifications in the cascade of intracellular events coupled to muscarinic acetylcholine receptor (mAChR) activation in brain. For this purpose, enzymatic activities were measured on normoxically incubated frontal cortical slices from mice exposed to hypobaric hypoxia for 72 hr. 2. We found that hypoxia induced alterations in several cerebral enzymatic basal activities: it increased nitric oxide synthase (NOS), but it decreased both membrane protein kinase C (PKC) and phospholipase C activities. 3. The mAChR agonist carbachol was found to increase phosphoinositide hydrolysis to greater values in hypoxic tissues than those found in normoxic conditions. Furthermore, a greater translocation of PKC in response to carbachol was observed in hypoxic tissues than in normoxic ones. 4. Besides, carbachol induced a drastic reduction of NOS activity in hypoxic brains, at concentrations that stimulated this enzyme activity in normoxic preparations. In the latter, inhibition is obtained only with high concentrations of the cholinergic muscarinic agonist. 5. These results pointed to a carbachol-mediated mAChR hyperactivity induced by hypoxic insult. 6. The possibility that these effects would account for a compensatory mechanism to diminish NOS hyperactivity, probably protecting for NO neurotoxic action in hypoxic brain, is also discussed.

Experimental evidence pointing to the bidirectional interaction between the immune system and the thyroid axis.

Among the many examples of neuroendocrine-immune system interactions the relationship between the thyroid axis and the immune function has yet to be clearly established. Here we studied the influence of thyroid hormones on the course of an alloimmune response. Murine T(3) and T(4) levels were found to be increased a few days after the immunization of mice with allogeneic lymphoid cells. Besides in vivo treatment with T(4) was shown to increase alloantibody titers during the early stages of alloimmunization and to enforce lymphoid proliferation in vitro in a mixed lymphocyte reaction. Conversely, lowering thyroid hormone seric levels by propylthiouracil treatment, negatively modulates the humoral and cellular alloimmune responses. The evidence here points to the existence of a bidirectional communication between both systems. The possibility that the antigenic challenge would increase the thyroid gland activity thus leading to a positive modulatory action upon the immune response is also discussed.

Chronic stress influences the immune system through the thyroid axis.

The aim of the present work was to analyze the effect of chronic stress on thyroid axis and its influence on the immune response. For this purpose a murine model of chronic stress was developed to evaluate and to correlate thyroid hormone levels with humoral alloimmune response. Results show a reduction in serum levels of thyroid hormones, specially a significant decrease in serum levels of triiodotyronine (T3) in stressed animals. On the other hand, alloimmunization was not able to induce an early increment in T3 and thyroxine (T4) levels as it was previously reported in normal animals. In addition, lower titers of alloantibodies were obtained in animals under stress conditions as compared to normal mice. The sustitutive T4 treatment in stressed animals increased significantly alloantibody production as well as the early increment in thyroid hormones after antigenic challenge. These findings suggest that chronic stress induces an alteration of the function of thyroid axis that alters the immune response.