Iron excess in rice: from phenotypic changes to functional genomics of WRKY transcription factors.

Iron (Fe) is an essential microelement for all living organisms playing important roles in several metabolic reactions. Rice (Oryza sativa L.) is commonly cultivated in paddy fields, where Fe goes through a reduction reaction from Fe3+ to Fe2+. Since Fe2+ is more soluble, it can reach toxic levels inside plant cells, constituting an important target for studies. Here we aimed to verify morphological changes of different rice genotypes focusing on deciphering the underlying molecular network induced upon Fe excess treatments with special emphasis on the role of four WRKY transcription factors. The transcriptional response peak of these WRKY transcription factors in rice seedlings occurs at 4 days of exposition to iron excess. OsWRKY55-like, OsWRKY46, OsWRKY64, and OsWRKY113 are up-regulated in BR IRGA 409, an iron-sensitive genotype, while in cultivars Nipponbare (moderately resistant) and EPAGRI 108 (resistant) the expression profiles of these transcription factors show similar behaviors. Here is also shown that some cis-regulatory elements known to be involved in other different stress responses can be linked to conditions of iron excess. Overall, here we support the role of WRKY transcription factors in iron stress tolerance with other important steps toward finding why some rice genotypes are more tolerant than others.

The effects of pharmacist interventions on adult outpatients with cancer: A systematic review.

WHAT IS KNOWN AND OBJECTIVES: Most antineoplastic drugs are highly toxic and have low therapeutic indexes, which can result in drug-related problems. In this context, pharmacist interventions may play an important role in the success of the treatment. The objective of this study was to examine the effects of pharmacist interventions on adult outpatients with cancer using antineoplastic drugs. METHODS: A literature search was performed using PubMed, ISI Web of Science and LILACS databases from January 1990 to April 2016, using MeSH terms or text words related to pharmacist interventions, cancer and outpatient care. Studies published in English, Portuguese or Spanish on the effects of pharmacist interventions in outcome measures in adult outpatients with cancer were included. Two independent authors performed study selection and data extraction with a consensus process. The articles were analysed according to previously established criteria, such as country, study design, setting, population, type of cancer, description of the intervention and control groups, outcomes, main conclusions and study limitations. RESULTS AND DISCUSSION: A total of 874 records were identified, of which 11 satisfied the inclusion criteria. The studies were conducted mainly in the United States and included patients aged >50 years. Most studies had a before-after design. Pharmacist interventions primarily included educating and counselling patients on the management of adverse events. Rates of nausea and vomiting control, medication adherence and patient satisfaction were the most common outcome measures; a significant benefit in these parameters as a result of pharmacist interventions was noted in most studies. WHAT IS NEW AND CONCLUSION: The findings from this systematic review indicate that pharmacist interventions can improve outcome measures in outpatients with cancer. However, the collective quality of the studies was poor and gaps identified indicate that further research is needed to provide more robust results.

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.

In vitro and in vivo experiments with iron oxide nanoparticles functionalized with DEXTRAN or polyethylene glycol for medical applications: magnetic targeting.

In this research work, DEXTRAN- and polyethylene glycol (PEG)-coated iron-oxide superparamagnetic nanoparticles were synthetized and their cytotoxicity and biodistribution assessed. Well-crystalline hydrophobic Fe3 O4 SPIONs were formed by a thermal decomposition process with d = 18 nm and σ = 2 nm; finally, the character of SPIONs was changed to hydrophilic by a post-synthesis procedure with the functionalization of the SPIONs with PEG or DEXTRAN. The nanoparticles present high saturation magnetization and superparamagnetic behavior at room temperature, and the hydrodynamic diameters of DEXTRAN- and PEG-coated SPIONs were measured as 170 and 120 nm, respectively. PEG- and DEXTRAN-coated SPIONs have a Specific Power Absorption SPA of 320 and 400 W/g, respectively, in an ac magnetic field with amplitude of 13 kA/m and frequency of 256 kHz. In vitro studies using VERO and MDCK cell lineages were performed to study the cytotoxicity and cell uptake of the SPIONs. For both cell lineages, PEG- and DEXTRAN-coated nanoparticles presented high cell viability for concentrations as high as 200 µg/mL. In vivo studies were conducted using BALB/c mice inoculating the SPIONs intravenously and exposing them to the presence of an external magnet located over the tumour. It was observed that the amount of PEG-coated SPIONs in the tumor increased by up to 160% when using the external permanent magnetic as opposed to those animals that were not exposed to the external magnetic field.

Biodistribution of the boron carriers boronophenylalanine (BPA) and/or decahydrodecaborate (GB-10) for Boron Neutron Capture Therapy (BNCT) in an experimental model of lung metastases.

BNCT was proposed for the treatment of diffuse, non-resectable tumors in the lung. We performed boron biodistribution studies with 5 administration protocols employing the boron carriers BPA and/or GB-10 in an experimental model of disseminated lung metastases in rats. All 5 protocols were non-toxic and showed preferential tumor boron uptake versus lung. Absolute tumor boron concentration values were therapeutically useful (25-76ppm) for 3 protocols. Dosimetric calculations indicate that BNCT at RA-3 would be potentially therapeutic without exceeding radiotolerance in the lung.

A new quantitative method to determine the uptake of SPIONs in animal tissue and its application to determine the quantity of nanoparticles in the liver and lung of Balb-c mice exposed to the SPIONs.

We propose a new method for determining the quantity of superparamagnetic iron oxide nanoparticles (Fe3O4, SPIONs) embedded in animal tissue using magnetization measurements. With this method, the smallest detectable quantity of magnetite nanoparticles in a tissue sample is -1 microg. We showed that this method has proved being efficient. In this study, we focused in determining the quantity of SPION confined in lung and liver tissue of mice injected with -13 nm magnetite superparamagnetic nanoparticles. Furthermore, the method allowed us to detect the magnetite nanoparticles present in animal tissues without letting the natural iron ions present in the tissue or blood interfere with the measurements.

Heme oxygenase 1 (HO-1) challenges the angiogenic switch in prostate cancer.

Prostate cancer (PCa) is the second leading cause of cancer-associated death in men. Once a tumor is established it may attain further characteristics via mutations or hypoxia, which stimulate new blood vessels. Angiogenesis is a hallmark in the pathogenesis of cancer and inflammatory diseases that may predispose to cancer. Heme oxygenase-1 (HO-1) counteracts oxidative and inflammatory damage and was previously reported to play a key role in prostate carcinogenesis. To gain insight into the anti-tumoral properties of HO-1, we investigated its capability to modulate PCa associated-angiogenesis. In the present study, we identified in PC3 cells a set of inflammatory and pro-angiogenic genes down-regulated in response to HO-1 overexpression, in particular VEGFA, VEGFC, HIF1α and α5ß1 integrin. Our results indicated that HO-1 counteracts oxidative imbalance reducing ROS levels. An in vivo angiogenic assay showed that intradermal inoculation of PC3 cells stable transfected with HO-1 (PC3HO-1) generated tumours less vascularised than controls, with decreased microvessel density and reduced CD34 and MMP9 positive staining. Interestingly, longer term grown PC3HO-1 xenografts displayed reduced neovascularization with the subsequent down-regulation of VEGFR2 expression. Additionally, HO-1 repressed nuclear factor κB (NF-κB)-mediated transcription from an NF-κB responsive luciferase reporter construct, which strongly suggests that HO-1 may regulate angiogenesis through this pathway. Taken together, these data supports a key role of HO-1 as a modulator of the angiogenic switch in prostate carcinogenesis ascertaining it as a logical target for intervention therapy.

Ion transport in tumors under electrochemical treatment: in vivo, in vitro and in silico modeling.

The electrochemical treatment of cancer (EChT) consists in the passage of a direct electric current through two or more electrodes inserted locally in the tumor tissue. The extreme pH changes induced have been proposed as the main tumor destruction mechanism. Here, we study ion transport during EChT through a combined modeling methodology: in vivo modeling with BALB/c mice bearing a subcutaneous tumor, in vitro modeling with agar and collagen gels, and in silico modeling using the one-dimensional Nernst-Planck and Poisson equations for ion transport in a four-ion electrolyte. This combined modeling approach reveals that, under EChT modeling, an initial condition with almost neutral pH evolves between electrodes into extreme cathodic alkaline and anodic acidic fronts moving towards each other, leaving the possible existence of a biological pH region between them; towards the periphery, the pH decays to its neutral values. pH front tracking unveils a time scaling close to t(1/2), signature of a diffusion-controlled process. These results could have significant implications in EChT optimal operative conditions and dose planning, in particular, in the way in which the evolving EChT pH region covers the active cancer cells spherical casket.

Inhibition of invasion and metastasis by glypican-3 in a syngeneic breast cancer model.

Glypican-3 (GPC3), a proteoglycan bound to the cell membrane through a GPI anchor, is widely expressed in the embryo but down regulated in most adult tissues, with some exceptions as mammary cells. GPC3 is involved in the regulation of cell proliferation and survival in specific cell types. LM3, a murine mammary tumor cell line unable to express GPC3, was stably transfected with the rat GPC3 gene to analyze its role in tumor progression. Upon injection into syngeneic BALB/c mice LM3-GPC3 clones showed less local invasiveness and developed fewer spontaneous and experimental lung metastasis than controls. GPC3-expressing cells were more sensitive to apoptosis induced by serum depletion, exhibited a delay in the first steps of spreading and were less motile than controls. On the other hand, LM3-GPC3 cells were significantly more adherent to FN than control ones. We observed that GPC3 transfectants presented a higher expression of E-cadherin and beta-catenin, molecules whose down regulation has been associated with tumor progression. Exogenous TGF-beta increased MMP-9 activity in both control and GPC3-expressing cells, but did not modulate MMP-2. Contrarily, GPC3 expression prevented the increase of MMP-2 activity induced by IGF-II. Our results suggest that GPC3 has a protective role against mammary cancer progression.

In vivo evaluation of three different 99mTc-labelled radiopharmaceuticals for sentinel lymph node identification.

This work was designed to compare sentinel lymph node (SLN) uptake of 99mTc-labelled human serum albumin colloid (99mTc-HSAC), 99mTc-labelled antimony sulphur colloid (99mTc-SC) and a 99mTc-labelled dextran 70 solution (99mTc-Dx) and their selectivity in the identification of this node in the right rear footpad (RRF) of normal mice and tumour bearing mice. Radiopharmaceutical uptake in the SLN (popliteal lymph node) and the lumbar lymph node (LLN), the second lymphatic node station from RRF, were measured at different time points post-intradermal or intratumoural injection into the RRF of NIH normal mice and of Balb/c mice harbouring the murine mammary tumour M2. 99mTc-HSAC uptake in the SLN was significantly higher than LLN uptake. The 99mTc-SC demonstrated high uptake in SLN, but accumulation in LLN was also high. 99mTc-Dx showed low uptakes in both SLN and LLN. The intradermal injection resulted in a more effective radiopharmaceutical accumulation in SLN than did the intratumoural inoculation. Data also show that increments in tumour volume reduced radiopharmaceutical uptake in the SLN. Our results show that 99mTc-HSAC exhibits the highest uptake in the SLN combined with the smallest amounts of radiopharmaceutical passing through to the LLN. Therefore, 99mTc-HSAC appears to be the best radiopharmaceutical for sentinel node detection.

Characterization of a fibroblastoid mammary carcinoma cell line (LM2) originated from a mouse adenocarcinoma.

We established and characterized a new mammary tumor cell line, LM2, derived from M2 mammary adenocarcinoma which spontaneously appeared in a BALB/c female mouse. The LM2 cell line has been maintained in culture for more than 40 passages and grows as poorly differentiated elongated cells. Ultrastructural and immunocytochemistry analysis revealed characteristic features of adenocarcinoma. Cytogenetic studies showed that LM2 cells are fundamentally hypotetraploid. They express metalloproteinases (MMP) and show high levels of plasminogen activator type urokinase (uPA). They were sensitive to nitric oxide (NO)-mediated cytotoxicity when NO derived from an exogenous donor. In vivo, although LM2 cells were able to grow in the lungs, they could not metastasize to the same target organ from s.c. primary tumors. The LM2 mouse mammary adenocarcinoma cell line is a suitable model to examine different aspects of tumor biology, in particular those related to the different pathways involved in the metastatic cascade and in the cytotoxicity mediated by NO.

The time of tumor cell division and death depends on the site of growth.

We show here, for the first time, in two very different murine tumors, a mammary one (ectoderm) and a lung one (endoderm), that: tumors have day/night differences of spontaneous apoptosis additional to the well-known circadian rhythm of mitosis. The times of maximal and minimal mitosis and apoptosis changed for a tumor cell line when growing in different organs (as metastasis) or anatomical sites. Both tumor lines, have identical circadian curves when growing in a specific organ or anatomical site. The peaks of apoptosis match with the valleys of mitosis and vice versa.

A synthetic, nitrogenated antimetastatic and anti-angiogenic compound with low toxicity in vivo.

The design of more effective therapies for metastatic disease involves development of new compounds able to specifically block the malignant process. We demonstrated previously that a new synthetic nitrogenated compound 3'-1-chloroethyl-2,3-dihydro-1H-imidazo-(2, 1-i)-purine-4-ium-7-yl-3'-deoxy-1',5', 6'-tri-O-(methylsulfonyl)-muco-inositol chloride (DIC) had an anti-proliferative activity on tumor cells in vitro. In the present work we demonstrate that DIC induces apoptosis on the LM3 murine mammary adenocarcinoma cell line in vitro and has anti-angiogenic activity in vivo. We also evaluated toxicity, biodistribution and anti-neoplastic properties of DIC in vivo. Toxicity studies allowed us to establish the LD50 (750 mg/kg body weight). Administration of 250 mg/kg/day (LD10) for 6 days did not cause overt toxic effects. Biodistribution assays revealed that DIC was rapidly eliminated (60% at t=10 min), although it accumulated in tumor tissue at higher concentrations than in other tissues. Daily s.c. treatment with DIC (LD10) for 24 days significantly reduced the number of spontaneous lung metastases. These results suggest that DIC has the ability of impairing the metastatic development by inhibiting angiogenesis and inducing apoptosis on tumor cells.

[Possible role of the immune system in lead peripheral polyneuropathy. Case report]. / Possível intermediação do sistema imune na polineuropatia periférica plúmbica. Relato de caso.

This is a case report of a twenty-five years old man who developed, due to lead intoxication, a severe axonal peripheral predominantly motor neuropathy, after a shotgun injury. The projectile was retained in the right hip. Before this diagnosis had been done he was treated with corticosteroids in immunosuppressive doses and showed an improvement, but he had worsened at each attempt to interrupt the drug. Because he had also other signs of lead intoxication, such as abdominal cramps, severe anemia and seizures it was search for the blood levels of lead that was 101.2 micrograms/dl. The patient was treated with calcium disodium edetate and surgical removal of lead fragments. After that he had a good outcome with no need of corticosteroids. It is emphasized the possible relevance of the immune system on the mechanism of plumbic intoxication and the importance of the withdrawal of the lead material retained in joints.

Specific biodetection of a murine spontaneous mammary carcinoma with labelled anti-human blood group-A monoclonal antibody.

The expression of cell surface antigens of the spontaneous transplantable M3-murine tumour was studied by means of the monoclonal antibody (MAb) B2C114 which recognizes the human blood group-A carbohydrate antigen. Following radioiodination the MAb retained their immunoreactivity and demonstrated a significantly higher in vitro binding with isolated M3-tumour cells as compared with a control antibody. B2C114 revealed 10(6) antigenic sites per cell, with a constant affinity of 5.1 x 10(9)/M. Biodistribution studies showed that B2C114 discriminated between malignant tumour and mouse normal tissues. Radioimmunodetection of Balb/c mice bearing s.c. M3-tumour showed that tumour was specifically defined without subtraction 1 day after injection of the radiolabelled antibody.

Slower growth of tumours in sympathetically denervated murine skin.

In order to examine tumour growth in sympathetically denervated murine skin, two breast cancer tumour lines were employed, i.e. M3 tumours, of a relatively high local growth and low metastatic capacity, and MM3-LN tumours, that grew locally at a slower rate but disseminated early to the lung. Mice subjected to unilateral superior cervical ganglionectomy or sham-operation 2 weeks earlier were used. M3 or MM3-LN tumours were implanted in the ipsilateral ear to the surgical procedure. Tumour size was assessed every 2-6 days, starting from the 7th day after tumour implantation. Growth of M3 and MM3-LN tumours was significantly slowed by a previous sympathetic denervation of the skin territory. There were no significant differences in the number or size of pulmonary metastases at autopsy between mice subjected to ganglionectomy or to sham-operation. Ganglionectomy increased significantly ipsilateral submaxillary lymph node ornithine decarboxylase activity by 62% and decreased noradrenaline content to 8% of the innervated contralateral lymph node. The present results indicate a local inhibitory modulation of tumour growth by the sympathetic nervous system.

In vivo selection and characterization of a murine mammary tumor subline with high potential for spontaneous lymph node metastasis.

A transplantable mammary adenocarcinoma, grown in Balb/c mice, with a marked enhancement in its draining lymph node metastatic ability (MM3LN), was obtained through an in vivo procedure from a variant tumor moderately metastatic to lymph nodes (MM3). Both MM3 and MM3LN presented a similar latency and tumor growth rate and reached the same tumor mean diameter at death. MM3LN tumor-bearing mice exhibited a larger mean survival time. The new variant showed a 2.5-fold higher incidence of tumor-draining lymph node metastases than MM3 line, with no differences in the incidence of lung metastases. Morphology as well as cytogenetic and in vitro adhesion properties were studied in order to characterize the new subline. This murine tumor model has potential application in the study of the metastatic process in lymphoid tissue.

Characterization of a new murine tumor variant with different in vivo behavior selected by its adhesive properties.

The purpose of the study was to determine whether the selection by adhesion to fibronectin (FN) also selects for cells with different tumorigenic and metastatic abilities. M3 murine mammary adenocarcinoma cells with moderate metastatic potential were seeded on FN-coated plastic substrates. Non-adherent cells were removed at 30 min, and the adherent ones were expanded in monolayer culture. Selected tumor cells were then harvested and inoculated sc into syngeneic mice. The selection procedure was repeated three times. After the third cycle, tumors were further maintained by sc trocar transplantation, and the variant obtained was called M3Ad. Although the in vitro selection was adhesion to FN substrate, the in vitro adhesion behavior of the variant M3Ad was identical to that of the parental tumor M3. However, the cytogenetic profile and the in vivo behavior indicated that M3Ad differed from M3. The distribution of the chromosome number of M3Ad cells revealed a lower mode and mean than of M3 cells. Moreover, the M3Ad variant exhibited a shorter latency, a higher growth rate and a lower incidence of spontaneous lung metastases. However, it produced significantly more and larger lung colonies than M3 after iv injection.

Inmunorregulación del crecimiento tumoral en un sistema murino / Immunoregulation of tumor growth in a murine model

Estudiamos la regulación del crecimiento tumoral y metastásico tanto por el propio tumor como por el sistema inmune, en un modelo murino. Sobrenadantes de cultivo de esplenocitos de portadores de tumor exacerban el crecimiento del propio tumor. Esta actividad desaparece luego de la cirugía tumoral pero con una cinética diferente dependendo de si el tumor es precoz o avanzado al tiempo de la cirugía. Las poblaciones esplénicas involucradas en la exacerbación varían durante el crecimiento del tumor mientras que las responsables de inducción de angiogénesis son siempre de naturaleza T. Con respecto de la autorregulación tumoral, encontramos que diferentes formas de antígenos tumorales (extractos tumorales, sobrenadantes de cultivo de células tumorales, células tumorales formolizadas) aumentan la diseminación metastásica, pero esta actividad está mediada por el sistema inmune del huésped. Por el contrario, sobrenadantes de cultivo de células tumorales no inducen exacerbación del tumor primario. Las células tumorales fueron tratadas con un modificador de membrana con la expectativa de alterar la respuesta inmune antitumoral