The differential sensitivity of the hypothalamic-hypophysial-ovarian axis to 5-hydroxytryptophan alters the secretion of estradiol.

Serotonin [5-hydroxytryptamine (5-HT)] modulates ovarian function. The precursor of 5-HT, 5-hydroxytryptophan (5-HTP), has been used to treat depression. However, the effects of 5-HTP on ovarian and reproductive physiology remain unknown. In this research, we analysed the impact of 5-HTP on the monoaminergic system and its interactions with the reproductive axis and ovarian estradiol secretion when administered by distinct routes. Female rats 30 days of age were injected with 5-HTP i.p. (100 mg/kg), into the ovarian bursa (1.5 µg/40 µL) or into the median raphe nucleus (20 µg/2.5 µL) and were killed 60 or 120 min after injection. As controls, we used rats of the same age injected with vehicle (0.9% NaCl). Monoamine, gonadotrophin and steroid ovarian hormone concentrations were measured. The injection of 5-HTP either i.p. or directly into the ovarian bursa increased the concentrations of 5-HT and the metabolite 5-hydroxyindole-3-acetic acid in the ovary. For both routes of administration, the serum concentration of estradiol increased. After i.p. injection of 5-HTP, the concentrations of luteinizing hormone were decreased and follicle-stimulating hormone increased after 120 min. Micro-injection of 5-HTP into the median raphe nucleus increased the concentrations of 5-HT in the anterior hypothalamus and dopamine in the medial hypothalamus after 120 min. Our results suggest that the administration of 5-HTP either i.p. or directly into the ovarian bursa enhances ovarian estradiol secretion.

Reactive oxygen species downregulate dystroglycans in the megakaryocytes of rats with arterial hypertension.

Hypertension is a multifactorial disease characterized by vascular and renal dysfunction, cardiovascular remodeling, inflammation, and fibrosis, all of which are associated with oxidative stress. We previously demonstrated cellular reactive oxygen species (ROS) imbalances may impact the structural and biochemical functions of blood cells and reported downregulation of ß-dystroglycan (ß-Dg) and overexpression of the epithelial sodium channel (ENaC) contribute to the pathophysiology of hypertension. In this study, we aimed to determine the expression of dystroglycans (Dg) and ENaC in platelet progenitors (megakaryocytes) and their surrounding niches. Thin sections of bone marrow from 5- and 28-week-old spontaneous hypertensive rats (SHR) were compared to age-matched normotensive rats (WKY). Cytometry and immunohistochemical assays demonstrated an oxidative environment in SHR bone marrow, characterized by high levels of myeloperoxidase and 3-nitrotyrosine and downregulation of peroxiredoxin II. In addition, transmission electron micrography and confocal microscopy revealed morphological changes in platelets and Mgks from SHR rats, including swollen mitochondria. Quantitative qRT-PCR assays confirmed downregulation of Dg mRNA and immunohistochemistry and western-blotting validated low expression of ß-Dg, mainly in the phosphorylated form, in Mgks from 28-week-old SHR rats. Moreover, we observed a progressive increase in ß-1 integrin expression in Mgks and extracellular matrix proteins in Mgk niches in SHR rats compared to WKY controls. These results indicate accumulation of ROS promotes oxidative stress within the bone marrow environment and detrimentally affects cellular homeostasis in hypertensive individuals.

Current and novel approaches in the pharmacological treatment of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most lethal malignant tumours worldwide. The mortality-to-incidence ratio is up to 91.6% in many countries, representing the third leading cause of cancer-related deaths. Systemic drugs, including the multikinase inhibitors sorafenib and lenvatinib, are first-line drugs used in HCC treatment. Unfortunately, these therapies are ineffective in most cases due to late diagnosis and the development of tumour resistance. Thus, novel pharmacological alternatives are urgently needed. For instance, immune checkpoint inhibitors have provided new approaches targeting cells of the immune system. Furthermore, monoclonal antibodies against programmed cell death-1 have shown benefits in HCC patients. In addition, drug combinations, including first-line treatment and immunotherapy, as well as drug repurposing, are promising novel therapeutic alternatives. Here, we review the current and novel pharmacological approaches to fight HCC. Preclinical studies, as well as approved and ongoing clinical trials for liver cancer treatment, are discussed. The pharmacological opportunities analysed here should lead to significant improvement in HCC therapy.

Characterization of the rat pituitary capsule: Evidence that the cerebrospinal fluid filled the pituitary cleft and the inner side of the capsule.

In humans, the pituitary gland is covered by a fibrous capsule and is considered a continuation of the meningeal sheath. However, in rodents some studies concluded that only the pars tuberalis (PT) and pars nervosa (PN) are enwrapped by the pia mater, while others showed that the whole gland is covered by this sheath. At PT the median eminence subarachnoid drains cerebrospinal fluid (CSF) to its cisternal system representing a pathway to the hypothalamus. In the present study we examined the rat pituitary capsule to elucidate its configuration, its physical interaction with the pituitary border and its relationship with the CSF. Furthermore, we also revisited the histology of the pituitary cleft and looked whether CSF drained in it. To answer such questions, we used scanning and transmission electron microscopy, intracerebroventricular infusion of Evan´s blue, fluorescent beads, and sodium fluorescein. The latter was measured in the pars distalis (PD) and various intracranial tissues. We found a pituitary capsule resembling leptomeninges, thick at the dorsal side of the pars intermedia (PI) and PD, thicker at the level of PI in contiguity with the PN and thinner at the rostro-ventral side as a thin membrane of fibroblast-like cells embedded in a fibrous layer. The capsule has abundant capillaries on all sides. Our results showed that the CSFs bathe between the capsule and the surface of the whole gland, and ciliate cells are present in the pituitary border. Our data suggest that the pituitary gland intercommunicates with the central nervous system (CNS) through the CSF.

The Combination Sorafenib-raloxifene-loratadine as a Novel Potential Therapeutic Approach Against Human Liver Cancer.

BACKGROUND/AIM: Liver cancer is one of the malignancies with the highest mortality-to-incidence ratio worldwide. Therefore, novel therapeutic approaches are urgently needed. Combination therapy and drug repurposing can improve the response of the patients to therapy in several cancers. The aim of the present study was to merge these two strategies and evaluate whether the two-drug- or three-drug- combination of sorafenib, raloxifene, and loratadine improves the antineoplastic effect on human liver cancer cells in comparison to the single-drug effect. MATERIALS AND METHODS: The human liver cancer cell lines HepG2 and HuH7 were studied. The effect of sorafenib, raloxifene, and loratadine on the metabolic activity was determined using the MTT assay. The inhibitory concentrations (IC20 and IC50) were calculated from these results and used in the drug-combination experiments. Apoptosis and cell survival were studied by flow cytometry and using the colony formation assay, respectively. RESULTS: In both cell lines, sorafenib, raloxifene, and loratadine in two-drug and three-drug combinations significantly reduced metabolic activity and significantly increased the percentage of apoptotic cells compared to the single-drug effect. In addition, all the combinations significantly reduced the colony-forming capacity in the HepG2 cell line. Surprisingly, the effect of raloxifene on apoptosis was similar to that observed using the combinations. CONCLUSION: The triple combination sorafenib-raloxifene-loratadine may be a novel promising approach in the treatment of liver cancer patients.

Physiological changes of growth hormone during lactation in pup rats artificially reared.

During the lactation period, rat pups are fed by the dam, and the patterns of mother-pup interaction change during this period. Additionally, there are changes in feeding; first, mother´s milk is the only food needed for sustenance, and later, it is combined with solid food and water. GH serum concentrations depend on both maternal-pup interaction and energy metabolism. In the artificial rearing (AR) procedure, pups are deprived of mother-pup interaction, and the feeding pattern is controlled. This rearing paradigm has been used in rats to analyze the effects of maternal deprivation on social behavior. In the present study, we analyzed the variation in GH, acylated ghrelin and IGF-1 serum concentrations throughout the lactation period in AR pups. At pnd7, the maternal rearing (MR) pups responded to a 4 h fast with a drop in GH serum concentration, which is a well-known response to maternal deprivation. GH serum levels in the AR pups did not change, suggesting an adaptation phenomenon. A dopamine inhibitory effect of GH secretion was observed in pnd7 cultured somatotropes, suggesting dopamine regulation of GH secretion at this age. Acylated ghrelin serum levels in the AR pups showed an inverted pattern compared to that in the MR pups, which was related to the artificial feeding pattern. IGF-1 serum levels were lower in the AR pups than in MR pups, which was associated with hepatic GH resistance and with low Igf1 mRNA expression at pnd7. Interestingly, at pnd14, both pup groups showed high hepatic Igf1 mRNA expression but low IGF-1 serum levels, and this was inverted at pnd21. However, serum glucose levels were lower in the AR pups at pnd14 but reached the same levels as the MR pups at pnd21. Moreover, hepatomegaly and higher hepatic GH-receptor levels were observed in the AR pups at pnd21, which was in agreement with an absence of a solid food meal. During AR, the pups lost the maternal interaction-stimulated GH secretion, which correlated with lower IGF-1 serum levels during the first week of postnatal development. Later, the AR pups exhibited hepatic responses, in order to satisfy the metabolic demand for the normal weaning, with low carbohydrates levels in their meal.

Differential regulation of human Eag1 channel expression by serum and epidermal growth factor in lung and breast cancer cells.

Oncogenic ether à-go-go-1 (Eag1) potassium channels are overexpressed in most primary human solid tumors. Low oxygen and nutrient/growth factor concentrations play critical roles in tumorigenesis. However, the mechanisms by which tumor cells survive and proliferate under growth factor-depleted conditions remain elusive. Here, we investigated whether serum-deprived conditions and epidermal growth factor (EGF) regulate Eag1 expression in human lung and breast cancer cells. The human cancer cell lines A549 and MCF-7 (from the lungs and breast, respectively) were obtained from the American Type Culture Collection and cultured following the manufacturer's recommendations. Eag1 gene and protein expression were studied by real-time PCR and immunocytochemistry, respectively. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and ERK1/2 phosphorylation was investigated by Western blot. Serum-deprived conditions increased Eag1 mRNA and protein expression in both cell lines. This Eag1 upregulation was prevented by EGF and the ERK1/2 inhibitor U0126 in only lung cancer cells; vascular endothelial growth factor did not prevent Eag1 upregulation. Our results suggest that Eag1 may act as a survival and mitogenic factor under low-serum and nutrient conditions and may be a clinical target during the early stages of tumor development.

Early social isolation provokes electrophysiological and structural changes in cutaneous sensory nerves of adult male rats.

Sensory and social deprivation from the mother and littermates during early life disturbs the development of the central nervous system, but little is known about its effect on the development of the peripheral nervous system. To assess peripheral effects of early isolation, male rat pups were reared artificially in complete social isolation (AR); reared artificially with two same-age conspecifics (AR-Social); or reared by their mothers and with littermates (MR). As adults, the electrophysiological properties of the sensory sural (SU) nerve were recorded. We found that the amplitude and normalized area (with respect to body weight) of the compound action potential (CAP) response provoked by single electrical pulses of graded intensity in the SU nerves of AR animals were shorter than the CAP recorded in SU nerves from MR and AR-Social animals. The slope of the stimulus-response curve of AR SU nerves was smaller than that of the other nerves. The histological characterization of axons in the SU nerves was made and showed that the myelin thickness of axons in AR SU nerves was significant lower (2-7µm) than that of the axons in the other nerves. Furthermore, the area and axon diameter of SU nerves of both AR and AR-Social animals were significant lower than in MR animals. This is the first report to show that maternal and littermate deprivation by AR disturbs the development of the myelination and electrophysiological properties of axons in the SU nerve; the replacement of social cues prevents most of the effects.

Expression of Eag1 K+ channel and ErbBs in human pituitary adenomas: cytoskeleton arrangement patterns in cultured cells.

Pituitary adenomas can invade surrounded tissue, but the mechanism remains elusive. Ether à go-go-1 (Eag1) potassium channel and epidermal growth factor receptors (ErbB1 and ErbB2) have been associated to invasive phenotypes or poor prognosis in cancer patients. However, cells arrange their cytoskeleton in order to acquire a successful migration pattern. We have studied ErbBs and Eag1 expression, and cytoskeleton arrangements in 11 human pituitary adenomas. Eag1, ErbB1 and ErbB2 expression were studied by immunochemistry in tissue and cultured cells. The cytoskeleton arrangement was analyzed in cultured cells by immunofluorescence. Normal pituitary tissue showed ErbB2 expression and Eag1 only in few cells. However, Eag1 and ErbB2 were expressed in all the tumors analyzed. ErbB1 expression was observed variable and did not show specificity for a tumor characteristic. Cultured cells from micro- and macro-adenomas clinically functional organize their cytoskeleton suggesting a mesenchymal pattern, and a round leucocyte/amoeboid pattern from invasive clinically silent adenoma. Pituitary tumors over-express EGF receptors and the ErbB2 repeated expression suggests is a characteristic of adenomas. Eag 1 was express, in different extent, and could be a therapeutic target. The cytoskeleton arrangements observed suggest that pituitary tumor cells acquire different patterns: mesenchymal, and leucocyte/amoeboid, the last observed in the invasive adenomas. Amoeboid migration pattern has been associated with high invasion capacity.

Differences in cell migration of cultured pituitary cells from infantile and adult rats: participation of the extracellular matrix and epidermal growth factor.

During early postnatal development in the rat, the tissue architecture of the pituitary gland shows changes, revealing an intense migration process of the cells. The aim of this work was to examine anterior pituitary cell migration over type I and III collagen as well as type IV collagen, of cultured pituitary cells from infantile rats and adult rats, and the participation of the epidermal growth factor in this process. Differences in cell migration rate over these two types of collagen substrates were observed at both ages, and all in all, three times more cells migrated over type I/III collagen than over type IV collagen. These data show the migration-promoting role of type I/III collagen for pituitary cells. Furthermore, when infantile cells were challenged to migrate over bovine serum albumin, the migration rate diminished, and, on the contrary, adult cell migration was higher. However, over collagen, infantile cells increased their migration rate with epidermal growth factor stimulation and adult cells showed a decrease in migration when the growth factor was in the medium. During migration, pituitary cells associated and arranged in clusters. This behavior increased in the presence of epidermal growth factor in the infantile cultures. Moreover, epidermal growth-factor-stimulated infantile cells formed larger aggregates. Adult cells also showed associative behavior, but more cells were observed isolated than in cluster arrangements and the growth factor did not induce changes in this behavior. Results showed a difference in the response of cell migration and cell association capacity to epidermal growth factor after migration of infantile and adult pituitary cells. With these observations we propose that epidermal growth factor is a cell regulator of the pituitary tissue re-arrangement process during the infantile period.