The shift in GH3 cell shape and cell motility is dependent on MLCK and ROCK.

Cytoskeletal organization, actin-myosin contractility and the cell membrane together regulate cell morphology in response to the cell environment, wherein the extracellular matrix (ECM) is an indispensable component. Plasticity in cell shape enables cells to adapt their migration mode to their surroundings. GH3 endocrine cells respond to different ECM proteins, acquiring different morphologies: a rounded on collagen I-III (C I-III) and an elongated on collagen IV (C IV). However, the identities of the molecules that participate in these responses remain unknown. Considering that actin-myosin contractility is crucial to maintaining cell shape, we analyzed the participation of MLCK and ROCK in the acquisition of cell shape, the generation of cellular tension and the cell motility mode. We found that a rounded shape with high cortical tension depends on MLCK and ROCK, whereas in cells with an elongated shape, MLCK is the primary protein responsible for cell spreading. Further, in cells with a slow and directionally persistent motility, MLCK predominates, while rapid and erratic movement is ROCK-dependent. This behavior also correlates with GTPase activation. Cells on C I-III exhibited higher Rho-GTPase activity than cells on C IV and vice versa with Rac-GTPase activity, showing a plastic response of GH3 cells to their environment, leading to the generation of different cytoskeleton and membrane organizations and resulting in two movement strategies, rounded and fibroblastoid-like.

Anterior and intermediate pituitary tissues express claudin 4 in follicle stellate cells and claudins 2 and 5 in endothelial cells.

Follicle-stellate cells are pituitary non-granular cells that are arranged between secretory cells or organized in follicles with small lumens. Cells from the follicles exhibit the typical phenotype of a transporting epithelium, including apical microvilli with a cilium and tight junctions. Freeze-fracture electron microscopy images show that the tight junctions consist of 5-7 anastomosing strands and that cultured follicle-stellate cells develop a trans-epithelial electrical resistance characteristic of "tight" epithelia. Here, we investigate the molecular composition of the tight junction from follicle stellate cells. We found that the rat anterior pituitary lobe expresses mRNAs for claudins 2, 4 and 5; the proteins of all these claudins are observed in the anterior lobe, whereas the intermediate lobe expresses claudins 2 and 5 and the posterior lobe contains only claudin 5. Follicle-stellate cells, identified by their protein marker S100ß, expresses claudin 4 in the apical membrane, in co-localization with dipeptidyl-peptidase and near acetylated ß-tubulin. Claudin 4 partially co-localizes with E-cadherin, indicating that a fraction of the protein is located in the basolateral domain. Follicle-stellate-enriched cell cultures develop patches of polygonal cells expressing claudin 4 and E-cadherin, encircled by extensive monolayers of fusiform cells. Claudin 2 stains specifically blood vessels, identified by claudin 5 and VE-cadherin labels. Thus, follicles in the anterior pituitary consist of "tight" epithelia that can carry out intense vectorial transport, together with a high cation movement in blood vessels, possibly related to the ion requirements of excitable secretory cells for hormone secretion.

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.

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 invasion mode of GH(3) cells is conditioned by collagen subtype, and its efficiency depends on cell-cell adhesion.

The adaptation of GH(3) cells to different microenvironments is a consequence of a partial compromise with the tumor phenotype. A collagen type IV enriched microenvironment favors an invasive phenotype and increases the substrate adhesion capacity, whereas it decreases the phosphorylation of the regulatory myosin light chain and the aggregation capacity. In contrast, the higher internal tension and increased aggregation capacity induced by collagen type I/III are factors that reduce the invasion rate. Our results show, for the first time, the importance of collagen subtypes in determining the migratory strategy: collagen I/III favors mesenchymal-like motility, whereas collagen type IV induces an ameboid-type displacement. The reciprocal modulation of the myosin light chain kinase and the Rho-kinase determines the invasive capacity through changes in tissue cohesion, extracellular matrix affinity, regulatory myosin light chain phosphorylation and spatial distribution. The collagen subtype determines which of the mechano-transduction signaling pathways will regulate the tensional homeostasis and affect the invasion ability as well as the preferred migration strategy of the cells.

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.

Effect of extracellular matrix on adhesion, viability, actin cytoskeleton and K+ currents of cells expressing human ether à go-go channels.

Ether à go-go (EAG) potassium channels possess oncogenic properties and have gained great interest as research tools for cancer detection and therapy. Besides, EAG electrophysiological properties are regulated through the cell cycle and determined by cytoskeletal interactions. Thus, because of the pivotal role of extracellular matrix (ECM) and cytoskeleton in cancer progression, we studied the effect of ECM components on adhesion, viability, actin organization and EAG currents in wild-type CHO cells (CHO-wt) and cells expressing human EAG channels (CHO-hEAG). At short incubation times, adhesion and viability of CHO-hEAG cells grown on collagen, heparin or poly-lysine were lower than CHO-wt cells, however, only CHO-hEAG sustained growing under total serum starvation. CHO-hEAG cells grown on poly-lysine did not organize their cytoskeleton but when grown on collagen or fibronectin displayed lamellipodia and stress fibers, respectively. Interestingly, EAG expressing cells displayed special actin structures suggesting a dynamic actin cytoskeleton, such structures were not exhibited by wild-type cells. EAG current density was significantly lower in cells grown on collagen at short incubation times. Finally, we studied potential associations between hEAG channels and integrins or actin filaments by confocal microscopy. No association between beta1-integrins and hEAG channels was found, however, a very strong co-localization was observed between hEAG channels and actin filaments, supported by immunoblot experiments in which hEAG channels were found in the insoluble fraction (associated to cytoskeleton). Our results suggest ECM components as potential modulators of oncogenic human-EAG expressing cells and emphasize the relationship between potassium channels, cytoskeleton, ECM and cancer.

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells.

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

GH3 tumor pituitary cell cytoskeleton and plasma membrane arrangement are determined by extracellular matrix proteins: implications on motility, proliferation and hormone secretion.

The extracellular matrix (ECM) influences different physiological and pathophysiological aspects of the cell. The ECM consists in a complex network of macromolecules with characteristic biochemical properties that allow cells to sense their environments inducing different signals and changing cell behavior. The purpose of the present study was to evaluate the participation of different ECM proteins in cell morphology and its implication on motility, proliferation and hormone secretion in GH3 cells, a tumor pituitary cell. GH3 cells were cultured with a defined medium on collagens I/III and IV, fibronectin and laminin. GH3 cells express α2 integrin subunit de novo. The cells responded to the ECM proteins with differentiated cell surface morphologies and membrane protrusions. A rounded shape with small membrane blebs, weak substrate adhesion and high motility was observed in cells on C I/III and fibronectin, while on C IV and laminin cells were viewed elongated and adhered. Differences on actin cytoskeleton, cytoskeletal-associated vinculin and phospho-MLC showed that ECM proteins determine the cytoskeleton organization. Cell proliferation showed dependency on the ECM protein, observing a higher rate in cells on collagen I/III. Prolactin secretion was higher in cells with small blebs, but an unchangeable response to EGF was obtained with the ECM proteins, suggesting is a consequence of cortical actin arrangement. We ascribe the functional differences of the GH3 cells to the cytoskeletal organization. Overall, the data showed that ECM plays a critical role in GH3 cells modulating different cellular comportment and evidenced the importance of the ECM composition of pituitary adenomas.

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.

Role of extracellular matrix-cell interaction and epidermal growth factor (EGF) on EGF-receptors and actin cytoskeleton arrangement in infantile pituitary cells.

Epidermal growth factor (EGF) induces changes in cell morphology, actin cytoskeleton, and adhesion processes in cultured infantile pituitary cells. The extracellular matrix, through integrin engagement, collaborates with growth factors in cell signaling. We have examined the participation of collagen I/III and collagen plus fibronectin in the EGF response of infantile pituitary cells with respect to their cell morphology and actin cytoskeleton. As a comparison, we have used poly-lysine as a substrate. Infantile cells elicit the EGF response when they are associated with extracellular matrix proteins, but no response can be obtained with poly-lysine as the substrate. Cells acquire a flattened shape and organize their actin filaments and vinculin as in focal adhesions. Because the EGF receptor (EGFR) is linked to the actin cytoskeleton in other cells structuring a microdomain in cell signaling, we have investigated this association and substrate adhesion participation in infantile pituitary cells. The proportion of EGFR associated with the actin cytoskeleton is approximately 31%; no difference has been observed between the substrates used. Cells in suspension show actin-associated EGFR, suggesting an association independent of cell adhesion. However, no colocalization of EGFRs with actin fibers has been observed, suggesting an indirect association. Compared with beta(1)-integrin, which is linked to actin fibers through structural proteins, EGFR binds more strongly with the actin cytoskeleton. This study thus shows cell adhesion dependence on the EGF effect in the actin cytoskeleton arrangement; this is probably favored by the actin fiber/EGFR association that facilitates the cell signaling pathways for actin cytoskeleton organization in infantile pituitary cells.