Thioholgamide A, a New Anti-Proliferative Anti-Tumor Agent, Modulates Macrophage Polarization and Metabolism.

Natural products represent powerful tools searching for novel anticancer drugs. Thioholgamide A (thioA) is a ribosomally synthesized and post-translationally modified peptide, which has been identified as a product of Streptomyces sp. MUSC 136T. In this study, we provide a comprehensive biological profile of thioA, elucidating its effects on different hallmarks of cancer in tumor cells as well as in macrophages as crucial players of the tumor microenvironment. In 2D and 3D in vitro cell culture models thioA showed potent anti-proliferative activities in cancer cells at nanomolar concentrations. Anti-proliferative actions were confirmed in vivo in zebrafish embryos. Cytotoxicity was only induced at several-fold higher concentrations, as assessed by live-cell microscopy and biochemical analyses. ThioA exhibited a potent modulation of cell metabolism by inhibiting oxidative phosphorylation, as determined in a live-cell metabolic assay platform. The metabolic modulation caused a repolarization of in vitro differentiated and polarized tumor-promoting human monocyte-derived macrophages: ThioA-treated macrophages showed an altered morphology and a modulated expression of genes and surface markers. Taken together, the metabolic regulator thioA revealed low activities in non-tumorigenic cells and an interesting anti-cancer profile by orchestrating different hallmarks of cancer, both in tumor cells as well as in macrophages as part of the tumor microenvironment.

The glucocorticoid-induced leucine zipper mediates statin-induced muscle damage.

Statins, the most prescribed class of drugs for the treatment of hypercholesterolemia, can cause muscle-related adverse effects. It has been shown that the glucocorticoid-induced leucine zipper (GILZ) plays a key role in the anti-myogenic action of dexamethasone. In the present study, we aimed to evaluate the role of GILZ in statin-induced myopathy. Statins induced GILZ expression in C2C12 cells, primary murine myoblasts/myotubes, primary human myoblasts, and in vivo in zebrafish embryos and human quadriceps femoris muscle. Gilz induction was mediated by FOXO3 activation and binding to the Gilz promoter, and could be reversed by the addition of geranylgeranyl, but not farnesyl, pyrophosphate. Atorvastatin decreased Akt phosphorylation and increased cleaved caspase-3 levels in myoblasts. This effect was reversed in myoblasts from GILZ knockout mice. Similarly, myofibers isolated from knockout animals were more resistant toward statin-induced cell death than their wild-type counterparts. Statins also impaired myoblast differentiation, and this effect was accompanied by GILZ induction. The in vivo relevance of our findings was supported by the observation that gilz overexpression in zebrafish embryos led to impaired embryonic muscle development. Taken together, our data point toward GILZ as an essential mediator of the molecular mechanisms leading to statin-induced muscle damage.

Developmental toxicity of the common UV filter, benophenone-2, in zebrafish embryos.

Benozophenone (BP) type UV filters are extensively used in the personal care products to provide protection against the harmful effects of UV radiation. BPs are one of the primary components in the UV filter family, in which benophenone-2 (BP2) is widely used as a UV filter reagent in the sunscreen. Humans used these personal care products directly on skin and the chemicals will be washed away to the water system. BP2 has been identified as one of the endocrine disruptor chemicals, which can inference the synthesis, metabolism, and action of endogenous hormones. Environmentally, it has been found to contaminate water worldwide. In this study, we aimed to unfold the possible developmental toxicology of this chemical. Zebrafish are used as the screening model to perform in situ hybridization staining to investigate the effects of BP2 on segmentation, brain regionalization, and facial formation at four developmental stages (10-12 somite, prim-5, 2 and 5 days post-fertilization). Results showed 40 µM (9.85 mg L-1) or above BP2 exposure in zebrafish embryos for 5 days resulted in lipid accumulation in the yolk sac and facial malformation via affecting the lipid processing and the expression of cranial neural crest cells respectively. To conclude, the study alarmed its potential developmental toxicities at high dosage exposure.

Triclosan (TCS) exposure impairs lipid metabolism in zebrafish embryos.

Triclosan (TCS) is an active antimicrobial ingredient used in many household products, such as skin creams and toothpaste. It is produced in high volumes, and humans are directly exposed to it and dispose it on a daily basis. TCS has been found to contaminate water worldwide. This study aimed to understand the potential developmental and metabolic abnormalities caused by TCS exposure by using zebrafish as the experimental model. Four developmental stages (70-85% epiboly, 10-12 somite, prim-5, and 5dpf) were selected to perform in situ hybridization staining to investigate the effects of TCS on dorsal ventral patterning, segmentation, brain development, and organ formation. Results showed, in terms of developmental toxicology, that neither phenotypic nor molecular changes were found after 5 days of 250µg/L TCS exposure. However, such dosage of TCS exposure resulted in lipid droplet accumulation in the yolk sac, which might due to the deregulated mRNA expression level of beta-oxidation transcripts. This study showed that 250µg/L TCS exposure does not affect normal embryogenesis or organogenesis; however, there are concerns regarding possible impairment of lipid metabolism.

Early embryogenesis in zebrafish is affected by bisphenol A exposure.

Exposure of a developing embryo or fetus to endocrine disrupting chemicals (EDCs) has been hypothesized to increase the propensity of an individual to develop a disease or dysfunction in his/her later life. Although it is important to understand the effects of EDCs on early development in animals, sufficient information about these effects is not available thus far. This is probably because of the technical difficulties in tracing the continuous developmental changes at different stages of mammalian embryos. The zebrafish, an excellent model currently used in developmental biology, provides new insights to the field of toxicological studies. We used the standard whole-mount in situ hybridization screening protocol to determine the early developmental defects in zebrafish embryos exposed to the ubiquitous pollutant, bisphenol A (BPA). Three stages (60-75% epiboly, 8-10 somite, and prim-5) were selected for in situ screening of different molecular markers, whereas BPA exposure altered early dorsoventral (DV) patterning, segmentation, and brain development in zebrafish embryos within 24 hours of exposure.

Dexamethasone (DEX) induces Osmotic stress transcription factor 1 (Ostf1) through the Akt-GSK3ß pathway in freshwater Japanese eel gill cell cultures.

Osmosensing and osmoregulatory processes undertaken in gills of euryhaline fish are coordinated by integrative actions of various signaling molecules/transcriptional factors. Considerable numbers of studies report the hyper- and hypo-osmoregulatory functions of fish gills, by illustrating the process of gill cell remodeling and the modulation of the expression of ion channels/transporters. Comparatively mechanistic information relayed from signal integration to transcriptional regulation in mediating gill cell functions has not yet been elucidated. In this study we demonstrate the functional links from cortisol stimulation, to Akt activation, to the expression of the transcriptional factor, Ostf1. Using the synthetic glucocorticoid receptor agonist, dexamethasone (DEX), Ostf1 expression is found to be activated via glucocorticoid receptor (GR) and mediated by the Akt-GSK3ß signaling pathway. Pharmacological experiments using kinase inhibitors reveal that the expression of Ostf1 is negatively regulated by Akt activation. The inhibition of PI3K or Akt activities, by the specific kinase inhibitors (wortmannin, LY294002 or SH6), stimulates Ostf1 expression, while a reduction of GSK3ß activity by LiCl reduces Ostf1 expression. Collectively, our report for the first time indicates that DEX can induce Ostf1 via GR, with the involvement of the Akt-GSK3ß signaling pathway in primary eel gill cell cultures. The data also suggest that Ostf1 may play different roles in gill cell survival during seawater acclimation.

Osmotic stress transcription factor 1b (Ostf1b) promotes migration properties with the modulation of epithelial mesenchymal transition (EMT) phenotype in human embryonic kidney cell.

Osmoregulation is an essential mechanism for euryhaline fish. Gill cells undergo rapid mechanism to maintain the cellular homeostasis during osmotic stress. Reports have suggested that gill cells may be able to migrate between primary filament and secondary lamella during seawater acclimination. However, the factor that can trigger such process is not well-known. Previously, we identified the osmotic stress transcription factor 1b (Ostf1b) in medaka and found that it is an early hypertonic responsive gene and can activate the c-Jun N-terminal kinase (JNK) pathway. In this report, we aim to know if Ostf1b plays the role in the migration. Ostf1b was ectopic expressed in the human embryonic kidney cell line (HEK293) to understand the Ostf1b function. Results clearly demonstrated that Ostf1b could constitutively activate the Rho kinase 1 (ROCK1) and myosin light chain 2 (MLC2) signalling pathway that promotes cell migration, epithelial mesenchymal transition (EMT) and cytoskeletal dynamics through stress fibre formation. The study supports the notion of cell migration and cytoskeleton rearrangement theories in osmoregulation.

Medaka osmotic stress transcription factor 1b (Ostf1b/TSC22D3-2) triggers hyperosmotic responses of different ion transporters in medaka gill and human embryonic kidney cells via the JNK signalling pathway.

Eukaryotic cells undergo rapid regulatory processes to maintain cellular homeostasis upon osmotic stress. In fishes, gill epithelial cells play main roles in these processes. Although osmoregulatory functions of fish gills have been well studied, little is known about the underlying mechanisms, particularly the hypertonic-induced signalling pathways during osmotic stress. This study reports for the first time on the osmo-sensing signal cascade that related to the medaka osmotic stress transcription factor 1 (Ostf1), a hypertonic induced immediate early gene, under hypertonic stress. Quantitative real-time PCR showed the rapid increase of Ostf1 in gill after transfer of medaka from fresh water to 50% seawater; particularly Ostf1b whose mRNA expression increased to 4 folds at 0.5h and reached to 10 folds at 6h after the transfer. The in vivo knockdown of Ostf1b profoundly inhibited SEK and JNK phosphorylation, but not p38 and ERK phosphorylation in the medaka gill tissue. To further investigate the possible role of Ostf1b in the JNK pathway, Ostf1b was ectopically expressed in HEK293 cells. Results indicated that Ostf1b is a downstream target of SEK and JNK and exerts a positive feedback loop on the JNK signalling pathway via activation of GCK and/or MLK3 proteins. Additionally, MAPK inhibitors experiments suggested that activation of the JNK pathway by hypertonicity is involved in the maintenance of Ostf1b stability, which in turn provides continuous stimulation of GCK for JNK phosphorylation. Lastly, changes in transcription levels of different water/ion transporters were found in knockdown or ecoptic over-expression of Ostf1b in medaka gills and human embryonic kidney cells, suggesting the role of Ostf1b in modulation of critical water channel/ion transporters during osmotic stress.

Modulation of ion transporter expression in gill mitochondrion-rich cells of eels acclimated to low-Na(+) or-Cl(-) freshwater.

In this study, we aimed to establish an experimental model to study the role of the gill mitochondrion-rich cells (MRCs) of freshwater fish in Na(+) uptake and to examine the effect of adjusting external Na(+) and Cl(-) ions on selected ion transporters in gill MRCs. Japanese eels (Anguilla japonica) acclimated to deionized (DI) water for 2 weeks were transferred directly to (a) ion-supplemented artificial freshwater (AF), (b) Na(+) -deficient AF, or (c) Cl(-) -deficient AF for 2 days. The effects of the transfer on the expression levels of ion transporters in isolated gill cells were investigated. Our data demonstrated that the 2-day acclimation in ion-supplemented AF, Na(+) -deficient AF, or Cl(-) -deficient AF led to a significant increase in serum osmolarity attributed mainly to an increase in serum Na(+) and/or Cl(-) levels when compared with DI-acclimated eel. Significant inductions of V-type H(+) -ATPase (V-H(+) -ATPase) and cotransporter (NBC1) mRNA expression in gill MRCs were detected in AF-acclimated fish. In fish acclimated to Na(+) -deficient AF, mRNA expression levels of V-H(+) -ATPase, NBC1, and Na(+) /H(+) -exchanger-3 (NHE3) were significantly increased in MRCs. Fish acclimated to Cl(-) -deficient AF showed no observable change in expression levels of ion transporters in gill MRCs. In addition, expression levels of ion transporters in pavement cells were stable throughout the 2-day experiments. These data indicate that the level of Na(+) in freshwater is important for altering the mRNA expression of ion transporters in gill MRCs, which supports the notion that gill MRCs play important roles in freshwater Na(+) uptake.

The deubiquitylating enzyme Cops6 regulates different developmental processes during early zebrafish embryogenesis.

Zebrafish cops6 encodes a putative deubiquitylating enzyme (DUB) that belongs to the JAMM family. It consists of 297 amino acids and includes the Mov34/MPN/PAD-1 (PF01398) domain. Ubiquitylation is involved in many cellular processes and deconjugation of ubiquitin-modified substrates is important to maintain a sufficient amount of free ubiquitin in the cell. Here, we report our findings regarding the general function of the cops6 gene, as a continuation of our previous studies involving DUB knockdown screening. We have found that cops6 plays different roles in early embryonic development in the zebrafish, including dorsoventral patterning, convergent extension movement and brain formation. In addition, our findings indicate that cops6 plays an anti-apoptotic role during segmentation. Overall, the present study that consolidates our previous work on zebrafish DUB genes, corroborates the hypothesis of multi-functional roles for DUB genes during development.

Genome-wide loss-of-function analysis of deubiquitylating enzymes for zebrafish development.

BACKGROUND: Deconjugation of ubiquitin and/or ubiquitin-like modified protein substrates is essential to modulate protein-protein interactions and, thus, signaling processes in cells. Although deubiquitylating (deubiquitinating) enzymes (DUBs) play a key role in this process, however, their function and regulation remain insufficiently understood. The "loss-of-function" phenotype studies can provide important information to elucidate the gene function, and zebrafish is an excellent model for this goal. RESULTS: From an in silico genome-wide search, we found more than 90 putative DUBs encoded in the zebrafish genome belonging to six different subclasses. Out of them, 85 from five classical subclasses have been tested with morpholino (MO) knockdown experiments and 57 of them were found to be important in early development of zebrafish. These DUB morphants resulted in a complex and pleiotropic phenotype that, regardless of gene target, always affected the notochord. Based on the huC neuronal marker expression, we grouped them into five sets (groups I to V). Group I DUBs (otud7b, uchl3 and bap1) appear to be involved in the Notch signaling pathway based on the neuronal hyperplasia, while group IV DUBs (otud4, usp5, usp15 and usp25) play a critical role in dorsoventral patterning through the BMP pathway. CONCLUSION: We have identified an exhaustive list of genes in the zebrafish genome belonging to the five established classes of DUBs. Additionally, we performed the corresponding MO knockdown experiments in zebrafish as well as functional studies for a subset of the predicted DUB genes. The screen results in this work will stimulate functional follow-up studies of potential DUB genes using the zebrafish model system.

Effect of osmotic shrinkage and hormones on the expression of Na+/H+ exchanger-1, Na+/K+/2Cl- cotransporter and Na+/K+ -ATPase in gill pavement cells of freshwater adapted Japanese eel, Anguilla japonica.

It is well-known that gill epithelial cells are important in fish osmoregulation. However, studies on the effect of osmotic stress on the direct cellular responses of the gill epithelial cells are limited. In this paper, we aimed to determine the effects of osmotic hypertonicity, hormones and cellular signaling molecules on the expression of ion transporters in the cultured primary freshwater pavement cells (PVCs), prepared from freshwater-adapted eels (Anguilla japonica). Our data demonstrated that the hypertonic (500 mOsmol l(-1)) treatment of the isolated PVCs induced cell shrinkage, followed by regulatory volume increase (RVI). Application of blockers (i.e. ouabain, bumetanide and EIPA) demonstrated that Na+/K+ -ATPase, Na+/K+/2Cl- cotransporter (NKCC) and Na+/H+ exchanger-1 (NHE-1) were involved in RVI. Western blot analysis of the hypertonic-treated cells revealed a significant induction of NHE-1, NKCC and, alpha and beta subunits of Na+/K+ -ATPase. In nonshrunken cultured PVCs, we found that dexamethasone and dibutyryl cAMP treatments significantly stimulated the expression levels of the three ion transporters. Both prolactin and insulin-like growth factor-1, can only induce the expression of NKCC. The effect of thyroid hormone (T3) and dibutyryl cGMP was negligible. In this study, the induction of ion transporter expression was found to be post-transcriptionally regulated as no significant change in mRNA levels was detected. This observation implies that the regulation is rapid and is probably induced via nongenomic actions.

Characterization of ion channel and transporter mRNA expressions in isolated gill chloride and pavement cells of seawater acclimating eels.

Ion channels and transporters (i.e. cystic fibrosis transmembrane regulator (CFTR), inward rectifier potassium channel (eKir), Na/K-ATPase, Na/K/Cl2 co-transporter (NKCC), aquaporin-3 (AQP-3), and Na/H exchanger-1 (NHE-1)) are known to be expressed in gill epithelia of teleost fish. Owing to the anatomical complexity of gill structures, their temporal expression profile in seawater acclimating gill pavement (PVCs) and chloride cells (CCs) are limited. In this study, we isolated the gill PVCs and CCs from seawater acclimating Japanese eels to address the issue. In the gill epithelia of freshwater adapted eels, CCs expressed the highest mRNA and/or protein levels of Na/K-ATPase, NKCC, and eKir as demonstrated by real-time PCR and/or immunohistochemical staining. AQP-3 mRNA was highly expressed in freshwater PVCs and its protein was in general expressed in all gill cells. The NHE-1 transcripts were expressed in similar levels in both PVCs and CCs. CFTR mRNA transcript was almost undetectable in all the freshwater gill cell samples. Seawater acclimation induced the transcript and/or protein levels of Na/K-ATPase, NKCC, CFTR, and eKir in CCs. The upregulation and the coexpression of these transporters in CCs suggested their cohort function in mediating Na+, K+, and Cl- transport. The expression of CFTR was found to be tightly regulated as its expression was restricted only in "seawater CCs". AQP-3 transcript and protein levels in PVCs reduced significantly during the acclimation. Interestingly immunocytochemical (ICC) staining of seawater gill epithelia revealed that AQP-3 immunoreactivities were mainly localized in seawater CCs. In the acclimation, there was no significant reduction of NHE-1 mRNA in both PVCs and CCs, however its protein level dropped significantly in the seawater condition. The present study is the first to demonstrate the activation of the mRNA transcripts for the ion channels and transporters in isolated gill CCs during seawater acclimation. The activating mechanism is found to be confined primarily in CCs. These results indicated that in addition to the increase in size and number of CCs, the molecular remodeling and the functional plasticity of CCs were essential in the ion transport process during seawater acclimation.