Doble synergetic anticancer activity through a combined chemo-photodynamic therapy and bioimaging of a novel Cas-ZnONPs all-in-one system.

A strategy for cancer treatment was implemented, based on chemo-photodynamic therapy, utilizing a novel formulation, low-cost system called Cas-ZnONPs. This system consisted of the incorporation of Casiopeina III-ia (CasIII-ia), a hydrophilic copper coordination compound with well-documented anti-neoplastic activity, on Zinc oxide nanoparticles (ZnONPs) with apoptotic activity and lipophilicity, allowing them to permeate biological barriers. Additionally, ZnONPs exhibited fluorescence, with emission at different wavelengths depending on their agglomeration and enabling real-time tracking biodistribution. Also, ZnONPs served as a sensitizer, generating reactive oxygen species (ROS) in situ. In in vitro studies on HeLa and MDA-MB-231 cell lines, a synergistic effect was observed with the impregnated CasIII-ia on ZnONPs. The anticancer activity had an increase in cellular inhibition, depending on the dose of exposure to UV-vis irradiation. In in vivo studies utilized zebrafish models for xenotransplanting stained MDA-MB-231 cells and testing the effectiveness of Cas-ZnONPs treatment. The treatment successfully eliminated cancer cells, both when combined with Photodynamic Therapy (PDT) and when used alone. However, a significantly higher concentration (50 times) of Cas-ZnONPs was required in the absence of PDT. This demonstrates the potential of Cas-ZnONPs in cancer treatment, especially when combined with PDT.

Betaglycan sustains HGF/Met signaling in lung cancer and endothelial cells promoting cell migration and tumor growth.

Persistent HGF/Met signaling drives tumor growth and dissemination. Proteoglycans within the tumor microenvironment might control HGF availability and signaling by affecting its accessibility to Met (HGF receptor), likely defining whether acute or sustained HGF/Met signaling cues take place. Given that betaglycan (BG, also known as type III TGFß receptor or TGFBR3), a multi-faceted proteoglycan TGFß co-receptor, can be found within the tumor microenvironment, we addressed its hypothetical role in oncogenic HGF signaling. We found that HGF/Met promotes lung cancer and endothelial cells migration via PI3K and mTOR. This effect was enhanced by recombinant soluble betaglycan (solBG) via a mechanism attributable to its glycosaminoglycan chains, as a mutant without them did not modulate HGF effects. Moreover, soluble betaglycan extended the effect of HGF-induced phosphorylation of Met, Akt, and Erk, and membrane recruitment of the RhoGEF P-Rex1. Data-mining analysis of lung cancer patient datasets revealed a significant correlation between high MET receptor, HGF, and PREX1 expression and reduced patient survival. Soluble betaglycan showed biochemical interaction with HGF and, together, they increased tumor growth in immunocompetent mice. In conclusion, the oncogenic properties of the HGF/Met pathway are enhanced and sustained by GAG-containing soluble betaglycan.

Adenoviral Vector Codifying for TNF as a Co-Adjuvant Therapy against Multi-Drug-Resistant Tuberculosis.

Mycobacterium tuberculosis is the main causal agent of pulmonary tuberculosis (TB); the treatment of this disease is long and involves a mix of at least four different antibiotics that frequently lead to abandonment, favoring the surge of drug-resistant mycobacteria (MDR-TB), whose treatment becomes more aggressive, being longer and more toxic. Thus, the search for novel strategies for treatment that improves time or efficiency is of relevance. In this work, we used a murine model of pulmonary TB produced by the MDR-TB strain to test the efficiency of gene therapy with adenoviral vectors codifying TNF (AdTNF), a pro-inflammatory cytokine that has protective functions in TB by inducing apoptosis, granuloma formation and expression of other Th1-like cytokines. When compared to the control group that received an adenoviral vector that codifies for the green fluorescent protein (AdGFP), a single dose of AdTNF at the chronic active stage of the disease produced total survival, decreasing bacterial load and tissue damage (pneumonia), which correlated with an increase in cells expressing IFN-γ, iNOS and TNF in pneumonic areas and larger granulomas that efficiently contain and eliminate mycobacteria. Second-line antibiotic treatment against MDR-TB plus AdTNF gene therapy reduced bacterial load faster within a week of treatment compared to empty vector plus antibiotics or antibiotics alone, suggesting that AdTNF is a new potential type of treatment against MDR-TB that can shorten second-line chemotherapy but which requires further experimentation in other animal models (non-human primates) that develop a more similar disease to human pulmonary TB.

Genistein-mediated thermogenesis and white-to-beige adipocyte differentiation involve transcriptional activation of cAMP response elements in the Ucp1 promoter.

Genistein is an isoflavone present in soybeans and is considered a bioactive compound due to its widely reported biological activity. We have previously shown that intraperitoneal genistein administration and diet supplementation activates the thermogenic program in rats and mice subcutaneous white adipose tissue (scWAT) under multiple environmental cues, including cold exposure and high-fat diet feeding. However, the mechanistic insights of this process were not previously unveiled. Uncoupling protein 1 (UCP1), a mitochondrial membrane polypeptide responsible for dissipating energy into heat, is considered the most relevant thermogenic marker; thus, we aimed to evaluate whether genistein regulates UCP1 transcription. Here we show that genistein administration to thermoneutral-housed mice leads to the appearance of beige adipocyte markers, including a sharp upregulation of UCP1 expression and protein abundance in scWAT. Reporter assays showed an increase in UCP1 promoter activity after genistein stimulation, and in silico analysis revealed the presence of estrogen (ERE) and cAMP (CRE) response elements as putative candidates of genistein activation. Mutation of the CRE but not the ERE reduced genistein-induced promoter activity by 51%. Additionally, in vitro and in vivo ChIP assays demonstrated the binding of CREB to the UCP1 promoter after acute genistein administration. Taken together, these data elucidate the mechanism of genistein-mediated UCP1 induction and confirm its potential applications in managing metabolic disorders.

Fluorescent Probe for in Vivo Partitioning into Dynamic Lipid Droplets Enables Monitoring of Water Permeability-Induced Edema.

Lipid droplets (LDs) are intracellular organelles found in most cell types from adipocytes to cancer cells. Although recent investigations have implicated LDs in numerous diseases, the current available methods to monitor them in vertebrate models rely on static imaging using fluorescent dyes, limiting the investigation of their rapid in vivo dynamics. Here, we report a fluorophore chemistry approach to enable in vivo LD dynamic monitoring using a Nernstian partitioning mechanism. Interestingly, the effect of atorvastatin and osmotic treatments toward LDs revealed an unprecedented dynamic enhancement. Then, using a designed molecular probe with an optimized response to hydration and LD dynamics applied to Zebrafish developing pericardial and yolk-sac edema, which represents a tractable model of a human cardiovascular disease, we also provide a unique dual method to detect disease evolution and recovery.

Betaglycan promoter activity is differentially regulated during myogenesis in zebrafish embryo somites.

BACKGROUND: Betaglycan, also known as the TGFß type III receptor (Tgfbr3), is a co-receptor that modulates TGFß family signaling. Tgfbr3 is upregulated during C2C12 myoblast differentiation and expressed in mouse embryos myocytes. RESULTS: To investigate tgfbr3 transcriptional regulation during zebrafish embryonic myogenesis, we cloned a 3.2 kb promoter fragment that drives reporter transcription during C2C12 myoblasts differentiation and in the Tg(tgfbr3:mCherry) transgenic zebrafish. We detect tgfbr3 protein and mCherry expression in the adaxial cells concomitantly with the onset of their radial migration to become slow-twitch muscle fibers in the Tg(tgfbr3:mCherry). Remarkably, this expression displays a measurable antero-posterior somitic gradient expression. CONCLUSIONS: tgfbr3 is transcriptionally regulated during somitic muscle development in zebrafish with an antero-posterior gradient expression that preferentially marks the adaxial cells and their descendants.

CD24+CD44+CD54+EpCAM+ gastric cancer stem cells predict tumor progression and metastasis: clinical and experimental evidence.

BACKGROUND: Gastric cancer (GC) is a leading cause of cancer-related deaths worldwide. Specific and thorough identification of cancer cell subsets with higher tumorigenicity and chemoresistance, such as cancer stem cells (CSCs), could lead to the development of new and promising therapeutic targets. For better CSC identification, a complete or extended surface marker phenotype is needed to provide increased specificity for new cell targeting approaches. Our goal is to identify and characterize a putative extended phenotype for CSCs derived from patients with GC before treatment, as well as to evaluate its clinical value. In addition, we aim to ensure that cells with this phenotype have stemness and self-renewal capabilities. METHODS: This is a cohort study including 127 treatment-naïve patients with GC who attended the Instituto Nacional de Cancerología. Multiparametric flow cytometry analysis was performed to determine the extended phenotype of cells derived from gastric biopsies. The tumorigenic capability of cells identified in patients was assessed in a zebrafish model. RESULTS: CD24+CD44+CD54+EpCAM+ cells were present in all treatment-naïve patients included, with a median abundance of 1.16% (0.57-1.89%). The percentage of CD24+CD44+CD54+EpCAM+ cells was categorized as high or low using 1.19% as the cutoff for the CD24+CD44+CD54+EpCAM+ cell subset. Additionally, a higher TNM stage correlated with a higher percentage of CD24+CD44+CD54+EpCAM+ cells (Rho coefficient 0.369; p < 0.0001). We also demonstrated that a higher percentage of CD24+CD44+CD54+EpCAM+ cells was positively associated with metastasis. The metastatic potential of these cells was confirmed in a zebrafish model. Ultimately, under our conditions, we conclude that CD24+CD44+CD54+EpCAM+ cells are true gastric cancer stem cells (GCSCs). CONCLUSION: The CD24+CD44+CD54+EpCAM+ cells present in tissue samples from patients are true GCSCs. This extended phenotype results in better and more specific characterization of these highly tumorigenic cells. The relative quantification of CD24+CD44+CD54+EpCAM+ cells has potential clinical value, as these cells are associated with metastatic disease, making their presence an additional prognostic marker and possibly a target for the design of new antineoplastic treatments in the era of precision oncology. Overall, the extended CD24+CD44+CD54+EpCAM+ phenotype of GCSCs could support their isolation for the study of their stemness mechanisms, leading to the identification of better molecular targets for the development of both new therapeutic approaches such as oncoimmunotherapy and new diagnostic and clinical prognostic strategies for GC.

Mitochondria-Assisted Photooxidation to Track Singlet Oxygen at Homeostatic Membrane Microviscosity.

Using intracellular-controlled photochemistry to track dynamic organelle processes is gaining attention due to its broad applications. However, most of the employed molecular probes usually require toxic photosensitizers and complex bioanalytical protocols. Here, the synthesis and performance of two new subcellular probes (MitoT1 and MitoT2) are described. The probes undergo photooxidation in the damaged tissue of zebrafish, a model system for tissue regeneration studies. Using high-resolution confocal microscopy and fluorescence spectroscopy, we combine the mentioned photoinduced interconversion at the homeostatic membrane viscosity to track singlet oxygen activity selectively. The continuous and real-time biosensing method reported here provides a new approach for simultaneously detecting endogenous singlet oxygen and viscosity status.

Immune Regulatory Effect of Osteopontin Gene Therapy in a Murine Model of Multidrug Resistant Pulmonary Tuberculosis.

Tuberculosis (TB) has been for many years a major public health problem since treatment is long and sometimes ineffective favoring the increase of multidrug-resistant mycobacteria (MDR-TB). Gene therapy is a novel and effective tool to regulate immune responses. In this study we evaluated the therapeutic effect of an adenoviral vector codifying osteopontin (AdOPN), a molecule known for their roles to favor Th1 and Th17 type-cytokine expression which are crucial in TB containment. A single dose of AdOPN administration in BALB/c mice suffering late progressive pulmonary MDR-TB produced significant lower bacterial load and pneumonia, due to higher expression of IFN-γ, IL-12, and IL-17 in coexistence with increase of granulomas in number and size, resulting in higher survival, in contrast with mice treated with the control adenovirus that codify the green fluorescent protein (AdGFP). Combined therapy of AdOPN with a regimen of second line antibiotics produced a better control of bacterial load in lung during the first days of treatment, suggesting that AdOPN can shorten chemotherapy. Taken together, gene therapy with AdOPN leads to higher immune responses against TB infection, resulting in a new potential treatment against pulmonary TB that can co-adjuvant chemotherapy.

The HGR motif is the antiangiogenic determinant of vasoinhibin: implications for a therapeutic orally active oligopeptide.

The hormone prolactin acquires antiangiogenic and antivasopermeability properties after undergoing proteolytic cleavage to vasoinhibin, an endogenous prolactin fragment of 123 or more amino acids that inhibits the action of multiple proangiogenic factors. Preclinical and clinical evidence supports the therapeutic potential of vasoinhibin against angiogenesis-related diseases including diabetic retinopathy, peripartum cardiomyopathy, rheumatoid arthritis, and cancer. However, the use of vasoinhibin in the clinic has been limited by difficulties in its production. Here, we removed this barrier to using vasoinhibin as a therapeutic agent by showing that a short linear motif of just three residues (His46-Gly47-Arg48) (HGR) is the functional determinant of vasoinhibin. The HGR motif is conserved throughout evolution, its mutation led to vasoinhibin loss of function, and oligopeptides containing this sequence inhibited angiogenesis and vasopermeability with the same potency as whole vasoinhibin. Furthermore, the oral administration of an optimized cyclic retro-inverse vasoinhibin heptapeptide containing HGR inhibited melanoma tumor growth and vascularization in mice and exhibited equal or higher antiangiogenic potency than other antiangiogenic molecules currently used as anti-cancer drugs in the clinic. Finally, by unveiling the mechanism that obscures the HGR motif in prolactin, we anticipate the development of vasoinhibin-specific antibodies to solve the on-going challenge of measuring endogenous vasoinhibin levels for diagnostic and interventional purposes, the design of vasoinhibin antagonists for managing insufficient angiogenesis, and the identification of putative therapeutic proteins containing HGR.

Chordacentrum mineralization is delayed in zebrafish betaglycan-null mutants.

BACKGROUND: The Transforming Growth Factor ß (TGFß) family is a group of related proteins that signal through a type I and type II receptors. Betaglycan, also known as the type III receptor (Tgfbr3), is a coreceptor for various ligands of the TGFß family that participates in heart, liver and kidney development as revealed by the tgfbr3-null mouse, as well as in angiogenesis as revealed by Tgfbr3 downregulation in morphant zebrafish. RESULTS: Here, we present CRISPR/Cas9-derived zebrafish Tgfbr3-null mutants, which exhibited unaltered embryonic angiogenesis and developed into fertile adults. One reproducible phenotype displayed by these Tgfbr3-null mutants is delayed chordacentra mineralization, which nonetheless does not result in vertebral abnormalities in the adult fishes. We also report that the canonical TGFß signaling pathway is needed for proper chordacentra mineralization and that Tgfbr3 absence decreases this signal in the notochordal cells responsible for this process. CONCLUSION: Betaglycan's "ligand presentation" function contributes to the optimal TGFß signaling required for zebrafish chordacentra mineralization.

Neotropical Rattlesnake (Crotalus simus) Venom Pharmacokinetics in Lymph and Blood Using an Ovine Model.

The most abundant protein families in viper venoms are Snake Venom Metalloproteases (SVMPs), Snake Venom Serine Proteases (SVSPs) and Phospholipases (PLA2s). These are primarily responsible for the pathophysiology caused by the bite of pit-vipers; however, there are few studies that analyze the pharmacokinetics (PK) of whole venom (WV) and its protein families. We studied the pathophysiology, PK profile and differential absorption of representative toxins from venom of Neotropical Rattlesnake (Crotalus simus) in a large animal model (ovine). Toxins studied included crotoxin (the main lethal component), which causes moderate to severe neurotoxicity; SVSPs, which deplete fibrinogen; and SVMPs, which cause local tissue damage and local and systemic hemorrhage. We found that Whole Venom (WV) was highly bioavailable (86%) 60 h following intramuscular (IM) injection, and extrapolation suggests that bioavailability may be as high as 92%. PK profiles of individual toxins were consistent with their physicochemical properties and expected clinical effects. Lymph cannulated animals absorbed 1.9% of WV through lymph during the first 12 h. Crotoxin was minimally detectable in serum after intravenous (IV) injection; however, following IM injection it was detected in lymph but not in blood. This suggests that crotoxin is quickly released from the blood toward its tissue targets.

Vasoinhibin generation and effect on neuronal apoptosis in the hippocampus of late mouse embryos.

Morphological and behavioral evidence suggests that vasoinhibin is present in the central nervous system (CNS), triggering neuroendocrine and behavioral responses to stress. Moreover, vasoinhibin reduces neuronal survival and differentiation of primary sensory neurons of the peripheral nervous system. To address the functional role played by vasoinhibin at the CNS, and to better understand the underlying mechanisms involved in its actions, we treated primary cultured hippocampal neurons obtained from embryonic day 16 (E16) mice with a human recombinant vasoinhibin. We examined the resulting cellular changes, focusing on neuronal cell death, and explored the local generation of vasoinhibin within the hippocampus. Our results show that vasoinhibin significantly reduced neuronal cell density and increased immunoreactive activated caspase-3 and TUNEL-positive staining at 72, 16, and 24 h, respectively. Furthermore, vasoinhibin increased the expression of proapoptotic genes BAX, BAD, BIM, and PUMA and decreased that of the antiapoptotic gene BCL-2 at 24 h, as assessed by quantitative real-time reverse transcription-polymerase chain reaction. Vasoinhibin effects were blocked by coincubation with a vasoinhibin antibody or with prolactin. Immunoreactive bands consistent with vasoinhibin were observed in hippocampal extracts by Western blot analysis, and a prolactin standard was cleaved to vasoinhibin by a hippocampal lysate in a heat- and cathepsin D inhibitor pepstatin A-dependent fashion. Taken together, these data support the notion that vasoinhibin is locally produced by cathepsin D within the embryonic mouse hippocampus, a brain region that plays a critical role in emotional regulation, resulting in decreased neuronal cell viability via the activation of the intrinsic apoptosis pathway.

Structural Adaptation in Its Orphan Domain Engenders Betaglycan with an Alternate Mode of Growth Factor Binding Relative to Endoglin.

Betaglycan (BG) and endoglin (ENG), homologous co-receptors of the TGF-ß family, potentiate the signaling activity of TGF-ß2 and inhibin A, and BMP-9 and BMP-10, respectively. BG exists as monomer and forms 1:1 growth factor (GF) complexes, while ENG exists as a dimer and forms 2:1 GF complexes. Herein, the structure of the BG orphan domain (BGO) reveals an insertion that blocks the region that the endoglin orphan domain (ENGO) uses to bind BMP-9, preventing it from binding in the same manner. Using binding studies with domain-deleted forms of TGF-ß and BGO, as well as small-angle X-ray scattering data, BGO is shown to bind its cognate GF in an entirely different manner compared with ENGO. The alternative interfaces likely engender BG and ENG with the ability to selectively bind and target their cognate GFs in a unique temporal-spatial manner, without interfering with one another or other TGF-ß family GFs.

Unravelling the modus-operandi of chromenylium-cyanine fluorescent probes: a case study.

Small-molecule fluorescent probes having optimized optical properties, such as high photostability and brightness, local microenvironment sensitivity and specific subcellular localizations, are increasingly available. Although the basis for designing efficient fluorophores for bioimaging applications is well established, implementing an improvement in a given photophysical characteristic always tends to compromise another optical property. This problem has enormous consequences for in vivo imaging, where ensuring a specific localization and precise control of the probe response is challenging. Herein we discuss a fluorescent probe, CC334, as a case study of the chromenylium-cyanine family that commonly exhibits highly complex photophysical schemes and highly interfered bioanalytical responses. By an exhaustive and concise analysis of the CC334 optical responses including detailed spectroscopic calibrations, steady-state microenvironment effects, ultrafast photophysics analysis and computational studies, we elucidate a new strategy to apply the probe in the singlet oxygen reactive oxygen species (1O2-ROS) monitoring using in vitro and in vivo models. The probe provides a new avenue for designing fluorescent probes to understand the dynamic behavior of subcellular environments.

Vasoinhibin Suppresses Nerve Growth Factor-Induced Differentiation and Survival of PC12 Pheochromocytoma Cells.

BACKGROUND: Vasoinhibin, a protein derived from prolactin, regulates various vascular functions including endothelial cell survival. Of note, vasoinhibin is present in the central nervous system, where it triggers neuroendocrine and behavioral responses to stress. Moreover, vasoinhibin compromises nerve growth factor (NGF)-induced neurite outgrowth in primary sensory neurons of the peripheral nervous system. Nonetheless, information on the functions of vasoinhibin in developing neurons remains limited. The present study explored whether vasoinhibin affects the neurotrophic actions of NGF by measuring the cell differentiation and survival of PC12 pheochromocytoma cells. METHODS: The effects of recombinant or lentiviral vector-transduced human vasoinhibin were tested on differentiating PC12 cells. Neurite outgrowth was quantified by measuring their length and density. The MTT assay was employed to assess cell viability, and ELISA was used to quantify DNA fragmentation as an index of apoptosis. Phosphorylated Akt and ERK1/2 were analyzed by Western blotting. RESULTS: The addition of a human recombinant vasoinhibin, and the transduction of a lentiviral vector carrying a human vasoinhibin sequence, significantly reduced NGF-induced neurite outgrowth, cell survival, and phosphorylation of Akt and ERK1/2, and increased DNA fragmentation and caspase 3 activation in PC12 cells. CONCLUSIONS: Vasoinhibin downregulates NGF-induced differentiation and survival of PC12 cells, blocking tropomyosin receptor kinase A-triggered signaling pathways and increasing apoptosis. These results establish that vasoinhibin interaction with NGF and other neurotrophins may be critical in mediating pathways involved in neuronal survival and differentiation.

Author Correction: Dual contribution of TRPV4 antagonism in the regulatory effect of vasoinhibins on blood-retinal barrier permeability: diabetic milieu makes a difference.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Dual contribution of TRPV4 antagonism in the regulatory effect of vasoinhibins on blood-retinal barrier permeability: diabetic milieu makes a difference.

Breakdown of the blood-retinal barrier (BRB), as occurs in diabetic retinopathy and other chronic retinal diseases, results in vasogenic edema and neural tissue damage, causing vision loss. Vasoinhibins are N-terminal fragments of prolactin that prevent BRB breakdown during diabetes. They modulate the expression of some transient receptor potential (TRP) family members, yet their role in regulating the TRP vanilloid subtype 4 (TRPV4) remains unknown. TRPV4 is a calcium-permeable channel involved in barrier permeability, which blockade has been shown to prevent and resolve pulmonary edema. We found TRPV4 expression in the endothelium and retinal pigment epithelium (RPE) components of the BRB, and that TRPV4-selective antagonists (RN-1734 and GSK2193874) resolve BRB breakdown in diabetic rats. Using human RPE (ARPE-19) cell monolayers and endothelial cell systems, we further observed that (i) GSK2193874 does not seem to contribute to the regulation of BRB and RPE permeability by vasoinhibins under diabetic or hyperglycemic-mimicking conditions, but that (ii) vasoinhibins can block TRPV4 to maintain BRB and endothelial permeability. Our results provide important insights into the pathogenesis of diabetic retinopathy that will further guide us toward rationally-guided new therapies: synergistic combination of selective TRPV4 blockers and vasoinhibins can be proposed to mitigate diabetes-evoked BRB breakdown.

TßRIII is induced by TCR signaling and downregulated in FoxP3+ regulatory T cells.

TGF-ß type III receptor (TßRIII) is a co-receptor for TGFß family members required for high-affinity binding of these ligands to their receptors, potentiating their cellular functions. TGF-ßs, Bone Morphogenetic Proteins (BMP2/4) and Inhibins/Activins regulate different checkpoints during T cell differentiation. We have previously reported that TßRIII modulates T cell development by protecting developing thymocytes from apoptosis, however the role of this co-receptor in peripheral lymphocytes still remains elusive. Here we describe a detailed characterization of TßRIII expression in murine and human lymphocyte subpopulations demonstrating that this co-receptor is significantly expressed in T but not B lymphocytes and among them, preferentially expressed on naïve and central memory T cells. TßRIII was upregulated after TCR stimulation, in parallel to other early activation markers. In contrast, natural and induced Tregs downregulated TßRIII in association with FoxP3 upregulation. Finally, anti-TßRIII blocking experiments demonstrated that TßRIII promotes TGFß-dependent iTreg conversion in vitro, and suggest that this co-receptor may be involved in modulating peripheral T cell tolerance and could be considered as a potential target to boost T cell immune responses.