：The epidermal growth factor receptor (EGFR) family contains four transmembrane tyrosine kinases (EGFR1/ErbB1, Her2/ErbB2, Her3/ErbB3 and Her4/ErbB4) and 13 secreted polypeptide ligands. EGFRs are ...overexpressed in many solid tumors, including breast, pancreas, head-and-neck, prostate, ovarian, renal, colon, and non-small-cell lung cancer. Such overexpression produces strong stimulation of downstream signaling pathways, which induce cell growth, cell differentiation, cell cycle progression, angiogenesis, cell motility and blocking of apoptosis.The high expression and/or functional activation of EGFRs correlates with the pathogenesis and progression of several cancers, which make them attractive targets for both diagnosis and therapy. Several approaches have been developed to target these receptors and/or the EGFR modulated effects in cancer cells. Most approaches include the development of anti-EGFRs antibodies and/or small-molecule EGFR inhibitors. This review presents the state-of-the-art and future prospects of targeting EGFRs to treat breast cancer.
Abstract Although siRNAs have surpassed expectations in experiments to alter gene expression in vitro , the lack of an efficient in vivo delivery system still remains a challenge in siRNA ...therapeutics development and has been recognized as a major hurdle for clinical applications. In this paper we describe an inorganic nanoparticle-based delivery system that is readily adaptable for in vivo systems. Layered double hydroxide (LDH) nanoparticles, a family of inorganic crystals, tightly bind, protect, and release siRNA molecules and deliver them efficiently to mammalian cells in vitro . The uptake of siRNA-loaded LDH nanoparticles occurs via endocytosis, whereby the nanoparticles dissolve due to the low pH in the endosome, thereby aiding endosomal escape into the cytoplasm. The influence of LDH nanoparticles on cell viability and proliferation is negligible at concentrations ≤0.050 mg mL−1 , and a pronounced down-regulation of protein expression upon LDH mediated siRNA transfection of HEK293T cells is observed.
Triple-negative breast cancer (TNBC) is a group of heterogeneous and refractory breast cancers with the absence of estrogen receptor (ER), progesterone receptor (PgR) and epidermal growth factor ...receptor 2 (HER2). Over the past decade, antibody drug conjugates (ADCs) have ushered in a new era of targeting therapy. Since the epidermal growth factor receptor (EGFR) and epithelial cell adhesion molecule (EpCAM) are over expressed on triple-negative breast cancer, we developed novel ADCs by conjugating benzylguanine (BG)-modified monomethyl auristatin E (MMAE) to EpCAM- and EGFR-specific SNAP-tagged single chain antibody fragments (scFvs). Rapid and efficient conjugation was achieved by SNAP-tag technology. The binding and internalization properties of scFv-SNAP fusion proteins were confirmed by flow cytometry and fluorescence microscopy. The dose-dependent cytotoxicity was evaluated in cell lines expressing different levels of EGFR and EpCAM. Both ADCs showed specific cytotoxicity to EGFR or EpCAM positive cell lines via inducing apoptosis at a nanomolar concentration. Our study demonstrated that EGFR specific scFv-425-SNAP-BG-MMAE and EpCAM-specific scFv-EpCAM-SNAP-BG-MMAE could be promising ADCs for the treatment of TNBC.
Key points
Newborn mice produce ultrasonic vocalization to communicate with their mother.
The neuronal glycine transporter (GlyT2) is required for efficient loading of synaptic vesicles in ...glycinergic neurons.
Mice lacking GlyT2 develop a phenotype that resembles human hyperekplexia and the mice die in the second postnatal week.
In the present study, we show that GlyT2‐knockout mice do not acquire adult ultrasonic vocalization‐associated breathing patterns.
Despite the strong impairment of glycinergic inhibition, they can produce sufficient expiratory airflow to produce ultrasonic vocalization.
Because mouse ultrasonic vocalization is a valuable read‐out in translational research, these data are highly relevant for a broad range of research fields.
Mouse models are instrumental with respect to determining the genetic basis and neural foundations of breathing regulation. To test the hypothesis that glycinergic synaptic inhibition is required for normal breathing and proper post‐inspiratory activity, we analysed breathing and ultrasonic vocalization (USV) patterns in neonatal mice lacking the neuronal glycine transporter (GlyT2). GlyT2‐knockout (KO) mice have a profound reduction of glycinergic synaptic currents already at birth, develop a severe motor phenotype and survive only until the second postnatal week. At this stage, GlyT2‐KO mice are smaller, have a reduced respiratory rate and still display a neonatal breathing pattern with active expiration for the production of USV. By contrast, wild‐type mice acquire different USV‐associated breathing patterns that depend on post‐inspiratory control of air flow. Nonetheless, USVs per se remain largely indistinguishable between both genotypes. We conclude that GlyT2‐KO mice, despite the strong impairment of glycinergic inhibition, can produce sufficient expiratory airflow to produce ultrasonic vocalization.
Key points
Newborn mice produce ultrasonic vocalization to communicate with their mother.
The neuronal glycine transporter (GlyT2) is required for efficient loading of synaptic vesicles in glycinergic neurons.
Mice lacking GlyT2 develop a phenotype that resembles human hyperekplexia and the mice die in the second postnatal week.
In the present study, we show that GlyT2‐knockout mice do not acquire adult ultrasonic vocalization‐associated breathing patterns.
Despite the strong impairment of glycinergic inhibition, they can produce sufficient expiratory airflow to produce ultrasonic vocalization.
Because mouse ultrasonic vocalization is a valuable read‐out in translational research, these data are highly relevant for a broad range of research fields.
Previously, we described the dysregulation of serotonin (5-HT) receptor subtype 5b (5-ht
) in a mouse model of Rett syndrome (RTT). 5-ht
has not been extensively studied, so we set out to ...characterize it in more detail. Unlike common cell surface receptors, 5-ht
displays no membrane expression, while receptor clusters are located in endosomes. This unusual subcellular localization is at least in part controlled by glycosylation of the N-terminus, with 5-ht
possessing fewer glycosylation sites than related receptors. We analyzed whether the localization to endosomes has any functional relevance and found that 5-ht
receptors can specifically interact with 5-HT
receptors and retain them in endosomal compartments. This interaction reduces 5-HT
surface expression and is mediated by interactions between the fourth and fifth trans-membrane domain (TMD). This possibly represents a mechanism by which 5-ht
receptors regulate the activity of other 5-HT receptor.
Rhythmic breathing movements originate from a dispersed neuronal network in the medulla and pons. Here, we demonstrate that rhythmic activity of this respiratory network is affected by the ...phosphorylation status of the inhibitory glycine receptor α3 subtype (GlyRα3), which controls glutamatergic and glycinergic neuronal discharges, subject to serotonergic modulation. Serotonin receptor type 1A-specific (5-HTR1A-specific) modulation directly induced dephosphorylation of GlyRα3 receptors, which augmented inhibitory glycine-activated chloride currents in HEK293 cells coexpressing 5-HTR1A and GlyRα3. The 5-HTR1A-GlyRα3 signaling pathway was distinct from opioid receptor signaling and efficiently counteracted opioid-induced depression of breathing and consequential apnea in mice. Paradoxically, this rescue of breathing originated from enhanced glycinergic synaptic inhibition of glutamatergic and glycinergic neurons and caused disinhibition of their target neurons. Together, these effects changed respiratory phase alternations and ensured rhythmic breathing in vivo. GlyRα3-deficient mice had an irregular respiratory rhythm under baseline conditions, and systemic 5-HTR1A activation failed to remedy opioid-induced respiratory depression in these mice. Delineation of this 5-HTR1A-GlyRα3 signaling pathway offers a mechanistic basis for pharmacological treatment of opioid-induced apnea and other breathing disturbances caused by disorders of inhibitory synaptic transmission, such as hyperekplexia, hypoxia/ischemia, and brainstem infarction.
•Glycine receptor alpha3 knockout mice do not show a breathing phenotype.•Alpha 3 subunit of glycine receptors appears to be involved in the irregular breathing rhythm of Mecp2-deficent ...mice.•Despite, survival of mice is not improved.
The glycine receptor α3 subunit is known to be a target for cAMP/PKA-mediated phosphorylation and regulation. Mice that lack this subunit are apparently normal but the 5-HT1A-receptor mediated modulation of respiratory network activity is disturbed. Since the intracellular cAMP-concentration is reduced in mice that lack the transcriptional modulator methyl-CpG-binding protein 2 (MeCP2) gene, we aimed to test if the α3 subunit of the glycine receptor is involved in the development of the breathing phenotype of MeCP2-deficient mice (Mecp2−/y). Therefore, we generated a double knock-out mouse line that lacks both the Mecp2 gene as well as the gene (Glra3) for the α3 subunit of the ionotropic glycine receptor. As compared to WT and Glra3−/− mice, both Mecp2−/y mice and Mecp2−/y; Glra3−/− mice (DKO) showed a slower respiratory rate and a tendency towards higher numbers of apneas. Interestingly, the irregularity of the breathing was significantly reduced in DKO as compared to Mecp2−/y littermates. In the light of the unaltered survival of DKO mice, however, the contribution of the glycine receptor α3 subunit for development and progression of the breathing disturbances in the mouse model of Rett syndrome appears to be only of minor relevance.
Mutations in the transcription factor methyl-CpG-binding protein 2 (MeCP2) cause the neurodevelopmental disorder Rett syndrome (RTT). Besides many other neurological problems, RTT patients show ...irregular breathing with recurrent apneas or breath-holdings. MeCP2-deficient mice, which recapitulate this breathing phenotype, show a dysregulated, persistent expression of G-protein-coupled serotonin receptor 5-ht
(
) in the brainstem. To investigate whether the persistence of 5-ht
expression is contributing to the respiratory phenotype, we crossbred MeCP2-deficient mice with 5-ht
-deficient mice to generate double knockout mice (
;
). To compare respiration between wild type (WT),
and
;
mice, we used unrestrained whole-body plethysmography. While the breathing of MeCP2-deficient male mice (
) at postnatal day 40 is characterized by a slow breathing rate and the occurrence of prolonged respiratory pauses, we found that in MeCP2-deficient mice, which also lacked the 5-ht
receptor, the breathing rate and the number of pauses were indistinguishable from WT mice. To test for a potential mechanism, we also analyzed if the known coupling of 5-ht
receptors to G
proteins is altering second messenger signaling. Tissue cAMP levels in the medulla of
mice were decreased as compared to WT mice. In contrast, cAMP levels in
;
mice were indistinguishable from WT mice. Taken together, our data points towards a role of 5-ht
receptors within the complex breathing phenotype of MeCP2-deficient mice.
The octamer-binding transcription factor 4 (Oct4) was originally described as a marker of embryonic stem cells. Recently, the role of Oct4 as a key regulator in pluripotency was shown by its ability ...to reprogram somatic cells in vitro, either alone or in concert with other factors. While artificial induction of pluripotency using transcription factors is possible in mammalian cell culture, it remains unknown whether a potential natural transfer mechanism might be of functional relevance in vivo. The stem cell based regeneration of deer antlers is a unique model for rapid and complete tissue regeneration in mammals and therefore most suitable to study such mechanisms. Here, the transfer of pluripotency factors from resident stem cell niche cells to differentiated cells could recruit more stem cells and start rapid tissue regeneration.
We report on the ability of STRO-1(+) deer antlerogenic mesenchymal stem cells (DaMSCs) to transport Oct4 via direct cell-to-cell connections. Upon cultivation in stem cell expansion medium, we observed nuclear Oct4 expression in nearly all cells. A number of these cells exhibit Oct4 expression not only in the nucleus, but also with perinuclear localisation and within far-ranging intercellular connections. Furthermore, many cells showed intercellular connections containing both F-actin and α-tubulin and through which transport could be observed. To proof that intercellular Oct4-transfer has functional consequences in recipient cells we used a co-culture approach with STRO-1(+) DaMSCs and a murine embryonic fibroblast indicator cell line (Oct4-GFP MEF). In this cell line a reporter gene (GFP) under the control of an Oct4 responsive element is only expressed in the presence of Oct4. GFP expression in Oct4-GFP cells started after 24 hours of co-culture providing evidence of Oct4 transfer from STRO-1(+) DaMSCs to Oct4-GFP MEF target cells.
Our findings indicate a possible mechanism for the expansion of a resident stem cell niche by induction of pluripotency in surrounding non-niche cells via transfer of transcription factors through intercellular connections. This provides a new approach to explain the rapid annual antler regrowth.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mutations in methyl-CpG-binding protein 2 (MECP2) gene have been shown to manifest in a neurodevelopmental disorder that is called Rett syndrome. A typical problem that occurs during development is a ...disturbance of breathing. To address the role of inhibitory neurons, we generated a mouse line that restores MECP2 in inhibitory neurons in the brainstem by crossbreeding a mouse line that expresses the Cre-recombinase (Cre) in inhibitory neurons under the control of the glycine transporter 2 (GlyT2, slc6a5) promotor (GlyT2-Cre) with a mouse line that has a floxed-stop mutation of the Mecp2 gene (Mecp2 (stop/y)). Unrestrained whole-body-plethysmography at postnatal day P60 revealed a low respiratory rate and prolonged respiratory pauses in Mecp2 (stop/y) mice. In contrast, GlyT2-Cre positive Mecp2 (stop/y) mice (Cre(+) ; Mecp2 (stop/y)) showed greatly improved respiration and were indistinguishable from wild type littermates. These data support the concept that alterations in inhibitory neurons are important for the development of the respiratory phenotype in Rett syndrome.
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