1-Chromonyl-5-Imidazolylpentadienone Demonstrates Anti-Cancer Action against TNBC and Exhibits Synergism with Paclitaxel.

Curcumin has been well studied for its anti-oxidant, anti-inflammatory, and anti-cancer action. Its potential as a therapy is limited due to its low bioavailability and rapid metabolism. To overcome these challenges, investigators are developing curcumin analogs, nanoparticle formulations, and combining curcumin with other compounds or dietary components. In the present study, we used a 1-chromonyl-5-imidazolylpentadienone named KY-20-22 that contains both the pharmacophore of curcumin and 1,4 benzopyrone (chromone) moiety typical for flavonoids, and also included specific moieties to enhance the bioavailability. When we tested the in vitro effect of KY-20-22 in triple-negative breast cancer (TNBC) cell lines, we found that it decreased the cell survival and colony formation of MDA-MB-231 and MDA-MB-468 cells. An increase in mitochondrial reactive oxygen species was also observed in TNBC cells exposed to KY-20-22. Furthermore, KY-20-22 decreased epithelial-mesenchymal formation (EMT) as evidenced by the modulation of the EMT markers E-cadherin and N-cadherin. Based on the fact that KY-20-22 regulates interleukin-6, a cytokine involved in chemotherapy resistance, we combined it with paclitaxel and found that it synergistically induced anti-proliferative action in TNBC cells. The results from this study suggested that 1-chromonyl-5-imidazolylpentadienone KY-20-22 exhibited anti-cancer action in MDA-MB-231 and MDA-MB-468 cells. Future studies are required to evaluate the anti-cancer ability and bioavailability of KY-20-22 in the TNBC animal model.

Direct evidence of bradycardic effect of omega-3 fatty acids acting on nucleus ambiguus.

Docosahexaenoic acid (DHA) an omega-3 polyunsaturated fatty acid, is an agonist of FFA1 receptor. DHA administration reduces the heart rate via unclear mechanisms. We examined the effect of DHA on neurons of nucleus ambiguus that provide the parasympathetic control of heart rate. DHA produced a dose-dependent increase in cytosolic Ca2+ concentration in cardiac-projecting nucleus ambiguus neurons; the effect was prevented by GW1100, a FFA1 receptor antagonist. DHA depolarized cultured nucleus ambiguus neurons via FFA1 activation. Bilateral microinjection of DHA into nucleus ambiguus produced bradycardia in conscious rats. Our results indicate that DHA decreases heart rate by activation of FFA1 receptor on cardiac-projecting nucleus ambiguus neurons.

Direct evidence of intracrine angiotensin II signaling in neurons.

The existence of a local renin-angiotensin system (RAS) in neurons was first postulated 40 years ago. Further studies indicated intraneuronal generation of ANG II. However, the function and signaling mechanisms of intraneuronal ANG II remained elusive. Since ANG II type 1 receptor (AT1R) is the major type of receptor mediating the effects of ANG II, we used intracellular microinjection and concurrent Ca(2+) and voltage imaging to examine the functionality of intracellular AT1R in neurons. We show that intracellular administration of ANG II produces a dose-dependent elevation of cytosolic Ca(2+) concentration ([Ca(2+)]i) in hypothalamic neurons that is sensitive to AT1R antagonism. Endolysosomal, but not Golgi apparatus, disruption prevents the effect of microinjected ANG II on [Ca(2+)]i. Additionally, the ANG II-induced Ca(2+) response is dependent on microautophagy and sensitive to inhibition of PLC or antagonism of inositol 1,4,5-trisphosphate receptors. Furthermore, intracellular application of ANG II produces AT1R-mediated depolarization of hypothalamic neurons, which is dependent on [Ca(2+)]i increase and on cation influx via transient receptor potential canonical channels. In summary, we provide evidence that intracellular ANG II activates endolysosomal AT1Rs in hypothalamic neurons. Our results point to the functionality of a novel intraneuronal angiotensinergic pathway, extending the current understanding of intracrine ANG II signaling.

Expression of estrogen receptor GPR30 in the rat spinal cord and in autonomic and sensory ganglia.

The G protein-coupled receptor GPR30 has recently been identified as a nonnuclear estrogen receptor. Reverse transcriptase-polymerase chain reaction revealed expression of GPR30 mRNA in varying quantities in the rat spinal cord, dorsal root ganglia, nodose ganglia, trigeminal ganglia, hippocampus, brain stem, and hypothalamus. Immunohistochemical studies that used a rabbit polyclonal antiserum against the human GPR30 C-terminus revealed a fine network of GPR30-immunoreactive (irGPR30) cell processes in the superficial layers of the spinal cord; some of which extended into deeper laminae. A population of neurons in the dorsal horn and ventral horn were irGPR30. Dorsal root, nodose, and trigeminal ganglionic neurons displayed varying intensities of irGPR30. Positively labeled neurons were detected in the major pelvic ganglion, but not in the superior cervical ganglion. A population of chromaffin cells in the adrenal medulla was irGPR30, so were cells of the zona glomerulosa. Double-labeling the adrenal medulla with GPR30 antiserum and tyrosine hydroxylase antibody or phenylethanolamine-N-methyltransferase antiserum revealed that irGPR30 is expressed in the majority of tyrosine hydroxylase-positive chromaffin cells. Last, some of the myenteric ganglion cells were irGPR30. Tissues processed with preimmune serum resulted in no staining. Voltage-sensitive dye imaging studies showed that the selective GPR30 agonist G-1 (1, 10, and 100 nM) depolarized cultured spinal neurons in a concentration-dependent manner. Collectively, our result provides the first evidence that GPR30 is expressed in neurons of the dorsal and ventral horn as well as in sensory and autonomic neurons, and activation of GPR30 by the selective agonist G-1 depolarizes cultured spinal neurons.

Nesfatin-1: distribution and interaction with a G protein-coupled receptor in the rat brain.

Nesfatin-1 is a recently identified satiety molecule detectable in neurons of the hypothalamus and nucleus of solitary tract (NTS). Immunohistochemical studies revealed nesfatin-1-immunoreactive (irNEF) cells in the Edinger-Westphal nucleus, dorsal motor nucleus of vagus, and caudal raphe nuclei of the rats, in addition to the hypothalamus and NTS reported in the initial study. Double-labeling immunohistochemistry showed that irNEF cells were vasopressin or oxytocin positive in the paraventricular and supraoptic nucleus; cocaine-amphetamine-regulated transcript or tyrosine hydroxylase positive in arcuate nucleus; cocaine-amphetamine-regulated transcript or melanin concentrating hormone positive in the lateral hypothalamus. In the brainstem, irNEF neurons were choline acetyltransferase positive in the Edinger-Westphal nucleus and dorsal motor nucleus of vagus; tyrosine hydroxylase positive in the NTS; and 5-hydroxytryptamine positive in the caudal raphe nucleus. The biological activity of rat nesfatin-1 (1-82) (100 nm) was assessed by the Ca(2+) microfluorometric method. Nesfatin-1 elevated intracellular Ca(2+) concentrations [Ca(2+)](i) in dissociated and cultured hypothalamic neurons. The response was prevented by pretreating the cells with pertussis toxin (100 nm) or Ca(2+)-free solution and by a combination of the L-type and P/Q-type calcium channel blocker verapamil (1 microm) and omega-conotoxin MVIIC (100 nm). The protein kinase A inhibitor KT 5720 (1 microm) suppressed nesfatin-1-induced rise in [Ca(2+)](i). The result shows that irNEF is distributed to several discrete nuclei in the brainstem, in addition to the hypothalamus and NTS reported earlier, and that the peptide interacts with a G protein-coupled receptor, leading to an increase of [Ca(2+)](i), which is linked to protein kinase A activation in cultured rat hypothalamic neurons.

Distribution and characterization of estrogen receptor G protein-coupled receptor 30 in the rat central nervous system.

The G protein-coupled receptor 30 (GPR 30) has been identified as the non-genomic estrogen receptor, and G-1, the specific ligand for GPR30. With the use of a polyclonal antiserum directed against the human C-terminus of GPR30, immunohistochemical studies revealed GPR30-immunoreactivity (irGPR30) in the brain of adult male and non-pregnant female rats. A high density of irGPR30 was noted in the Islands of Calleja and striatum. In the hypothalamus, irGPR30 was detected in the paraventricular nucleus and supraoptic nucleus. The anterior and posterior pituitary contained numerous irGPR30 cells and terminal-like endings. Cells in the hippocampal formation as well as the substantia nigra were irGPR30. In the brainstem, irGPR30 cells were noted in the area postrema, nucleus of the solitary tract, and dorsal motor nucleus of the vagus; a cluster of cells were prominently labeled in the nucleus ambiguus. Tissue sections processed with pre-immune serum showed no irGPR30, affirming the specificity of the antiserum. G-1 (100 nM) caused a large increase of intracellular calcium concentrations [Ca(2+) ](i) in dissociated and cultured rat hypothalamic neurons, as assessed by microfluorometric Fura-2 imaging. The calcium response to a second application of G-1 showed a marked homologous desensitization. Our result shows a high expression of irGPR30 in the hypothalamic-pituitary axis, hippocampal formation, and brainstem autonomic nuclei; and the activation of GPR30 by G-1 is associated with a mobilization of calcium in dissociated and cultured rat hypothalamic neurons.

Smooth muscle-associated protein 8: distribution and biological activity in the rat brain.

With the use of an antiserum directed against the human smooth muscle-associated protein 8 (SMAP8) fragment SMAP8(98-138), Western blot and immunohistochemical studies revealed SMAP8 expression in the rat brain. A band with a molecular size of about 45 kDa was detected in tissues from the rat hypothalamus and a weaker band from the cortex. SMAP8 immunoreactivity (irSMAP8) was detected in neurons of the hypothalamic paraventricular, supraoptic, and supraoptic retrochiasmatic nuclei; a few irSMAP8 cells were scattered in the zona incerta as well as the cerebral cortex. Immunoreactive cell processes were detected mostly in the internal layer of the median eminence. Double labeling the hypothalamic sections with SMAP8 and vasopressin (VP) or oxytocin (OT) antiserum revealed that a population of VP- and OT-immunoreactive neurons expressed irSMAP8. The biological activity of SMAP8 in rat central neurons was assessed by the calcium microfluorimetric Fura-2 method. SMAP8 (100 nM) elevated cytosolic calcium concentrations [Ca2+]i in a population of dissociated and cultured rat hypothalamic neurons; the response was eliminated in Ca2+-free saline. This is the first evidence of irSMAP8 in a population of VP/OT-containing hypothalamic neurons in the rat, and the peptide is biologically active in hypothalamic neurons, as evidenced by mobilization of extracellular Ca2+.

Insulin-like peptide 5: expression in the mouse brain and mobilization of calcium.

Insulin-like peptide 5 (INSL5) mRNA was detected in the mouse hypothalamus by RT-PCR. Immunohistochemical studies using an antiserum against the mouse INSL5 peptide revealed INSL5-immunoreactive (irINSL5) neurons in the paraventricular, supraoptic, accessory secretory, and supraoptic retrochiasmatic nuclei and immunoreactive cell processes in the internal layer of the median eminence. In the pituitary, irINSL5 was detected in terminal-like elements of the posterior lobe and in cells of the anterior lobe. Double-labeling experiments showed that irINSL5 is expressed in vasopressin-, but not oxytocin-containing neurons. INSL5 (100 nm) administered to dissociated and cultured mouse hypothalamic neurons elevated cytosolic calcium concentrations [Ca(2+)](i), as assessed by the microfluorimetric fura-2 method. In a Ca(2+)-free medium, INSL5 induced in dissociated neurons an increase of [Ca(2+)](i), which was sensitive to the endoplasmic reticulum calcium pump inhibitor thapsigargin (1 microm) and the IP(3) receptor blocker 2-aminoethoxydiphenyl borate (100 microm) or xestospongin C (5 microm). Our result provides the first evidence that INSL5 is expressed in a population of cells in the mouse hypothalamus and pituitary and that it elevates [Ca(2+)](i) by a mechanism involving both Ca(2+) influx and Ca(2+) release from intracellular stores. The concentration of irINSL5 in the hypothalamic-pituitary axis suggests a neuroendocrine function of this insulin superfamily member.

Neuropeptide B immunoreactivity in the central nervous system of the rat.

Neuropeptide B (NPB) is a recently identified endogenous ligand for the orphan G protein-coupled receptors GPR7 and GPR8. NPB mRNA is expressed in the human, rat, and mouse brain. With the use of an antiserum directed against the rat NPB, immunoreactivity to NPB (irNPB) was detected in several discrete areas of the hypothalamus and midbrain. In the hypothalamus, irNPB cells were present in the medial preoptic area and nucleus, ventromedial preoptic nucleus, retrochiasmatic nucleus, paraventricular hypothalamic nucleus, supraoptic nucleus, accessory neurosecretory nuclei, periventricular hypothalamic nucleus, dorsomedial hypothalamic nucleus, supraoptic retrochiasmatic nucleus, lateral hypothalamic area, posterior hypothalamic area, dorsal hypothalamic area, and zona incerta. A few irNPB perikarya were noted in the arcuate nucleus, whereas a dense network of nerve fibers was present in the median eminence. In the midbrain, irNPB somata were noted in the substantia nigra (compact, reticular, medial, and lateral parts), paranigral nucleus, ventral tegmental area, interfascicular nucleus, and dorsal raphe nucleus. Neurons in the Edinger-Westphal were strongly labeled. Labeled cells were not detected in the cortex, medulla oblongata, and spinal cord; few lightly labeled cells were occasionally seen in the hippocampus. Double labeling the hypothalamic sections with NPB antiserum and vasopressin or oxytocin antibody revealed that a population of vasopressin- but not oxytocin-immunoreactive cells was irNPB. Tyrosine hydroxylase-positive neurons in the midbrain, presumably dopaminergic, were irNPB. The distribution of irNPB neurons in several areas of the hypothalamus and midbrain together with the colocalization with vasopressin or tyrosine hydroxylase suggests that the peptide may subserve neuroendocrine, autonomic, and motor functions.

Neuropeptide W-immunoreactivity in the hypothalamus and pituitary of the rat.

Neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30) are 23- and 30-amino acid peptides recently isolated from the porcine hypothalamus. Immunohistochemical studies using a rabbit polyclonal antiserum against the rat NPW23 peptide revealed a limited distribution in the rat brain. NPW23-immunoreactive (irNPW) cells were detected in the paraventricular nucleus (PVH), mainly in the parvocellular division, supraoptic nucleus (SO), accessory neurosecretory nuclei, dorsal and lateral hypothalamic areas, perifornical nucleus, arcuate nucleus, and anterior and posterior pituitary; whereas, irNPW fibers were noted in the PVH and SO, retrochiasmatic nucleus, dorsal and lateral hypothalamic areas, median eminence, amygdala, and posterior pituitary. The pattern of distribution of irNPW in the hypothalamus corroborates a possible role of NPW on prolactin release and feeding behavior reported by others.

Beacon/ubiquitin-like 5-immunoreactivity in the hypothalamus and pituitary of the mouse.

Beacon is a 73-amino acid peptide encoded by a novel gene in the hypothalamus of Israeli sand rat Psammomys obesus. Reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemical techniques were used to investigate the presence of beacon mRNA and the distribution of beacon-immunoreactivity (irBC) in the hypothalamus of ICR mice. RT-PCR experiments revealed beacon mRNA in the mouse hypothalamus. Using a rabbit polyclonal antiserum directed against the synthetic C-terminal peptide fragment (47-73), irBC was detected in the mouse hypothalamus and pituitary. In the hypothalamus, irBC was concentrated in perikarya of the supraoptic (SO), paraventricular (PVH) and accessory neurosecretory nuclei and in cell processes of the median eminence and pituitary stalk. In the pituitary, irBC was noted mainly in the posterior lobe. Double-labeling the hypothalamic sections with guinea-pig vasopressin-antiserum or mouse monoclonal oxytocin-antibody and beacon-antiserum revealed that <30% of vasopressin-immunoreactive neurons and nearly all oxytocin-immunoreactive neurons in the PVH and SO were irBC. The result shows the presence of beacon mRNA in the mouse hypothalamus, and the distribution of irBC is distinctively different from that reported in the hypothalamus of Psammomys obesus, but similar to that of the Sprague-Dawley rats described in our earlier study. More interestingly, Blast search uncovered a 73-amino acid peptide, human ubiquitin-like 5, which has the same exact sequence as beacon. Thus, irBC observed in the mouse brain could be that of ubiquitin-like 5.

Apelin-immunoreactivity in the rat hypothalamus and pituitary.

With the use of an antiserum against human apelin-36, apelin-immunoreactivity (irAP) was detected in neurons and cell processes of the supraoptic nucleus (SO), paraventricular nucleus (PVH), accessory neurosecretory nuclei (Acc) and suprachiasmatic nucleus. Strongly labeled cells/processes were noted in the internal layer of the median eminence, infundibular stem, anterior and posterior pituitary. Double-labeling the sections with goat polyclonal neurophysin I-antiserum and rabbit polyclonal apelin-antiserum revealed a population of magnocellular neurons in the PVH, SO and Acc expressing both irAP and neurophysin I-immunoreactivity (irNP), the latter being a marker of oxytocin-containing neurons. By inference, the AP-positive but irNP-negative magnocellular neurons could be vasopressin-containing. The presence of irAP in certain hypothalamic nuclei and pituitary suggests that the peptide may be a signaling molecule released from the hypothalamic-hypophysial axis.

Beacon-like immunoreactivity in the hypothalamus of Sprague-Dawley rats.

Distribution of the novel peptide beacon in the hypothalamus of Sprague-Dawley rats was examined by immunohistochemical methods. Beacon-immunoreactive (irBC) neurons were found in the paraventricular, supraoptic, and accessory neurosecretory nuclei, and intensely labeled fibers in the median eminence and infundibulo-pituitary stalk. Scattered cells and/or fibers were noted in the suprachiasmatic nucleus, arcuate nucleus, retrochiasmatic area, lateral and medial preoptic area, as well as anterior and lateral hypothalamic area. The wide distribution of irBC in the hypothalamus of Sprague-Dawley rats suggests that the peptide may influence, in addition to a proposed role in feeding, a multitude of biological activities associated with the hypothalamic-pituitary axis.