Antineoplastic effects of melatonin on a rare malignancy of mesenchymal origin: melatonin receptor-mediated inhibition of signal transduction, linoleic acid metabolism and growth in tissue-isolated human leiomyosarcoma xenografts

Abstract: Melatonin provides a circadian signal that regulates linoleic acid (LA)-dependent tumor growth. In rodent and human cancer xenografts of epithelial origin in vivo, melatonin suppresses the growth-stimulatory effects of linoleic acid (LA) by blocking its uptake and metabolism to the mitogenic agent, 13-hydroxyoctadecadienoic acid (13-HODE). This study tested the hypothesis that both acute and long-term inhibitory effects of melatonin are exerted on LA transport and metabolism, and growth activity in tissue-isolated human leiomyosarcoma (LMS), a rare, mesenchymally-derived smooth muscle tissue sarcoma, via melatonin receptor-mediated inhibition of signal transduction activity. Melatonin added to the drinking water of female nude rats bearing tissue-isolated LMS xenografts and fed a 5% corn oil (CO) diet caused the rapid regression of these tumors (0.17 ± 0.02 g/day) versus control xenografts that continued to grow at 0.22 ± 0.03 g/day over a 10-day period. LMS perfused in situ for 150 min with arterial donor blood augmented with physiological nocturnal levels of melatonin showed a dose-dependent suppression of tumor cAMP production, LA uptake, 13-HODE release, extracellular signal-regulated kinase (ERK 1/2), mitogen activated protein kinase (MEK), Akt activation, and [³H]thymidine incorporation into DNA and DNA content. The inhibitory effects of melatonin were reversible and preventable with either melatonin receptor antagonist S20928, pertussis toxin, forskolin, or 8-Br-cAMP. These results demonstrate that, as observed in epithelially-derived cancers, a nocturnal physiological melatonin concentration acutely suppress the proliferative activity of mesenchymal human LMS xenografts while long-term treatment of established tumors with a pharmacological dose of melatonin induced tumor regression via a melatonin receptor-mediated signal transduction mechanism involving the inhibition of tumor LA uptake and metabolism.     

Phosphoinositide-generated second messengers in cardiac signal transduction

Hokin and Hokin were the first to demonstrate that tissue inositol phospholipid (phosphoinositide, PI) turnover was increased by hormone treatment. Twenty years later, Michell published a seminal review in which he suggested a relationship between stimulated inositol phospholipid metabolism and Ca²⁺ mobilization. The biochemical link between these two events was subsequently identified by Berridge and colleagues as inositol trisphosphate (InsP₃), a Ca²⁺-mobilizing ligand that is formed by the breakdown of phosphatidylinositol bisphosphate (PIP₂). The other product of inositol phospholipid hydrolysis, diacylglycerol, activates a Ca²⁺-sensitive phospholipid-dependent protein kinase, protein kinase C, which has been considered as a potential regulator of cardiac ion channels, inotropic state, and gene expression. This review summarizes our current state of knowledge concerning the formation of phosphoinositide-generated second messengers in cardiac cells and their potential role in mediating functional responses in the myocardium.     

Chimeric Galphaq subunits can distinguish the long form of the Xenopus Mel1c melatonin receptor from the mammalian mt1 and MT2 melatonin receptors

The family of melatonin receptors is composed of the mt1, MT2, and Mel1c subtypes. The Mel1c is further divided into one long and two short isoforms. A recent study has shown that, unlike mt1 and MT2, the long form of Mel1c is incapable of activating the pertussis toxin-insensitive G16. Here we used three well-characterized Galphaq chimeras to explore the coupling specificity of the melatonin receptors. The qi5, qo5, and qz5 chimeras can link numerous Gi-coupled receptors to the stimulation of phosphoinositide-specific phospholipase C. Both mt1 and MT2 receptors interacted productively with the Galphaq chimeras, while the long form of Mel1c was totally ineffective. Among the Galphaq chimeras, qo5 was less efficiently coupled to the melatonin receptors. Such differential coupling is best explained by structural differences between the melatonin receptors as well as among the Galphaq chimeras. Since the long form of Mel1c receptor possesses an exceptionally large C-terminal tail, we tested the ability of four melatonin receptor C-terminal tail chimeras (Chi 1-4) to interact with the Galphaq chimeras. The presence of the large C-terminal tail of Mel1c in Chi 1 and Chi 3 markedly hindered their coupling to the Galphaq chimeras. On the other hand, the attachment of either the mtl or MT2 C-terminal tail to a Mel1c backbone produced chimeras (Chi 2 and Chi 4) that were capable of activating the Galphaq chimeras. These findings suggest the involvement of C-terminal regions of melatonin receptors in the recognition of G proteins.     

Microtubules modulate melatonin receptors involved in phase-shifting circadian activity rhythms: in vitro and in vivo evidence

Abstract:  MT1 melatonin receptors expressed in Chinese hamster ovary (CHO) cells remain sensitive to a melatonin re-challenge even following chronic melatonin exposure when microtubules are depolymerized in the cell, an exposure that normally results in MT1 receptor desensitization. We extended our findings to MT2 melatonin receptors using both in vitro and in vivo approaches. Using CHO cells expressing human MT2 melatonin receptors, microtubule depolymerization prevents the loss in the number of high potency states of the receptor when compared to melatonin-treated cells. In addition, microtubule depolymerization increases melatonin-induced PKC activity but not PI hydrolysis via Gi proteins similar to that shown for MT1Rs. Furthermore, microtubule depolymerization in MT2-CHO cells enhances the exchange of GTP on Gi-proteins using a photoaffinity analog of GTP. To test whether microtubules are capable of modulating melatonin-induced phase-shifts, microtubules are depolymerized specifically within the suprachiasmatic nucleus of the hypothalamus (SCN) of the Long Evans rat and the efficacy of melatonin to phase shift their circadian activity rhythms was assessed and compared to animals with intact SCN microtubules. We find that microtubule depolymerization in the SCN using either Colcemid or nocodazole enhances the efficacy of 10 p m melatonin to phase-shift the activity rhythms of the Long Evans rat. No enhancement occurs in the presence of β-lumicolchicine, the inactive analog of Colcemid. Taken together, these data suggest that microtubule dynamics can modulate melatonin-induced phase shifts of circadian activity rhythms which may explain, in part, why circadian disturbances occur in individuals afflicted with diseases associated with microtubule disturbances.     

Identification of signal transduction pathways involved in the formation of platelet subpopulations upon activation

Platelets are formed elements of blood. Upon activation, they externalize phosphatidylserine, thus accelerating membrane‐dependent reactions of blood coagulation. Activated platelets form two subpopulations, only one of which expresses phosphatidylserine. This study aimed to identify signalling pathways responsible for this segregation. Gel‐filtered platelets, intact or loaded with calcium‐sensitive dyes, were activated and labelled with annexin V and antibodies, followed by flow cytometric analysis. Calcium Green and Fura Red dyes were compared, and only the latter was able to detect calcium level differences in the platelet subpopulations. Phosphatidylserine‐positive platelets produced by thrombin had stably high intracellular calcium level; addition of convulxin increased and stabilized calcium level in the phosphatidylserine‐negative subpopulation. PAR1 agonist SFLLRN also induced calcium rise and subpopulation formation, but the resulting platelets were not coated with alpha‐granule proteins. Adenylatecyclase activator forskolin inhibited phosphatidylserine‐positive platelets formation several‐fold, while its inhibitor SQ22536 had no effect. This suggests that adenylatecyclase inactivation is necessary, but not rate‐limiting, for subpopulation segregation. Inhibition of mitogen‐activated protein kinase kinase (U0126) and glycoprotein IIb‐IIIa (Monafram^®) was without effect, whereas inhibitors of phosphatidylinositol 3‐kinase (wortmannin) and Src tyrosine kinase (PP2) decreased the procoagulant subpopulation threefold. These data identify the principal signalling pathways controlling platelet heterogeneity.     

Cytokines and signal transduction

Many studies have characterized the role of growth factors in multiple myeloma (MM) pathogenesis and have derived novel therapies to improve patient outcome based upon targeting cytokines and their signaling cascades both in the MM cell and in the bone-marrow (BM) microenvironment. These cytokines include interleukin 6 (IL-6), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta), stromal cell-derived factor 1alpha (SDF-1alpha), IL-21, B-cell stimulating factor 3 (BSF-3) and fibroblast growth factor (FGF). These cytokines are secreted from stromal cells (SCs), endothelial cells and/or osteoclasts, and promote MM cell growth, survival and migration, as well as paracrine cytokine secretion and angiogenesis in the BM milieu. Thus inhibition of signaling cascades induced by these cytokine provides rationale for a therapeutic option for MM.     

敲除AT1a基因对血管紧张素II受体介导的信号传导作用

目的研究敲除血管紧张素Ⅱ受体亚型（ＡＴ１ａ）基因对血管紧张素Ⅱ（ＡｎｇⅡ）信号传导的影响,明确ＡＴ１ａ在血管功能调节中的作用。方法应用缺乏ＡＴ１ａ基因小鼠的主动脉血管平滑肌细胞（ＶＳＭＣ）,采用钙荧光分析技术,观察Ｇ蛋白受体偶联和酪氨酸激酶相关的钙离子信号传导通路的变化。结果敲除ＡＴ１ａ基因,并不影响ＡｎｇⅡ介导的ＶＳＭＣ钙增加,应用Ｇ蛋白拮抗剂和酪氨酸激酶抑制剂均能显著抑制ＡｎｇⅡ反应。结论敲除ＡＴ１ａ基因,其他ＡＴ１受体亚型能起明显的代偿作用,ＡＴ１ａ受体亚型受Ｇ蛋白和酪氨酸激酶信号传导通路共同调节。     

Microvesicle-mediated exocytosis of glutamate is a novel paracrine-like chemical transduction mechanism and inhibits melatonin secretion in rat pinealocytes

Abstract: Mammalian pinealocytes are neuroendocrine cells that synthesize and secrete melatonin, these processes being positively controlled by norepinephrine derived from innervating sympathetic neurons. Previously, we showed that pinealocytes contain a large number of microvesicles (MVs) that specifically accumulate L -glutamate through a vesicular glutamate transporter and contain proteins for exocytosis such as synaptobrevin 2 (VAMP2). These findings suggested that the MVs are counterparts of synaptic vesicles and are involved in paracrine-like chemical transduction in the pineal gland. Here, we show that pinealocytes actually secrete glutamate upon stimulation by KC1 in the presence of Ca²⁺ at 37°C. The ability of glutamate secretion disappeared when the cells were incubated at below 20°C. Loss of the activity was also observed on successive stimulation, but it was recovered after 12 hr incubation. A low concentration of cadmium chloride or ω-conotoxin GVIA inhibited the secretion. Botulinum neurotoxin E cleaved synaptic vesicle-associated protein 25 (SNAP-25) and thus inhibited the secretion. The released L -glutamate stimulated pinealocytes themselves via glutamate receptor(s) and inhibited norepinephrine-stimulated melatonin secretion. These results strongly suggest that pinealocytes are glutaminergic paraneurons, and that the glutaminergic system regulates negatively the synthesis and secretion of melatonin. The MV-mediated paracrine-like chemical transduction seems to be a novel mechanism that regulates hormonal secretion by neuroendocrine cells.     

Evidence that membrane-bound G protein-coupled melatonin receptors MT1 and MT2 are not involved in the neuroprotective effects of melatonin in focal cerebral ischemia

Melatonin is synthesized and released by the pineal gland in a circadian rhythm, and many of its peripheral actions are mediated via membrane MT1 and MT2 receptors. Apart from its metabolic functions, melatonin is a potent neuroprotective molecule owing to its antioxidative actions. The roles of MT1 and MT2 in the neuroprotective effects of melatonin and cell signaling after cerebral ischemia remain unknown. With the use of MT1 and MT2 knockout (mt1/2(-/-) ) mice treated with melatonin, we evaluated brain injury, edema formation, inducible nitric oxide synthase (iNOS) activity, and signaling pathways, including CREB, ATF-1, p21, Jun kinase (JNK)1/2, p38 phosphorylation, resulting from ischemia/reperfusion injury. We show that the infarct volume and brain edema do not differ between mt1/2(-/-) and wild-type (WT) animals, but melatonin treatment decreases infarct volume in both groups and brain edema in WT animals after middle cerebral artery occlusion. Notably, melatonin's neuroprotective effect was even more pronounced in mt1/2(-/-) animals compared to that in WT animals. We also demonstrate that melatonin treatment decreased CREB, ATF-1, and p38 phosphorylation in both mt1/2(-/-) and WT mice, while p21 and JNK1/2 were reduced only in melatonin-treated WT animals in the ischemic hemisphere. Furthermore, melatonin treatment lowered iNOS activity only in WT animals. We provide evidence that the absence of MT1 and MT2 has no unfavorable effect on ischemic brain injury. In addition, the neuroprotective effects of melatonin appear to be mediated through a mechanism independent of its membrane receptors. The underlying mechanism(s) should be further studied using selective melatonin receptor agonists and antagonists.     

肝纤维化中肝星状细胞内主要信号转导通路

肝纤维化是肝脏对各种慢性刺激进行损伤修复反应时,以胶原为主的细胞外基质(ECM)在肝内大量沉积的病理过程.活化的肝星状细胞(HSC)是肝纤维化时产生ECM的主要细胞.细胞因子、氧化应激以及ECM的改变等外部因素通过一定的细胞内信号转导通路激活HSC.了解HSC活化的信号转导通路能从根本上为治疗肝纤维化提供更多更有效的思路和方法.目前研究较多的信号途径有TGF-β/Smad通路、MAPK通路、PI3K通路、JAK/STAT通路、NF-κB通路、过氧化物酶体增殖物激活受体通路等.本文简要综述了肝纤维化时HSC中主要的细胞内信号转导通路.     

C-terminal domains within human MT1 and MT2 melatonin receptors are involved in internalization processes

Abstract:  Melatonin, a molecule implicated in a variety of diseases, including cancer, often exerts its effects through G-protein-coupled melatonin receptors, MT₁ and MT₂. In this study, we sought to understand further the domains involved in the function and desensitization patterns of these receptors through site-directed mutagenesis. Two mutations were constructed in the cytoplasmic C-terminal tail of each receptor subtype: (i) a cysteine residue in the C-terminal tail was mutated to alanine, thus removing a putative palmitoylation site, and a site possibly required for normal receptor function (MT₁C7.72A and MT₂C7.77A) and (ii) the C-terminal tail in the MT₁ and MT₂ receptors was truncated, removing the putative phosphorylation and β-arrestin binding sites (MT₁Y7.64 and MT₂Y7.64). These mutations did not alter the affinity of 2-[¹²⁵I]-iodomelatonin binding to the MT₁ or MT₂ receptors. Using confocal microscopy, it was determined that the putative palmitoylation site (cysteine residue) did not play a role in receptor internalization; however, this residue was essential for receptor function, as determined by 3',5'-cyclic adenosine monophosphate (cAMP) accumulation assays. Truncation of the C-terminal tail of both receptors (MT₁Y7.64 and MT₂Y7.64) inhibited internalization as well as the cAMP response, suggesting the importance of the C-terminal tail in these receptor functions.     

In vitro autoradiographical localization of melatonin binding sites in the caprine brain

The recent development of a specific 2-[125I]-iodo-melatonin ligand has led to the identification of 125I-melatonin binding sites in the brains of numerous mammalian species. The present study reports the localization of 125I-melatonin binding sites in the brain of the dairy goat. Six previously untreated female goats, aged 5-7 years, were culled under natural light between 0900 and 1100. Brains and pituitaries were immediately dissected out and frozen on dry ice. Both transverse and sagittal sections of frozen brain were cut 20 microns thick and thaw-mounted onto gelatin-coated slides. Three consecutive sections were cut at intervals throughout the brain, mounted onto three slides, labeled A, B, and C, and thusly treated: (A) incubated for 2 hr at room temperature in a 50 pM solution of 125I-melatonin; (B) incubated for 2 hr at room temperature in a 50 pM solution of 125I-melatonin plus 1 microM cold melatonin; (C) fixed in Clarke's fluid and stained with toluidine blue. After incubation, A (specific) and B (nonspecific) slides were washed three times in ice-cold Tris-HCl buffer (pH 7.7), air-dried, exposed to an X-ray film for 2 weeks at -20 degrees C, and then fixed and stained. Specific 125I-melatonin binding sites were found in the pars tuberalis (PT), the area of the suprachiasmatic nucleus (SCN), preoptic area (POA), fornix/mediolateral septal areas, hippocampus, and the cerebral cortex. 125I-melatonin did not bind in the hindbrain, midbrain, neurohypophysis, pars intermedia or pars distalis of the adenohypophysis, or the pineal.     

Comparative aspects of the pineal/melatonin system of poikilothermic vertebrates

Abstract: The pineal gland of poikilothermic vertebrates originates as an evagination from the diencephalic roof between the habenular and the posterior commissures, and associates with a parapineal organ to form the so-called pineal complex. The pinealocytes may be photosensitive, secretory or intermediate cells between both. Melatonin, the indoleamine secreted by the pineal, exhibits a circadian secretory rhythm that conveys environmental information to the organism. The peak melatonin secretion occurs during the night, although there are a few examples of an increase in indoleamine secretion during the day. Melatonin is also synthesized in other sites such as the retina, and it has been found in many invertebrates and unicellular organisms. The rhythmic secretory pattern of melatonin is responsible for many biological rhythms exhibited by lower vertebrates. These rhythms are abolished by pinealectomy in some species, but not in others, suggesting the existence of an extra-pineal pacemaker. The photoperiod and the temperature (especially in reptiles) are the main environmental factors affecting the secretory rhythm of melatonin. Poikilothermic vertebrates exhibit a circadian rhythmic color change, with nocturnal blanching, usually related to melatonin secretion. In amphibians, melatonin exhibits a potent skin lightening activity. However, in fishes and reptiles the melatonin effects vary with the species, the developmental stage, and the pigment cell location. Melatonin also exerts inhibitory or excitatory activity on the amphibian reproductive system, regulation of circadian locomotory activity in reptiles, and modulation of the amphibian metamorphosis. Melatonin has also a modulatory effect on the response of target cells to different hormones and high concentrations or prolonged exposure to the indoleamine may cause autodesensitization in various tissues. Binding sites of melatonin have been detected in the central nervous system and peripheral tissues of various vertebrates. The relative potencies of melatonin analogues demonstrated two subtypes of melatonin receptors (ML-1 and ML-2). A transmembrane melatonin receptor has been cloned from Xenopus laevis melanophores; it belongs to the family of the G protein-coupled receptors and exhibits 85% homology with the mammalian nervous system receptor. Melatonin binding sites in the nucleus of many cell types and its potent intracellular anti-oxidant action suggest mechanisms of action other than through the G-protein coupled receptor.     

Effect of chronic growth hormone treatment on insulin signal transduction in rat tissues

Growth hormone (GH) is known to produce insulin resistance, but the exact molecular mechanism remains unclear. We have chronically treated rats with GH and observed that the levels of insulin receptor in the liver or muscle were similar in both the GH-treated and non-treated rats. Insulin-stimulated receptor autophosphorylation was unaltered in the liver, but was reduced in the muscle of rats treated with GH. Insulin receptor substrate-1 (IRS-1) and phosphatidylinositol (PI) 3-kinase protein levels decreased in the liver but not muscle of GH-treated rats. There was no change in hepatic and muscle IRS-2 concentrations. A common finding in liver and muscle was the decrease in IRS-1 and IRS-2 tyrosine phosphorylation associated with a reduction in the interaction between these substrates and PI 3-kinase. These data suggest that changes in the early steps of insulin signal transduction may have a role in the insulin resistance observed in rats exposed to an excess of GH.     

Microtubule network is required for insulin-induced signal transduction and actin remodeling

Both microtubule and actin are required for insulin-induced glucose uptake. However, the roles of these two cytoskeletons and their relationship in insulin action still remain unclear. In this work, we examined the morphological change of microtubule/actin and their involvement in insulin signal transduction using rat skeletal muscle cells. Insulin rapidly led to microtubule clustering from ventral to dorsal surface of the cell. Microtubule filaments were rearranged to create space where new actin structures formed. Disruption of microtubule prevented insulin-induced actin remodeling and distal insulin signal transduction, with reduction in surface glucose transporter isoform 4 (GLUT4) and glucose uptake. Though microtubule mediated actin remodeling through PKCζ, reorganization of microtubule depended on tyrosine phosphorylation of insulin receptor, the mechanism is different from insulin-induced actin remodeling, which relied on the activity of PI3-kinase and PKCζ. We propose that microtubule network is required for insulin-induced signal transduction and actin remodeling in skeletal muscle cells.