RESUMO
Serotonin (5-hydroxytryptamine, 5-HT) and its receptors play important roles in the development and progression of malignant tumors. The effect of the 5-HT receptor 1D (HTR1D), a member of the serotonin receptor family, on gastric cancer (GC) is not clear. Analysis of clinical data has shown that high expression of HTR1D was associated with poor prognosis in patients with GC and was an independent risk factor for reduced overall survival (OS) and disease-free survival (DFS). The present study assessed the effects of HTR1D knockdown and the HTR1D inhibitor GR127935 on the biological behavior of GC cells, which both impaired the proliferation and migration of GC cells. RNA sequencing showed that GR127935 inhibited tumor progression by limiting DNA replication and the cell cycle, inducing ferroptosis, and affecting tumor metabolism. Taken together, these findings showed that HTR1D has a potent oncogenic effect on GC and may provide a novel therapeutic target.
Assuntos
Receptor 5-HT1D de Serotonina/metabolismo , Serotonina , Neoplasias Gástricas , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Receptor 5-HT1D de Serotonina/genética , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Antagonistas do Receptor 5-HT1 de Serotonina , Neoplasias Gástricas/patologiaRESUMO
Competing endogenous RNAs (ceRNAs) are a newly discovered class of molecular regulators involved in many diseases, especially tumors. Therefore, exploration of the potential ceRNA regulatory network regarding the occurrence and development of pancreatic cancer will provide a new theoretical basis for its diagnosis and treatment. Based on the above background, we applied a bioinformatics approach to mine the public database The Cancer Genome Atlas (TCGA) and performed a series of subsequent molecular biology assays to confirm the hypothesis that HOXA10-AS/ miR-340-3p/HTR1D axis could modulate the malignant progression of pancreatic cancer. Here, our present study demonstrated that the expression level of HTR1D, positively correlated with the level of lncRNA HOXA10-AS and negatively associated with the level of miR-340-3p, was significantly increased in pancreatic cancer cell lines (PCs) compared with that in normal HPDE6-C7 cells. Knocking down HTR1D obviously inhibited the proliferation and migration of PCs and promoted apoptosis by upregulating p-AKT. Elevated miR-340-3p blocked the progression of pancreatic cancer by downregulating HTR1D. Lessened level of lncRNA HOXA10-AS reduced the sponging of miR-340-3p, resulting in an increase of miR-340-3p and a subsequent decrease of HTR1D to ultimately suppress the malignant biological behaviors of cancer. These data illustrated that the HOXA10-AS/miR-340-3p/HTR1D ceRNA axis acted a crucial part in the malignant biological behavior of pancreatic cancer in an AKT-dependent manner.
Assuntos
MicroRNAs , Neoplasias Pancreáticas , RNA Longo não Codificante , Receptor 5-HT1D de Serotonina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Proteínas Homeobox A10 , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transdução de Sinais , Neoplasias PancreáticasRESUMO
Status epilepticus (SE) is a life-threatening neurologic disorder that can be as both cause and consequence of neuroinflammation. In addition to previous reports on anti-inflammatory property of the anti-migraine medication sumatriptan, we have recently shown its anticonvulsive effects on pentylenetetrazole-induced seizure in mice. In the present study, we investigated further (i) the effects of sumatriptan in the lithium-pilocarpine SE model in rats, and (ii) the possible involvement of nitric oxide (NO), 5-hydroxytryptamin 1B/1D (5-HT1B/1D ) receptor, and inflammatory pathways in such effects of sumatriptan. Status epilepticus was induced by lithium chloride (127 mg/kg, i.p) and pilocarpine (60 mg/kg, i.p.) in Wistar rats. While SE induction increased SE scores and mortality rate, sumatriptan (0.001-1 mg/kg, i.p.) improved it (P < 0.001). Administration of the selective 5-HT1B/1D antagonist GR-127935 (0.01 mg/kg, i.p.) reversed the anticonvulsive effects of sumatriptan (0.01 mg/kg, i.p.). Although both tumor necrosis factor-α (TNF-α) and NO levels were markedly elevated in the rats' brain tissues post-SE induction, pre-treatment with sumatriptan significantly reduced both TNF-α (P < 0.05) and NO (P < 0.001) levels. Combined GR-127935 and sumatriptan treatment inhibited these anti-inflammatory effects of sumatriptan, whereas combined non-specific NOS (L-NAME) or selective neuronal NOS (7-nitroindazole) inhibitors and sumatriptan further reduced NO levels. In conclusion, sumatriptan exerted a protective effect against the clinical manifestations and mortality rate of SE in rats which is possibly through targeting 5-HT1B/1D receptors, neuroinflammation, and nitrergic transmission.
Assuntos
Lítio/toxicidade , Óxido Nítrico/fisiologia , Pilocarpina/toxicidade , Receptor 5-HT1B de Serotonina/fisiologia , Receptor 5-HT1D de Serotonina/fisiologia , Estado Epiléptico/tratamento farmacológico , Sumatriptana/uso terapêutico , Animais , Modelos Animais de Doenças , Masculino , Óxido Nítrico/análise , Ratos , Ratos Wistar , Índice de Gravidade de Doença , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/mortalidade , Sumatriptana/farmacologia , Fator de Necrose Tumoral alfa/análiseRESUMO
BACKGROUND: Hepatocellular Carcinoma (HCC) is a primary liver cancer with high mortality. Paeoniflorin is a pinane monoterpene picroside with anti-tumor effect isolated from Chinese peony root and white peony root. OBJECTIVE: The study was conducted to investigate the underlying mechanism of Paeoniflorin (PF) regulating Hepatocellular Carcinoma (HCC) progression via 5-hydroxytryptamine receptor 1D (5-HT1D). METHODS: HepG2 and SMMC-7721 hepatoma cells were treated with different concentrations of PF (0, 5, 10, 20 µM). Cell proliferation, apoptosis, migration, and invasion were examined by CCK-8 and colony formation assays, flow cytometry, wound healing assay, and transwell assay, respectively. RTqPCR assay was used to detect the expression level of 5-HT1D, and Western blot assay was used to detect the expressions of 5-HT1D and Wnt/ß-catenin pathway-related proteins. RESULTS: With the increase in PF concentration, the mRNA levels of 5-HT1D in HepG2 and SMMC- 7721 hepatoma cells were decreased in a dose-dependent manner, and the proliferation, colony formation, migration and invasion ability of cells were gradually weakened, while the apoptosis rate was gradually increased. Overexpression of 5-HT1D significantly promoted the proliferation, colony formation, migration and invasion of HepG2 and SMMC-7721 cells, and increased the expression of Wnt/ß-catenin pathway-related proteins, ß -actenin, survivin, C-myc, and Cyclin D1. Furthermore, 5-HT1D overexpression could reverse the effect of PF on hepatoma cells and inhibit the expressions of Wnt/ß-catenin pathway-related proteins. CONCLUSION: PF may inhibit the progression of HCC by blocking Wnt/ß-catenin pathway expression through downregulating 5-HT1D.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Glucosídeos/farmacologia , Monoterpenos/farmacologia , Receptor 5-HT1D de Serotonina/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , RNA Mensageiro/metabolismo , Receptor 5-HT1D de Serotonina/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismoRESUMO
This study was planned to evaluate the effects of sumatriptan, 5-HT1B/1D receptors agonist, on ischaemia/reperfusion injury in bilateral testes after unilateral testicular torsion/detorsion in rats. Male Wistar rats (n = 42) were allocated into a sham-operated group, a control group and treatment groups which were injected sumatriptan (0.1, 0.3 and 1 mg/kg), GR-127935 (0.01 mg/kg)-5-HT1B/1D receptors antagonist-and sumatriptan (0.1 mg/kg) + GR-127935 (0.01 mg/kg). Torsion was induced for 1 hr by rotating right testis 7200 in the clockwise direction, and after 7 days of detorsion, bilateral orchiectomy was conducted. While the level of TNF-α rose in testicular tissue after inducing torsion/detorsion, sumatriptan injection notably lowered TNF-α level in ipsilateral (torted) and contralateral (nontorted) testes (p < 0.001). Moreover, after inducing testicular torsion/detorsion, SOD activity was decreased, whereas administration of sumatriptan significantly increased SOD activity in bilateral testes (p < 0.001). After induction of torsion/detorsion, macroscopic and histological analyses also showed severe damages which were improved by sumatriptan injection. Interestingly, co-administration of sumatriptan with GR-127935 reversed the beneficial impacts of sumatriptan on macroscopic appearance, microscopic pattern and biochemical markers. It is concluded that sumatriptan presumably via stimulation of 5-HT1B/1D receptors decreased inflammation, oxidative stress and deteriorations induced by ischaemia/reperfusion injury following testicular torsion/detorsion.
Assuntos
Traumatismo por Reperfusão/tratamento farmacológico , Agonistas do Receptor 5-HT1 de Serotonina/administração & dosagem , Torção do Cordão Espermático/complicações , Sumatriptana/administração & dosagem , Testículo/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Humanos , Masculino , Oxidiazóis/administração & dosagem , Estresse Oxidativo/efeitos dos fármacos , Piperazinas/administração & dosagem , Ratos , Ratos Wistar , Receptor 5-HT1B de Serotonina/metabolismo , Receptor 5-HT1D de Serotonina/metabolismo , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/patologia , Antagonistas da Serotonina/administração & dosagem , Superóxido Dismutase/metabolismo , Testículo/patologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Serotonin and its receptors have been shown to play critical regulatory roles in cancer biology. Nevertheless, the contributions of 5-hydroxytryptamine 1D (5-HT1D), an indispensable member of the serotonergic system, to hepatocellular carcinoma (HCC) remain unknown. The present study demonstrated that the 5-HT1D expression level was significantly up-regulated in HCC tissues and cell lines. The 5-HT1D expression level was closely correlated with unfavorable clinicopathological characteristics. Survival analyses show that elevated 5-HT1D expression level predicts poor overall survival and high recurrence probability in HCC patients. Functional studies revealed that 5-HT1D significantly promoted HCC proliferation, epithelial-mesenchymal transition, and metastasis in vitro and in vivo. Mechanistically, 5-HT1D could stabilize PIK3R1 by inhibiting its ubiquitin-mediated degradation. The interaction between 5-HT1D and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) enhanced the expression of FoxO6 through the PI3K/Akt signaling pathway; FoxO6 could also be directly transcriptionally activated by 5-HT1D in an Akt-independent manner. MicroRNA-599 was found to be an upstream suppressive modulator of 5-HT1D. Additionally, 5-HT1D could attenuate tryptophan hydroxylase 1 expression through the PI3K/Akt/cut-like homeobox 1 axis in HCC. Conclusion: Herein, we uncovered the potent oncogenic effect of 5-HT1D on HCC by interacting with PIK3R1 to activate the PI3K/Akt/FoxO6 pathway, and provided a potential therapeutic target for HCC.
Assuntos
Carcinoma Hepatocelular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Receptor 5-HT1D de Serotonina/metabolismo , Animais , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , China/epidemiologia , Transição Epitelial-Mesenquimal , Feminino , Células Hep G2 , Humanos , Neoplasias Hepáticas Experimentais/mortalidade , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Metástase Neoplásica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Triptofano Hidroxilase/metabolismoRESUMO
G-protein-coupled receptors (GPCRs) play a major role in psychiatric disorders and are the targets of several current therapeutic approaches in this field. A number of studies have now shown that GPCRs can assemble as high molecular weight homo- and hetero-oligomers, which could affect ligand binding, intracellular signalling or trafficking. This information could be critical in design of new drugs to treat neurological and psychiatric disorders. This chapter describes a sequential co-immunoprecipitation and immunoblot protocol for determining oligomerisation of the 5-hydroxytryptamine (HT)1A receptor with other GPCRs in co-transfected HEK-293 cells.
Assuntos
Western Blotting/métodos , Cromatografia de Afinidade/métodos , Imunoprecipitação/métodos , Receptores Acoplados a Proteínas G/análise , Células HEK293 , Humanos , Oligopeptídeos , Multimerização Proteica , Proteínas Proto-Oncogênicas c-myc , Receptor 5-HT1A de Serotonina/análise , Receptor 5-HT1A de Serotonina/química , Receptor 5-HT1B de Serotonina/análise , Receptor 5-HT1B de Serotonina/química , Receptor 5-HT1D de Serotonina/análise , Receptor 5-HT1D de Serotonina/química , Receptores Acoplados a Proteínas G/química , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/química , TransfecçãoRESUMO
Amongst the side effects of triptans, a substantial percentage of patients experience injection site pain and tenderness, the underlying mechanism of which is unknown. We found that the dose range from 10fg to 1000ng (intradermal) of sumatriptan induced a complex dose-dependent mechanical hyperalgesia in male rats, with distinct peaks, at 1pg and 10ng, but no hyperalgesia at 1ng. In contrast, in females, there was 1 broad peak. The highest dose (1000ng) did not produce hyperalgesia in either sex. We evaluated the receptors mediating sumatriptan hyperalgesia (1pg, 1 and 10ng). In males, the injection of an antagonist for the serotonin (5-HT) receptor subtype 1B (5-HT1B), but not 5-HT1D, markedly inhibited sumatriptan (1pg)-induced hyperalgesia, at 10ng a 5-HT1D receptor antagonist completely eliminated hyperalgesia. In contrast, in females, the 5-HT1D, but not 5-HT1B, receptor antagonist completely blocked sumatriptan (1pg and 10ng) hyperalgesia and both 5-HT1B and 5-HT1D receptor antagonists attenuated hyperalgesia (1ng) in females, which is GPR30 estrogen receptor dependent. While selective 5-HT1D or 5-HT1B, agonists produce robust hyperalgesia in female and male rats, respectively, when co-injected the hyperalgesia induced in both sexes was attenuated. Mechanical hyperalgesia induced by sumatriptan (1pg and 10ng) is dependent on the G-protein αi subunit and protein kinase A (PKA), in IB4-positive and negative nociceptors. Understanding the mechanisms responsible for the complex dose dependence for triptan hyperalgesia may provide useful information for the design of anti-migraine drugs with improved therapeutic profiles.
Assuntos
Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Receptor 5-HT1B de Serotonina/metabolismo , Receptor 5-HT1D de Serotonina/metabolismo , Caracteres Sexuais , Sumatriptana/toxicidade , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Feminino , Hiperalgesia/tratamento farmacológico , Masculino , Nociceptores/efeitos dos fármacos , Nociceptores/metabolismo , RNA Mensageiro/metabolismo , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Agonistas do Receptor 5-HT1 de Serotonina/toxicidade , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia , TatoRESUMO
Although recent studies have implicated serotonin 5-HT1B/D receptors in the nociceptive sensitivity of primary afferent neurons, the underlying molecular and cellular mechanisms remain unclear. In this study, we identified a novel functional role of the 5-HT1D receptor subtype in regulating A-type potassium (K(+)) currents (IA) as well as membrane excitability in small trigeminal ganglion (TG) neurons. We found that the selective activation of 5-HT1D, rather than 5-HT1B, receptors reversibly increased IA, while the sustained delayed rectifier K(+) current was unaffected. The 5-HT1D-mediated IA increase was associated with a depolarizing shift in the voltage dependence of inactivation. Blocking G-protein signaling with pertussis toxin or by intracellular application of a selective antibody raised against Gαo or Gß abolished the 5-HT1D effect on IA. Inhibition of protein kinase A (PKA), but not of phosphatidylinositol 3-kinase or protein kinase C, abolished the 5-HT1D-mediated IA increase. Analysis of phospho-p38 (p-p38) revealed that activation of 5-HT1D, but not 5-HT1B, receptors significantly activated p38, while p-ERK and p-JNK were unaffected. The p38 MAPK inhibitor SB203580, but not its inactive analogue SB202474, and inhibition of B-Raf blocked the 5-HT1D-mediated IA response. Functionally, we observed a significantly decreased action potential firing rate induced by the 5-HT1D receptors; pretreatment with 4-aminopyridine abolished this effect. Taken together, these results suggest that the activation of 5-HT1D receptors selectively enhanced IA via the Gßγ of the Go-protein, PKA, and the sequential B-Raf-dependent p38 MAPK signaling cascade. This 5-HT1D receptor effect may contribute to neuronal hypoexcitability in small TG neurons.
Assuntos
Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Neurônios/metabolismo , Canais de Potássio/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Receptor 5-HT1D de Serotonina/metabolismo , Gânglio Trigeminal/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Membrana Celular/efeitos dos fármacos , Subunidades beta da Proteína de Ligação ao GTP , Subunidades gama da Proteína de Ligação ao GTP , Ativação do Canal Iônico/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos ICR , Modelos Biológicos , Neurônios/efeitos dos fármacos , Receptor 5-HT1B de Serotonina , Agonistas do Receptor 5-HT1 de Serotonina/farmacologia , Sumatriptana/farmacologiaRESUMO
Fluoxetine (FLX), a selective serotonin reuptake inhibitor is an antidepressant in the treatment of mood disorders. Its impact on reproductive processes is incompletely known. The present study analyzed the reproductive effects of FLX in prepubertal female rats. Two experiments were conducted. First (acute administration), 30-day-old female rats were injected intraperitoneally with 5mg/kg of fluoxetine-hydrochloride, and were terminated 24, 48 or 72h after the treatment. Second (subchronic administration), FLX was injected on days 30-33 of age, and the animals were terminated the day of first estrus. In acute treatment estradiol concentration increased to 72h. In subchronic treatment increased serotonin concentration in ovaries and decreased the number of ova shed. An increase in number of atretic follicles and oocyte fragmentation was observed in these animals. The results suggest that FLX acts on the ovary or hypothalamus-pituitary axis resulting in modifications of the follicular development and ovulation.
Assuntos
Fluoxetina/toxicidade , Oócitos/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Ovário/efeitos dos fármacos , Ovulação/efeitos dos fármacos , Inibidores Seletivos de Recaptação de Serotonina/toxicidade , Serotonina/metabolismo , Fatores Etários , Animais , Feminino , Hormônios Esteroides Gonadais/sangue , Hibridização Genética , Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Sistema Hipotálamo-Hipofisário/metabolismo , Oócitos/metabolismo , Oócitos/patologia , Folículo Ovariano/metabolismo , Folículo Ovariano/patologia , Ovário/metabolismo , Ovário/patologia , Ovário/fisiopatologia , Ratos Long-Evans , Ratos Wistar , Receptor 5-HT1D de Serotonina/efeitos dos fármacos , Receptor 5-HT1D de Serotonina/genética , Receptor 5-HT1D de Serotonina/metabolismo , Maturidade Sexual , Fatores de TempoRESUMO
Serotonin [5-hydroxytryptamine (5-HT)] is involved in both motor and sensory functions in hollow organs, especially in the gastrointestinal tract. However, the involvement of 5-HT in visceral sensation of the urinary bladder remains unknown. Because distention-induced ATP release from the urothelium plays an essential role in visceral sensation of the urinary bladder, we investigated the regulation of urothelial ATP release by the 5-HT signaling system. RT-PCR and immunohistochemical analyses of the urothelium revealed specific expression of 5-HT1D and 5-HT4 receptors. The addition of 5-HT did not affect urothelial ATP release without bladder distention, but it significantly reduced distention-induced ATP release by physiological pressure during urine storage (5 cmH2O). The inhibitory effect of 5-HT on distention-elicited ATP release was blocked by preincubation with the 5-HT1B/1D antagonist GR-127935 but not by the 5-HT4 antagonist SB-204070. mRNA encoding tryptophan hydroxylase 1 was detected in the urinary bladder by nested RT-PCR amplification, and l-tryptophan or the selective serotonin reuptake inhibitor citalopram also inhibited ATP release, indicating that 5-HT is endogenously synthesized and released in the urinary bladder. The addition of GR-127935 significantly enhanced the distention-elicited ATP release 40 min after distention, whereas SB-204070 reduced the amount of ATP release 20 min after distention. These data suggest that 5-HT4 facilitates the distention-induced ATP release at an earlier stage, whereas 5-HT1D inhibits ATP release at a later stage. The net inhibitory effect of 5-HT indicates that the action of 5-HT on the urothelium is mediated predominantly by 5-HT1D.
Assuntos
Trifosfato de Adenosina/metabolismo , Receptor 5-HT1D de Serotonina/metabolismo , Receptores 5-HT4 de Serotonina/metabolismo , Serotonina/farmacologia , Bexiga Urinária/metabolismo , Urotélio/metabolismo , Animais , Citalopram/farmacologia , Dioxanos/farmacologia , Masculino , Camundongos , Oxidiazóis/farmacologia , Piperazinas/farmacologia , Piperidinas/farmacologia , Antagonistas da Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Bexiga Urinária/efeitos dos fármacos , Urotélio/efeitos dos fármacosRESUMO
Overexpression of 5-hydroxytryptamine (5-HT) in human cancer contributes to tumor metastasis, but the role of 5-HT receptor family in cancer has not been thoroughly explored. Here, we report overexpression of 5-HT(1D) receptor (5-HT(1D)R) was associated with Wnt signaling pathway and advanced tumor stage. The underlying mechanism of 5-HT(1D)R-promoted tumor invasion was through its activation on the Axin1/ß-catenin/MMP-7 pathway. In an orthotopic colorectal cancer mouse model, we demonstrated that a 5-HT(1D)R antagonist (GR127935) effectively inhibited tumor metastasis through targeting Axin1. Furthermore, in intestinal epithelium cells, we observed that 5-HT(1D)R played an important role in cell invasion via Axin1/ß-catenin/MMP-7 pathway. Together, our findings reveal an essential role of the physiologic level of 5-HT(1D)R in pulmonary metastasis of colorectal cancer.
Assuntos
Proteína Axina/metabolismo , Neoplasias Colorretais/metabolismo , Metaloproteinase 7 da Matriz/metabolismo , Receptor 5-HT1D de Serotonina/metabolismo , beta Catenina/metabolismo , Animais , Proteína Axina/genética , Western Blotting , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Células HT29 , Humanos , Imuno-Histoquímica , Masculino , Metaloproteinase 7 da Matriz/genética , Camundongos Nus , Pessoa de Meia-Idade , Metástase Neoplásica , Oxidiazóis/farmacologia , Piperazinas/farmacologia , Receptor 5-HT1D de Serotonina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Antagonistas da Serotonina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/genéticaRESUMO
Pancreatic ductal adenocarcinoma is characterized by extensive local tumor invasion, metastasis and early systemic dissemination. The vast majority of pancreatic cancer (PaCa) patients already have metastatic complications at the time of diagnosis, and the death rate of this lethal type of cancer has increased over the past decades. Thus, efforts at identifying novel molecularly targeted therapies are priorities. Recent studies have suggested that serotonin (5-HT) contributes to the tumor growth in a variety of cancers including prostate, colon, bladder and liver cancer. However, there is lack of evidence about the impact of 5-HT receptors on promoting pancreatic cancer. Having considered the role of 5-HT-1 receptors, especially 5-HT1B and 5-HT1D subtypes in different types of malignancies, the aim of this study was to investigate the role of 5-HT1B and 5-HT1D receptors in PaCa growth and progression and analyze their potential as cytotoxic targets. We found that knockdown of 5-HT1B and 5-HT1D receptors expression, using specific small interfering RNA (siRNA), induced significant inhibition of proliferation and clonogenicity of PaCa cells. Also, it significantly suppressed PaCa cells invasion and reduced the activity of uPAR/MMP-2 signaling and Integrin/Src/Fak-mediated signaling, as integral tumor cell pathways associated with invasion, migration, adhesion, and proliferation. Moreover, targeting 5-HT1B and 5-HT1D receptors down-regulates zinc finger ZEB1 and Snail proteins, the hallmarks transcription factors regulating epithelial-mesenchymal transition (EMT), concomitantly with up-regulating of claudin-1 and E-Cadherin. In conclusion, our data suggests that 5-HT1B- and 5-HT1D- mediated signaling play an important role in the regulation of the proliferative and invasive phenotype of PaCa. It also highlights the therapeutic potential of targeting of 5-HT1B/1D receptors in the treatment of PaCa, and opens a new avenue for biomarkers identification, and valuable new therapeutic targets for managing pancreatic cancer.
Assuntos
Regulação Neoplásica da Expressão Gênica , Pâncreas/metabolismo , RNA Interferente Pequeno/genética , Receptor 5-HT1B de Serotonina/genética , Receptor 5-HT1D de Serotonina/genética , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Claudina-1/genética , Claudina-1/metabolismo , Transição Epitelial-Mesenquimal/genética , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Integrinas/genética , Integrinas/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Pâncreas/patologia , RNA Interferente Pequeno/metabolismo , Receptor 5-HT1B de Serotonina/metabolismo , Receptor 5-HT1D de Serotonina/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Transdução de Sinais , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ensaio Tumoral de Célula-Tronco , Homeobox 1 de Ligação a E-box em Dedo de Zinco , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Pancreatic ductal adenocarcinoma is characterized by extensive local tumor invasion, metastasis and early systemic dissemination. The vast majority of pancreatic cancer (PaCa) patients already have metastatic complications at the time of diagnosis, and the death rate of this lethal type of cancer has increased over the past decades. Thus, efforts at identifying novel molecularly targeted therapies are priorities. Recent studies have suggested that serotonin (5-HT) contributes to the tumor growth in a variety of cancers including prostate, colon, bladder and liver cancer. However, there is lack of evidence about the impact of 5-HT receptors on promoting pancreatic cancer. Having considered the role of 5-HT-1 receptors, especially 5-HT1B and 5-HT1D subtypes in different types of malignancies, the aim of this study was to investigate the role of 5-HT1B and 5-HT1D receptors in PaCa growth and progression and analyze their potential as cytotoxic targets. We found that knockdown of 5-HT1B and 5-HT1D receptors expression, using specific small interfering RNA (siRNA), induced significant inhibition of proliferation and clonogenicity of PaCa cells. Also, it significantly suppressed PaCa cells invasion and reduced the activity of uPAR/MMP-2 signaling and Integrin/Src/Fak-mediated signaling, as integral tumor cell pathways associated with invasion, migration, adhesion, and proliferation. Moreover, targeting 5-HT1B and 5-HT1D receptors down-regulates zinc finger ZEB1 and Snail proteins, the hallmarks transcription factors regulating epithelial-mesenchymal transition (EMT), concomitantly with up-regulating of claudin-1 and E-Cadherin. In conclusion, our data suggests that 5-HT1B- and 5-HT1D-mediated signaling play an important role in the regulation of the proliferative and invasive phenotype of PaCa. It also highlights the therapeutic potential of targeting of 5-HT1B/1D receptors in the treatment of PaCa, and opens a new avenue for biomarkers identification, and valuable new therapeutic targets for managing pancreatic cancer.
Assuntos
Neoplasias do Colo/secundário , Pâncreas/patologia , Neoplasias Pancreáticas/patologia , Interferência de RNA , Receptor 5-HT1B de Serotonina/genética , Receptor 5-HT1D de Serotonina/genética , Linhagem Celular Tumoral , Proliferação de Células , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Quinase 1 de Adesão Focal/metabolismo , Humanos , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Pâncreas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Receptor 5-HT1B de Serotonina/metabolismo , Receptor 5-HT1D de Serotonina/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismoRESUMO
Tryptophan 2,3-dioxygenase (TDO) is expressed in endometrium and catabolizes tryptophan, a precursor in the biosynthesis of serotonin. Tryptophan metabolism is an important mechanism for regulation of serotonin levels. Preimplantation mouse embryos are known to express serotonin receptors, specifically the 5-HT1D and 5-HT7 serotonin receptor subtypes. Here we demonstrate that Hoxa10 regulates endometrial TDO expression and improves embryo viability through increased serotonin production. Transfection of pcDNA-Hoxa10 to the murine uterus increased total TDO expression. In vitro, epithelial cell TDO expression was decreased after transfection with Hoxa10. Decreased glandular TDO in response to HOXA10 may augment serotonin production by increasing tryptophan availability. Conversely, stromal TDO expression increased with constitutive Hoxa10 expression. In mice, epithelial serotonin was increased in response to constitutive expression of Hoxa10. Embryo quality was impaired after treatment with Hoxa10 antisense. Blockade of serotonin receptors 1D and 7 also resulted in impaired embryo development, indicating an essential role for Hoxa10 induction of TDO and subsequent serotonin production in embryo development. Transfection of pcDNA-TDO also decreased the number of T cells in the endometrial stroma. We have shown a novel mechanism by which HOXA10 regulates endometrial TDO expression. In the endometrial stroma, HOXA10 increases TDO mRNA, which may increase tryptophan catabolism, allowing for immune tolerance at the time of embryo implantation. In endometrial glands, HOXA10 decreases TDO mRNA leading to increased serotonin that in turn acts to promote normal embryo development.
Assuntos
Embrião de Mamíferos/metabolismo , Proteínas de Homeodomínio/metabolismo , Receptor 5-HT1D de Serotonina/metabolismo , Receptores de Serotonina/metabolismo , Triptofano Oxigenase/metabolismo , Animais , Linhagem Celular , DNA Antissenso/genética , DNA Antissenso/metabolismo , Implantação do Embrião/imunologia , Embrião de Mamíferos/citologia , Embrião de Mamíferos/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Endométrio/imunologia , Endométrio/metabolismo , Feminino , Proteínas Homeobox A10 , Proteínas de Homeodomínio/genética , Humanos , Tolerância Imunológica , Camundongos , Especificidade de Órgãos , RNA Mensageiro/metabolismo , Distribuição Aleatória , Antagonistas do Receptor 5-HT1 de Serotonina/farmacologia , Antagonistas da Serotonina/farmacologia , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Triptofano Oxigenase/genética , Útero/citologia , Útero/metabolismoRESUMO
Although Interferon-alpha (IFN-alpha, CAS 9008-11-1) is a powerful drug in treating several viral infections and certain tumors, a considerable amount of neuropsychiatric side-effects such as depression and anxiety are an unavoidable consequence. Combination with the selective serotonin (5-HT) reuptake inhibitor (SSRI) fluoxetine (CAS 56296-78-7) significantly improved the situation. However, the potential 5-HT(1A) receptor- and 5-HT(1B) receptor-signals involved in the antidepressant effects are still unclear. The effects of 5-HT(1A) receptor- and 5-HT(1B) receptor signals were analyzed by using the mouse forced swimming test (FST), a predictive test of antidepressant-like action. The present results indicated that (1) fluoxetine (administrated intragastrically, 30 mg/kg; not subactive dose: 15 mg/kg) significantly reduced IFN-alpha-induced increase of the immobility time in the forced swimming test; (2) 5-HT(1A) receptor- and 5-HT(1B) receptor ligands alone or in combination had no effects on IFN-alpha-induced increase of the immobility time in the FST; (3) surprisingly, WAY 100635 (5-HT(1A) receptor antagonist, 634908-75-1) and 8-OH-DPAT(5-HT(1A) receptor agonist, CAS 78950-78-4) markedly enhanced the antidepressant effect of fluoxetine at the subactive dose (15 mg/kg, i. g.) on the IFN-alpha-treated mice in the FST. Further investigations showed that fluoxetine combined with WAY 100635 and 8-OH-DPAT failed to produce antidepressant effects in the FST. (4) Co-application of CGS 12066A (5-HT(1B) receptor agonist, CAS 109028-09-3) or GR 127935 (5-HT(1B/1D) receptor antagonist, CAS 148642-42-6) with fluoxetine had no synergistic effects on the IFN-alpha-induced increase of immobility time in FST. (5) Interestingly, co-administration of GR 127935, WAY 100635 and fluoxetine significantly reduced the IFN-alpha-induced increase in immobility time of FST, being more effective than co-administration of WAY 100635 and fluoxetine. All results suggest that (1) compared to the 5-HT(1B) receptor, the 5-HT(1A) receptor signal plays the dominant role in improving the anti-immobility effect of fluoxetine in the IFN-alpha-induced depression; (2) combination of the 5-HT(1A) antagonist with subactive fluoxetine can be helpful in IFN-alpha-induced depression treatment.
Assuntos
Depressão/induzido quimicamente , Depressão/psicologia , Interferon-alfa/farmacologia , Receptor 5-HT1A de Serotonina/efeitos dos fármacos , Receptor 5-HT1B de Serotonina/efeitos dos fármacos , Receptor 5-HT1D de Serotonina/efeitos dos fármacos , Natação/psicologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Buspirona/farmacologia , Fluoxetina/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos ICR , Atividade Motora/efeitos dos fármacos , Oxidiazóis/farmacologia , Piperazinas/farmacologia , Piridinas/farmacologia , Quinoxalinas/farmacologia , Agonistas do Receptor 5-HT1 de Serotonina , Antagonistas do Receptor 5-HT1 de Serotonina , Antagonistas da Serotonina/farmacologia , Agonistas do Receptor de Serotonina/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
The relationship between serotonin (5-HT) and major depressive disorder (MDD) has been extensively studied but certain aspects are still ambiguous. Given the evidence that 5-HT neurotransmission is reduced in depressed subjects, it is possible that one or more of the 5-HT regulators may be altered in the dorsal raphe nucleus (DR) of depressed subjects. Candidates that regulate 5-HT synthesis and neuronal activity of 5-HT neurons include intrinsic regulators such as tryptophan hydroxylase 2, 5-HT autoreceptors, 5-HT transporter and transcription factors, as well as afferent regulators such as estrogen and brain-derived neurotrophic factor. The present study was designed to quantify mRNA concentrations of the above 5-HT regulators in an isolated population of 5-HT-containing DR neurons of MDD subjects and gender-matched psychiatrically normal control subjects. We found that mRNA concentrations of the 5-HT1D receptor and the transcription factors, NUDR and REST, were significantly increased in DR-captured neurons of female MDD subjects compared to female control subjects. No significant differences were found for the transcripts in male MDD subjects compared to male controls. This study reveals sex-specific alterations in gene expression of the pre-synaptic 5-HT1D autoreceptors and 5-HT-related transcription factors, NUDR and REST, in DR neurons of women with MDD.
Assuntos
Transtorno Depressivo Maior/patologia , Regulação da Expressão Gênica/fisiologia , Neurônios/metabolismo , Núcleos da Rafe/patologia , Serotonina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ligação a DNA , Feminino , Humanos , Lasers , Masculino , Microdissecção/métodos , Pessoa de Meia-Idade , Proteínas Nucleares/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Receptor 5-HT1D de Serotonina/genética , Receptor 5-HT1D de Serotonina/metabolismo , Receptor trkB/genética , Receptor trkB/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Serotonina/genética , Fatores Sexuais , Fatores de Transcrição , Triptofano Hidroxilase/metabolismoRESUMO
We studied the role of several serotonin (5-HT) and histamine receptors in the regulation of human umbilical artery (HUA) contractility. Among the 5-HT agonists used, only the 5-HT( 2A) and 5HT(1B/D) agonists contracts HUA. The 5-HT-induced contractions were fully inhibited by ketanserin (5-HT(2A) antagonist). The 5-HT( 7)-activation also relaxes and increases intracellular cyclic adenosine monophosphate (cAMP). Among the histamine receptor agonists, only betahistine (H(1) agonist) induced significant contractile effect. Histamine-induced contraction was partially relaxed by pyrilamine (H(1) antagonist). Betahistine-induced contraction was partially blocked by dimaprit (H( 2) agonist) and by the H(3) agonist when a low concentration of forskolin is present. Both, H(2) and H(3) agonists increased the cAMP intracellular levels in HUA smooth muscle. These findings show that in HUA, 5-HT(2A)- and 5-HT(1B/1D)-activation lead to vasoconstriction and 5-HT(7)-activation induces vasorelaxation. Concerning histamine receptors, H(1)-activation induces contraction and H(2)- and H(3)-activation lead to vasorelaxation.
Assuntos
Receptores Histamínicos/metabolismo , Receptores de Serotonina/metabolismo , Artérias Umbilicais/metabolismo , Vasoconstrição , Vasodilatação/efeitos dos fármacos , AMP Cíclico/metabolismo , Antagonistas de Dopamina/farmacologia , Feminino , Agonistas dos Receptores Histamínicos/farmacologia , Antagonistas dos Receptores Histamínicos/farmacologia , Humanos , Gravidez , RNA Mensageiro/análise , Receptor 5-HT1B de Serotonina/metabolismo , Receptor 5-HT1D de Serotonina/metabolismo , Receptor 5-HT2A de Serotonina/metabolismo , Receptores Histamínicos/efeitos dos fármacos , Receptores Histamínicos/genética , Receptores Histamínicos H1/metabolismo , Receptores Histamínicos H2/metabolismo , Receptores Histamínicos H3/metabolismo , Receptores de Serotonina/efeitos dos fármacos , Receptores de Serotonina/genética , Agonistas do Receptor de Serotonina/farmacologia , Artérias Umbilicais/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasoconstritores/farmacologia , Vasodilatadores/farmacologiaRESUMO
The diagnosis of migraine can sometimes be difficult because some patients do not fulfill the International Headache Society's criteria for migraine. Hence, an accurate and reliable diagnostic marker for migraine is required. In this study, lymphocytes were used to establish Epstein-Barr virus (EBV)-immortalized lymphoblast cell lines, which were then analyzed using a differential cRNA microarray analysis. The gene expression results were validated using real-time polymerase chain reaction. Gene expression profiling identified 15 genes as being differentially expressed in lymphoblasts originating from patients diagnosed as having migraine with aura (MA). One-fifth of these genes were associated with cytoskeletal proteins. The expressions of seven genes increased significantly by more than 50% of the value in the controls, while the expressions of eight genes decreased significantly by more than 50% of the value in the controls. We also verified that the expression of alpha-fodrin, which was 1 of the 15 genes that were differentially expressed in lymphoblasts originating from patients with MA, increased after cortical spreading depression in an animal model. Thus, alpha-fodrin might play an important role in the pathophysiology of migraine, possibly serving as a migraine biomarker.
Assuntos
Enxaqueca com Aura/diagnóstico , Enxaqueca com Aura/genética , Adulto , Animais , Biomarcadores , Encéfalo/fisiologia , Proteínas de Transporte/genética , Linhagem Celular Transformada , Transformação Celular Viral , Depressores do Sistema Nervoso Central/farmacologia , Depressão Alastrante da Atividade Elétrica Cortical/efeitos dos fármacos , Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Feminino , Perfilação da Expressão Gênica , Hipocalcina/metabolismo , Humanos , Linfócitos/fisiologia , Masculino , Camundongos , Proteínas dos Microfilamentos/genética , Reação em Cadeia da Polimerase , Cloreto de Potássio/farmacologia , RNA Mensageiro/metabolismo , Receptor 5-HT1D de Serotonina/metabolismoRESUMO
In this study, we investigated adhesion-induced changes in cellular responses to serotonin 5-HT(1D) and purinergic P2Y receptor stimulation. We demonstrated that detachment of LTK-8 cells increased 5-HT(1D) receptor-mediated intracellular Ca(2+) and extracellular signal regulated kinase (ERK) phosphorylation responses without affecting the adenylate cyclase response. Additionally, detachment enabled 5-HT(1D) receptor stimulation to inhibit P2Y receptor-induced [Ca(2+)](i) mobilization. Such a cross talk between the two receptor systems was not observed in attached cells. P2Y receptor-induced Ca(2+) response was insensitive to adhesion state of the cells, while ERK phosphorylation response was enhanced upon detachment. Integrity of the actin cytoskeleton did not appear to play a role in adhesion sensitivity of 5-HT(1D)-mediated responses, as treatment of attached cells with cytochalasin D did not mimic detachment-induced effects. Effects of detachment were reversed immediately after re-attachment of the suspended cells on poly-l-lysine coated cover slips, suggesting that the involvement of integrins or focal adhesion complexes is unlikely. Taken collectively, our results demonstrate that not only cellular responses induced by different G protein-coupled receptors, but also different responses induced by a particular G protein-coupled receptor, can be affected differentially by the adhesion status of cells. This suggests an important role for cell adhesion in controlling the coupling of a single G protein-coupled receptor to different intracellular responses.