Orexins are hypothalamic peptides that regulate food intake, wakefulness, the reward system and energy metabolism. Recent studies have demonstrated the ability of orexins to promote a robust apoptosis and subsequent inhibition of cell growth in various types of cancer cells. The present study was conducted to investigate the effects of orexin A on the survival of human gastric cancer cells, SGC‑7901, and the possible mechanisms. SGC‑7901 cells were exposed to various concentrations of orexin A in vitro in the presence or absence of the orexin receptor 1 (OX1R) antagonist (SB334867), extracellular signal‑regulated kinases 1 and 2 (ERK1/2) antagonist (U0126) or a combination of the two antagonists. The amount of cell proliferation, viability and apoptosis, caspase‑8 and caspases‑9 activities, OX1R protein expression and ERK1/2 protein levels were determined. The expression of OX1R in SGC‑7901 cells was observed. Orexin A (10-10 to 10-6 M) stimulated SGC‑7901 cell proliferation and viability, reduced the pro‑apoptotic activity of caspase‑9 and protected the cells from apoptosis in a dose‑dependent manner. Additionally, ERK1/2 phosphorylation was stimulated by orexin A (10-10 to 10-6 M). However, the OX1R antagonist SB334867 (10-6 M), ERK1/2 antagonist U0126 (30 µM) or the combination of antagonists blocked the effects of orexin A to a certain extent. These results suggest that stimulation of OX1R induces the growth of SGC‑7901 gastric cancer cells through activation of ERK1/2 signaling pathway. These findings add a new dimension to the biological activities of orexin, which may have important implications in health and disease, in particular gastric cancer.
Our aim is to investigate the role of the AKT/PKB (protein kinase B) signaling pathway acting via orexin receptor 1 (OX1R) and the effects of orexin A (OXA) on cell proliferation in the insulin-secreting beta-cell line (INS-1 cells). Rat INS-1 cells were exposed to different concentrations of OXA in vitro and treated with OX1R antagonist (SB334867), PI3K antagonist (wortmannin), AKT antagonist (PF-04691502), or negative control. INS-1 amount of cell proliferation, viability and apoptosis, insulin secretion, OX1R protein expression, caspase-3 activity, and AKT protein levels were determined. We report that OXA (10−10 to 10−6 M) stimulates INS-1 cell proliferation and viability, reduces the proapoptotic activity of caspase-3 to protect against apoptotic cell death, and increases insulin secretion. Additionally, AKT phosphorylation was stimulated by OXA (10−10 to 10−6 M). However, the OX1R antagonist SB334867 (10−6 M), the PI3K antagonist wortmannin (10−8 M), the AKT antagonist PF-04691502 (10−6 M), or the combination of both abolished the effects of OXA to a certain extent. These results suggest that the upregulation of OXA-OX1R mediated by AKT activation may inhibit cell apoptosis and promote cell proliferation in INS-1 cells. This finding provides functional evidence of the biological actions of OXA in rat insulinoma cells.
These results demonstrate that ORA activation of OX(1)R up-regulates 3β-HSD expression and testosterone production via the ERK1/2 and p38 MAPKs signaling pathways in primary rat Leydig cells.
Orexin A and B are multifunctional neuropeptides that are involved in the regulation of food intake, energy metabolism, glucose regulation and wakefulness. They signal through two G-protein‑coupled receptors (GPCR): orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Previous studies have shown that orexins interact with PI3K/AKT signaling pathways through OX1R-coupling in other cell types, but are seldom involved in hepatocytes. In the present study, reverse transcription (RT)-PCR and western blot analysis revealed that OX1R mRNA expression and activation in rat hepatocytes in vitro were upregulated by exogenous orexin A (10(-10) to 10(-6) M) in a dose-dependent manner. The result showed that orexin A affects increasing cell proliferation and protects cells from apoptosis. Additionally, inhibition studies showed that orexin A induced forkhead box O1 (FoxO1) and mammalian target of rapamycin 1 (mTORC1) phosphorylation, while OX1R antagonist (SB334867, 10(-6) M), AKT antagonist (PF-04691502, 10(-6) M), Foxo1 inhibitor (AS1842856, 10(-6) M) or mTORC1 inhibitor (everolimus, 10(-5) M) blocked these effects of orexin A. The results of the present study showed a possible effect of orexin A on cell apoptosis in regulating Foxo1 and mTORC1 through the OX1R/PI3K/AKT signaling pathway in rat hepatocytes.
Abstract. Orexin-A is a regulatory peptide involved in the regulation of food intake, sleep-wakefulness, and it has various endocrine and metabolic functions. It orchestrates diverse central and peripheral processes through the stimulation of two G-protein coupled receptors, orexin receptor type 1 (OX 1 receptor) and orexin receptor type 2 (OX 2 receptor). In this study, human adrenocortical cells (NCI-H295R cells) were incubated with various concentrations of orexin-A (10 -10 to 10 -6 M) in vitro, and the mRNA and protein expression of OX 1 receptor was determined in the cells. In addition, NCI-H295R cells treated with 10 -6 M orexin-A were then treated with or without OX 1 receptor specific antagonist (SB334867), AKT antagonist (PF-04691502), or a combination of both. Subsequently, cell proliferation, the cortisol content in the medium and the mRNA and protein expression expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) were analyzed. The activity of the AKT signaling pathway was also determined in the NCI-H295R cells. We observed that the increase in the mRNA and protein expression of OX 1 receptor was orexin-A concentration-dependent, with 10 -6 M orexin-A exerting the most potent effect. Orexin-A enhanced cell proliferation and cortisol production, and increased the mRNA and protein expression of 3β-HSD in the NCI-H295R cells; however, these effects were partly blocked by the OX 1 receptor antagonist, the AKT antagonist and the combination of both. Furthermore, orexin-A significantly increased the phosphorylation of AKT, with the levels of total AKT protein remaining unaltered. This effect was blocked in the presence of PF-04691502 (10 -6 M), SB334867 (10 -6 M) and the combination of both. On the whole, our data demonstrate that the effects of orexin-A on the survival and function of human adrenocortical cells are mediated through the AKT signaling pathway.
Numerous studies have demonstrated the ability of orexin-A to regulate adrenocortical cells through the mitogen-activated protein kinase signaling pathway. In the present study, human H295R adrenocortical cells were exposed to orexin‑A (10‑10-10‑6 M), with orexin receptor type 1 (OX1 receptor) antagonist SB334867 or AKT antagonist PF‑04691502. It was found that orexin‑A stimulated H295R cell proliferation, reduced the pro‑apoptotic activity of caspase‑3 to protect against apoptotic cell death and increased cortisol secretion. Furthermore, phospho‑AKT protein was increased by orexin‑A. SB334867 (10‑6 M) and PF‑04691502 (10‑6 M) abolished the effects of orexin‑A (10‑6 M). These results suggested that the orexin‑A/OX1 receptor axis has a significant pro-survival function in adrenal cells, which is mediated by AKT activation. Further studies investigating the effects of orexin-A-upregulation may further elucidate the diverse biological effects of orexin-A in adrenal cells.
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