Effects of erythropoietin and methylprednisolone on AQP4 expression in astrocytes.

Methylprednisolone sodium succinate (MPSS) has been suggested as a treatment for spinal cord injury (SCI), but its use has been limited due to its adverse effects. Erythropoietin (EPO) has been suggested as a promising candidate for limiting SCI in mammals. The aim of the present study was to investigate the effects of EPO in combination with MPSS on astrocytes following ischemic injury in vitro. Astrocytes were isolated from the cerebral cortex of postnatal day 3 Sprague­Dawley rats and cultured in vitro. Astrocyte ischemic injury was induced by oxygen and glucose deprivation for 4 h, and reperfusion was simulated by subsequent culture under normoxic conditions. The effects of EPO and MPSS on the expression of aquaporin­4 (AQP4) were investigated. Ischemic astrocytes were treated with EPO (10 U/ml), MPSS (10 µg/ml), or EPO (10 U/ml) in combination with MPSS (10 µg/ml) during reperfusion. The cell viability of astrocytes was assessed using an MTT assay. The mRNA and protein expression levels of AQP4 were determined using reverse transcription­quantitative polymerase chain reaction and western blot analysis, respectively. The role of the protein kinase C (PKC) signaling pathway in the molecular mechanisms underlying the effects of EPO and MPSS was also investigated. The present results demonstrated that following treatment with EPO and MPSS, the mRNA expression levels of AQP4 were upregulated and cell viability was enhanced. EPO and MPSS effectively inhibited the oxygen and glucose deprivation­mediated downregulation of AQP4 following reperfusion. In addition, the combined treatment with EPO and MPSS exhibited higher AQP4 expression levels and cell viability compared with each treatment alone. Finally, the effects of EPO and MPSS on AQP4 expression were partially reversed by pretreatment with the PKC inhibitor Ro 31­8220. The present study indicated that EPO and MPSS had a synergistic effect on AQP4 expression following reperfusion, and suggest that they may be combined in the treatment of SCI.

SIRT1 inhibition by melatonin exerts antitumor activity in human osteosarcoma cells.

Melatonin, the main secretory product of the pineal gland, has potent antitumor activity against various types of cancer. However, the molecular mechanisms underlying the effects of melatonin remain largely unknown. SIRT1, a conserved nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase, has been implicated in modulating transcriptional silencing and cell survival and plays a key role in carcinogenesis through the deacetylation of important regulatory proteins. In this study, we assessed the antitumor activity of melatonin against human osteosarcoma cells (9607 cell line) and explored the role of SIRT1 in the activity of melatonin. Melatonin treatment resulted in strong antitumor activity, as evidenced not only by reductions in tumor cell vitality, adhesion ability, migration ability and glutathione (GSH) levels but also by increase in the apoptotic index and reactive oxygen species. Additionally, melatonin treatment down-regulated SIRT1 and up-regulated acetylated-p53. Sirtinol (a known SIRT1 inhibitor) and SIRT1 siRNA further enhanced the antitumor activity of melatonin, while SRT1720 (a known SIRT1 activator) attenuated the antitumor activity of melatonin. In summary, melatonin is a potent inhibitor of osteosarcoma cell growth that targets SIRT1 signaling, and the inhibition of SIRT1 signaling is a novel mechanism of action for melatonin during therapeutic intervention in osteosarcoma.