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Small interfering RNA-directed knockdown of S100A4 decreases proliferation and invasiveness of osteosarcoma cells

Osteosarcoma is the most common osteogenic malignant tumor characterized by a high level of malignancy, relapse, metastasis and poor prognosis. S100A4 has been implicated in the proliferation, cell cycle progression, and metastasis of many malignant tumors, although the roles of S100A4 in osteosarcoma have not been documented. This study showed that the expression of S100A4 was found in two osteosarcoma cell lines MG-63 and U-2OS, and in 70.7% of osteosarcoma clinical tissues, and the expression was correlated with the expression of CD44V6. In addition, transfection with S100A4 siRNA significantly reduced the proliferation and the invasiveness of MG-63 cells. Furthermore, S100A4 siRNA down-regulated the expression of CD44 and MMP2, suggesting that S100A4 may promote the proliferation, invasion and metastasis of osteosarcoma cells by regulating the expression of other proteins that are crucial in modulating cell-ECM adhesion and facilitating ECM degradation. Therefore, siRNA-directed knockdown of S100A4 may represent a viable clinical therapy for osteosarcoma.

Temporary loss of perivascular aquaporin-4 in white matter after the spinal cord ischemic injury of rats.

The study was performed to investigate whether the ischemic gray matter and white matter show distinct patterns of aquaporin-4 (AQP4) expression in the reperfusion phase using an in-vivo transient spinal cord ischemia model in rats. We investigated to the time course of AQP4 expression at the blood–spinal cord interface by the quantitative immunogold and western blots methods. The results showed that disruption of AQP4 anchoring at the perivascular membrane did not lead to a net loss of protein. This is the first systematic and extensive study fully showing AQP4 expression dynamics after transient spinal cord ischemia and the findings are of major clinical and experimental interest.

Mdivi-1 Inhibits Astrocyte Activation and Astroglial Scar Formation and Enhances Axonal Regeneration after Spinal Cord Injury in Rats

After spinal cord injury (SCI), astrocytes become hypertrophic, and proliferative, forming a dense network of astroglial processes at the site of the lesion. This constitutes a physical and biochemical barrier to axonal regeneration. Mitochondrial fission regulates cell cycle progression; inhibiting the cell cycle of astrocytes can reduce expression levels of axon growth-inhibitory molecules as well as astroglial scar formation after SCI. We therefore investigated how an inhibitor of mitochondrial fission, Mdivi-1, would affect astrocyte proliferation, astroglial scar formation, and axonal regeneration following SCI in rats. Western blot and immunofluorescent double-labeling showed that Mdivi-1 markedly reduced the expression of the astrocyte marker glial fibrillary acidic protein (GFAP), and a cell proliferation marker, proliferating cell nuclear antigen, in astrocytes 3 days after SCI. Moreover, Mdivi-1 decreased the expression of GFAP and neurocan, a chondroitin sulfate proteoglycan. Notably, immunofluorescent labeling and Nissl staining showed that Mdivi-1 elevated the production of growth-associated protein-43 and increased neuronal survival at 4 weeks after SCI. Finally, hematoxylin-eosin staining, and behavioral evaluation of motor function indicated that Mdivi-1 also reduced cavity formation and improved motor function 4 weeks after SCI. Our results confirm that Mdivi-1 promotes motor function after SCI, and indicate that inhibiting mitochondrial fission using Mdivi-1 can inhibit astrocyte activation and astroglial scar formation and contribute to axonal regeneration after SCI in rats.

The effect of aminoguanidine on compression spinal cord injury in rats

The current study was performed to investigate the effect of aminoguanidine (AG) on spinal cord injury (SCI) in rat. AG (75, 150 and 300mg/kg, i.p. respectively ) was administered to rats immediately following SCI. It was found that AG (150mg/kg) significantly reduced spinal cord water content and improved motor function, however, AG at the doses of 75 and 300mg/kg had no effect. Compared to SCI group without treatment, AG at the dosage of 150mg/kg induced a reduction in the permeability of blood-spinal cord barrier (BSCB) after injury 48h (from 59.8+/-5.5microl/g to 39.8+/-3.8microl/g), a 38% decrease of Malondialdehyde (MDA) values and a 1-fold increase of the Glutathione (GSH) levels at 12h after SCI. And the expression of inducible nitric oxide synthase (iNOS) protein reached a peak at 24h after injury, which was significantly attenuated by treatment with AG (150mg/kg). In addition, the expression of AQP4 protein was down-regulated by the treatment of AG (150mg/kg) at 24h after SCI, and the changes still lasted at 48h after injury. Our results indicated that AG could induce spinal cord edema clearance and improve motor function, which could be correlated with antioxidative property, the down-regulation of iNOS and AQP4 protein expression after SCI.

Acetyl-l-carnitineamelioratesmitochondrial damage and apoptosis following spinal cord injury in rats

Acetyl-l-carnitine (ALC) facilitates the entry and exit of fatty acids from mitochondria and plays an essential role in energy metabolism. Although ALC is known to exert neuroprotective effects in multiple neurological diseases, its effects on spinal cord injury (SCI)-induced mitochondrial impairments and apoptosis remain unclear. In this study, we aimed to evaluate the putative effects of ALC on mitochondrial dysfunction and apoptosis induced by SCI in a rodent model. Our results indicate that SCI elicits dynamic alternations in the expression of mitochondria-related proteins. Transmission electron microscopy analysis showed that ALC administration abrogated key ultrastructural abnormalities in mitochondria at 24h after SCI by maintaining mitochondrial length, reducing the number of damaged mitochondria, and reversing mitochondrial score (P<0.05 compared with SCI group). In addition, ALC administration maintained the mitochondrial membrane potential and mitochondrial Na(+)-K(+)-ATPase activity following SCI (P<0.05 compared with SCI group). ALC administration reversed the downregulation of mitofusin 1 (Mfn1), Mfn2, Bcl-2, and the upregulation of dynamin-related protein 1 (Drp1), mitochondrial fission 1 (Fis1), Bcl-2-associated X protein (Bax) and cytosol cytochrome c (cyto-CytC) induced by SCI (P<0.05 compared with SCI group). Finally ALC administration greatly reduced the percentage of apoptotic cells compared with the SCI group (P<0.01). In conclusion, our findings demonstrated that ALC ameliorated SCI-induced mitochondrial structural alternations, mitochondrial dysfunction, and apoptosis.

Netrin-1 Improves Functional Recovery through Autophagy Regulation by Activating the AMPK/mTOR Signaling Pathway in Rats with Spinal Cord Injury

Autophagy is an process for the degradation of cytoplasmic aggregated proteins and damaged organelles and plays an important role in the development of SCI. In this study, we investigated the therapeutic effect of Netrin-1 and its potential mechanism for autophagy regulation after SCI. A rat model of SCI was established and used for analysis. Results showed that administration of Netrin-1 not only significantly enhanced the phosphorylation of AMP-activated protein kinase (AMPK) but also reduced the phosphorylation of mammalian target of rapamycin (mTOR) and P70S6K. In addition, the expression of Beclin-1 and the ratio of the light-chain 3B-II (LC3B-II)/LC3B-I in the injured spinal cord significantly increased in Netrin-1 group than those in SCI group. Moreover, the ratio of apoptotic neurons in the anterior horn of the spinal cord and the cavity area of spinal cord significantly decreased in Netrin-1 group compared with those in SCI group. In addition, Netrin-1 not only preserved motor neurons but also significantly improved motor fuction of injured rats. These results suggest that Netrin-1 improved functional recovery through autophagy stimulation by activating the AMPK/mTOR signaling pathway in rats with SCI. Thus, Netrin-1 treatment could be a novel therapeutic strategy for SCI.

The Role of Netrin-1 in Improving Functional Recovery through Autophagy Stimulation Following Spinal Cord Injury in Rats

Our previous findings indicated that treatment with Netrin-1 could improve functional recovery through the stimulation of autophagy, by activating the AMP-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway in rats following spinal cord injury (SCI). However, the underlying mechanisms were not elucidated. The purpose of this study was to investigate the underlying mechanisms by which Netrin-1 promotes autophagy and improves functional recovery after SCI. Following controlled SCI in Sprague-Dawley rats, we observed that the autophagic flux in neurons was impaired, as reflected by the accumulation of light chain 3-II (LC3-II)-positive and LC3-positive autophagosomes (APs), accompanied by the accumulation of the autophagic substrate, Sequestosome 1 (SQSTM1; also known as p62). Our results showed that treatment with Netrin-1 increases the levels of the lysosomal protease cathepsin D (CTSD) and lysosomal-associated membrane protein 1 (LAMP1), through the regulation of the nuclear localization of Transcription factor EB (TFEB) via the AMPK/mTOR signaling pathway. In addition, this enhancement of lysosomal biogenesis correlated strongly with the restoration of autophagic flux, inhibition of neural apoptosis and improved functional recovery. Suppression of lysosomal biogenesis via the inhibition of the nuclear translocation of TFEB by Compound C abolished this restoration of autophagic flux and the functional recovery effects of Netrin-1 following SCI. Taken together, these results indicate that Netrin-1 enhances lysosomal biogenesis by regulating the nuclear translocation of TFEB via the AMPK/mTOR signaling pathway. Furthermore, the enhancement of lysosomal biogenesis by Netrin-1 following SCI promotes autophagic flux and improves functional recovery in rats. Thus, the regulation of lysosomal biogenesis by modulating the nuclear localization of TFEB might be a novel approach for the treatment of SCI.

Therapeutic potential of spinal GLP-1 receptor signaling

HighlightsWe summarized the expression of GLP‐1R and the innervation of PPG neurons in the spinal cord.Stimulation of GLP‐1R within the spinal cord promotes neuroprotection in specific neurodegenerative and traumatic spinal cord disorders.Activating GLP‐1R in spinal microglia cells suppresses nociceptive sensitization. &NA; GLP‐1 signaling pathway has been well studied for its role in regulating glucose homeostasis, as well as its beneficial effects in energy and nutrient metabolism. A number of drugs based on GLP‐1 have been used to treat type 2 diabetes mellitus. GLP‐1R is expressed in multiple organs and numerous experimental studies have demonstrated that GLP‐1 signaling pathway exhibits pro‐survival functions in various disorders. In the central nervous system, stimulation of GLP‐1R produces neuroprotective effects in specific neurodegenerative disorders, such as Alzheimers disease and Parkinsons disease. The preproglucagon neurons located in the brainstem can also produce GLP‐1. GLP‐1 analogs have a long‐acting effect and are able to pass the blood‐brain barrier, which probably extends the therapeutic efficacy of GLP‐1R activation. Neurodegenerative or traumatic conditions can damage the spinal cord and result in motor and sensory dysfunction. Evidence supports that GLP‐1R activation in the spinal cord possesses beneficial effects and significant therapeutic potential. Herein, we review studies that have focused on GLP‐1 and the spinal cord, and summarize the expression of GLP‐1R and the innervation of PPG neurons in the spinal cord, as well as the potential therapeutic benefits of GLP‐1R activation.

Cell division cycle 20 promotes cell proliferation and invasion and inhibits apoptosis in osteosarcoma cells

ABSTRACT Cdc20 (cell division cycle 20 homologue) has been reported to exhibit an oncogenic role in human tumorigenesis. However, the function of Cdc20 in osteosarcoma (OS) has not been investigated. In the current study, we aim to explore the role of Cdc20 in human OS cells. Multiple approaches were used to measure cell growth, apoptosis, cell cycle, migration and invasion in OS cells after depletion of Cdc20 or overexpression of Cdc20. We found that down-regulation of Cdc20 inhibited cell growth, induced apoptosis and triggered cell cycle arrest in OS cells. Moreover, Cdc20 down-regulation let to inhibition of cell migration and invasion in OS cells. Consistently, overexpression of Cdc20 in OS cells promoted cell growth, inhibited apoptosis, enhanced cell migration and invasion. Mechanistically, our Western blotting results showed that overexpression of Cdc20 reduced the expression of Bim and p21, whereas depletion of Cdc20 upregulated Bim and p21 levels in OS cells. Altogether, our findings demonstrated that Cdc20 exerts its oncogenic role partly due to regulation of Bim and p21 in OS cells, suggesting that targeting Cdc20 could be useful for the treatment of OS.