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Effects of combining methylprednisolone with rolipram on functional recovery in adult rats following spinal cord injury

Methylprednisolone (MP) has been widely used as a standard therapeutic agent for the treatment of spinal cord injury (SCI). Because of its controversial beneficial effects, the combination of MP and other pharmacological agents aimed at enhancing functional recovery is desirable. The phosphodiesterase 4 (PDE4) inhibitor rolipram has been implicated in promotion of regeneration due to elevating cAMP. In the present study, we sought to determine the effects of MP and rolipram, administered in combination, after spinal cord injury (SCI) in adult rats. Here we show that in vitro administration of rolipram and MP significantly increased neuron survival and promoted neurite outgrowth of neurons on the inhibitory substrate CSPGs by upregulation of MMP-2 expression; in vivo administration of rolipram and MP inhibited CSPG expression and increase CSPG digestion after rat SCI. Rolipram and MP combining treatment promoted significant neuroprotection through reduced motoneuron death, minimized lesion cavity, and increased regeneration of lesioned corticospinal tract (CST) axons beyond the lesion site after SCI. Enhanced functional recovery was also observed. Overall, our study strongly suggested that the combination treatment of MP and rolipram may represent a promising strategy for clinically applicable pharmacological therapy for rapid initiation of neuroprotection after SCI.

Glucocorticoid receptor β regulates injury-mediated astrocyte activation and contributes to glioma pathogenesis via modulation of β-catenin/TCF transcriptional activity

Astrocytes react to central nervous system (CNS) injury and participate in gliotic responses, imparting negative, as well as positive effects on axonal regeneration. Despite the considerable biochemical and morphological changes astrocytes undergo following insult, and the known influence of steroids on glial activation, details surrounding glucocorticoid receptor expression and activity are lacking. Such mechanistic information is essential for advancing and enhancing therapies in the treatment of CNS injuries. Using an in vitro wound-healing assay, we found glucocorticoid receptor β (GRβ), not GRα, is upregulated and acts as a regulator of gliosis after injury. In addition, our results suggest that GRβ interacts with β-catenin and is a necessary component for proliferation and migration in both injured astrocytes and glioma cells. Further analysis indicated GRβ/β-catenin interaction as a key modulator of astrocyte reactivity through sustained Wnt/β-catenin/TCF signaling in its dominant-negative effect on GRα mediated trans-repression by a GSK-3β-independent manner. These findings expand our knowledge of the mechanism of GRβ action in promoting astrocyte proliferation and migration following injury and in glioma. This information furthers our understanding the function of glucocorticoid receptor in CNS injury and disease, as well as in the basic biochemical responses astrocytes undergo in response to injury and glioma pathogenesis.

The effect of glucosylceramide synthase on P-glycoprotein function in K562/AO2 leukemia drug-resistance cell line

Previous work from our laboratory demonstrated that glucosylceramide synthase (GCS) and multidrug resistance 1 gene (MDR1) are co-overexpressed in drug-resistant leukemia cells. We hypothesized that GCS and MDR1 may interact. In this study, we used RNA interference (RNAi) to silence the GCS or MDR1 gene in K562/AO2 drug-resistant cells. The sensitivity of cells to different treatments with doxorubicin was evaluated. We used Taqman probe fluorescence real-time quantitative PCR, and detected expression of GCS and MDR1 mRNAs in different interfering groups. Intracellular mean fluorescence intensity (MFI), which represents rhodamine123 (rh123) retention, was determined by flow cytometry (FCM). An MTT cytotoxicity assay showed that the 50% inhibition concentration (IC50) of doxorubicin of K562/AO2 cells (138.25 ± 3.75 µg/ml) was significantly higher than that of K562 drug-sensitive cells (2.125 ± 0.125 µg/ml), and that IC50 was evidently lower in K562/AO2 cells, whether it was transfected with a small interfering RNA (siRNA) targeting GCS (GCSsiRNA) or one targeting MDR1 (MDR1siRNA). Compared with untreated K562/AO2 cells, the inhibition rates of GCS mRNA in the cells transfected with GCSsiRNA for 9 and 36 h were 56.67 ± 9.29% (p < 0.05) and 74 ± 6.38% (p < 0.05), respectively. Interestingly, the expression of MDR1 mRNA was also inhibited to 51.7 ± 4.5% (p < 0.05) 36 h after transfection with GCSsiRNA, but there was no significant difference in MDR1 expression at 9 h post-transfection in cells treated with GCSsiRNA and a negative control. It is well known that rh123 retention in cells results from an efflux function of P-glycoprotein (P-gp). In K562 cells, rh123 retention was much higher than in K562/AO2 cells (p < 0.01). We also noted that rh123 retention in the K562/AO2 cells transfected with GCSsiRNA for 48 h was significantly higher than in the negative control group. In conclusion, we show in the present study that inhibition of the GCS gene affects the expression of MDR1 mRNA and P-gp function.

Glutamine synthetase functions as a negative growth regulator in glioma

Our recent study demonstrated that glutamine synthetase (GS) may not only serve as a glutamate-converting enzyme in glial cells, but may also function as a regulator of astrocyte migration after injury. In this report, we showed that GS expression increased in cultured rat C6 glioma cells that underwent long-term serially propagation. The stable overexpression of GS in C6 glioma cells resulted in growth arrest and motility suppression; however the stable knockdown of GS resulted in motility enhancement. In correlation with cell aggregation, N-cadherin levels increased at sites of cell–cell contact in C6 cells overexpressing GS, and decreased in C6 cells with stable GS knockdown; total N-cadherin expression levels remained unchanged in these cells. In addition, levels of p21, a potent cyclin-dependent kinase inhibitor, increased, while cyclin D1 levels decreased in C6 cells overexpressing GS. Our additional studies showed that N-cadherin-mediated cell–cell contacts were implicated in GS-induced cell growth arrest and impairment of cell migration, as evidenced by the inhibition of GS on cell growth and motility by the neutralizing anti-N-cadherin monoclonal antibody (GC-4 mAb). Collectively, these observations suggest a novel mechanism of growth regulation by GS that involves N-cadherin mediated cell–cell contact.

Glucocorticoid Receptor β Acts as a Co-activator of T-Cell Factor 4 and Enhances Glioma Cell Proliferation

We previously reported that glucocorticoid receptor β (GRβ) regulates injury-mediated astrocyte activation and contributes to glioma pathogenesis via modulation of β-catenin/T-cell factor/lymphoid enhancer factor (TCF/LEF) transcriptional activity. The aim of this study was to characterize the mechanism behind cross-talk between GRβ and β-catenin/TCF in the progression of glioma. Here, we reported that GRβ knockdown reduced U118 and Shg44 glioma cell proliferation in vitro and in vivo. Mechanistically, we found that GRβ knockdown decreased TCF/LEF transcriptional activity without affecting β-catenin/TCF complex. Both GRα and GRβ directly interact with TCF-4, while only GRβ is required for sustaining TCF/LEF activity under hormone-free condition. GRβ bound to the N-terminus domain of TCF-4 its influence on Wnt signaling required both ligand- and DNA-binding domains (LBD and DBD, respectively). GRβ and TCF-4 interaction is enough to maintain the TCF/LEF activity at a high level in the absence of β-catenin stabilization. Taken together, these results suggest a novel cross-talk between GRβ and TCF-4 which regulates Wnt signaling and the proliferation in gliomas.

Association of leptin, visfatin, apelin, resistin and adiponectin with clear cell renal cell carcinoma

Although an association between obesity and the occurrence of renal cell carcinoma (RCC) has been identified, the mechanism by which obesity functions to increase this risk of cancer remains unclear. Leptin, visfatin, apelin, resistin and adiponectin are peptide hormones secreted by adipocytes; it is considered that these may affect RCC development by exerting effects on proliferation, cell growth and inflammation. The aim of the present study was to investigate the association between the aforementioned adipokine genes and clear cell RCC (CC-RCC). The GSE6344 dataset was downloaded from the Gene Expression Omnibus database, and the relative expression levels of the adipokine genes were analyzed. To verify the results of the mRNA microarray, 77 paired samples of CC-RCC and corresponding adjacent normal tissue were allocated into two groups. The extraction of total RNA was conducted, and the mRNA expression of adipokine genes was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The data from the GSE6344 dataset indicated that the expression of visfatin and apelin was upregulated (P<0.0001 and P<0.01, respectively), and adiponectin was downregulated (P<0.001) in the CC-RCC tissues compared with the adjacent normal tissues. The data from RT-qPCR demonstrated that visfatin and resistin gene expression was increased (P<0.01 and P<0.05, respectively) in the CC-RCC tissues. Furthermore, the mRNA expression level of leptin and adiponectin in the adjacent normal tissue was higher than those in the cancer tissue (P<0.01). The current study verifies that visfatin and adiponectin are associated with an increased risk of CC-RCC, which presents further insights into the molecular mechanisms of CC-RCC tumorigenesis.

Glucosylceramide synthase promotes Bcl-2 expression via the ERK signaling pathway in the K562/A02 leukemia drug-resistant cell line

Multidrug resistance (MDR) to chemotherapeutic agents is a major obstacle to curative treatment of cancer. In various types of cancers, overexpression of glucosylceramide synthase (GCS) has been observed to be associated with MDR, thus making GCS a target for reversal of resistance. Our previous work demonstrated that GCS and Bcl-2 are co-overexpressed in the K562/A02 leukemia multidrug-resistant cell line compared with its sensitive counterpart, K562. In the present study, we investigated the effects of GCS on apoptosis in K562/A02 and the associated molecular mechanisms. Our results indicate that the inhibition of GCS caused downregulation of Bcl-2 as well as apoptosis enhancement in response to ADM via the ERK pathway, while JNK or p38 MAPK signaling appeared to play less significant roles in the regulation of apoptosis and MDR in K562/A02 cells. Targeting GCS by siRNA also enhanced ceramide accumulation, which is involved in GCS knockdown-induced inhibition of ERK activation and Bcl-2 expression levels.