Differentiation in Neuroblastoma: Diffusion-Limited Hypoxia Induces Neuro-Endocrine Secretory Protein 55 and Other Markers of a Chromaffin Phenotype

Background: Neuroblastoma is a childhood malignancy of sympathetic embryonal origin. A high potential for differentiation is a hallmark of neuroblastoma cells. We have previously presented data to suggest that in situ differentiation in tumors frequently proceeds along the chromaffin lineage and that decreased oxygen ( hypoxia) plays a role in this. Here we explore the utility of Neuro-Endocrine Secretory Protein 55 ( NESP55), a novel member of the chromogranin family, as a marker for this process.Methodology/Principal Findings: Immunohistochemical analyses and in situ hybridizations were performed on human fetal tissues, mouse xenografts of human neuroblastoma cell lines, and on specimens of human neuroblastoma/ganglioneuroma. Effects of anaerobic exposure on gene expression by cultured neuroblastoma cells was analyzed with quantitative real-time PCR. Fetal sympathetic nervous system expression of NESP55 was shown to be specific for chromaffin cell types. In experimental and clinical neuroblastoma NESP55 immunoreactivity was specific for regions of chronic hypoxia. NESP55 expression also correlated strikingly with morphological evidence of differentiation and with other chromaffin-specific patterns of gene expression, including IGF2 and HIF2 alpha. Anaerobic culture of five neuroblastoma cell lines resulted in an 18.9-fold mean up-regulation of NESP55.Conclusions/Significance: The data confirms that chronic tumor hypoxia is a key microenvironmental factor for neuroblastoma cell differentiation, causing induction of chromaffin features and NESP55 provides a reliable marker for this neuronal to neuroendocrine transition. The hypoxia-induced phenotype is the predominant form of differentiation in stroma-poor tumors, while in stroma-rich tumors the chromaffin phenotype coexists with ganglion cell-like differentiation. The findings provide new insights into the biological diversity which is a striking feature of this group of tumors

l-Tetrahydropalmatine, an Active Component of Corydalis yanhusuo W.T. Wang, Protects against Myocardial Ischaemia-Reperfusion Injury in Rats

l-Tetrahydropalmatine (l-THP) is an active ingredients of Corydalis yanhusuo W.T. Wang, which protects against acute global cerebral ischaemia-reperfusion injury. In this study, we show that l-THP is cardioprotective in myocardial ischaemia-reperfusion injury and examined the mechanism. Rats were treated with l-THP (0, 10, 20, 40 mg/kg b.w.) for 20 min before occlusion of the left anterior descending coronary artery and subjected to myocardial ischaemia-reperfusion (30 min/6 h). Compared with vehicle-treated animals, the infarct area/risk area (IA/RA) of l-THP (20, 40 mg/kg b.w.) treated rats was reduced, whilst l-THP (10 mg/kg b.w.) had no significant effect. Cardiac function was improved in l-THP-treated rats whilst plasma creatine kinase activity declined. Following treatment with l-THP (20 mg/kg b.w.), subunit of phosphatidylinositol 3-kinase p85, serine473 phosphorylation of Akt and serine1177 phosphorylation of endothelial NO synthase (eNOS) increased in myocardium, whilst expression of inducible NO synthase (iNOS) decreased. However, the expression of HIF-1α and VEGF were increased in I30 minR6 h, but decreased to normal level in I30 minR24 h, while treatment with l-THP (20 mg/kg b.w.) enhanced the levels of these two genes in I30 minR24 h. Production of NO in myocardium and plasma, activity of myeloperoxidase (MPO) in plasma and the expression of tumour necrosis factor-α (TNF-α) in myocardium were decreased by l-THP. TUNEL assay revealed that l-THP (20 mg/kg b.w.) reduced apoptosis in myocardium. Thus, we show that l-THP activates the PI3K/Akt/eNOS/NO pathway and increases expression of HIF-1α and VEGF, whilst depressing iNOS-derived NO production in myocardium. This effect may decrease the accumulation of inflammatory factors, including TNF-α and MPO, and lessen the extent of apoptosis, therefore contributing to the cardioprotective effects of l-THP in myocardial ischaemia-reperfusion injury