Schisandrin B Inhibits Vascular Endothelial Growth Factor Expression In Anoxic Human Glioma Cells

Fructus Schisandrae(Schisandra chinensis,FS),a traditional Chinese medicine, has been commonly studied because of its therapeutic and immunomodulatory functions and less toxicity.The compound abstract from FS attracts extra attention in recent years.Fructus schisandrae-containing compounds are known to increase the production of granulocyte-macrophage colony-stimulating factor(GM-CSF),possess anti-inflammatory effects,and induce apoptosis,as reported by previous study. Schisandrin B(Sch B),a lignin compound isolated from FS,molecular weight 400.46 delton,is an effective antioxidant.Vascular endothelial growth factor(VEGF),also termed vascular permeability factor(VPF),was first found by Senger in tumor cells excretion and can potentiate high vascular permeability,which cause exosmosis of protein in tumor vessels.It is well known now that VEGF,as a growth factor specific for endothelial cells,is a critical regulator of vasculogenesis and angiogenesis.Hypoxia and hypoxia-regulated transcription factor,hypoxia-inducible factor(HIF),are the most important inducer of VEGF expression.Neoplasms of the intracranial components of the central nervous system include the cerebral hemispheres,basal ganglia,hypothalamus,thalamus,brain stem,and cerebellum.Brain neoplasms are subdivided into primary(originating from brain tissue) and secondary(i.e.,metastatic) forms.Primary neoplasms are subdivided into benign and malignant forms.Malignant gliomas are the most common primary brain neoplasms.Currently,surgery and radiation therapy are applicated to the treatment of patients with glioblastoma multiforme.The highly infiltrative nature of glioma cells that invade into surrounding brain parenchyma always cause the failure of therapy and the recurrence of tumor.Glioma progression is strongly dependent on the development of a new vascular network that occurs primarily by angiogenesis. Vascular endothelial growth factor(VEGF) is the dominant pro-angiogenic factor up-regulated by hypoxia-inducible factor -1α(HIF-1α).Antiangiogenic therapies targeting VEGF are presently being developed,but the results remain palliative.Based on the potential effects of FS on inhibition of carcinoma cell proliferation, we hypothesized that one of its most effective compounds,Schisandrin B,has similar influence on tumor cells through anti-angiogenic pathways.In present study,we investigated the possible effect of Schisandrin B on VEGF expression in human glioma cells under hypoxia and its relation with HIF-1α.Object:To address the effect of Schisandrin B(Sch B) on the tumor proliferation suppression,expression of vascular endothelial growth factor(VEGF) and its relation with hypoxia-inducible factor(HIF) in human glioma cells exposed to hypoxia.Method:Athymic nude mice bearing U251 transplant tumor were used to test the inhibitory efficacy of Sch B.Cytotoxic effects of Sch B on multiple U251 cells was evaluated by MTT colorimetry.U251 cells were treated with Sch B under normoxia or hypoxia,the levels of VEGF and HIF-1αmRNA and protein were analyzed by reverse transcription-polymerase chain reaction(RT-PCR),real-time quantitative PCR and enzyme-linked immunosorbent assay(ELISA) or western blotting.Result: 1.Intraperitoneal injection of Sch B significantly inhibited the tumor growth in athymic nude mice bearing U251 transplant tumor,while the treatment of SchB showed no effects on cultured U251 proliferation.2.Effect of Sch B on VEGF expression under hypoxiaThe levels of VEGF mRNA and protein were low during whole experiment in U251 cells under normoxia.VEGF mRNA was up-regulated under hypoxia condition throughout 24 h period,and this increase of VEGF mRNA were partly suppressed after 1 h Sch Bpre-treatment,which was correspondingly followed by hysteretic VEGF protein suppression in 12 and 24 h.The results of real-time quantitative PCR showed a concentration-dependent VEGF mRNA suppression after Sch B treatment under 12 h hypoxia,accompanied by decreasing secretion of VEGF protein from U251 24 h after hypoxia treatment.Sch B had no significantly effect on U251 cell, however it showed antitumor effect in vivo.Hypoxic treatment increased vascular endothelial growth factor(VEGF) mRNA and protein expression in U251 glioma cells.Pre-treating the cell with Sch B could partially inhibit hypoxia-induced VEGF over-expression via down-regulating HIF-1αin time- and concentration-dependent manners.3.Effect of Sch B on HIF-1αexpression under hypoxiaHypoxia caused a significantly increase expression of HIF-1αmRNA in U251. Pre-treated the cells with Sch B could partially suppressed this hypoxia-induced overexpression of HIF-1αmRNA in a concentration-dependent manner,while under normoxia,Sch B had no effect on the expression of HIF-1αmRNA.At the same time, an increasing of HIF-1αprotein content was detected in anoxic cell extracts under hypoxia,while it was also partially suppressed by Sch B treatment.Conclusion:Hypoxic treatment increased vascular endothelial growth factor (VEGF) mRNA and protein expression in U251 glioma cells.Pre-treating the cell with Sch B could partially inhibit hypoxia-induced VEGF over-expression via down-regulating HIF-1αmRNA and protein expressions in a time- and dose-dependent manners.These findings offer a novel explanation for the antioxidant effect of Sch B on antioxidant related diseases especially tumors.