1. Establishment Of A Method For Screening Cysteinyl Leukotrienes Receptor 1 Antagonists 2. Dihydroartemisinin Inhibits Proliferation And Induces Apoptosis In Rat Glioma C6 Cells

AIM: Cysteinyl leukotrienes (CysLTs) are important inflammatory mediators metabolized from arachidonic acid through 5-lipoxygenase pathway. CysLTs play roles in periphery inflammatory diseases, and also in the pathophysiological processes of the central nervous system diseases, such as stroke, trauma and brain tumors. The effects of CysLTs are mediated by two receptors, CysLT₁ and CysLT₂ receptor. It has been reported that CysLTs activate their receptors and exert responses through releasing free Ca²⁺ from the intracellular storage pool. We have found that CysLTi receptor is involved in cerebral ischemia. There are several selective CysLT₁ receptor antagonists but they possess non-receptor effect, so that more compounds with CysLT₁ receptor antagonizing activity should be investigated. In addition, selective CysLT₂ receptor antagonists are still lack, so its responses are little known. The purpose of this study is to establish a cell model and screening methods to find new CysLT₁ receptor antagonists, which will provide a basis for discovery of new compounds and development of new drugs.METHODS: We chose differentiated human histocytic lymphoma cells (dU937 cells) as the cell model, which express a high density of CysLT₁ receptors. We detected the increased intracellular Ca²⁺ concentration induced by leukotriene D₄ (LTD₄), an agonist for CysLT₁ receptor, as an indicator for detecting CysLT₁ receptor antagonizing activity of the compounds. CysLT₁ receptor antagonists, pranlukast and montelukast,were used as the standard compounds to control the screening. The protective activity of the screened compounds was further investigated on oxygen-glucose deprivation (OGD)-induced injury in human umbilical vein endothelial cells (EA.hy926 cells).RESULTS: dU937 cells expressed a high level of CysLT₁ receptor protein. A positive compound ATP at 5×10^-5M increased intercellular Ca²⁺ concentration by 30% in dU937 cells; LTD₄ at 10^-7M also increased the Ca²⁺ signal by 56% of ATP. Selective CysLT₁ receptor antagonists, pranlukast and montelukast, inhibited LTD4-increased Ca²⁺, with IC50 values of 9.2 ×10^-8M and 7.5 ×10^-8M, respectively. The newly synthesized compounds, D_Xw-1, 10, 11, 12, 13 and 19, inhibited the Ca²⁺ signal; the IC50 values were from 2.2 ×10^-8 M to 2.4 ×10^-7M and the maximal inhibition (Emax) were 19-23%. D_Xw 10, 11 and 12 at 10^-6M did not show any toxic effect in EA.hy926 cells; however, they did not affect OGD-induced injury in EA.hy926 cells. In contrast, pranlukast at 10(-6)M showed the protective effect on OGD-induced injury.CONCLUSION:1. dU937 cells can be used as cell model for the responses of CysLT₁ receptor. In this cell model, measurement of intracellular Ca²⁺ concentration and the method for preliminary screening of CysLT₁ receptor antagonists are established.2. Selective CysLT₁ receptor antagonists, pranlukast and montelukast, can inhibit LTD4-increased intercellular Ca²⁺ in dU937 cells, and attenuate OGD-induced injury in endothelial cells. Compounds, D_Xw-1, 10, 11, 12, 13 and 19 can inhibit the LTD₄-increased Ca²⁺ in dU937 cells, indicating the CysLT₁ receptor antagonizing-like activity. However, Dxw-10, 11 and 12 did not show any effect on OGD-induced injury in endothelial cells. Therefore, whether these compounds are the CysLT₁ receptor antagonists needs to be further investigated.Part 2Dihydroartemisinin inhibits proliferation and induces apoptosis in rat glioma C6 cellsABSTRACTAIM: Astrocytoma is the most common tumor in central nervous system without any efficient therapy. Until now, the clinical therapies for glioblastomas consist of surgical resection, irradiation and chemotherapy. But the prognosis is always poor. Artemisinin and its analogs have been used widely as antimalaria drugs and considered to be safe drugs without obvious side effects. It was reported that artemisinin and its derivatives are toxic to tumor cells in vitro and in vivo, but safe to normal cells. Dihydroartemisinin (DHA) is the main active metabolite of artemisinins in vivo. The antineoplasmic activity of DHA was stronger than artesunate and artemininin. Moreover, because it can pass through the blood brain barrier, therefore, DHA may be efficient to brain tumor. Our study was aimed to determine whether dihydroartemisin (DHA) affect the proliferation and apoptosis of rat C6 glioma cells.METHODS: DHA (1-125μmol/L) was added into the cultured C6 cells for 24, 48 and 72 h. The cell proliferation and viability were determined by trypan blue exclusion assay and 3-(4,5-dimethylthiazol-2yl)-2,5dipheyl tetrazolium bromide (MTT) reduction assay. The apoptosis was detected by Hoechst 33342 staining. The intracellular reactive oxygen species (ROS) was measured by H2DCFDA oxidative reaction.RESULTS:It shown that DHA 5-125 μmol/L inhibited the proliferation of C6 cells in concentration- and time-dependent manners, the IC50 at 48 h was 23.4 μmol/L. 5 μmol/L DHA induced apoptosis of C6 cells in 24 h (P < 0.05), the rate of apoptosis cells achieved 30% in 48 h (P < 0.01) , while 25μmol/L was 50%. DHA increased theintracellular ROS (P< 0.01) , 1-fold by 5 μmol/L, 2-fold by 25 μmol/L, and more than 4-fold by 125 μmol/L. CONCLUSION:1. DHA inhibits the proliferation and induces the apoptosis of glioma cells; its cytotoxic effect may result from the increase of intercellular ROS.2. DHA can kill tumor cells and pass through the blood brain barrier, therefore, therefore, it may be effective therapeutic agent for the treatment of glioma.