Dihydroartemisinin exerts cytotoxic effects and inhibits hypoxia inducible factor-1alpha activation in C6 glioma cells.

Artemisinin and its analogue dihydroartemisinin exert cytotoxic effects in some kinds of cancer cell lines. Here we determined whether dihydroartemisinin inhibits the growth and induces apoptosis of rat C6 glioma cells. We found dihydroartemisinin (5-25 microM) inhibited the growth and induced apoptosis of C6 cells in a concentration- and time-dependent manner; however, it was much less toxic to rat primary astrocytes. Dihydroartemisinin (5-25 microM) also increased the generation of reactive oxygen species in C6 cells. These effects of dihydroartemisinin were enhanced by ferrous ions (12.5-100 microM) and reduced by the iron chelator deferoxamine (25-200 microM). Immunoblotting analysis revealed that dihydroartemisinin (5-25 microM) significantly reduced hypoxia- and deferoxamine-induced expression of hypoxia inducible factor-1alpha and its target gene protein, vascular endothelial growth factor, in C6 cells. The results showed that dihydroartemisinin exerts a selective cytotoxic effect on C6 cells by increasing the reactive oxygen species and inhibiting hypoxia inducible factor-1alpha activation.

[Dihydroartemisinin inhibits proliferation and induces apoptosis of rat glioma C6 cells].

OBJECTIVE: To determine the effects of dihydroartemisinin (DHA) on the proliferation and apoptosis of rat glioma C6 cells. METHODS: DHA (1~ 125 micromol/L) was added into the cultured C6 cells and incubated 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, 5 diphenyl tetrazolium bromide (MTT) reduction assay. The apoptosis was detected by Hoechst 33342 staining. The intracellular reactive oxygen species (ROS) was measured by H(2)DCFDA oxidative reaction. RESULTS: DHA 5 ~ 125 micromol/L inhibited the proliferation of C6 cells in concentration- and time-dependent manners, the IC50 at 48 h was 23.4 micromol/L. DHA 5 ~ 25 micromol/L induced C6 cell apoptosis (P<0.05), and 5 ~ 125 micromol/L increased the intracellular ROS (P<0.01). CONCLUSION: DHA inhibits the proliferation and induces the apoptosis of C6 cells; its cytotoxic effect may result from the increase of intercellular ROS.

[Transfection with 5-lipoxygenase/green fluorescence protein for evaluating injury-induced 5-lipoxygenase translocation to the nuclear membrane in PC12 cells].

OBJECTIVE: To evaluate the translocation of 5-lipoxygenase (5-LOX)) after injuries by transfection with green fluorescence protein (GFP)/5-LOX in PC12 cells. METHODS: PC12 cells were stably transfected with pEGFP-C2/5-LOX (GFP/5-LOX) or pEGFP-C2 vectors (control). After treatment with oxygen-glucose deprivation (OGD), H(2)O(2) or NMDA, GFP/5-LOX localization in the cells was observed under a fluorescence microscope. Wild-type 5-LOX was determined by immunostaining after the treatment. RESULT: In the GFP/5-LOX-transfected cells, GFP/5-LOX was primarily localized in the nucleus; while in the GFP-transfected cells, GFP was localized in both the cytoplasm and nucleus. After OGD and H(2)O(2) treatments, GFP/5-LOX was translocated to the nuclear membrane in 50.6 % and 57.7% cells respectively. However, after NMDA treatment or in GFP-transfected cells, no translocation was observed. Wild-type 5-LOX was distributed in the nuclei and cytoplasm, and all the 3 treatments induced 5-LOX translocation to the nuclear membrane. CONCLUSION: In the PC12 cells stably transfected with GFP/5-LOX, GFP/5-LOX is primarily distributed in the nuclei; the OGD-, H(2)O(2)- and NMDA-induced 5-LOX translocation exhibits different properties.

Baicalin attenuates oxygen-glucose deprivation-induced injury via inhibiting NMDA receptor-mediated 5-lipoxygenase activation in rat cortical neurons.

The flavonoid baicalin exerts neuroprotective effects but the mechanism is not fully clarified. On the other hand, 5-lipoxygenase (5-LOX) activation is involved in ischemic neuronal injury. In this study, we determined whether baicalin protects rat cortical neurons against oxygen-glucose deprivation (OGD)-induced ischemic-like injury, if so, whether this effect relates to 5-LOX activation. After the neurons were injured by 1.5-h OGD and 24-h recovery, their viability reduced and necrosis occurred; these injuries were attenuated by baicalin (1 and 5microM) as well as caffeic acid (a 5-LOX inhibitor, 5 and 25microM) and MK-801 (an NMDA receptor antagonist, 1-10microM). OGD-induced 5-LOX translocation to the nuclear envelope as detected by immunoblotting, immunocytochemistry and 5-LOX transfection; this translocation was inhibited by baicalin (5microM) and MK-801 (5microM) but not by caffeic acid (5microM). During 0.5- to 2-h recovery after 1.5-h OGD, the production of 5-LOX metabolites, cysteinyl leukotrienes, was increased; this increased production was inhibited by baicalin and MK-801, while both the increased and baseline production were inhibited by caffeic acid. In addition baicalin and MK-801, not caffeic acid, inhibited glutamate-induced elevation of intracellular calcium. These results indicate that baicalin attenuates ischemic-like injury in the neurons, and this effect partly relates to the inhibition of NMDA receptor-mediated 5-LOX activation.

Activation of 5-lipoxygenase after oxygen-glucose deprivation is partly mediated via NMDA receptor in rat cortical neurons.

5-Lipoxygenase (5-LOX) is the enzyme metabolizing arachidonic acid to produce pro-inflammatory leukotrienes. We have reported that 5-LOX is translocated to the nuclear envelope after ischemic-like injury in PC12 cells. In the present study, we determined whether 5-LOX is activated (translocation and production of leukotrienes) after oxygen-glucose deprivation (OGD) in primary rat cortical neurons; if so, whether this activation is mediated by NMDA receptor. After OGD, 5-LOX was translocated to the nuclear envelope as detected by immunoblotting, immunostaining and green fluorescent protein-5-LOX transfection. 5-LOX metabolites, cysteinyl-leukotrienes (CysLTs) but not leukotriene B4, in the culture media were increased 0.5-1.5 h after recovery. Similarly, NMDA (100 microm) also induced 5-LOX translocation, and increased the production of CysLTs during 0.5-1 h NMDA exposure. Both OGD and NMDA reduced neuron viability. NMDA receptor antagonist MK-801 inhibited almost all the responses to OGD and NMDA; whereas 5-LOX activating protein inhibitor MK-886 and 5-LOX inhibitor caffeic acid inhibited the reduction of neuron viability and the production of CysLTs, but did not affect 5-LOX translocation. From these results, we conclude that OGD can activate 5-LOX in primary rat cortical neurons, and that this activation may be partly mediated via activating NMDA receptor.