A Study Of The Therapeutic Values Of SEH Inhibitor T-AUCB On Glioma

Objective: Glioblastoma is one of the most aggressive glial tumors with an averagelife expectancy less than one year. Current local control with surgery, radiation, andsystemic chemotherapy for glioblastoma remain the pillars. It is expected to developmore potent agents with low side effects for glioblastoma chemotherapy. sEH is aubiquitously expressed homodimeric enzyme which hydrolyzes a wide variety ofendogenous and exogenous epoxides. Most studies of sEH and its inhibitors wereinvolved in inflammatory and cardiovascular diseases, because the preferredsubstrates of sEH, epoxyeicosatrienoic acids (EETs), possess numerous beneficialeffects on inflammatory and cardiovascular events. In this study, we focused on amore potent and selective sEH inhibitor, t-AUCB, and investigated its effects onU251and U87human glioblastoma cell lines.Methods:1. The sEH inhibitor t-AUCB was a gift from Professor Bruce D.Hammock. U251and U87human glioblastoma cell lines were kindly provided byProfessor Kun Yao.2. Soluble epoxide hydrolase (sEH) activities in U251, U87andHepG2cells were measured using sEH activity assay kit.3. Cell growth assay waspreformed using the cell counting kit-8(CCK-8) from Dojindo Laboratories, and thecell-cycle and apoptosis analysis was performed using flow cytometry.4. The levelsof mRNA were detected using Reverse Transcription PCR (RT-PCR) and the proteinlevels were analyzed using western blotting.5. All data were analyzed by SPSS13.0,and expressed as mean±standard deviation (SD). Results at P<0.05were consideredsignificant.Results:1. t-AUCB efficiently inhibited sEH activities in U251and U87cells. For10 μM t-AUCB, In U251cells the sEH activity inhibition rate was86.3%, and in U87the rate was81.5%.2. t-AUCB suppressed U251and U87cell growth in aconcentration-dependent manner. In200μM t-AUCB treated U251and U87cells, thephosphorylation levels of p65started to increase after10min, reached to peak after30min and lasted for at least2h. NF-κB inhibitor PDTC completely abolishedt-AUCB-induced growth inhibition in both two glioblastoma cell lines, indicatingt-AUCB suppressed U251and U87cell growth by activating NF-κB-p65.3. t-AUCBinduced cell-cycle G0/G1phase arrest by regulating Cyclin D1and phosphorylatedCDC2levels.4.200μM t-AUCB can not induce significant apoptosis of U251orU87, indicating that U251and U87may resist t-AUCB-induced cell apoptosis.5.t-AUCB treatment induces significant phosphorylations of p38MAPK, MAPKAPK2and Hsp27in U251and U87cells. By using the p38MAPK inhibitor SB203580andHsp27inhibitor KRIBB3, we demonstrated that inhibition of either p38MAPK orHsp27can abolish the apoptosis resistance in U251and U87cells, indicating theactivation of p38MAPK-Hsp27pathway plays a key role in resistingt-AUCB-induced apoptosis.Conclusion: In this study we investigated the effects of t-AUCB, an improved sEHinhibitor, on human glioblastoma cells. We demonstrated t-AUCB suppressed U251and U87cell growth by activating NF-κB-p65and induced cell-cycle G0/G1phasearrest by regulating Cyclin D1and CDC2. Moreover, in U251and U87cells, theactivation of p38MAPK-Hsp27pathway plays a key role in resistingt-AUCB-induced apoptosis. Since t-AUCB possesses very well pharmacokineticproperties, we proposed it a potential agent for glioma chemotherapy, especially withthe combination of Hsp27inhibitor.