Abstract：
OBJECTIVE: To explore the potential mechanism of lysionotin in treating glioma.
METHODS: First, target prediction based on Bernoulli Naïve Bayes profiling and pathway enrichment was used to predict the biological activity of lysionotin. The binding between 5-lipoxygenase (5-LO) and lysionotin was detected by surface plasmon resonance (SPR) and molecular docking, and the inhibitory effects of lysionotin on 5-LO and proliferation of glioma were determined using enzyme inhibition assay in vitro and cell viability analysis, respectively. Furthermore, the pharmaceutical effect of lysionotin was explored by cell survival rate analysis and liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein expression, intracellular calcium ion concentration and cytoskeleton detection were revealed by Western blot, flow cytometry and fluorescence labeling, respectively.
RESULTS: Target prediction and pathway enrichment revealed that lysionotin inhibited 5-LO, a key enzyme involved in the arachidonic acid metabolism pathway, to inhibit the proliferation of glioma. Molecular docking results demonstrated that 5-LO can be binding to lysionotin through hydrogen bonds, forming bonds with His600, Gln557, Asn554, and His372. SPR analysis further confirmed the interaction between 5-LO and lysionotin. Furthermore, enzyme inhibition assay in vitro and cell survival rate analysis revealed that 50% inhibition concentration of lysionotin and the median effective concentration of lysionotin were 90 and 16.58 µmol/L, respectively, and the results of LC-MS/MS showed that lysionotin inhibited the production of 5S-hydroperoxy-eicosatetraenoic acid (P<0.05), and moreover, the LC-MS/MS results indicated that lysionotin can enter glioma cells well (P<0.01) and inhibit their proliferation. Western blot analysis demonstrated that lysionotin can inhibit the expression of 5-LO (P<0.05) and downstream leukotriene B4 receptor (P<0.01). In addition, the results showed that lysionotin affected intracellular calcium ion concentration by inhibiting 5-LO to affect the cytoskeleton, as determined by flow cytometry and fluorescence labeling.
CONCLUSIONS: Lysionotin binds to 5-LO could suppress glioma by inhibiting arachiodonic acid metabolism pathway.
METHODS: First, target prediction based on Bernoulli Naïve Bayes profiling and pathway enrichment was used to predict the biological activity of lysionotin. The binding between 5-lipoxygenase (5-LO) and lysionotin was detected by surface plasmon resonance (SPR) and molecular docking, and the inhibitory effects of lysionotin on 5-LO and proliferation of glioma were determined using enzyme inhibition assay in vitro and cell viability analysis, respectively. Furthermore, the pharmaceutical effect of lysionotin was explored by cell survival rate analysis and liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein expression, intracellular calcium ion concentration and cytoskeleton detection were revealed by Western blot, flow cytometry and fluorescence labeling, respectively.
RESULTS: Target prediction and pathway enrichment revealed that lysionotin inhibited 5-LO, a key enzyme involved in the arachidonic acid metabolism pathway, to inhibit the proliferation of glioma. Molecular docking results demonstrated that 5-LO can be binding to lysionotin through hydrogen bonds, forming bonds with His600, Gln557, Asn554, and His372. SPR analysis further confirmed the interaction between 5-LO and lysionotin. Furthermore, enzyme inhibition assay in vitro and cell survival rate analysis revealed that 50% inhibition concentration of lysionotin and the median effective concentration of lysionotin were 90 and 16.58 µmol/L, respectively, and the results of LC-MS/MS showed that lysionotin inhibited the production of 5S-hydroperoxy-eicosatetraenoic acid (P<0.05), and moreover, the LC-MS/MS results indicated that lysionotin can enter glioma cells well (P<0.01) and inhibit their proliferation. Western blot analysis demonstrated that lysionotin can inhibit the expression of 5-LO (P<0.05) and downstream leukotriene B4 receptor (P<0.01). In addition, the results showed that lysionotin affected intracellular calcium ion concentration by inhibiting 5-LO to affect the cytoskeleton, as determined by flow cytometry and fluorescence labeling.
CONCLUSIONS: Lysionotin binds to 5-LO could suppress glioma by inhibiting arachiodonic acid metabolism pathway.
摘要：
目的：探讨溶解素治疗脑胶质瘤的潜在机制。
方法：首先，基于BernoulliNaiveBayes分析和途径富集的靶标预测用于预测裂解素的生物活性。通过表面等离子体共振(SPR)和分子对接检测5-脂氧合酶(5-LO)与裂解素的结合,利用体外酶抑制试验和细胞活力分析，确定了裂解素对5-LO和胶质瘤增殖的抑制作用，分别。此外，通过细胞存活率分析和液相色谱-串联质谱（LC-MS/MS）研究了溶素的药理作用。蛋白质表达，Westernblot显示细胞内钙离子浓度和细胞骨架检测,流式细胞术和荧光标记，分别。
结果：目标预测和途径富集表明，溶解素抑制5-LO，参与花生四烯酸代谢途径的关键酶，抑制胶质瘤的增殖。分子对接结果表明，5-LO可以通过氢键与裂解素结合，与His600、Gln557、Asn554和His372形成债券。SPR分析进一步证实了5-LO与溶素之间的相互作用。此外，体外酶抑制试验和细胞存活率分析表明，50%抑制浓度的溶解素和中位有效浓度的溶解素分别为90和16.58μmol/L，分别,LC-MS/MS结果表明，溶素抑制了5S-氢过氧二十碳四烯酸的产生（P<0.05），而且,LC-MS/MS结果表明，溶解素可以很好地进入胶质瘤细胞（P<0.01）并抑制其增殖。Westernblot分析显示,细胞溶解素能抑制5-LO(P<0.05)和下游白三烯B4受体的表达(P<0.01)。此外,结果表明，细胞溶解素通过抑制5-LO影响细胞骨架，从而影响细胞内钙离子浓度，通过流式细胞术和荧光标记确定。
结论：Lysionotin结合5-LO可能通过抑制花生四烯酸代谢途径抑制胶质瘤。
方法：首先，基于BernoulliNaiveBayes分析和途径富集的靶标预测用于预测裂解素的生物活性。通过表面等离子体共振(SPR)和分子对接检测5-脂氧合酶(5-LO)与裂解素的结合,利用体外酶抑制试验和细胞活力分析，确定了裂解素对5-LO和胶质瘤增殖的抑制作用，分别。此外，通过细胞存活率分析和液相色谱-串联质谱（LC-MS/MS）研究了溶素的药理作用。蛋白质表达，Westernblot显示细胞内钙离子浓度和细胞骨架检测,流式细胞术和荧光标记，分别。
结果：目标预测和途径富集表明，溶解素抑制5-LO，参与花生四烯酸代谢途径的关键酶，抑制胶质瘤的增殖。分子对接结果表明，5-LO可以通过氢键与裂解素结合，与His600、Gln557、Asn554和His372形成债券。SPR分析进一步证实了5-LO与溶素之间的相互作用。此外，体外酶抑制试验和细胞存活率分析表明，50%抑制浓度的溶解素和中位有效浓度的溶解素分别为90和16.58μmol/L，分别,LC-MS/MS结果表明，溶素抑制了5S-氢过氧二十碳四烯酸的产生（P<0.05），而且,LC-MS/MS结果表明，溶解素可以很好地进入胶质瘤细胞（P<0.01）并抑制其增殖。Westernblot分析显示,细胞溶解素能抑制5-LO(P<0.05)和下游白三烯B4受体的表达(P<0.01)。此外,结果表明，细胞溶解素通过抑制5-LO影响细胞骨架，从而影响细胞内钙离子浓度，通过流式细胞术和荧光标记确定。
结论：Lysionotin结合5-LO可能通过抑制花生四烯酸代谢途径抑制胶质瘤。
