Objective:

2

To explore the potential mechanism of lysionotin in treating glioma.

Methods:

2

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:

2

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 t

he 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.

Conclusion:

2

Lysionotin binds to 5-LO could suppress glioma by inhibiting arachiodonic acid metabolism pathway.