Research On Anti-tumor Activity And Its Underlying Molecular Mechanism Of Novel Spermine Oxidase Small Molecule Inhibitor SI-4650

Objective Spermine Oxidase（SMO）is an amine oxidase which participates in the catabolism of polyamine and its abnormal expression is closely related to the occurrence and development of various diseases including tumors,thus becomes a new molecule target for tumor prevention and anti-tumor drug design.SI-4650（SMO Inhibitor-4650）is a new type of SMO small molecule inhibitor,which was newly designed and screened out by computer-assisted drug design technology combined with high-throughput virtual screening technology based on pharmacophore in our laboratry.Previous studies have pointed out that SI-4650 can inhibit SMO enzyme activity effeciently and has the pharmacological activity in killing human non-small cell lung cancer A549 cells.In this study,human osteosarcoma143B cells,human melanoma A375 cells and human glioma U87MG cells were used as research models to further investigate the effects of SI-4650 on the proliferation and migration of above three tumor cell lines and their underlying molecular mechanisms.Methods（1）MTT assay was used to analyze the effect of SI-4650 on proliferation ability of tumor cell;（2）PI-staining combined with flow cytometry（FCM）was used to analyze the effect of SI-4650 on cell cycle;（3）Chemiluminescence assay was used to determine the inhibitory effect of SI-4650 on SMO activity in tumor cells;（4）High performance liquid chromatography（HPLC）assay was performed to detect the contents of SI-4650 and polyamines in the tumor cells;（5）DCFH-DA probe combined with FCM assay was used to analyze the effect of SI-4650 on the accumulation of cellular reactive oxygen species（ROS）;（6）Inverted microscope was used to observe the effect of SI-4650 on the morphology of tumor cells;（7）Transwell assay was used to evaluate effect of SI-4650 on tumor cell migration ability;（8）PI/FITC-Annexin V double-staining combinded with FCM was used to detect the apoptosis/necrosis rate of tumor cells;（9）DIOC6（3）probe combinded with FCM was perfomed to evaluaste the effect of SI-4650 on the mitochondrial membrane potential of the tumor cells;（10）Inverted fluorescence microscopy/laser confocal microscopy（LSCM）were used to detect the effect of SI-4650 on autophagosome formation in tumor cells;（11）Changes of migration-,invasion-,apoptosis-and autophagy-related protein expression levels induced by SI-4650 in the tumor cells were detected by Western blot assay.Results（1）MTT results revealed that SI-4650 could inhibit the proliferation of 143B cells,A375 cells and U87MG cells significantly,and under 48 hours of treatment,its IC₅₀ for proliferation inhibition of the three tumor cells were（79.85±3.32）μmol/L,（90.48±6.99）μmol/L and（303.475±27.94）μmol/L respectively.What’s more,the inhibition rates of the cell proliferation were positively correlated with the concentration and treatment time of SI-4650;（2）SI-4650 could induce cell cycle arrestment and made cell cycle be blocked in S phase（143B cells,A375 cells）and G0/G1 phase（U87MG cells）respectively;（3）SI-4650could significantly inhibit SMO activity in three kinds of tumor cells in a concentration-dependent manner;（4）SI-4650 could get inside the tumor cells,effectively interfere with the polyamine metabolism and reduce the total polyamine content in tumor cells;（5）Treating the tumor cells with SI-4650 reduced the accumulation level of cellular reactive oxygen species;（6）SI-4650 could inhibit the migration ability of the three kinds of tumor cells significantly,which may be related to increasing cell adhesion,reducing cell motility and inhibiting tumor cell’s mesenchymal phenotype transformation;（7）After treated by SI-4650for 48 hours,the number of PI/Annexin-V double positive cells,the expression levels of c-Caspase 3 and c-PARP,and Bax/Bcl-2 ratio increased,but the mitochondrial membrane potential decreased significantly in the three kinds of tumor cells,suggesting that SI-4650can induce apoptosis of these tumor cells;（8）After treatment with SI-4650 for 48 hours,the intracellular GFP-LC3 fusion protein converted from diffuse distribution to spotted distribution,in the same time,the expression levels of autophagic activation protein Beclin-1 and autophagic tubulin LC3-Ⅱincreased significantly,and the expression level of P62protein was up-regulated（143B and A375 cells）or down-regulated（U87MG cells）;Pre-treatment with 3-MA partially antagonized the autophagy induction by SI-4650 in 143B cells,suggesting that SI-4650 can induce autophagic cell death effectively.Conclusion SI-4650 has a pharmacological activity against human osteosarcoma 143B cells,human melanoma A375 cells and human glioma U87MG cells,and its underlying mechanism may be related to interfering polyamine metabolism and inducing cell cycle arrest,as well as cell apoptosis and autophagy.The results of this study suggest that SI-4650has important potential application value in the treatment and research of the related tumors.