Small Molecule Inhibitor XYA-2 Inhibits The Growth And Metastasis Of Gastric Cancer By Targeting The STAT3 Signaling Pathway

Objective Gastric cancer is one of the leading causes of cancer-related deaths worldwide.Advanced gastric cancer has a high incidence and poor prognosis after traditional chemotherapy in China.The development of STAT3 inhibitors has been intensely pursued.This study aimed to elucidate the interaction between the XYA-2 and the STAT3 signaling pathway,and further clarify its mechanism and pharmacological activity.Methods A molecular docking and structure-based virtual screening assay was performed against an in-house library of natural products to identify small molecule inhibitors for STAT3.The direct interaction between XYA-2 and STAT3 protein was verified by SPR.Western blot was used to detect the phosphorylation level and downstream protein expression of the STAT3 signaling pathway.An immunofluorescence assay was performed on MGC803 and MKN28 to detect the nuclear translocation of STAT3.CCK8 assay was performed on gastric cancer cell lines MGC803,AGS,SGC7901,BGC823,MKN28,HGC27,and SNU-1 to detect cell proliferation.A colony formation experiment was performed on MGC803 and MKN28 to detect cell colony formation.A wound healing experiment was performed on MGC803 and MKN28 to detect cell migration.The MKN28 subcutaneous xenograft mouse model was constructed to monitor the effects of XYA-2 on tumor growth and bodyweight of mice.The MGC803 gastric cancer orthotopic mouse model was constructed to monitor the effects of XYA-2 on tumor growth,metastasis,and bodyweight of mice.H&E staining was performed on the main organs of both mouse models to observe the pathological changes.STAT3 knockdown MGC803 cell line was constructed to assess the role of STAT3 in XYA-2’s anti-gastric cancer activity.Results Molecular docking results predicted that XYA-2 may form hydrogen bonds with Ser611 and Ser613 sites in the key pY pocket of STAT3 SH2 domain.SPR results confirmed the direct interaction between XYA-2 and STAT3.XYA-2 significantly inhibited the phosphorylation of STAT3 in MGC803 and MKN28.XYA-2 significantly inhibited the nuclear translocation of STAT3 and subsequently inhibited the expression of the downstream protein c-Myc.Furthermore,XYA-2 inhibited STAT3 phosphorylation at Tyr705 in MGC803 orthotopic mouse model.XYA-2 significantly inhibited the proliferation of gastric cancer cell lines in a concentration-time-dependent manner,and the IC50 values of all cell lines were lower than 1 μM after 48 h and 72 h treatment of XYA-2.XYA-2 significantly inhibited the colony formation and migration of MGC803 and MKN28 cells in a concentration-dependent manner.XYA-2 significantly suppressed tumor growth in MKN28 subcutaneous xenograft mouse model and MGC803 orthotopic mouse model.Additionally,XYA-2 inhibited tumor metastasis of MGC803 orthotopic mouse model.No obvious in vivo side effects were observed.Knockdown of STAT3 reversed the inhibitory effect of XYA-2 on the proliferation of MGC803,indicating STAT3 plays an important role in anti-gastric cancer effects of XYA-2.Conclusion XYA-2 inhibits the phosphorylation and activation of STAT3 at Tyr705 by directly binding to the SH2 domain of STAT3,interferes with its dimerization and nuclear translocation,and further inhibits the expression of downstream protein c-Myc.XYA-2 inhibited cell proliferation,colony formation,and migration in vitro.XYA-2 suppressed tumor growth in MKN28 subcutaneous xenograft mouse model and suppressed tumor growth and metastasis in MGC803 orthotopic mouse model without obvious side effects.STAT3 plays an important role in the pharmacological effects of XYA-2.