PFK15 Exhibits Anti-tumor Effects Against Gastric Cancer And Its Potential Mechanisms Of Action

PFKFB3 is a key regulator of high glycolytic flux in cancers by catalyzing the synthesis of F2,6P2 which allosterically activates PFK-1,the rate-limiting enzyme of glycolysis The objective of this study was to investigate the antitumor activity of PFK15,a small molecule inhibitor of PFKFB3,against gastric cancer and to explore its potential mechanisms.The combined effects on angiogenesis of PFK15 with sorafenib were also tested.The protein expressions of PFKFB3 in gastric tumors and endothelial cells,and the on-target metabolic effects of PFK15 were first confirmed.Then the effects of PFK15 on proliferation,apoptosis and cell cycle progression in gastric cancer cells were evaluated by cytotoxicity and apoptosis assays and flow cytometry.Effects of PFK15 on cell migration and invasion were also measured by cytoskeleton assay,wound-healing and transwell invasion assay.In addition,the anti-angiogenic activities of PFK15 were examined by endothelial cell tube formation assays,endothelial cell spheroids vessel sprouting assay and matrigel plug assay.Two xenograft tumor models were used to verify the therapeutic effect of PFK15 in vivo.Western blot assay and immunohistochemical staining assay were used to detect the effects of PFK15 on protein expression levels and relative cell signaling pathways.Furthermore,combined effects of PFK15 with sorafenib on angiogenesis were evaluated.Results showed that PFKFB3 protein were overexpressed in gastric tumors and endothelial cells compared with the normal nude mouse stomach.PFK15 treatment dose-dependently reduced F2,6P2 levels and glucose uptake in gastric cancer cells and EA.hy926 cells,and the rescues experiment of F2,6P2 addition abolished the proliferation inhibition effect induced by PFK15 further supported the specificity of PFK15 against the PFKFB3.PFK15 inhibited cell proliferation,caused cell cycle arrest in G0/G1 phase by blocking the Cyclin-CDKs/Rb/E2F signaling pathway,and induced apoptosis through mitochondria pathway in gastric cancer cells.PFK15 also inhibited the the cell migration and invasion by decreasing the F-actin filaments numbers,disturbancing the cytoskeleton organization and altering cell adhesion related protein expressions.Tumor weights and tumor volume was significantly suppressed by PFK15 in AGS and MKN45 xenograft models.In addition,PFK15 significantly inhibited tube formation and vessel formation in Matrigel Plugs and impaired EC spheroid vessel sprouting.PFK15 also decreased the CD31 staining areas in AGS tumors by ICH analysis.Especially,PFK15 ampified the angiogenic effects of sorafenib,a VEGFR blocker in in vitro and in vivo models.Taken together,our findings indicate that PFK15 is a promising anticancer drug for treating gastric cancer by inhibiting cell proliferation and cell motility,inducing cell apoptosis,cell cycle block and anti-angiogenesis effects.And combination of anti-glycolysis inhibitor with VEGFR blocker may be an effective anticancer strategy and deserves further investigation.