Study On The Antiglioma Effect Of F2 Isolated From Grape Seeds And Its Mechanism

Gliomas are the most common and lethal tumor type in the brain.Based on the previous finding that FPR may represent a molecular target for the development of novel anti-glioma therapeutics,we established a drug screening platform taking FPR as a target and finally discovered the target compound F2 through a wide screen.The present study investigated the effect of oligomer procyanidins(F2)(F2,degree of polymerization 2-15),a natural fraction isolated from grape seeds on the biological behavior of glioblastoma cells.It was found for the first time that F2 in non-cytotoxic concentrations selectively inhibited U-87 cell chemotaxis/migration mediated by a G-protein coupled receptor formyl peptide receptor FPR,which is implicated in tumor cell invasion and metastasis.Further experiments indicated that F2 inhibited fMLF-induced U-87 cell calcium mobilization and MAP kinases ERK1/2 phosphorylation.Moreover,F2 attenuated the glioblastoma FPR expression,a new molecular target for glioma therapeutics,which has been shown to play important roles in glioma cells chemotaxis,proliferation and angiogenesis in addition to its promotion to tumor progression,but did not affect FPR mRNA expression in U-87 cells.To further explore the relationship and molecular mechanisms of F2 involved in FPR function,effects of F2 on FPR desensitization in cultured ETFR cells and human monocytes were investigated.It was found that F2 was not chemotactic for monocytes or ETFR cells and preincubation of ETFR cells with F2 did not reduce cell surface expression of FPR, suggesting that F2 is not a pure agonist for FPR.Furthermore,F2 potently inhibited ETFR cells response to fMLF.In addition,it was found that F2 induced calcium flux in human monocytes and reduced cell response to fMLF and MCP-1,however,fMLF and MCP-1 had little effect in reducing monocyte response to F2,suggesting that F2 is biologically active and is capable of inhibiting cell responses to chemoattractants.In conclusion,these results suggest that F2 is biologically active and could inhibit the function of G-protein coupled receptors used by chemoattractants.In addition,we found that F2 significantly inhibited the glioblastoma growth,with little cytotoxicity on normal cells,induced G2/M arrest and decreased mitochondrial membrane potential in U-87 cells.It also induced a non-apoptotic cell death phenotype resembling paraptosis in U-87 cells.Similarly,it was found that F2 markedly reduced the cell viability of human glioma cells U-251,Tj-899 and Tj-905,rat glioma cell line C6,human hepatoma cells Hep3B,human colonic cancer cell line colon-205 and human promyelocytic leukemia cells HL-60,suggesting its wide anti-tumor spectrum.We then further investigated the death mode of human glioblastma cells induced by F2 and gained insight into the nature of the signaling pathways activated by F2 in glioblastoma cells.Morphology studies revealed extensive cytoplasmic vacuolation in dying cells and no apoptotic body formation,membrane bleb formation or nuclear fragmentation,though some degree of chromatin condensation occurred.Characteristically,cell death triggered by F2 was accompanied by MAPKs activation,new protein synthesis and independent of caspases activation.Moreover,we demonstrated the involvement of calcium mobilization in F2-induced U-87 cell signaling.To clarify the mechanism underlying glioma cell death upon F2 exposure,the cytotoxicity of F2 in U-87 and C6 cancer cell lines was evaluated,and changes of mitochondrial transmembrane potential(MMP) and production of reactive oxygen species (ROS) in drug-treated cells were monitored.Moreover,morphological changes associated with F2-induced cells death were examined.The results showed that F2 induced a concentration-dependent increase in ROS production and decrease in mitochondrial membrane potential.Furthermore,pre-incubation with N-acetylcysteine(NAC) and rotenone,resulted in partial inhibition of F2-induced ROS generation and marked attenuation of cell death and the cytoplasmic vacuolization induced by F2 but not necessarily attenuation of the loss of MMP.In conclusion,ROS production was correlated with F2-induced glioma cell death as well as the cytoplasmic vacuolization formation. These results suggested that the increase in ROS level might be an important mechanism that might contribute to the cytotoxicity of F2 in glioma cells.It was also found that noncytotoxic concentrations of F2 were able to significantly induce the migration of U-87 cells to its higher concentrations,which helped to exert the cytotoxic effect of F2. In conclusion,our results suggest that F2 may be a promising candidate for the development of novel anti-tumor therapeutics.