The Research On Anti-Tumor Activity And Mechanisms Of FFJ-5

Objective: To explore the anti-tumor activity of FFJ-5 in vitro, one of structural modifications of mollugin, and the effect of FFJ-5 on EGFR-Akt-PKM2 signal pathways.Methods: MTT assay was used to detect the effects of FFJ-5 and mollugin on the cell viability of A549 and HepG2 cells; Flow cytometry was used to analyze the cell cycle of A549 and HepG2 cells; Hoechst 33342 / PI double staining was performed to detect the cell apoptosis of A549 and HepG2 cells. ATP Assay Kit was used to detect ATP production of A549 and HepG2 cells; The pH in A549 and HepG2 cells was measured by Confocal Scanning Laser Microscopy after incubating BCECF- AM(p H fluorescent probe); The levels of proteins expression of PKM-2, EGFR, p-EGFR, Akt, p-Akt and PARP, Cleaved PARP, Caspase3, and Cleaved Caspase3 proteins expression were determined by western blot; The change of mitochondrial membrane potential(MMP) was analyzed using Rh123 staining with Confocal Scanning Laser Microscopy.Results:(1) MTT results showed that FFJ-5 possessed significantly anti-cancer activity. The IC50 of Mollugin is 7.8 times and 5.6 times of FFJ-5 in A549 and HepG2 cells respectively.(2) Flow cytometry results showed that FFJ-5 could arrest cells cycle in G2 / M phase in A549 and HepG2 cells respectively.(3) Hochst 33342 / PI double staining results showed that FFJ-5 could induce cell apoptosis at a dose-dependent manner.(4) Western blot results showed that in A549 and HepG2 cells, FFJ-5 was able to down-regulate the expression of PKM2, EGFR, p-EGFR, Akt and p-Akt proteins at a dose-dependent manner.(5) ATP Assay results showed that FFJ-5 could decrease ATP production in A549 and HepG2 cells.(6) FFJ-5 could decrease the intracellular pH in cancer cells.(7) Rh123 results showed that FFJ-5 could significantly decrease mitochondrial membrane potential at a dose-dependent manner in A549 and HepG2 cells.(8) Western blot results also showed that FFJ-5 was able to increase the expression of Cleaved PARP, Cleaved Caspase3 protein at a dose-dependent manner in A549 and HepG2 cells.Conclusion:(1)FFJ-5 could significantly inhibit the growth and proliferation in human cancer cells, and arrest cell cycle in G2 / M phase.(2)FFJ-5 decreased ATP production and intracellular pH level in human cancer cells by inhibiting PKM2 expression.(3) FFJ- 5 could inhibit proliferation and induce apoptosis in human cancer cells by inhibiting EGFR-Akt- PKM2 signal pathways.