FFJ-3 Regulates The Growth Of Cancer Cells Through Inhibiting The EGFR-PI3K/Akt-PKM2 Pathway

Objective: To explore the anti-tumor activity and mechanism of FFJ-3, one of structural modifications of Mollugin.Methods: MTT assay was used to detect the effect of FFJ-3 on HepG2, MCF7, A549 cell proliferation; The effect of FFJ-3 on cell apoptosis was performed by Hoechst 33342/PI double staining. Cell cycle was detected with flow cytometry; EGFR, p-EGFR, Akt, p-Akt, PKM-2 and PARP, Cleaved PARP, Caspase3, Cleaved Caspase3 protein expression were determined by western blot; The regulatory roles of PI3K/Akt pathway on PKM2 was detected in MCF cells treated with LY294002 or insulin with western blot and real-time PCR. Rh123 staining was used to investigate the changes of mitochondrial membrane potential.Results:(1) MTT results showed that FFJ-3 inhibited Hep G2, MCF7 and A549 cell proliferation at a concentration-dependent manner. The IC50 values of HepG2, MCF7 and A549 cells were 26.46±0.20 μmol?L-1, 26.99 ± 0.25 μmol?L-1, 33.93 ± 3.74 μmol?L-1 respectively.(2) Hochst 33342 / PI double staining results showed that FFJ-3 could does-dependently increase cell apoptosis rate of HepG2 MCF7 and A549 cells and obvious morphological characteristics of apoptosis was observed.(3) Flow cytometry results showed that FFJ-3 could arrest cell cycle in G2 / M phase and G1 phase in HepG2 and A549 cell respectively.(4) Western blot results showed that FFJ-3 was able to down-regulate the expression of EGFR, p-EGFR, Akt, p-Akt, PKM2 protein in HepG2, MCF7 and A549 cells at a dose-dependent manner.(5) Western blot results showed that LY294002 could up-regulate the protein expression of Akt,down-regulate p-Akt and PKM2 at a time and dose dependent manner; insulin could decrease the expression of Akt protein, increase the expression of p-Akt and PKM-2 protein at a time and dose dependent manner. The results suggested PI3K/Akt pathway can regulate the expression of PKM2 protein.(6) Real-time PCR results showed that LY294002 and insulin can not affect the PKM2 mRNA level.(7) Rh123 results showed that FFJ-3 could significantly reduce mitochondrial membrane potential in HepG2, A549 and MCF7 cells at a dose-dependent manner.(8) Western blot results also showed that FFJ-3 was able to increase the expression of Cleaved PARP, Cleaved Caspase3, Bax protein and decrease Bcl-2 protein at a dose-dependent manner.Conclusion:(1)FFJ-3 had obvious antitumor activity.(2)FFJ-3 inhabited cell proliferation via EGFR-PI3K/Akt-PKM2 signaling pathway.(3)FFJ-3 induced cell apoptosis through the mitochondrial apoptosis pathway.