Cell Cycle Arrest And Apoptosis Induced By (5-hydroxy-4-oxo-4H-chromene-3-yl)-benzoyl Hydrazone In HepG2 Cells

Subject: To investigate the mechanism of proliferation inhibition and apoptosis effect on HepG2 hepatoma cells induced by a novel flavonoid complex, named as (5-hydroxy-4-oxo-4H-chromene-3-yl)-benzoyl hydrazone (L1b for shot).Method: HepG2 cells were treated with L1b at different concentrations and different periods. The proliferation inhibition effect evaluated by SRB method and colony forming test. Then observing the morphology change under phase-contrast microscope. DNA damages were detected by electrophoresis. Analyzing the proliferation cycle by flow cytometry. Apoptosis effect was detected by fluorescence microscope via AO/EB double stain. And electron transmission microscope was used to reveal the apoptosis effect also. Western blot analysis was performed to determine the contents of MDM2, P53 and P21.Result: L1b can suppress the HepG2 and L02 cells proliferation dominantly in vitro,and the IC₅₀ values of HepG2 cells exposed to L1b for 24h, 32h and 48h were 109.67, 67.11, 31.18μg/ml, while the values of L02 cells were 90.38, 56.36, 30.48μg/ml. The HepG2 cells' colony forming ability was suppressed by L1b significantly. Inhibited proliferation and abnormal morphology were seen under the phase contrast microscope. Electrophoresis revealed that L1b can cause the DNA damage after 24h and 48h treatment. Flow cytometric analysis through PI stain showed L1b can arrest cell cycle at G₁ phase & G₂ phase, and increase the percentage of apoptosis up to 10.3%. The photos gained from fluorescence microscope and electron transmission microscope showed apoptosis induced by L1b. Under the stress of L1b, the contents of P53 and P21 were increasing significantly, while the content of MDM2 was stable.Conclusion: L1b can suppress the HepG2 and L02 cells proliferation and colony forming ability significantly in vitro. L1b arrest cell cycle at G1 phase & G2 phase via P53-P21 pathway. L1b induce HepG2 cells apoptosis via increasing the expression of p53 gene.