4-hydroxyderricin Promotes Apoptosis And Cell Cycle Arrest In Hepatocellular Cells Through Regulating PI3K/AKT/mTOR Pathway

Objective: Abnormal activation of phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/AKT/m TOR)signaling pathway is closely related to the occurrence of liver cancer cells,providing targeted therapy for its potential targets.At present,the main chalcone active ingredient 4-hydroxydrisin(4-HD)in Angelica keiskei,recognized as a new food ingredient in China,regulates the PI3K/AKT/m TOR signaling pathway and participates in the anti-liver cancer effect.However,its mechanism has not been reported yet.This study aims to study the effects of4-HD on inducing apoptosis and cell cycle arrest of liver cancer cells by regulating the PI3K/AKT/m TOR signaling pathway to inhibit the proliferation.Method: First,the inhibitory effect of 4-HD on human liver cancer cells Hep G2 and Huh7 was verified.Different concentrations of 4-HD(20 μM and 40 μM)were incubated with Huh7 and Hep G2 cells for different periods of time(24 h and 48 h).The CCK-8method was performed to detect the effect of 4-HD on the viability of liver cancer cells.The wound healing and Transwell(Matrigel)experiments were employed to detect the changes of 4-HD on the migration and invasion of liver cancer cells.TUNEL experiment was utilized to verify liver cancer cells death.Flow cytometry was used to analyze the apoptosis and cycle changes of HCC cells treated with 4-HD.Western blot experiment was carried out to detect the expression of apoptosis protein,cyclin and PI3K/AKT/m TOR signaling pathway protein after 4-HD treatment.Immunofluorescence experiment was implemented to verify the expression of signaling pathway protein p-AKT.The inhibitory effect of 4-HD on PI3K/AKT/m TOR signaling pathway was further assessed by the combination of PI3 K specific inhibitor LY294002.The CCK-8experiment was employed to screen the appropriate 4-HD concentration.Western blot and immunofluorescence assays were carried out to detect the effect of the inhibitor combination group on PI3K/AKT/m TOR signaling pathway in liver cancer cells.The wound healing and Transwell(Matrigel)experiments were implemented to detect the migration and invasion ability of HCC cells after treated with inhibitor combination group.Flow cytometry was used to analyze the cell cycle arrest and apoptosis of HCC cells treated with inhibitor combined with treatment group,and Western blot was utilized to verify the results.Each experiment was repeated three times independently,and SPSS17.0 software was used for statistical analysis.Results: The results of CCK-8 show that 4-HD inhibited the proliferation of liver cancer cells in a dose-dependent manner.Compared with the normal group,the proliferation of Huh7 and Hep G2 was significantly surpressed by 4-HD(20 μM and 40 μM)after treatment for 48 h(**p<0.01).Wound healing experiment results show that with the increase of 4-HD dose,the cell scratch distance was decreased.Transwell experiment results show that,compared with the normal group,the number of cells entering the lower chamber of Transwell was decreased significantly after 4-HD treatment(**p<0.01),and the inhibitory effect was dose-dependent,indicating that 4-HD can effectively attenuate the invasion ability of Huh7 and Hep G2 cells.The results of flow cytometry show that4-HD at 20 μM and 40 μM on Huh7 and Hep G2 cells for 48 h can significantly induce HCC cells apoptosis(**p<0.01)and cause cell cycle arrest.Hep G2 cells were blocked in G2 phase,while Huh7 cells were blocked in G1 phase.Western blot results confirm that4-HD significantly increased the activation of apoptosis-related proteins caspase-3 and PARP,and decreased the protein expression of cyclin in a dose-dependent manner(**p<0.01).Western blot results also show that the protein expressions of PI3 K,p-PI3 K,p-AKT,and p-m TOR were decreased,while the results of immunofluorescence experiments reflect that p-AKT expression was decreased,suggesting that 4-HD caused the down-regulation of PI3K/AKT/m TOR signaling pathway.CCK-8 assay show that the viability of cells cultured in combination with 4-HD and LY294002 was significantly lower than that in the 4-HD group alone.The results of wound healing and Transwell experiments show that LY294002 weakened the migration and invasion ability of 4-HD on HCC cells.The western blot results show that the protein expressions of PI3 K,p-PI3 K,p-AKT,and p-m TOR in the 4-HD+LY294002 group were significantly lower than those in the 4-HD group alone.The results of flow cytometry show that,compared with the4-HD group,the 4-HD+LY294002 group significantly induced cell apoptosis and cell cycle arrest.Moreover,western blot results show that 4-HD+LY294002 group up-regulated the expression of pro-apoptotic related proteins,and down-regulated the expression of cyclin,compared with 4-HD group.Conclusion: 4-HD regulates the PI3K/AKT/m TOR signaling pathway to induce apoptosis and cell cycle arrest to affect the proliferation,apoptosis,migration and invasion of HCC cells.And the inhibitory effect of 4-HD on HCC cells was dose-dependent.The results of this study will provide new insights for in-depth exploration of the mechanism of 4-HD inhibiting liver cancer.