Effect Of Oleandrin On Proliferation And Invasion Of HepG2 Hepatoma Cells

Objectives:Hepatocellular Carcinoma(HCC)accounts for 85%-90% of primary liver cancer,and it is one of the most threatening cancers to human health worldwide.At present,the treatment of liver cancer is mainly surgical resection,local ablation,combined with chemotherapy and radiotherapy.Due to the lack of typical clinical manifestations in the early stage of liver cancer,most of the patients have entered the middle and late stages when they are found,resulting in a low prognosis and survival rate of liver cancer patients.Oleander is a common ornamental shrub,widely distributed in tropical and subtropical areas.Oleandrin is a cardiac glycoside compound extracted from Oleander,which has a strong effect on the heart.Evidence from recent studies has highlighted the potential of its anti-cancer properties,including but not limited to many different types of cancers such as breast cancer,lung cancer,pancreatic cancer,prostate cancer,colorectal cancer.It remains unclear whether oleandrin has anti-HCC function and which regulatory mechanisms are involved.Therefore,the aim of this study is to investigate the effects of oleandrin on the proliferation,migration,invasion,apoptosis and cell cycle of human hepatocellular carcinoma Hep G2 cells,and to explore the possible mechanism of oleandrin inducing apoptosis and regulating cell proliferation of human hepatocellular carcinoma Hep G2 cells.Methods:The effect of oleandrin on the proliferation of Hep G2 cells was detected by CCK-8 method.The IC50 value of oleandrin on Hep G2 cells was calculated,and the corresponding drug concentration was selected for subsequent experiments.Cell clone formation assay was used to test the effect of oleandrin on the proliferation of Hep G2 cells.Cell scratch assay was used to verify the effect of oleandrin on Hep G2 cell migration.Transwell assay was used to verify the effect of oleandrin on Hep G2 cell invasion.The apoptotic cells in the Hep G2 cells treated with oleandrin were labeled by terminal deoxynucleotidyl transferase-mediated dut P Nick end labeling(Tunel)and observed under a fluorescence microscope.The effect of oleandrin on the apoptosis of Hep G2 cells was detected by flow cytometry.Western blot was used to detect the expression of apoptotic proteins and cyclin in Hep G2 cells with and without oleandrin treatment.Results:1.First,the IC50 value of oleandin in the treatment of Hep G2 cells was determined to be 23.78 n M,and the following experiments were established to use 20 n M,25 n M and 30 n M concentrations of oleandin to treat Hep G2 cells.CCK-8 results showed that the OD values of the experimental groups were lower than those of the control group,and showed an inverse concentration dependence,that is,the cell survival rate of the experimental group was lower than that of the control group.2.The results of cell colony formation assay showed that the cell clusters formed in the experimental group were less than those in the control group,and with the increase of oleandrin concentration,the cell clusters formed by cell proliferation were less and smaller.3.The results of cell scratch test showed that the scratch healing rate of the experimental group was significantly lower than that of the control group,and with the increase of oleandrin concentration,the scratch healing rate of the experimental group was lower,that is,the cell migration rate of the experimental group was lower than that of the control group.4.The results of Transwell experiment showed that Hep G2 cells in the control group and the experimental group could pass through the matrix glue,but with the increase of oleandrin concentration,fewer and fewer Hep G2 cells passed through the matrix glue,that is,the cell invasion rate of the experimental group was lower than that of the control group.5.The results of in situ terminal apoptosis assay and flow cytometry showed that the number of apoptotic cells in the experimental group was more than that in the control group,and it was concentration-dependent,that is,the apoptosis rate of the experimental group was higher than that of the control group.6.Western blot results showed that the expression levels of apoptosis-related proteins Bim,Bax,Caspase-3,Caspase-8 and Caspase-9 in Hep G2 cells in the experimental group were higher than those in the control group,and the expression levels increased with the increase of drug concentration.The expression level of Bcl-2protein was lower than that of the control group,and the expression level decreased with the increase of the concentration.The expression levels of cell cycle-related proteins Cyclin-D1 and Cyclin-D3 in Hep G2 cells in the experimental group were lower than those in the control group,and the expression levels decreased with the increase of the concentration.Conclusions:1.Oleandrin inhibited the proliferation,migration and invasion of Hep G2 cells in a dose-dependent manner;Oleandrin can induce apoptosis of Hep G2 cells.2.Oleandrin could promote the expression of apoptosis-related proteins Caspase-3,Caspase-8,Caspase-9,Bax,Bim and inhibit the expression of Bcl-2 in Hep G2 cells.3.Oleandrin prevented the transition of Hep G2 cells from G1 phase to S phase by inhibiting the expression of Cyclin-D1 and Cyclin-D3,resulting in the arrest of Hep G2 cells in G1 phase,thereby inhibiting the proliferation of Hep G2 cells.