The Mechanism Of Human Hepatocellular Carcinoma Cell Line SK-Hep-1 Proliferation,Apoptosis And Autophagy Was Induced By Pine Pollen

Objective: Pine Pollen has pharmacological effects such as protecting organs,regulating metabolism and enhancing immunity,however,its antineoplastic effect and mechanism is still unclear.In this study,the effects of Pine Pollen on the proliferation,apoptosis,cell cycle and autophagy of human hepatocellular carcinoma cell line SK-Hep-1 cells were observed,in order to further explore the anti-hepatoma effect of Pine Pollen and its mechanism,so as to provide theoretical and experimental basis for the clinical application of Pine Pollen.Methods: SK-Hep-1 cells were cultured in vitro,and were randomly divided into 4groups,including one control group(0 mg/m L Pine Pollen),and three experimental groups(30,45,60 mg/m L Pine Pollen).(1)The inhibition rates of SK-Hep-1 cells after interventing by different concentrations of Pine Pollen in different time(12,24,48 and 72 h)were detected by cell proliferation/toxicity test kit(CCK-8).(2)After 2 weeks of Pine Pollen interaction,the effects of Pine Pollen on SK-Hep-1 cells clone and proliferation were observed by flat cloning experiment.(3)Morphological change of SK-Hep-1 cells was observed by phase contrast microscope after Pine Pollen treatment for 48 h.(4)Hoechst 33258 fluorescence staining was used to detect the morphological change of apoptosis,and annexin V-FITC/ PI double staining combined with flow cytometry was used to observe the apoptosis.(5)Flow cytometry was used to detect change in cell cycle of SK-Hep-1 cells.(6)The expression levels of autophagy-related genes Beclin-1,LC3-?,and P62 m RNA were detected by RT-PCR.The expression levels of autophagy-related proteins Beclin-1,LC3-?,and P62 were detected by Western blot.(7)SPSS 23.0 statistical software was used for data processing.The measurement of data was expressed as the mean±standard deviation(mean±SD).Theinter-group comparison was compared using one-way ANOVA or non-parametric test,indicating that the difference was statistically significant in P <0.05.Results:(1)CCK-8 test showed that Pine Pollen was significantly inhibited the proliferation of SK-Hep-1 cells in a time and dose-dependent manner.(2)Flat cloning experiments showed that the number of colony formation decreased with the increase of Pine Pollen concentration.(3)The phase contrast microscope showed that Pine Pollen was significantly inhibited the proliferation of SK-Hep-1 cells.(4)Hoechst 33258 fluorescent staining showed that Pine Pollen was significantly facilitated cell apoptosis,in addition,Annexin V-FITC/ PI double staining showed that compared with the control group,the apoptosis rate of SK-Hep-1 cells increased significantly in different Pine Pollen concentration intervention groups(P <0.05 or P <0.01),and the apoptosis rate increased with the increase of drug concentration.(5)Cell cycle experiments showed that Pine Pollen blocked SK-Hep-1cells cycle in G1 phase.Besides,the cell proportion in S and G2 phase also decreased.(6)RT-PCR analysis showed that after treatment by Pine Pollen(30,45,60 mg/m L),the m RNA expression of Beclin-1,LC3-? was significantly increased than that in the control group(P<0.05 or P <0.01),while P62 was decreased(P <0.05 or P <0.01).The expression of autophagy-related proteins detecting by Western blot also reached the same conclusion,which further demonstrated the effects of Pine Pollen on autophagy on human hepatocellular carcinoma cell liner SK-Hep-1 cells.Conclusion:(1)Pine Pollen significantly reduced SK-Hep-1 cells proliferation capacity in a dose-dependent and time-dependent manner.Pine Pollen facilitated cell apoptosis and blocked cell cycle in G1 phase.(2)Pine Pollen enhanced autophagy on SK-Hep-1 cells and facilitate autophagy on human hepatocellular carcinoma cell line strains SK-Hep-1 cells.Its antineoplastic mechanism maybe to increase the expression of Beclin-1 and LC3-? on SK-Hep-1 cells,and reduce the expression of P62,and play an anti-hepatoma role by inducing autophagy.