Study On The Effect Of Ginsenoside 24-OH-PD From Red Ginseng Induces Apoptosis In CCRF-CEM Cells

Objective:Acute T-lymphocytic leukemia(T-ALL)is a malignant tumor disease with the highest incidence rate in childhood.Ginsenosides are the main effective components in many rare Chinese medicinal materials.Ginsenoside 24-hydroxy-ginsengdiol(24-OH-PD),extracted from red ginseng,strongly inhibits the growth of human T-cell acute lymphoblastic leukaemia(T-ALL)CCRF-CEM cells.Thus,we aimed at investigating the mechanism underlying this inhibition.Methods:Cell viability of CCRF-CEM cells treated with different concentrations was determined using the Cell Counting Kit-8 assay(CCK-8).The CCRF-CEM cell-bearing NOD/SCID mouse model was used to verify the effect of 24-OH-PD in the treatment of T-ALL in vivo.We equally analysed pathways related to 24-OH-PD in CCRF-CEM cells using RNA-Seq analysis.Cell apoptosis of CCRF-CEM cells were determined using Hoechst 33342/Annexin V-FITC/PI staining and flow cytometry,respectively.The activity of caspase3 and caspase9 was detected by enzyme activity detection kits.The expression of differentially expressed genes(DEGs)Caspase-3,Caspase-8,Caspase-9,Bax and P53 was determined through quantitative reverse-transcription PCR assays(q RT-PCR).The levels of apoptosis-related proteins Caspase-3,Caspase-8,Caspase-9,Bax,Bcl-2,AIF,Cytc and P53 were determined through Western blotting.Caspase-3 and Caspase-9 activities were detected by enzyme activity detection kit.Reactive oxygen species(ROS),mitochondrial membrane potential(ΔΨm),and mitochondrial permeability transition pore(m PTP)levels were determined using flow cytometry and laser scanning confocal microscopy.The changes of cell viability,apoptosis and ROS generation were determined using the antioxidant NAC.Results:Our results showed that 24-OH-PD significantly reduced the viability of CCRF-CEM cells in a concentration dependent manner.Meanwhile,xenograft animal studies have confirmed that 24-OH-PD can also significantly inhibit the development of T-ALL in vivo.The results of transcriptome analysis showed that the apoptosis-related pathways were significantly up-regulated in CCRF-CEM cells after 24-OH-PD treatment,and were closely related to mitochondrial pathways.The results of immunofluorescence and flow cytometry showed that 24-OH-PD could significantly induce CCRF-CEM cells apoptosis.The mitochondrial pathway was activated during this process.Flow cytometry and immunofluorescence results showed that MPTP was opened and ΔΨm was decreased after 24-OH-PD treatment.Further examination of the key genes in the mitochondrial apoptosis pathway showed that 24-OH-PD treatment increased the expression of pro-apoptotic protein Bax and decreased the expression of anti-apoptotic protein Bcl-2,resulting in the release of cytochrome c(Cytc)and apoptosis-inducing factor AIF from the mitochondria to the cytoplasm.Furthermore,it increases the expression level of Caspase family Caspase-3,Caspase-8 and Caspase-9,leading to cell apoptosis.Meanwhile,ROS level was increased,and the effects of 24-OH-PD on cell viability,apoptosis and ROS production were significantly reversed by pretreatment with antioxidant NAC,indicating that ROS-mediated oxidative stress is the main pathway of24-OH-PD-induced apoptosis in CCRF-CEM cells.Conclusion:Studies have shown that 24-OH-PD has a good anti-T-ALL activity and can significantly inhibit the tumor activity in vitro and in vivo without obvious side effects.The inhibitory effect of 24-OH-PD on T-ALL is mainly through the activation of the ROS-mediated endogenous mitochondria-dependent apoptotic pathway,triggering apoptosis in T-ALL cells.At the same time,the expression levels of Bax,Cytc,AIF and Caspase family Caspase-3,Caspase-8 and Caspase-9 were significantly up-regulated during the process.These results suggest that 24-OH-PD May be further developed as a candidate drug for clinical treatment of T-ALL patients,and this study has laid a solid theoretical foundation for its development.