| BackgroundLeukemia is a common hematologic disease with a high incidence in all kinds of tumors.Leukemia has the characteristics of uncontrolled proliferation and differentiation disorders,etc.At present,the most commonly used treatment for leukemia is still a side-effects method such as chemotherapy,and it hurts the patient physically and mentally.Therefore,exploring new treatments and medications can greatly help patients.Ginsenoside Rh2(Rh2)is one of the active ingredients extracted from the Panax ginseng,which has many pharmacological effects,such as inhibition of proliferation,induction of differentiation,regulation of immunity and promotion of apoptosis.Recent studies have shown that ginsenoside Rh2 has obvious pro-apoptotic effects on ovarian cancer,gastric cancer,liver cancer,leukemia and other tumor cells.However,its specific mechanism is not yet clear.Protein acetylation Modification is a universal,reversible and highly regulated post-translational modification of proteins.It is believed that protein acetylation is involved in the development of tumors and is a potential therapeutic target for tumors.Reconstruction of chromosome structure induced by histone acetylation plays a very important role in the regulation of gene expression.The acetylation modification of histone is regulated by histone acetylase(HAT)and histone deacetylase(HDAC).The role of HAT is to transfer the acetyl group of acetyl-Co A to the specific lysine residues in the amino terminal of histone,and increase the acetylation level of histones to activate gene transcription.HDAC's role is to deacetylate histones,whereas inhibiting gene transcription.Aberrant expression of the Wnt/?-catenin signaling pathway has been found in a variety of tumor cells.In the Wnt/?-catenin signaling pathway,Gsk-3? is a very important regulatory factor that targeting ?-catenin,and ?-catenin is a downstream effector in this pathway.Protein kinase B(Akt)is a pathway regulatory factor in the cytoplasm,which directly regulates Gsk-3? after its phosphorylation and activates Wnt/?-catenin signaling pathway.Akt/Gsk-3?/?-catenin signaling pathway has a wide range of functions,which can regulate cell proliferation,invasion,migration and apoptosis.In addition,studies have suggested that the Akt/Gsk-3?/?-catenin signaling pathway is also involved in various modification of histone,such as histone acetylation modification,which regulates chromosome structure and gene expression.ObjectiveTo explore the effect of ginsenoside Rh2 on proliferation and apoptosis of leukemia K562 and KG1 a cells and its specific mechanism.Then we study the effect of Ginsenoside Rh2 on the histone acetylation of leukemia K562 and KG1 a cells,which regulates the proliferation and apoptosisMethodsCCK-8 assay was used to detect the survival rate of leukemia K562 and KG1 a cells induced by ginsenoside Rh2 at concentrations of Control,30 ?M,and 60 ?M for 24 h,and the method was used to detect the effect of 5 ?M DEVD,5 ?M C646 and 0.1 ?M FK228 in combination with 60 ?M Rh2 on the proliferation of both cells.Flow cytometry(FCM)was used to detect the apoptotic rate of cells induced by ginsenoside Rh2 at concentrations of Control,30 ?M,and 60 ?M for 24 h,and as well as the effect of 5 ?M DEVD,5 ?M C646 and 0.1 ?M FK228 in combination with 60 ?M Rh2 on the apoptosis of both cells.The Caspase-3 kit was used to detect the effects of Control,30 ?M and 60 ?M ginsenoside Rh2 on the activity of Caspase-3 in leukemia K562 and KG1 a cells and as well as the effects of 5 ?M DEVD,5 ?M C646 and 0.1 ?M FK228 combined with 60 ?M Rh2.The acetylation of histone in leukemia K562 and KG1 a cells was detected by Western blot,including H3,Ac-H3,H4 and Ac-H4,and the expression of P300/CBP and HDAC1/2 was also detected.The expression of Caspase-3 signaling pathway and Akt/Gsk-3b/b-catenin signaling pathway associated protein was detected by Western blot,including Bax,Bcl-2,Caspase-3,p-Akt,Akt,Gsk-3b and b-catenin.Results1.Ginsenoside Rh2 could inhibit the proliferation of leukemia K562 and KG1 a cells,and with the increase of the concentration,the inhibitory effect is more obvious;2.Ginsenoside Rh2 could promote the activation and expression of Caspase-3 and up-regulate Bax while down-regulate Bcl-2 in leukemia K562 and KG1 a cells;3.The effect of ginsenoside Rh2 on inhibition of proliferation and pro-apoptosis in leukemia K562 and KG1 a cells was decreased after inhibiting Caspase-3 with DEVD;4.Ginsenoside Rh2 could increase acetylation levels of histone H3 and histone H4 in leukemia K562 and KG1 a cells;5.Ginsenoside Rh2 could promote the expression of P300/CBP and inhibit the expression of HDAC1/2 in leukemia K562 and KG1 a cells;6.Ginsenoside Rh2 could decrease p-Akt,while up-regulate the expression of GSK-3? and inhibit the expression of ?-catenin in leukemia K562 and KG1 a cells;7.After inhibiting the expression of P300/CBP by C646,the effect of ginsenoside Rh2 on inhibition of proliferation and pro-apoptosis in leukemia K562 and KG1 a cells were weakened,and the acetylated modification of histone H3 and histone H4 were reduced,whereas Akt/GSK-3?/?-catenin signaling pathway was activated while Caspase-3 signaling pathway was inhibited;8.After the expression of HDAC1/2 was activated by FK228,the inhibitory effect of ginsenoside Rh2 on the proliferation in leukemia K562 and KG1 a cells was increased,whereas the effect of apoptosis and the level of histone H3 and histone H4 acetylation were promoted,but Akt/GSK-3?/?-catenin signaling pathway was inhibited while Caspase-3 signaling pathway was activated.Conclusion1.Ginsenoside Rh2 has obvious inhibition of proliferation and pro-apoptosis effect in a dose-dependent way on leukemia K562 and KG1 a cells;2.The specific mechanism of ginsenoside Rh2 induced leukemia K562 and KG1 a cells apoptosis may be in the way of activating the Caspase-3 signaling pathway through the Akt/Gsk-3b/b-catenin signaling pathway;3.Ginsenoside Rh2 may regulate P300/CBP and HDAC1/2 through the Akt/Gsk-3b/b-catenin signaling pathways that further regulate histone acetylation modification in leukemia K562 and KG1 a cells,and subsequently inhibiting cell proliferation and promote apoptosis. |
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