Ginsenoside Rh2Induces Apoptosis In Human Erythroleukemia K562Cells Through The Inhibition Of Histone Deacetylases

Leukemia is a malignant clonal disease of the hematopoietic stemcells, which is characterized by uncontrolled proliferation ofhematopoietic cells, maturation blocked and the normal process ofapoptosis occurring disorders, resulting in a large number of abnormal cellproliferation and differentiation. According to reports, the incidence ofleukemia in a variety of tumors accounts for sixth place in China.Currently, there are various methods for the treatment of leukemia, such asradiation therapy, chemotherapy, targeted therapy, immunotherapy, andstem cells transplantation. But these therapies have serious side effects,easy to generate drug resistance and some expensive. Therefore, weeagerly have to find effective anti-leukemia drugs.Because of their high anti-tumor activity and low toxicity, it makestraditional Chinese medicines and their active ingredients have moreattention by people. Ginseng saponins Rh2[20(S)-ginsenoside Rh2, Rh2(S)] is one of the effective components of ginseng, which could inhibitproliferation, induce differentiation and promote apoptosis in a variety of tumor cells, such as ovarian cancer, gastric cancer, liver cancer andleukemia[1-4]. It had been reported that the activation and abnormalexpression of histone deacetylase (HDAC) was related to the occurrenceof cancer[5].In recent years, more researchers have attracted by the histonedeacetylase for cancer therapy. It was developed that the histonedeacetylase inhibitor was a new, efficient and low toxicity of anticancerdrug, and its molecular mechanism is similar with Rh2(S), which mainlyinhibit proliferation, induce apoptosis and cell cycle arrest[6-7]. We considerthat the anti-tumor effects of Rh2(S) may be associated with histonedeacetylase. Therefore, in this study human erthroleukemia cell lineK562act as experimental object to explore the effects of Rh2(S) on theproliferation, apoptosis and histone deacetylase of K562cell, to observethe epigenetic modification of Rh2(S) and to provide the experimentalevidence for its clinical application in this study.ObjectiveTo investigate the effects of ginsenoside Rh2(S) on the proliferationinhibition, cell cycle and apoptosis of human erthroleukemia cell lineK562, and to explore the effects of Rh2(S) on histone acetylation of K562cells from the aspects of epigenetic regulation and the regulatorymechanisms of histone modifying enzymes.Methods The density of K562cells in logarithmic phase was regulated to5×108L-1and incubated with10,20,40,60,80μmol/L of Rh2(S). Theblank control group is performed in RPMI1640with0.1%DMSO; Theinhibition of cell proliferation was examined by CCK-8assay; Thechanges of cell cycle distribution and apoptosis were detected by flowcytometry (FCM); The cell morphological changes of apoptosis wereobserved by hoechst staining; Chemical colorimetry assay was used tomeasure the activity of histone modification enzymes in cells; Thechanges of histone acetylation level, expressions of histone deacetylaseprotein and expressions of key enzymes on signaling pathway, which isassociated with HDAC, were examined by western blot analysis. Thelocalization of HDAC6protein was identified by immunofluorescence.Results1. CCK-8showed that ginsenoside Rh2(S) can effectively inhibit theproliferation of K562cells in vitro, which exhibits a dose-dependentmanner at range of10~80μmol/L Rh2(S). And the inhibition rate reached apeak with the dose of60μmol/L at48h.2. Flow cytometry indicated that Rh2(S)(60μmol/L) could arrestK562cells in G0/G1phase. The apoptosis rate of K562cells were(8.09±0.86)%,(9.44±0.53)%,(22.80±2.16)%after induced by20,40,60μmol/L Rh2(S), which showed statistically significant difference (P＜ 0.05) compared with the control group(2.63±0.14)%.3. Hoechst staining revealed that Rh2(S) could induce apoptosis andcells in each group which treated with Rh2(S) had chromatin condensationgathered and typical apoptotic morphological changes.4. Chemical colorimetry assay indicated that40~60μmol/L Rh2(S)could reduce the activity of HDAC in K562cells; however60μmol/L Rh2(S) would improve the activity of HAT.5. Western blot showed that the expressions of Bax, CleavedCaspase-3, p16, p21, p-p38, p-JNK proteins were increased indose-dependent manner after induced by Rh2(S), and expressions of Bcl-2,Cyclin D1, CDK4, HDAC1, HDAC2, HDAC6, p-ERK proteins weredecreased (P all <0.05), expressions of HDAC3, HDAC4, p38, JNK, ERKproteins were no significant difference (P all>0.05).6. Immunofluorescence revealed that the expression of HDAC6waslocated in the cytoplasm and reduced after Rh2(S) treatment.Conclusion1. Ginsenoside Rh2(S) can effectively inhibit the proliferation ofK562cells in a dose-and time-dependent manner in vitro.2. Ginsenosides Rh2(S) can induce cell cycle arrest in G0/G1phaseand apoptosis of K562cells.3. Ginsenosides Rh2(S) can inhibit the activity of HDAC, and increase the activity of HAT and reduce the expressions of HDAC1,HDAC2, HDAC6protein, thus contributing to increase levels of histoneacetylation and expression changes of key protein in signaling pathwayrelated downstream of HDAC in K562cells, which may be associated tothe inhibition of cell proliferation and the induction of cell apoptosis.