Ginsenoside(s)Rh2 Induces Autophagy And Apoptosis In Human Erythroleukemia Cells Through HDAC6

Leukemia is a grave harm to people’s life and health of hematopoietic system malignant tumor. It is also one of the top ten high incidences of malignant tumors which is characterized by uncontrolled proliferation of hematopoietic cells, maturation blocked and the normal process of apoptosis occurring disorders, resulting in a large number of abnormal cell proliferation and differentiation. So far, the treatment of leukemia is still mainly adopted the traditional high-dose chemotherapy, not only costly, and the side effects are very large, especially the damage to immune system and hematopoietic system, high recurrence rate. Therefore, an efficient anti-leukemia drug is urgently needed.Ginseng, which the main medicinal ingredient is ginsenoside, is a traditional Chinese herbal medicine and has high medicinal value in the treatment of cancer. Ginseng saponins Rh2 [20(S)-ginsenoside Rh2, Rh2(S)] is one of the effective components of ginseng, which had high anti-tumor activity and low toxicity. It could inhibit proliferation, induce differentiation and promote apoptosis on a variety of tumor cells in ovarian cancer, gastric cancer, liver cancer and leukemia.Recent studies have shown that Histone deacetylases(HDACs) anomaly expression in blood malignant tumor which is a close relationship between the occurrence and development with malignant tumor, and the inhibition of HDACs can be a new strategy in tumour therapy. HDAC6 is special histone deacetylases, which could participate not only on histone acetylation in the nuclear but also on non-histone acetylation in the cytoplasm and high expression in leukemia cells. So we believe that the inhibition of abnormal expression of HDAC6 become a key strategy for the treatment of tumor.The cell death has three types: necrosis, apoptosis and autophagic cell death. The abnormality of autophagy is closely related to the occurrence and development of tumor. It can affect the process of tumor from different biological behavior, including cell cycle, proliferation, apoptosis, drug resistance, angiogenesis and tumor treatment. In recent years, it is found that the acetylation and deacetylation of proteins can affect the formation of cell autophagy, and then affect the proliferation and apoptosis of tumor cells, which provides a new idea for the treatment of cancer.Therefore, these experiment with leukemia K562 cells and KG1α as experimental object, to clarify the molecular mechanisms of(s)Rh2 affection leukemia cell proliferation, autophagy and apoptosis.ObjectiveTo investigate the effects of ginsenoside(s)Rh2 on the proliferation inhibition, autophagy and apoptosis of human leukemia K562 and KG1α cell lines, and to preliminary discuss the impact of(s)Rh2 on histone acetylation modify and the mechanism of HDAC6 regulating autophagy and apoptosis in K562 and KG1α cells.MethodsPart ⅠCCK-8 assay was used to screen the most effective ingredient on the proliferation of leukemia K562 and KG1α cell lines among total ginseng saponins, ginsenoside Rb1, Rg1, Re,(R)Rg3 and(s)Rh2. The effects of(s)Rh2 on autophagy and apoptosis were detected by acridine and MDC staining. The expression levels of the vital genes closely associated with autophagy were measured by RT-PCR, and the expression levels of the proteins closely associated with autophagy and apoptosis were detected by Western Blot. After joining autophagy inhibitors, using CCK-8 to test the proliferation activity of cells, cell apoptosis was measured by FCM, and Western Blot was usde to test the expressions of the proteins closely associated with autophagy and apoptosis.Part ⅡChemical colorimetry assay was used to measure the enzymatic activity of HAT and HDAC in cells. The expression changes of histone acetylation(H3K9), HDACs and the vital proteins closely associated with autophagy and apoptosis were examined by western blot and RT-PCR. The construct of lentivirus plasmid were used to transfect the leukemia cell line K562 and KG1α which interferenced HDAC6 gene, and then selected the stable expression of si HDAC6 cell lines. The stable expression of si HDAC6 K562 and KG1α cell lines treated by(s)Rh2, and the transfection blank plasmid K562 and KG1α cells lines were used as control group. The effects of(s)Rh2 on proliferation and apoptosis of K562 and KG1α cells which silencing HDAC6 were detected by trypan blue staining, FCM and Hoechest staining. The expression levels of HDAC6 and Hsp90 m RNA were tested by RT-PCR. The relationship of between HDAC6 and Hsp90, Beclin-1, cleaved Caspases3, LC3 A, LC3 B were detecteded by Western Blot.Part ⅢThe anti-tumor activity of(s)Rh2 was estimated by K562 xenograft model in vivo. The diameter and volumes of tumor were measured, the inhibitory rates of tumor were calculated, HE staining and immunohistochemistry were used to observe the cells apoptosis and the expressions of HDAC6, Hsp90 and cleaved Caspases-3. The enzymatic activity of HAT and HDAC xenograft were assessed by chemical colorimetry assay. The transcriptions of HDAC6, Hsp90 and the vital genes closely associated with autophagy were detected by RT-PCR. The expressions of HDAC1, HDAC2, HDAC3, HDAC4, HDAC5,HDAC6 proteins were detected by Western Blot.Results1. CCK-8 assay showed that(s)Rh2 had the most efficient inhibitory effect on leukemia K562 and KG1α cells in a dose-dependent manner.2. Flow cytometry test results showed that(s)Rh2(60 μmol/L) could induce the early apoptosis of K562 and KG1α cells(P<0.05). Western blot results showed that Bax and cleaved Caspases3 were increased in a dose-dependent manner after treated by(s)Rh2.3. Acridine and MDC staining revealed that(s)Rh2 increased the number of acidic autophagy vesicles and the levels of cell autophagy in cells. Western blot and RT-PCR results showed that(s)Rh2 could increase the expressions of the vital molecules closely associated with autophagy.4. The tested results of enzymatic activity showed that the high concentration of(s)Rh2(60 μmol/L) could improve the enzymatic activity of HAT and decrease the activity of HDAC in K562 and KG1α cells.5. Western blot showed that the expressions of HDAC1, HDAC2 and HDAC6 proteins were decreased, while increased the expression levels of Ac-H3 protein in dose-dependent manner after induced by(s)Rh2.6. Trypan blue staining results showed that the interference of HDAC6 gene could effectively inhibite the proliferation of K562 and KG1α cells, and increase the sensitivity of(s)Rh2 on K562 and KG1α cells.7. FCM and Hoechst staining results showed that the interference of HDAC6 gene improved apoptosis, and enhanced the apoptotic effect of(s)Rh2 on K562 and KG1α cells.8. Western blot results showed that(s)Rh2 induced autophagy and apoptotic by reducing the expressions of HDAC6 and Hsp90 proteins and enhancing the expressions of Beclin-1, LC3 A, LC3 B and cleaved Caspase-3.9. Ginsenoside(s)Rh2 could inhibit the growth of k562 xenograft, and the inhibition rate of(s)Rh2 was 53.10%. Ginsenoside(s)Rh2 could decrease the levels of HDAC activiy and significantly decrease the expression of HDAC1,HDAC2,HDAC6. The expression levels of three vital autophagy genes(Beclin-1, LC3 A and LC3B) were increased and Hsp90 was decreased after treated by Ginsenoside(s)Rh2. The study of histopathology showed ginsenoside(s)Rh2 could promote the apoptosis of tumor cells.Conclusion1. Ginsenoside(s)Rh2 could effectively inhibit the proliferation of leukemia cells in a dose- and time-dependent manner in vitro, and compared with other ginseng monomer had the best inhibition effectiveness.2. Ginsenosides(s)Rh2 could induce autophagy and apoptosis in leukemia cells.3. Ginsenosides(s)Rh2 could inhibit the enzymatic activity of HDAC, increase the activity of HAT and reduce the expressions of HDAC1, HDAC2, HDAC6 proteins, thus contributing to increase the levels of histone acetylation and expression changes of key proteins in signaling pathway related downstream of HDAC in K562 and KG1α cells, which may be associated to the inhibition of cell proliferation and the induction of cell autophagy apoptosis.4. After silencing the HDAC6 gene, ginsenosides(s)Rh2 inhibited the proliferation of human leukemia cells, and induced autophagy and apoptosis mainly through the Hsp90 pathways.5. The vivo experiments also proved that(s)Rh2 could induce autophagy and apoptosis in leukemia cells through HDAC6 and Hsp90.