Effects Of Ginsenoside RB1,RG1,RE On Proliferation Inhibition Of KG1α And K562 Cells And Signal Transduction Mechanism

Leukemia is a malignant haematopoietic tumor which threatens the health of humanity. It's pathogeny is due to abnormal proliferation and disdifferentiation of bone marrow haematopoietic stem cells. So far, traditional chemotherapy and radiotherapy are still the main methods, but they cause many side effects and high recurrence rate, and greatly damage the normal immunity and haematopoiesis system. More and more researches have shown that the traditional Chinese medicine may play an important role in the cancer therapy for its little side affection on human body and many other advantages. Therefore, finding natural traditional Chinese medicine which has the effect of inhibiting the proliferation or inducing apoptosis of leukemia cells becomes much attractive issue to treat leukemia.Ginseng has been extensively used in traditional Chinese medicine, ginsenosides are the major active components in ginseng and have a variety of biomedical efficacies, such as anti-tumor,promotting apoptosis and inducing differentiation activity. Of the ginsenosides, Rb1, Rg1, Re are isolated from ginseng, and antioxidation, promoting memory, enhanced anti-tumor activity of chemotherapy,etc.However, the molecular mechanisms underlying the anti-tumor activity ginsenosides have not been comprehensively investigated.there are two parts in the research:1. Effects of ginsenoside Rb1,Rg1,Re on proliferation of KG1αcells and signal transduction mechanism .2 . Effects of ginsenoside Rb1,Rg1,Re on proliferation of K562 cells and signal transduction mechanism .Our research aims to find the molecular target of ginsenoside inducing proliferation inhibition and apoptosis of leukemic cells, and to provide theoretical and experimental evidence for clinic treatment of haematologic disease.Part 1 : Effects of ginsenoside Rb1,Rg1,Re on proliferation of KG1αcells and signal transduction mechanismObjective:To investigate the influence of ginsenoside Rb1, Rg1 and Re on proliferation of KG1αcells and their molecular mechanism. Methods:1. experimental groups: Cells normal cultivated as control group; In ginsenoside Rb1 group cells were incubated with various concentration of Rb1 (Rb1, 20μmol/L,40μmol/L,80μmol/L,160μmol/L); ginsenoside Rg1 group (Rg1, 5μmol/L,10μmol/L,20μmol/L,40μmol/L,80μmol/L);ginsenoside Re group (Re, 30μmol/L,60μmol/L,90μmol/L,120μmol/L,150μmol/L). Cells in each group were collected for the following research after 24, 48 and 72 hours . 2. The proliferation of KG1αcells were examined at different times by MTT assay. 3. To determine the effect of ginsenoside Rb1 on KG1αcell cycle and apoptosis through flow cytometry (FCM). 4. To observe the change of KG1αcell morphology by Wright's staining. 5. p38, Phospho-p38, Erk, Phospho-Erk, JNK, Phospho–JNK, p53 and Phospho-p53 in KG1αcells were measured by western blotting at different times .Results:1,ginsenoside Rb1 can inhibit the proliferation of KG1αcells in a dose-dependent manner, which peaked at 48h(74.82%), and the value of IC50 was 120μmol/L . ginsenoside Rg1 can the proliferation of KG1αcells in a dose-dependent manner, which peaked at 48h (22.98%). The optimal concentration was 20μmol/L. However no prominent proliferation inhibition of KG1αcells was induced by ginsenoside Re. 2. As determined by FCM analysis, Rb1 may arrest the cells in G2/M phase , and promote apoptosis of KG1αcells. 3,As determined by Western blotting, the expression of p38 and Phospho-p38 were rapidly increased in KG1αcells treated with Rb1 for 48h, and Phospho-JNK protein was gradually reduced at time-dependent manner, whereas the expression level of JNK, Erk, Phospho-Erk remained unchanged. 4,The expression of p53, Phospho-p53 were increased in Rb1 treated cells at the times of 12h , 24h ,48h , 72h .Conclusion: Rb1 may inhibit the proliferation of KG1αcells and induce their apoptosis ex vivo partly via a p38 MAPK pathway.Part 2 : Effects of ginsenoside Rb1,Rg1,Re on proliferation of K562 cells and possible signal transduction molecule mechanismObjective:To investigate the influence of ginsenoside Rb1, Rg1 and Re on proliferation of K562 cells and their molecular mechanism.Methods:1. experimental groups: Cells normal cultivated as control group; In ginsenoside Rb1 group cells were incubated with various concentration of Rb1 (Rb1, 20μmol/L,40μmol/L,80μmol/L,160μmol/L); ginsenoside Rg1 group (Rg1, 5μmol/L,10μmol/L,20μmol/L,40μmol/L,80μmol/L);ginsenoside Re group (Re, 30μmol/L,60μmol/L,90μmol/L,120μmol/L,150μmol/L). Cells in each group were collected for the following research after 24, 48 and 72 hours . 2. the proliferation of K562 cells were examined at different times by MTT assay .3 . To determined the effect of the Rb1 and Rg1 on K562 cell cycle at 48h through flow cytometry (FCM). 4. p38 , Phospho-p38 ,Erk , Phospho-Erk , JNK , Phospho–JNK, JAK2 , Phospho- JAK2 , STAT5 and Phospho- STAT5 in K562 cells were measured by western blotting in Rb1 group( 160μmol/L) at different times .Results:1,ginsenoside Rb1 showed inhibitory effect on the K562 cells in a dose-dependent manner, which peaked at 48h ( 39.81%) , The optimal concentration was 160μmol/L. 2,ginsenoside Rg1 showed inhibitory effect on the K562 cells in a dose-dependent manner, which peaked at 48h(32.18%). The optimal concentration was 20μmol/L. 3,No prominent proliferation inhibition of KG1αcells was induced by ginsenoside Re. 4,As determined by FCM analysis, compared with contorled group , Rg1 arrested the cells in S phase , but cell cycle of Rb1 treated group was unchanged. 5,As determined by Western blotting , the expression of JAK2 were increased whereas Phospho-JAK2 down in K562 cells treated with Rb1 at time-dependent, and Phospho-STAT5 protein was rapidly down at 48h , whereas the expression level of STAT5 remained unchanged. 6,The expression of Phospho-Erk and Phospho-p38 were rapidly down in K562 cells treated with Rb1,respectively,at 48h,72h. But the expression level of Phospho-JNK, JNK, p38 and Erk in K562 cells remained unchanged.