Study On The Apoptosis Of K562 Cells Induced By Glucosamine Sulfate And Berbamine

Human leukemia is a common hematological malignant disease and the conventional treatment results have reached a plateau. Because most patients can not tolerate the side effects of chemotherapy drugs or leukemia cells are resistant to chemotherapy, the failure of treatment occurs. Inducing the apoptosis of leukemia cells is currently one of the important strategies for the treatment of leukemia, so it is urgent to find inductors of which have lower side effects and obvious curative effect. Studying the mechanism of the apoptosis of tumor deeply will lay a good foundation for treatment of malignant disease.Glucosamine sulfate(GS) is one of the major glucosamine derivatives and it is a very important raw materials in human body to synthetize viscosity polysaccharide,glycoprotein and glycolipid etc. A new pilot study revealed a new biological role of GS. And a research has found that glucosamine hydrochloride salt can induce K562 cells to macrophage differentiation, and K562 cells can be induced apoptosis by its sulfate. GS originates from natural biology and is safe. Its poisonous side effect is small. GS has higher bioavailability than the same kind product. GS has a very strong penetration force and can enter internal cell rapidly. In the field of blood system tumour, the reaserch of GS is comparatively few. It is reported that GS can activate Caspase-3 and reduce the level of Bcl-2 protein, and its role in inducing apoptosis is through mitochondrial pathway to down-regulate Bcl-2, change membrane potential,open permeability transition pore and activate Caspase-3.Berbamine(BBM) is a kind of bis-benzylisoquinoline alkaloids that was extracted from Chinese herb. BBM and its derivatives belong to calmodulin antagonist. Recently, many studies demonstrated that it could reverse the multidrug resistance of some leukemia cell lines and greatly inhibit the proliferation of a variety of tumor cell lines in vitro. BBM can significantly inhibit the growth of chronic myeloid K562 cells,Imatini resistance of K562 cells and primary leukemia cells, and induce the apoptosis of those cells, but the molecular mechanism is not yet very clear. Some studies reported that BBM caused the apparent level of Caspase-3 expression increase in the process that BBM induced the apoptosis of K562 cells. As the two drugs in the induction of apoptosis of K562 cells have certain crosspoint, we choose the two drugs in combination and explore whether the combination can enhance the effect of increase Caspase-3 expression level through the apoptosis pathway and futher strengthen the effect of induce K562 cells apoptosis.Survivin, a novel inhibitor of apoptosis, is located on 17q25. In these years, some studies have reported Survivin is faintly detected in fetal tissues,thymus gland and over-expresses in most human cancers such as leukemia, but not usually expresses in normal tissues. The up-regulation of Survivin expression predicts poor prognosis in human cancers. Survivin can inhibit apoptosis of cells, promote cell proliferation and play a role in cell caryocinesis. Survivin can abrogate the process of cell apoptosis induced by various stimuli through binding the terminal effectors Caspase-3 and Caspase-7 directly. So it is a new oncogene target for leukemia therapy.Caspases (cystein aspartate-specific proteases) is a high degree of homology protease, related with cytokine maturity and cell apoptosis. And Caspases play a key role in the apoptosis pathway. Caspases found so far have more than 14 members. Caspase-3 in which many normal cells or tumor cells were expressed, is the activation of caspases-reaction waterfall and an important effector molecule that can activate other caspases, known as the "amplifier" protease. Caspase-3, as the key enzyme of apoptosis cascade is considered to be the common checkpoint in the apoptosis pathway after cells were suffered from injury. Once apoptotic signaling pathway activation of caspases, it can cause the cellular protein degradation, and cells will be put to irreversible death.Cytochrome C(Cyt c) is a water-soluble protein in mitochondrial membrane between endomembrane and adventitia, and loosely connected with endomembrane. Apart from cellular aerobic respiration, Cyt c plays an important role in the activation of caspases and induction of apoptosis. When Cyt c moves into cytoplasm from mitochondria, it can combine with Apaf-1. Then it activates Caspase-3 and start apoptosis cascade that could trigger apoptosis. In this study we choose these three proteins closely linked to research the molecular mechanisms by GS and BBM induced apoptosis of K562 cells.ObjectiveThe aim of the trial is to study the molecular mechanisms of induce K562 cells apoptosis by the two drugs GS and BBM. First in order to find the optimum doses of the two drugs, we used different concentrations to detect the antiproliferation effects of K562 cells cultured by GS and BBM alone. Then we observed the apoptosis-induced effects in K562 cells cultured by GS and BBM alone and combination again with the two optimum doses which is good expremential information for drug combination in clinic therapy. We explored the related mechanisms in inducing apoptosis of K562 cells by GS and BBM alone and in combination through analyzing the expression of Cyt c,Survivin and Caspase-3 protein.MethodsK562 cells line were treated with different drug and different concentration. The condition of cell growth was analyzed by cell growth curve and morphological changes which were observed through cell count and inverted microscope. The optimum doses of the drugs in apoptosis-inducing effect were identified with MTT assay. Then observe the apoptosis-induced effects in K562 cells cultured by GS and BBM alone and combination again with the two optimum doses. Exponential phase of growth period of cell will be randomly divided into four groups: control(K562 cells+ 10%RPMI1640),experiment group 1 (K562 cells+10% RPMI1640+5.0mmol/L GS),experiment group 2( K562 cells + 10% RPMI1640+ 10mg/L BBM),experiment group 3(K562 cells+10% RPMI1640+5.0mmol/L GS+10mg/L BBM). Apoptosis, Caspase-3 and Survivin protein expression were analyzed by using flow cytometry respectively in the cultured K562 of each group. Western blot was performed to detect the release of Cyt c from chondriosome of each group.ResultsThe results of MTT indicate that 5.0mmol/L GS and 10mg/L BBM were the optimum doses needed to induce apoptosis of K562 cells, the group 3 significantly inhibited cell proliferation. The apoptosis rate results showed that the apoptosis rate of the experiment groups were obviously higher than the control group (P<0.01) . It was significantly improved to (71.09 + 1.24) % at 72h of the group 3, which showed a clear synergistic effect in combination. The positive expression rate of Caspase-3,Survivin were (78.46+0.12) %,(8.24+0.45) % respectively, the former was greatly higher than the control group(7.56±1.33) %( P<0.01), the reverse side of the latter was much lower than the control group (38.24±0.14) %( P<0.05). Western blot result showed that the Cyt c content in the endochylema of the group 3 was obviously higher than the control group at 72h (P<0.05) .Conclusions1. GS and BBM can inhibite proliferation of K562 cells alone. This effect of combination is also significantly enhanced.2. Detection of apoptotic rate suggested that the early apoptosis of K562 cells induced by combination group be significantly higher than that by alone group, so we can conclude that there is certain coordination in enhancing the role of apoptosis in K562 cells.3. The process contained the increase of Cyt c content in the cytoplasm, the down-regulation of Survivin expression and the up-regulation of Caspase-3 expression.4. The combination group can increase the apoptosis of K562 cells mainly through mitochondrial-caspase pathway.5. Moreover Survivin reduction can ease the inhibition of Caspase-3 and inhance the role of induce apoptosis of K562 cells.