| Background:Human leukemia is the most common hematological malignancy and the major leading cause of human death, even though various treatment strategies developed. In recent years, incidence and mortality of leukemia has been increasing. An interesting example is Agaricus blazei Murill, which is a mushroom native to Brazil, and is widely used among cancer patients as complementary and alternative medicine In recent years, After the in vivo and in vitro studies, the pharmacological effects of Agaricus blazei murill including antitumor, antiviral, anti-inflammatory, liver protection, antidiabetic, antihyperlipidemic, antiatherosclerosis, antiallergic and immunomodulating were found. Growing evidences had proved that some polysaccharides and polysaccharide-protein complexes isolated from A. blazei were bioactive principles responsible for treating and preventing cancer. However, up to now, antitumor effect and the exact mechanism of A. blazei polysacchairdes in models of human leukemia in vitro and in an in vivo xenograft mouse model have not been addressed in any of the foregoing studies. Bearing this in mind, we therefore investigated the effects of a polysaccharide from A. blazei on proliferation and apoptosis in HL-60 human promyelocytic leukaemia cells in vitro and further examined the anti-tumor effect in vivo.There are two main pathways of apoptosis signal transduction pathway:endogenous mitochondrial pathway and the death receptor pathway. Bcl-2 family proteins such as Bcl-2 proteins and pro apoptotic proteins Bax proteins play an important role in the regulation of apoptosis. In the presence of apoptotic stimuli, the expression of pro-apoptotic proteins Bax and/or BH3-only proteins (apoptosis initiator) increased, following which they bind to pro-survival Bcl-2 proteins to release Bax/Bak from inhibition. Free Bax and Bak form oligomers, leading to loss of the mitochondrial membrane potential and cytochrome c release from the intermembrane space of mitochondria to the cytoplasm, likely by forming a channel in OMM. The released cytochrome c activates the caspase cascade to induce apoptosis. The released cytochrome c forms the apoptosome with Apaf-1 and subsequently activates the initiator caspase, caspase-9, which cleaves and activates effector caspases, caspase-3 and caspase-7, leading to ultimate apoptotic cell death. Caspase-3 Caspase-3 -ADP-PARP ADP-ribose polymerase, which is one of the most active Poly substrates. PARP can be cleaved into 89kDa and 21kDa two peptides in the role of Caspase-3, the DNA does not have the ability to damage PRAP after shear, resulting in the increase of the activity of the PARP negative regulation, the degradation of the DNA induced apoptosis. Tumor apoptosis induced by mitochondrial pathway is one of the hot spots in tumor research.Objectives:1. To extract and purify polysaccharide from Agaricus blazei murill for the study of its medical value. The polysaccharide was monitored by the phenol-sulfuric acid assay.2. To explore specific mechanism of the inhibitory effect of ABP on HL-60 cells in vitro.3. To investigate whether or not ABP can affect HL-60 cells in vivo by injecting HL-60 cells by s.c. into the mice for generating leukemia tumor xenograft model, by calculating tumor inhibition (RI) ratio to determine the effect of polysaccharide.Methods:1. Polysaccharide purification:A polysaccharide from Agaricu blazei murill was extracted and purified through hot water extraction and ethanol precipitation, ion-exchange chromatography purification.2. The effect of Agaricus blazei on polysaccharide on HL-60 cells in vitro was studied.(1)The growth inhibitory effect of polysaccharide ABP on HL-60 cells was tested in vitro using MTT assay following exposure of cancer cells to ABP.(2) The HL-60 cells were incubated with different concentrations of ABP for 48 h, and then the occurrence of apoptotic cells were quantified by flow cytometry using annexin V/PI double-staining assay.(3) Caspase-3 activity was measured with the use of the caspase-3 colorimetric assay kit.3. Specific mechanism of Agaricus blazei polysaccharide inducing HL-60 cells apoptosis was studied.(1) We investigated the effect of ABP on the change of △ψm using a cationic dye Rhodamine 123, which can reflect the change of △ψm by means of flow cytometry.(2)We detected the changes of cytochrome c (Cyt-C), Bax, bcl-2 protein, poly ADP ribose polymerase (PARP) protein using Western blotting after HL-60 cells were treated with ABP for 48 h.4. The effect of ABP on HL-60 cells in vivo was studied through xenograft tumor-bearing mice. The xenograft tumor-bearing mice were randomly divided into five groups:one negative control group, three doses of ABP-treated groups and one positive control group. At the end of the experiment, animals were euthanized by cervical dislocation, and the solid tumors were picked up and weighed. The antitumor effect of ABP was certified by calculating the tumor inhibition ratio.Results:1.42.4g crude polysaccharide can be obtained from the fruit bodies of Agaricus blazei murill. The content of polysaccharide was 89.2% by the phenol-sulfuric assay.2. The data demonstrated that AMP had a specific suppressing effect on the growth of HL-60 cells in vitro by MTT test with a dose-dependent relationship.3. The percentage of Annexin V-FITC positive cells increased with the concentration of ABP applied, which indicated that ABP-induced growth suppression of HL-60 cells involves the induction of apoptosis. It is found that the rate of apoptosis increased with the increase of ABP concentration.4. Results showed that caspase-3 activity significantly increased as the concentration of ABP raised.5. The results demonstrated that treating HL-60 cells with ABP for 48 h resulted in a dose-dependent loss of the mitochondrial membrane potential.6. ABP induced cytochrome c release from the mitochondria into the cytosol in a concentration-dependent manner. ABP increased Bax protein expression and Bcl-2 protein can’t change significantly, resulting in an increase in the ratio of Bax/Bcl-2, promote the apoptosis of the cells.7. ABP treatment caused cleavage of PARP,116 kDa into 89 kDa fragment, which corresponded with the activation of caspase-3.8. Results showed ABP treatment did not alter body weight, but significantly decreased the tumor weight compared to controls. The results demonstrated that ABP could suppress tumor growth in vivo.Conclusions:1. Agaricus blazei polysaccharide can be obtained from the fruit bodies of Agaricus blazei through hot water extraction and ethanol precipitation, ion-exchange chromatography purification.2. This study demonstrated that ABP could inhibit the proliferation of HL-60 cells by induction of apoptosis. So ABP can suppress HL-60 cells in vitro.3. This study demonstrated that ABP could inhibit the proliferation of HL-60 cells by induction of apoptosis through activation of the mitochondrial-mediated intrinsic caspase pathway. It was found that the apoptotic effects of ABP are mediated through the dissipation of △ψm, release of cytochrome c from the mitochondria into the cytosol, activation of caspase-3, cleavage of PARP, and elevated the ratio of Bax/Bcl-2.4. ABP administration significantly inhibited tumor growth in vivo. It is found that the polysaccharides from Agaricus blazei Murill could effectively inhibit the growth of leukemia cells in nude mice tumor model, and inhibit the proliferation of leukemia cells. |
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