| Objective: Myeloid leukemia is a heterogeneous group of diseases in which the malignant clone arises from the uncontrolled proliferation of myelocytic progenitors in the bone marrow. Leukemia cells accumulate in bone and other hematopoietic tissue, infilltrate in other tissue andorgan, supress normal hematogenesis. The mortality of leukemia ranks 6th(male)and 8th(female) in overall mortality of adult malignant tumer, however, it ranks 1st in childen malignant tumer. Acute myeloid leukemia (AML) is the main kind of leukemia happening to adults, though a great deal of AML patients can reach complete remission with current treatment protocols, many individuals eventually relapse and the overall outcome has not improved in recent years. Chronic myeloid leukemia (CML), of which the median duration of chronic phase is 3–4 years, is found to have a higher morbidity in eastern countries especially in China than that in western countries, even with a lot of modern treatment protocols, many CML patients have eventually died of the subsequent blast crisis. Therefore, it is a permanent requirement to find new anti-leukemia drugs and effective therapies for the clinical treatment of myeloid leukemia. Acanthopanacis is the root and rhizome of Acanthopanaxsenticos, which is a kind of invigorative Chinese medicine. Several lines of evidence indicate that Acathopanassenticosus Polysaccharides (ASPS) plays an important role in regulating immunity, improving adaptive capbility and tolerance, and anti-aging. Recent data showed that Acanthopanacis can affect cancer cell perliferation, differentiation, apoptosis and matastasis.it play its role through improving immnity and direct cytotoxic to cancer cell. But, little was known about its molecular mechanism, till now.The aim of this expriment is to explore the effects of Acanthopanacis on the growth of leukemia cell line k562 and detect the morphological change of apoptosis, cell cycle and related proteins, like as p21, Bax and Bcl-2. It will provide a theoretical foundation in clarifying the mechanism of ASPS inhibiting the leukemia cells.Methods: 1 Cell culture: The K562 cells were cultured in RPMI-1640 medium which supplemented with 10% new born bovine serum medium, 100 IU/ml penicillin and 100ug/ml phytomycin with the environment of 37℃, 5% CO2, fully humidified atmosphere.2 IC50 assay: The microculture tetrazolium (MTT) was used to measure the inhibition ratios of K562 cell under different ASPS concentration: cells were seed in 96-well microplate, added different concentration of ASPS, measured the extinction value. Based on the formula: survival rate=(experiment extinction value / control extinction value)×100% to calculated the IC50.3 Hoechst 33258 staining was to measure the changes of cell apoptosis .: K562 cells treated with 170μg/ml ASPS for 48h were collected, washed with PBS, mixed with fluorescence reagent 0.5ml for 10 minutes.,400 cells was examined under high power lens of fluorescence microscope to observe morphological changes .4 FCM was used to measure the changes of cell cycle, apoptosis rate and related protein: For FCM analysis, K562 cells were treated with 170μg/ml ASPS for 48h. The cells were collected, washed with PBS, and fixed with 70% ethanol for 24h, stained with 50 mg/L PI for 30 mintes. 1×106 fixed cells were examined per experimental condition to mearsure the change of cell cycle, apoptosis rate and expression of apoptosis related protein Bax and Bcl-2.5 RT-PCR was used to measure the mRNA expression of Bcl-2, Bax, P21 and cyclind 1: Total RNA was isolated from harvested cells by Trizol. The reverse transcription reaction was performed by M-MLV and Random primer. After used the special primer to amplify the target gene, the agarose gel electrophoresis was carried out, and the photodensity of bands were acquired by image analysis system. The mRNA expression levels were obtained by compared with the photodensity of each item ofβ-action.Results: 1 Treatment with ASPS results in growth inhibition of K562cells in a dose dependent manner and the IC50 of ASPS is 170μg/ml.2 After cells treated with 170 nmol/L ASPS for 48h, marked morphological changes of cell apoptosis such as condensation of chromatin and nuclear fragmentations were found clearly using Hoechst 33258 staining .3 FCM showed ASPS increased the percentage of cells of G0/G1 phase( P<0.05)and decreased the percentage of cells of S phase (P<0.05)the k562 cell line in a dose-dependence manner, moreover, it increased rate of apoptosis in a dose-dependent manner( P<0.05).4 FCM also showed that ASPS can decrease the expression of apoptosis related protein Bcl-2 and increase the expression of Bax in the cells in a dose-dependence manner( P<0.05).5 RT-PCR results showed that ASPS can decrease the mRNA expression of CyclinD1,Bcl-2 ( P<0.05)and increase the expression of p21 and bax ( P<0.05), the ratio of bax and Bcl-2 increase significiantly in the k562 cells in a dose-dependence manner( P<0.05).Conclusion: Acathopanassenticosus Polysaccharides can inhabit the growth of human leukemia cell line k562, increase the percentage of cells in G0/G1 phase, decrease the percentage of cells in S phase, increase apoptosis rate. RT-PCR results suggested that ASPS can decrease the mRNA expression of CyclinD1,Bcl-2 and increase the expression of p21 and Bax, the ratio of Bax and Bcl-2 increase significiantly. Futher reserch suggested that ASPS decrease the expression of apoptosis related protein Bcl-2 and increase the expression of Bax ,which sugests that Bcl-2,Bax,CyclinD1,p21 may take part in the process of k562 cell growth arrest and apoptosis. It is suggest that ASPS may be a potential drug for leukemia theraby.
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