| ObjectiveCell cycle is the basic process in cell life. In the recent years, cyclin, cyc-lin dependent kinase and cyclin dependent kinase inhibitor have important effect in cell cycle modulation. P21WAF1 is a kind of cyclin dependent kinase inhibitor, it can be bind to several kinds of cyclin/CDK compounds and arrest the cell cycle.The occurrence and progress of cells'apoptosis were controlled by a series of gene,including carcinoma gene and anticarcinoma gene,such as Rb,bcl -2 and caspase. Rb is an inhibiting cancer gene, its expressing product can control repb'cation and transcription of DNA in nuclei, hence control the cell life progress . Bel - 2 expressing product is localized at mitochondrion endometrium, nuclear membrane and endoplasmic reticulum membrane, it can inhibit lipid peroxide , so inhibit apoptosis and prolong the cell life. Caspase is belong to cysteine protease, apoptosis of many kinds of cancer involves caspase activation and at last induces apoptosis.Bufalin is a component of bufadienolides in the traditional Chinese medicine Chan su, it can induce cell cycle arrest and apoptosis. Our previous work observed bufalin inhibited cell growth and induced apoptosis of human leukemia HL - 60, K562 and NB4, reinforcement by bufalin of all - trans retinoic acid -induced differentiation of acute promyelocytic leukemia cells in primary culture, arrested K562 in C2/M phase,OVCAR3 in M phase,and the expression of bcl -2 was downregulated and PKC II was activated during the apoptosis of HL -60 and the expression of WT1 was downregulated during apoptosis and differentiation of K562 induced by bufalin.In order to further study the mechanism of bufalin, we explored bufalin oncell cycle arrest and apoptosis in breast cancer MCF -7.Materials and MethodsMain materialsBufalin was purchased from sigma USA; Propidium iodide ( PI) was purchased from sigma Fluka corporation; Immunocytochemical mouse antihuman p21 ,pRb,bcl -2, caspase -9 antibody were purchased from Maixin_Bio;RPMI -1640 was purchased from GIBCO BRL USA,fetal bovine was purchased from Institute of Hematology CAMS.Cell cultureThe breast cancer cell line MCF -7 was cultured in RPMI -1640 supplemented with 10% (v/v) heated - inactived fetal bovine serum containing 12U/ ml gentamycin and incubated at 37 *C in 5% C02 atmosphere. In all experiments, cells were used in logarithmic growth phase.Assays for proliferation of MCF - 7 cell.Cell proliferation was determined by M'lT.Assays for cell cycle arrest and apoptosisTne morphological change was observed with microscope and dyed in Wright - Giemsa.Assay for cell cycle * Cell cycle distribution was determined by flow cytometric DNA analysis.ImmunocytochemistryThe expression of p21, pRb, bcl - 2, caspase - 9 protein was analyzed by immunocytochemistry.Statistical analysisTne data were analyzed by SPSS,Tne wilcoxon signed rank test was used to assess the significance of difference of the results.Results1. Inhibitory effect of bufalin on MCF -7 cell proliferationBufalin inhibited MCF - 7 cell proliferation in a time - and dose - dependent manner,and the IC50 of 24,48 hours were 80. 5nmol/L and 58. Snmol/ L, respectively.2. Effect of bufalin on cell cycle arrest and apoptosis in MCF - 7 cell. After treatment with 10 ~ lOOnmol/L bufalin for 0 ~ 48 hours, we observedthe morphologic changes at 24h, including more polyploid in cells. After treatment with lOOOnmol/L bufalin for 0 ~48h,we observed the membrane was intact , the nuclear broke into pieces, suggesting typical morphological characteristics of apoptosis appeared.3. Effect of bufalin on cell cycle arrestAfter treatment with 10 ~ lOOnmol/L bufalin for 0-48 hours, MCF - 7 cells arrested at G2/M phase determined by flow cytometry, The cells in G2/M increased from a control value of 17.5% to 37. 2% , which the later condition is lOOnmol/L bufalin for 48h,the ratio of G0/Gj cells gradually decreased.4. The expression of p21 ,pRb,caspase -9 protein were downregulated and bcl - 2 prot...
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