摘要
目的:研究大萼香茶菜甲素(MA)抑制白血病细胞株HL-60增殖,诱导细胞分化和凋亡作用,并对其作用机制进行初步探讨。
方法:将不同浓度的MA与HL-60细胞在体外培养,台盼蓝染色、MTT比色法观察对HL-60细胞增殖的抑制作用;细胞形态学、DNA含量、DNA梯度电泳及细胞周期分析、Annexin-V/PI双标记和Hoechst 33258荧光染色等分析细胞凋亡;通过细胞表面抗原CD11b、CD13、CD14,NBT试验和细胞形态学检测MA对HL-60细胞的分化作用;流式细胞术检测Bcl-2、Bax、P53、Fas和线粒体膜蛋白、线粒体跨膜电位(Δψm)的表达变化;RT-PCR检测Bcl-2、Bax、P53、caspase-3 mRNA变化。
结果:MA呈时效及量效性地抑制HL-60细胞的增殖和活力,8μg/ml MA作用HL-60细胞24～72h后,MTT法检测24h、48h、72h的细胞增殖抑制率分别为(52.9±0.3)%、(70.0±1.0)%和(75.3±1.2)%,均显著高于对照组(P＜0.01);24h、48h、72h的IC50分别为8.76、7.17、7.14μg/ml;HL-60细胞经MA作用后,大部分细胞阻滞于G0/1期,DNA片段化,出现典型的细胞形态改变,经4～20μg/ml的MA作用后,SubG1由(2.85±1.6)%上升至(51.7±5.9)%,与对照组(1.74±0.5)%相比具有显著性差异(P＜0.05),Annexin-V/PI标记升高,Hoechst染色后的凋亡细胞的特征性改变等均表明MA能诱导HL-60细胞凋亡;HL-60细胞经4-8μg/ml MA作用24h后,细胞发生部分分化,细胞表面CD11b表达增加,NBT阳性细胞增多。MA诱导HL-60细胞凋亡和分化过程中,Bcl-2表达无变化,但Bax表达显著增加,Bax/Bcl-2的比值升高,Fas、P53表达无变化。随MA作用的浓度增加,caspase-3表达升高、线粒体膜电位(Δψm)下降、而线粒体膜蛋白表达显著升高。
结论:MA能抑制HL-60细胞增殖和细胞活力、诱导HL-60细胞向粒系分化、促进细胞凋亡,其机制与上调bax基因和bax/bcl-2比值、caspase-3激活与线粒体膜电位显著下降等有关。
Objective:To study the effect of MA on proliferation inhibition, differentiation and apoptosis in HL-60 human leukemia cell line and explore its possible mechanisms.
Methods:Different concentration of MA and the different time of cultivation were used to treat HL-60 cell.The proliferation inhibition was analyzed by Trypan blue staining and MTr assay.Cell apoptosis was analyzed by cell morphology,DNA agarose gel electrophoresis,DNA content and cell cycle analyzation,Annexin-Ⅴ/PI,Hoechst 33258 fluorescence staining.The cell morphological analysis,expression of CD11b,CD13,CD14,were performed to evaluate differentiation of HL-60 cells.The expressions of bcl-2,bax,Fas, P53 and mitochondrial membrane protein were analyzed by flow cytometry, While the mitochondrial transmembrancepotential(ΔΨm) was labeled by dihydrorhodamin 123.RT-PCR method was used to study the bcl-2、bax、p53、caspase-3 mRNA levels.
Results:MA could inhibit HL-60 cell proliferation viability within a certain range of treating time and doses,with a 24h IC50 of 8.76μg/ml,48 h of 7.17μg/ml and 72h of 7.14μg/ml.A majority of HL-60 cells were arrested in G0/G1 phase.The HL-60 cells apoptosis was confirmed by type cell morphology,DNA fragment,sub-G1 phase and Annexin-Ⅴ/PI Labeling method with a time and dose related manner.The morphology of HL-60 cells cultured in the presence of 4-8μg/ml MA for 16hs was more mature with higher positive rate of NBT and up-regulated expressions of CD11b than those cultured without MA.The expression of bax was increased,and bcl-2、p53、fas were unchanged by the treatment of MA.MA could increase the expression of mitochondrial membrane protein and caspase-3 in a dose-dependent manner while theΔΨm was reduced.
Conclusion::MA can inhibit proliferation,induce differentiation and apoptosis of HL-60 cells.The mechanism may associate with its up-regulation of bax,open the mitochondrial permeabilitytransition pore and reduceΔΨm.
引文
1. Iovino CS, Camacho LH. Acute myeloid leukemia:a classification and treatment update.Clin J Oncol Nurs.2003;7:535-540.
2. Hagemeijer A,Garson OM,Kondo K.Fourth International Workshop on Chromosomes in Leukemia 1982:Translocation(8;21)(q22;q22)in acute nonlymphocytic leukemia.Cancer Genet Cytogenet.l984;11(3):284-287.
3. Miyoshi H, Shimizu K, Kozu T, et al.T(8;21) breakpoints on chromosome 21 in acute myeloid leukemia are clustered within a limited region of a single gene,AMLl.Proc Natl Acad Sci U S A.1991;88(23):10431-10434.
4. Erickson P, Gao J, Chang KS, et al. Identification of breakpoints in t(8;21) acute myelogenous leukemia and isolation of a fusion transcript,AML1/ETO,with similarity to Drosophila segmentation gene, runt. Blood. 1992;80(7):1825-1831.
5. Larson RA, Kondo K,Vardiman JW, et al. Evidence for a 15;17 translocation in every patient with acute promyelocytic leukemia.Am J Med 1984;76:827-841.
6. Hogge DE, Misawa S, Schiffer CA, et al. Promyelocytic blast crisis in chronic granulocytic leukemia with 15; 17 translocation.Leuk Res.l984;8(6):1019-1023.
7. De The H, Chomienne C, Lanotte M, et al.The t(15;17) translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor a gene to a novel transcribed locus.Nature.l990;347(6293):558-561.
8. Borrow J, Goddard AD, Sheer D, et al. Molecular analysis of acute promyelocytic leukemia breakpoint cluster region on chromosome 17.Science. 1990;249(4976):1577-80.
9. Kakizuka A,Miller WH Jr,Umesono K, et al. Chromosomal translocation t(15;17)in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcriptionfactor,PML.Cell. 1991;66(4):663-674.
10.Horlein AJ, Naar AM, Heinzel T, et al. Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor co-repressor.l995;377(6548):397-404.
11.Chen JD, Evans RM. A transcriptional co-repressor that interacts with nuclear hormone receptors.Nature.l995;377(6548):454-457.
12.Koken MH, Puvion-Dutilleul F, Guillemin MC, et al.The t(15;17) translocation alters a nuclear body in a retinoic acid-reversible fashion.EMBO J.1994;13(5):1073-1083.
13.Weis K, Rambaud S, Lavau C, et al. Retinoic acid regulates aberrant nuclear localization of PML-RAR alpha in acute promyelocytic leukemia cells.Cell.l994;76(2):345-356.
14.Dyck JA, Maul GG, Miller WH Jr, et al. A novel macromolecular structure is a target of the promyelocyte-retinoic acid receptoroncoprotein.Cell.1994;76(2):333-343.
15.Winton EF, Langston AA. Update in acute leukemia 2003:a risk adapted approach to acute myeloblastic leukemia in adults.Semin Oncol.2004;31:80-86.
16.Chen GQ, Shi XG, Tang W, et al. Use of arsenic trioxide(As2O3)in the treatment of acute promyelocytic leukemia(APL):I.As2O3 exerts dose-dependent dual effects on APL cells.Blood.l997;89:3345-3353.
17.Puccetti E,Ruthardt M. Acute promyelocytic leukemia:PML/RARalpha and the leukemic stem cell.Leukemia.2004;18:1169-1175.
18.Tallmann MS. Curative therapeutic approaches to APL.Ann Hematol.2004;83:S81-82.
19.McKenzie SB.Advances in understanding the biology and genetics of acute myelocytic leukemia.Clin Lab Sci.2005;18(1):28-37.
20. Kimby E, Nygren P, Glimelius B; SBU-group. A systematic overview of chemotherapy effects in acute myeloid leukaemia.Acta Oncol.2001;40:231-252.
21.Voliotis D, Diehl V. Challenges in treating hematologic malignancies.Semin Oncol.2002;29:30-39.
22. Frankel AE, Baer MR, Hogge DE, et al. Immunotherapy of acute myeloid leukemia.Curr Pharm Biotechnol.2001;2:209-215.
23.Hamblin TJ. Disappointments in treating acute leukemia in the elderly.N Engl J Med.l995;332:1712-1713.
24.Sun SY, Hail N Jr, Lotan R. Apoptosis as a novel target for cancer chemopreventionJ Natl Cancer Inst.2004;96:662-672.
25.Parmar S, Rundhaμgen LM, Boehlke L, et al. Phase Ⅱ trial of arsenic trioxide in relapsed and refractory acute myeloid leukemia,secondary leukemia and/or newly diagnosed patients at least65 years old.Leuk Res.2004;28:909-919.
26 程培元,林永乐,徐光漪.大萼香茶菜新二萜成分:大萼香茶菜甲素和乙素的结构.药学学报,1984;19(8),593-598
27 石浩;何山;何兰;潘远江.大萼香茶菜中的新二萜化合物,高等学校化学学报,2007;01:112-114
28.Koester SK,Roth P,Mikulka WR,et al.Monitoring early cellular responses in apoptosis isaided by the mitochondrial membrane protein-specific monoclon-al antibody APO2.7.Cytometry,1997;29:306-312
29 王先荣,王兆全,董金广.大萼香茶菜甲素和丙素的化学结构.植物学报,1984,26(4),425-437
30 桂明玉.尾叶香茶菜二萜类成分与抗癌活性研究[D];吉林大学;2006
31 Hlestrap Ap,Doran E,Gillespie JP,O'Toole A.Mitochondriaand cell death.Biochemi Soci Transac,2000;28:170-177
32 Shimizu.S,Konishi A,Kodama T,Tsujimoto Y.BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death.Proc Natl Acad Sci,2000;97:3100-3105
1 Downing JR. The AML1-ETO chimaeric transcription factor in acute myeloid leukemia: biology and clinical significance. Br J Haematol,1999,106:296-308.
2 Licht JD. AML1 and the AML1/ETO fusion protein in the pathogenesis of t (8;21) AML. Oncogene,2001;20:5660-5679
3 Rubnitz JE,Raimondi SC,Halbert AR,et al.Characteristics and outcome of t(8;21)-positive childhood acute myeloid leukemia: a single institution's experience. Leukemia,2002;16:2072-2077.
4 Yan M, Kanbe E,Peterson LF,et al.A previously unidentified alternatively spliced isoform of t(8;21) transcript promotes leukemogenesis.Nat Med,2006;12:945-949.
5 Li Huang,Lynne V, Abruzzo, et al. Acute myeloid leukemia associated with variant t(8;21) detected by conventional cytogenetic and molecular studies. Am J Clin Pathol 2006;125:267-272.
6 De Greef GE,Hagemeijer A,Morgan R,et al.Identical fusion transcript associated with different breakpoint in the AML1 gene in simple and variant t(8;21) acute myeloid leukemia.Leukemia.l995;9:282-287
7 Vieira L,Oliveira V,Ambrosio AP,et al.Translocation (8;17;15;21)(q22;q23;ql5;q22) in acute myeloid leukemia (M2): a four- way variant of t(8;21).Cancer Genet Cytogenet.2001;128:104-107.
8 Gamerdinger U,Teigler-Schlegel A,Pils S, et al.Cryptic chromosomal aberrations leading to an AML1-ETO rearrangement are frequently caused by small insertions.Genes Chromosomes Cancer.2003;36:261-272.
9 Kondo K,Sasaki M,Mikuni C.A complex translocation involving chromosomesl,8 and 21 in acute myeloblastic leukemia.Proc Jpn Acad.l978;54:21-24.
10 Oguma N, Misawa S,Oguma N,et al. A variant 8;21 translocation in acute myeloblastic leukemia. Am J Hematal.l983;15:391-396.
11 Testa JR,Misawa S,Oguma N,et al. Chromosomal alterations in acute leukemia patients studied with improved culture methods.Cancer Res.l985;45:430-434.
12 Rubnitz JE,Raimondi SC,Halbert AR,et al.Characteristics and outcome of t(8;21)-positive childhood acute myeloid leukemia: a single institution,s experience.leukemia.2002;16:2072-2077.
13 Golomb HM,Vardiman JW,Rowley JD, et al.Correlation of clinical findings with quinacrine-banded chromosomes in 90 adults with acute nonlymphocytic leukemia:an eight-year study(1990-1997).N Engl J Med.l978;299:613-619.
14 Minamihisamatsu M, Ishihara T. Translocation (8;21) and its variant in acute nonlymphocytic leukemia: the relative importance of chromosomes 8 and 21 to the genesis of the disease.Cancer Genet Cytogenet.l988;33:161-173.
15 Testa JR, Oguma N, Pollak A, et al. Near-tetraploid clones in acute leukemia. Blood,1983,61:71.
16 Abe R, Raza A, Preisler HD, et al. Chromosomes and causation of human cancer and leukemia. Cancer Genet Cytogenet,1985,14:45.
17 Yamamoto K, Nagata K, Kida A, et al. CD7+ near-tetraploid acute myeloblastic leukemia M2 with double t(8;21)(q22;q22) translocations and AML1/ETO rearrangements detected by fluorescence in situ hybridization analysis. Int J Hematol. 2001,74(3):316-321.
18 Xue Y, He J, Wang Y, et al. Secondary near-pentaploidy and/or near-tetraploidy characterized by the duplication of 8;21 translocation in the M2 subtype of acute myeloid leukemia. Int J Hematol. 2000,71(4):359-365.
19 Xue Y, Pan Y, Liu Zh, et al. Tetraploid or near-tetraploid clones characterized by two 8;21 translocation and other chromosomal abnormalities in two patients with acute myeloblastic leukemia. Cancer Genet Cytogenet, 1996,92:18-23.
20 Rubnitz E, Pui CH, Downing JP. The role of TEL genes in pediatric leukemias.Leukemia,1999,13:6-13.
21 Wang M, Wang L,Wang JX,et al.Analysis and identification of transcriptional repression domain of ETO. Chin J Hematol,2003,24:10-13.
22 Hiebert SW, Lutterbach B, Amann J.Role of co-repressors in transcriptional repression mediated by the t(8;21),t(16;21),t(12;21),and inv(16) fusion protein.Curr Opin Hematol,2001;8:197-200.
23 Amann JM, Nip J, Strom DK, et al.ETO,a target of t(8;21) in acute leukemia,makes distinct contacts with multiple histone deacetylases and binds mSin3A throμgh its oligomerization domain.Mol Cell Biol,2001;21:6470-6483.
24 Licht JD. AML1 and the AML1-ETO fusion protein in the pathogenesis of t(8;21) AML.Oncogene,2001;20:5660-5679.
25 Pabst T, Muceller BU, Harakawa N, et al.AML1-ETO down-regulates the granulocytic differentiation factor C/EBPalpha in t(8;21) myeloid leukemia.Nat Med,2001:7:444-451.
26 Vangala RK, Heiss-Neumann MS, Rangatia JS,et al.The myeloid master regulator transcription factor PU.1 is inactivated by AML1-ETO in t(8;21) myeloid leukemia.Blood,2003;101:270-277.
27 Jakubowiak A, Pouponnot C,Berguido F,et al.inhibition of the transforming growth factor beta 1 signaling pathway by the AML1/ETO leukemia-associated fusion protein.J Biol Chem,2000;275:40282-40287.
28 Linggi B,Muller-Tidow C,van-de Locht L,et al.The t(8;21) fusion protein ,AML1 ETO,specifically represses the transcription of the pl4(ARF) tumor suppressor in acute myeloid leukemia.Nat Med,2002;8:743-750.
29 Yan M, Burel SA,Peterson LF,et al.Deletion of an AML1-ET0 C-terminal NcoR/SMRT-interacting region strongly induces leukemia development.Proc Natl Acad Sci USA,2004;101:17186-17191.
30 Yan M,Kanbe E,Peterson LF,et al. A previously unidentified alternatively spliced isoform of t(8;21) transcript promotes leukemogeesis.Nat Med,2006;12:945-949.
31 Ibanez V,Sharma A,Buonamici S,et al. AML1-ETO decreases ETO-2(MTG16) interactions with nuclear receptor corepressor,an e feet that impairs granulocyte differentiation.CancerRes,2004,64:4547-4554.
32 Annika E,Michael F,Alexander K,et al.The fusion protein AML1-ETO in acute myeloid leukemia with,translocation t(8;21) induces c-jun protein expression via the proximal AP-1 site of the c-jun promoter in anindirect,JNK-dependent manner.Oncogene,2003,22:5646-5657.
33 Zada AA,Singh SM,Reddy VA,et al.Downregulation of c-Jun expression and cell cycle regulatory molecules in acute myeloid leukemia cells upon CD44 ligation.Oncogene,2003,22:2296-2308.
34 Thomas P,Beatrice U,Nari H,et al.AMLl-ETO downregulates the granulocytic differentiation factor C/EBPa in t(8;21) myeloid leukemia.Nat Med,2001,7:444-451.
35 Tanaka K,Tanaka T,Kurokawa M,et al.The AML1/ETO(MTG8) and AML1/Evi-1 leukemia-associated chimeric oncoproteins accumulate PEBP2b(CBFb) in the nucleus more efficiently than wild-type AMLl.Blood,1998,91:1688-1699.
36 Lutterbach B,Hiebert SW.Role of the transcription factor AML-1 in acute leukemia and hematopoietic differentiation.Gene,2000,245:223-235.
37 de Figueiredo LL,de Abreue Lima RS,Rego EM.Granulocyte colony-stimulating factor and leukemogenesis.Mediators Inflamm,2004,13:145-150.
38 Wang YY,Zhou GB,Yin T,et al.AMLl-ETO and C-KIT mutation/overexpression in t(8;21) leukemia: implication in stepwise leukemogenesis and reponse to Gleevec.Proc Natl Acad Sci USA.2005;102:1104-1109.
39 Sakakura C, Yamaguchi-Iwai Y, Satake M, et al.Growth inhibition and induction of differentiation of t(8;21) acute nyeloid leukemia cells by the DNA-binding domain of PEBP2 and the AML1/MTG8(ETO)-specfic antisense oligonucleotide. Proc Natl Acad Sci USA,1994;91:11723-11727.
40 Matsushita H, Kobayashi H, Mori S,et al. Ribozymes cleave the AML1/MTG8 fusion transcript and inhibit proliferation of leukemia cells with t(8;21).Biochem Biophys Res Commun,1995;215:431-437.
41 Matsushita H, Kizaki M, Kobayashi H, et al. Induction of apoptosis in myeloid leukaemic cells by ribozymes targeted against AML1/MTG8.Br J Cancer,1999;79:1325-1331.
42 Heidenreich O, Krauter J, Riehle H, et al.AML1/MTG8 oncogene suppression by small interfering RNAs supports myeloid differentiation of t(8;21)-positive leukemic cells.BLOOD,2003;101:3157-3163.
43 Zhou GB, Kang H, Wang L,et al. Oridonin,a diterpenoid extracted from medicinal herbs,targets AML1-ETO fusion protein and shows potent antitumor activity with low adverse effects on t(8;21) leukemia in vitro and in vito.Blood,2007;109:3441-3450.
44 Wang L, Zhao WL, Yan JS, et al.Eriocalyxin B induce apoptosis of t(8;21) leukemia cells through NF-kappaB and MAPK signaling pathways and triggers degradation of AML1-ETO oncoprotein in a caspase-3-dependent manner.Cell Death Differ,2006;14:306-317.
45 Tyler JK, Kadonaga JT. The "dark side" of chromatin remodeling:repressive effects on transcription.Cell,1999;99:443-446.
46 Redner RL,Wang J, Liu JM. Chromatin remodeling and leukemia:new therapeutic paradigms.Blood,1999;94:417-428.
47 Ferrara FF, Fazi F, Bianshini A, et al. Histone deacetylase-targeted treatment restores retinoic acid signaling and differentiation in acute myeloid leukemia.Cancer Res,2001;61:2-7.
48 Wang J, Saunthararajah Y, Redner RL, et al.Inhibitors of Histone deacetylase relieve ETO-mediated repression and induce differentiation of AML1-ET0 leukemia cells.Cancer Res.l999;59:2766-2769.