吴茱萸碱诱导人子宫颈癌HeLa细胞周期阻滞的机理研究
摘要
本文就吴茱萸碱诱导人子宫颈癌HeLa细胞周期阻滞的机理进行研究。使用浓度1μg/ml吴茱萸碱作用于HeLa细胞,0、4、8、12、16、20、24小时收集细胞流式测定:吴茱萸碱使HeLa细胞阻滞在G2/M期。通过对周期蛋白cyclinB1的mRNA研究发现,吴茱萸碱能强烈的抑制cyclinB1 mRNA的表达,进一步分析周期蛋白激酶抑制剂p21mRNA发现,吴茱萸碱能上调p21 mRNA的表达。提示吴茱萸碱诱导HeLa细胞周期阻滞可能与cyclinB1和p21的转录有关。
Cancer is a major killer of human health. The development of tumor is closely related to the disorders of normal cell cycle control mechanisms. Studies found the expression of cell cycle regulators,related to cell cycle changes, is disordered in many tumor cells. Therefore, take cell cycle regulators for targets and develop drugs , those provide a new program to cancer treatment.
Evodiamine is an alkaloid extracted from traditional Chinese medicine Wuzhuyu . From the laboratory basic research, evodiamine can induce cervical carcinoma HeLa cells to apoptosis and cycle arrest ,but the mechanism is still unclear. In this paper, we research the initial molecular mechanism of cell cycle arrest induced by evodiamine. We hope that we can have a deep understand of the anti-tumor effect of evodiamine, and provide some reference to its research , its development and cancer treatment .
First, evodiamine induced HeLa cells with the concentration of 0.1,1,10μg / ml ,then measured by crystal violet staining to verify its cytotoxicity. From the result ,we can know evodiamine has high cytotoxicity, and their cytotoxicity has time and concentration dependent obviously. Next, the concentration of 1μg/ml of evodiamine is selected to induced HeLa cells with 0,4,8,12,16,20,24 hours of proliferation. cell cycle of HeLa cells detected by flow cytometry was arrested at G2 / M phase. In order to understand the molecular mechanism of G2 / M arrest caused by evodiamine. the expression of cyclinB1mRNA was researched , and we found that the expression of cyclinB1mRNA was inhibited. p21mRNA expression was raised thought the further research
In summary, we have come to the preliminary conclusion: Evodiamine is up the expression of p21mRNA,then may lead to the increase of p21 protein, which mediated the inhibition of the expression of cyclinB1mRNA.And cyclinB1 protein expression may be inhibited, thus make Hela cells arrested in G2 / M phase.
Cancer is a major killer of human health. The development of tumor is closely related to the disorders of normal cell cycle control mechanisms. Studies found the expression of cell cycle regulators,related to cell cycle changes, is disordered in many tumor cells. Therefore, take cell cycle regulators for targets and develop drugs , those provide a new program to cancer treatment.
Evodiamine is an alkaloid extracted from traditional Chinese medicine Wuzhuyu . From the laboratory basic research, evodiamine can induce cervical carcinoma HeLa cells to apoptosis and cycle arrest ,but the mechanism is still unclear. In this paper, we research the initial molecular mechanism of cell cycle arrest induced by evodiamine. We hope that we can have a deep understand of the anti-tumor effect of evodiamine, and provide some reference to its research , its development and cancer treatment .
First, evodiamine induced HeLa cells with the concentration of 0.1,1,10μg / ml ,then measured by crystal violet staining to verify its cytotoxicity. From the result ,we can know evodiamine has high cytotoxicity, and their cytotoxicity has time and concentration dependent obviously. Next, the concentration of 1μg/ml of evodiamine is selected to induced HeLa cells with 0,4,8,12,16,20,24 hours of proliferation. cell cycle of HeLa cells detected by flow cytometry was arrested at G2 / M phase. In order to understand the molecular mechanism of G2 / M arrest caused by evodiamine. the expression of cyclinB1mRNA was researched , and we found that the expression of cyclinB1mRNA was inhibited. p21mRNA expression was raised thought the further research
In summary, we have come to the preliminary conclusion: Evodiamine is up the expression of p21mRNA,then may lead to the increase of p21 protein, which mediated the inhibition of the expression of cyclinB1mRNA.And cyclinB1 protein expression may be inhibited, thus make Hela cells arrested in G2 / M phase.
引文
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[3] Koepp D M, Schaefer L K, Ye X, et al. Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase [J]. Science,2001,294 (5540) : 173-177
[4] Bartlet R, Nurse P. Yeast as a model system for understanding the control of DNA replication in eukaryotes[J]. Bioessays, 1990,12:457-463
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[8] Reid B J , Hartwell L H. Regulation of mating in the cell cycle of S accharymyces cerevisiae[J]. J Cell Biol,1977,75:355-365
[9] Evans T, Rosenthal E T, Hunt T,et al . Cyclin: a protein specified by maternal mR NA in sea urchin eggs that is destroyed at each cleavage division[J].Cell,1983,33: 38 9 -3 96.
[10] Nurse P, Thuriaux P. Regulatory genes controlling mitosis in the fission yeast Schizosacchharomyces pombe[J] . Genetics,1980,96:627-637.、
[11] Simanis V, Hayles J,Nurse P. Control over the onset of DNA synthesis in fission yeast [J].Philos Trans R Soc Lond B Biol Sci,1987,317:507-516
[12] Simanis V,Nurse P. The cell cycle control gene cdc2+of fission yeast encodes a Protein k inase potentially regulated by phosphorylation[J]. Cell, 1986 ,45:261-268.
[13] Hunt T, Luca F C, Ruderman J V. The requirements for protein synthesis and degradation,and the contiol of destruction o f cyclins A and B in the meiotic and mitotic cell cycles of the clam embryo[J]. J Cell Biol ,1992,116: 707-724.
[14] HartwellL H, CulotiJ , PringleJ R , et al. Genetic control of the cell d ivision cycle in yeast [J].Science,1974,183:46-51
[15] Hunt T. Cyclins and their partners : from a simple idea to complicated reality[J] Semin Cell Biol,1991,2(4):213-222.
[16] Charles J S, James M R. CDK negative regulators of G1-phase inhibitors: positive and porgerssion[J] . Gene Dev,1999; 13:1501-1512.
[17] Chin L, Pomerantz J, DePinho R A. The INK4a/ARF tumor suppressor: one gene-two products-two pathways[J].T rends Biochem Scl, 1998;2 3(8):291-296.
[18] Shapiro G I, Edwards C D, Rollins B J. The physiology of p16( INK4A) -mediated G1 proliferative arrest[J]. Cell Biochem Biophys, 2000, 33 (2) : 189-197.
[19] Pestel R G, Albanese C, Reutens A T, et al. The cyclins and cyclic-dependent kinase Inhibitors in hormonal regulation of proliferation and differentiation[J]. Endocr Rev, 1999;2 0(4):5 01-534.
[20] Luo, Y, Hurwitz J, Massague J. Cell-cycle inhibition by independent CDK and PCNA binding domains in p21CIP1[J]. Nature,1995 375, 159–161
[21] Chen J, Jackson P K, Kirschner M W, et al. Separate domains of p21 involved in the inhibition of Cdk kinase and PCNA[J]. Nature 374, 386–388 (1995).
[22] Chen J, Saha P, Kornbluth S, et al. Cyclin-binding motifs are essential for thefunction of p21CIP1[J]. Mol Cell Biol,1996,. 16, 4673–4682,
[23] Zhu L, Harlow E, Dynlacht B D. p107 uses a p21CIP1-related domain to bind cyclin/cdk2 and regulate interactions with E2F[J]. Genes Dev, 1995,9,1740–1752
[24] Shiyanov P. p21 disrupts the interaction between cdk2 and the E2F–p130 complex[J]. Mol Cell Biol, 1996,16:737–744
[25] Saha P, Eichbaum Q, Silberman E D, et al. p21CIP1 and Cdc25A: competition between an inhibitor and an activator of cyclin dependent kinases[J]. Mol Cell Biol, 1997,17, 4338–4345
[26] Moldovan G L, Pfander B, Jentsch S. PCNA, the maestro of the replication fork[J]. Cell,2007,129:665–679.
[27] Gunter T, Pines J. cyclins and cancerⅡ:cyclin D and CDK inhibitors come of age[J]. Cel1,1994,79(6):573
[28] Sherr C J. Mammalian G1 cyclins[J].cell ,1993, 73:1069
[29] Sherr C J. G1 phase progression: cycling on cue[J] .cell ,1994,79:551
[30] Hirai H, Roussel M F, Kato J Y, et al. Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6[J].Mol Cell Biol,1995, 15(5):2672-2681
[31] Baldin V, Lukas J, Marcote M J, et al. Cyclin D1 is a nuclear protein required for cell cycle progression in G1[J].Genes Dev, 1993 ,7:812-821
[32] Nevins J R. E2F: a link between the Rb tumor suppressor protein and viral oncoproteins[J]. science,1992,258(5081):424-429
[33] Stteven J, Weintraub, Cheryl A, et al. Retinoblastoma protein switches the E2F site from positive to negative element [J].nature,1992,358:259-261
[34] Mark E, Ewen, Hayla K, et al. Functional interactions of the retinoblastoma protein with mammalian D-type cyclins[J] .cell,1993,73(3):487-497
[35] Nicholas B, Thangue L. DP and E2F proteins: components of a heterodimeric transcription factor implicated in cell cycle control Current Opinion in Cell [J].Biology, 1994,6(3):443-450
[36] Weinberg R A .The retinoblastoma protein and cell cycle control [J]. Cell, 1995,81:323-330
[37] Won K A, Reed S I. Activation of cyclin E/CDK2 is coupled to site-specific autophosphorylation and ubiquitin-dependent degradation of cyclin E[J]. EMBO J,1996,15(16): 4182–4193
[38] Polyak K L, Lee M H, Bromage H E, et al. Cloning of p27Kip1, a cyclin-dependent kinase inhibitor and a potential mediator of extracellular antimitogenic signals[J]. Cell,1994, 78(1):59-66
[39] Krek W, Ewen M E, Shirodkar S, et al. Negative regulation of the growth-promoting transcription factor E2F-1 by a stably bound cyclin A-dependent protein kinase[J]. Cell,1994,78(1):161-172
[40] Julio E, Madsen C P, Celis A, et al. Nielsena and Borbala Gessera Cyclin (PCNA, auxiliary protein of DNA polymeraseδ) is a central component of thepathway(s) leading to DNA replication and cell division[J]. FEBS letters,1987 ,220(1):1-7
[41] Sheer C J. Cancer cell cycles[J]. Science,1996, 274(5293):1672-1677
[42] Meijer L, Azzi L, Wang J Y. Cyclin B1 targets p34cdc2 for tyrosine phosphorylation[J]. EMBO,1991,10:1545–1554.
[43] Parker L L, Lee M S, Ogg S, et al. Cyclin promotes the tyrosine phosphorylation of p34cdc2 in a wee11 dependent manner[J]. EMBO ,1991,10:1255–1263.
[44] Toyoshima F k, Moriguchi T, Wada A, et al.Nuclear export of cyclin B1 and its possible role in the DNA damage-induced G2 checkpoint[J]. EMBO, 1998, 17(10):2728-2735
[45] Dunphy W G. The decision to enter mitosis[J]. Trends Cell Biol,1994,4: 202-207
[46] Sudakin V, Ganoth D, Dahan A, et al. The Cyclosome, a Large Complex Containing Cyclin-Selective Ubiquitin Ligase Activity, Targets Cyclins for Destruction at the End of Mitosis Molecular [J].Biology of the Cell,1995, 6: 185-198
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