植物多酚提高柔红霉素耐药白血病干细胞杀伤敏感性的研究
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摘要
目的探讨植物多酚提高柔红霉素耐药白血病干细胞杀伤敏感性、杀伤活性及其机制。方法分离出对柔红霉素耐药的白血病干细胞,应用Annexin V/PI和API等标记,通过流式细胞仪检测植物多酚刺激柔红霉素耐药的白血病干细胞的细胞凋亡及周期特异性;运用RT-PCR方法检查Caspase-3、Caspase-9活化情况;MTT检测细胞增殖活力;Transwell体外迁移实验检测细胞体外迁移和侵袭能力。结果处于静止期的耐药白血病干细胞对植物多酚凋亡诱导不敏感,G1期耐药白血病干细胞应用植物多酚诱导后出现了明显的细胞凋亡,提示植物多酚诱导的细胞凋亡主要在细胞周期的G1期发生。植物多酚诱导柔红霉素耐药的白血病干细胞的Caspase-3、Caspase-9 mRNA表达值显著高于对照组。Transwell结果显示,植物多酚诱导第7天体外迁移能力明显下降,细胞活力明显下降。结论植物多酚提高柔红霉素耐药白血病干细胞的杀伤敏感性与细胞周期相关,存在G1期细胞周期阻滞的周期特异性,且此过程中发生了caspase活化、参与凋亡,从而抑制了细胞活性及迁移侵袭能力。
Objective To study the plant polyphenols to enhance the sensitivity,cytotoxicity and mechanism of daunorubicin to leukemia stem cells. Methods The leukemia stem cells resistant to daunorubicin were isolated and identified by Annexin V/PI and API. The apoptosis and periodicity of leukotriene-resistant leukemia stem cells were detected by flow cytometry. The activation of Caspase-3 and Caspase-9 was detected by RT-PCR. Cell viability was detected by MTT assay. Transwell migration assay was used to detect cell migration and invasion in vitro. Results In the quiescent period,the resistant leukemia stem cells were not sensitive to the induction of apoptosis of plant polyphenols. The apoptosis of G1 phase resistant leukemia stem cells induced by plant polyphenols,suggested that plant polyphenol-induced apoptosis was mainly in cells The G1 phase of the cycle occurs. The expression of Caspase-3 and Caspase-9 mRNA in the leukemic stem cells induced by plant erythromycin-induced leukotriene was significantly higher than that of the control group. Transwell results showed that the migration ability of plant polyphenols induced in vitro was significantly enhanced and the cell viability decreased significantly. Conclusion The sensitivity of plant polyphenols to increase the cytotoxicity of daunorubicin-resistant leukemia stem cells is related to the cell cycle,and there is a period-specificity of G1 phase cell cycle arrest. Caspase activation occurs during this process and is involved in apoptosis,activity and migration ability.
Objective To study the plant polyphenols to enhance the sensitivity,cytotoxicity and mechanism of daunorubicin to leukemia stem cells. Methods The leukemia stem cells resistant to daunorubicin were isolated and identified by Annexin V/PI and API. The apoptosis and periodicity of leukotriene-resistant leukemia stem cells were detected by flow cytometry. The activation of Caspase-3 and Caspase-9 was detected by RT-PCR. Cell viability was detected by MTT assay. Transwell migration assay was used to detect cell migration and invasion in vitro. Results In the quiescent period,the resistant leukemia stem cells were not sensitive to the induction of apoptosis of plant polyphenols. The apoptosis of G1 phase resistant leukemia stem cells induced by plant polyphenols,suggested that plant polyphenol-induced apoptosis was mainly in cells The G1 phase of the cycle occurs. The expression of Caspase-3 and Caspase-9 mRNA in the leukemic stem cells induced by plant erythromycin-induced leukotriene was significantly higher than that of the control group. Transwell results showed that the migration ability of plant polyphenols induced in vitro was significantly enhanced and the cell viability decreased significantly. Conclusion The sensitivity of plant polyphenols to increase the cytotoxicity of daunorubicin-resistant leukemia stem cells is related to the cell cycle,and there is a period-specificity of G1 phase cell cycle arrest. Caspase activation occurs during this process and is involved in apoptosis,activity and migration ability.
引文
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[2]Steelman LS,Franklin RA,Abrams SL,et al.Roles of the Ras/Raf/MEK/ERK pathway in leukemia therapy[J].Leukemia,2011,25(7):1080-1094.
[3]Gerber JM1,Smith BD,Ngwang B,et al.A clinically relevant population of leukemic CD34(+)CD38(-)cells in acute myeloid leukemia[J].Blood,2012,119(15):3571-3577.
[4]Hsieh TC,Wu JM.Resveratrol:Biological and pharmaceutical properties as anticancer molecule[J].Biofactors,2010,36(5):360-369.
[5]Wong RS,Cheong SK.Leukemic stem cells:drug resistance,metastasis and therapeutic implications[J].Malays J Pathol,2012,34(2):77-88.
[6]Hoang VT,Zepeda-Moreno A,Ho AD.Identification of leukemia stem cells in acute myeloid leukemia and their clinical relevance[J].Biotechnol J,2012,7(6):779-788.
[7]Pollyea DA,Gutman JA,Gore L,et al.Targeting acute myeloid leukemia stem cells:a review and principles for the development of clinical trials[J].Haematologica,2014,99(8):1277-1284.
[8]Iwasaki H.Leukemia stem cell[J].Gan To Kagaku Ryoho,2014,41(3):280-284.
[9]Thomas X,Cannas G.Leukemia stem cells and new strategies to overcome resistance to therapy[J].Curr Stem Cell Res Ther,2010,5(3):277-86.
[10]Elliott A,Adams J,Al-Hajj M.The ABCs of cancer stem cell drug resistance[J].IDrugs,2010,13(9):632-635.
[11]Giménez-BonaféP,Tortosa A,Pérez-Tomás R.Overcomingdrug resistance by enhancing apoptosis of tumor cells[J].Curr Cancer Drug Targets,2009,9(3):320-340.
[12]Yanada M,Naoe T.Acute myeloid leukemia in older adults[J].Int J Hematol,2012,96(2):186-193.
[13]Ichim CV.Kinase-independent mechanisms of resistance of leukemia stem cells to tyrosine kinase inhibitors[J].Stem Cells Transl Med,2014,3(4):405-415.
[14]Lutz C,Hoang VT,Buss E,et al.Identifying leukemia stem cells--is it feasible and does it matter?[J].Cancer Lett,2013,338(1):10-14.
[15]Liang CZ,Zhang JK,Shi Z,et al.Matrine induces caspasedependent apoptosis in human osteosarcoma cells in vitro and in vivo through the upregulation of Bax and Fas/Fas L and downregulation of Bcl-2[J].Cancer Chemother Pharmacol,2012,69(2):317-331.