雄黄微生物浸出液通过线粒体途径诱导人急性髓系白血病细胞HL60细胞凋亡
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摘要
研究雄黄微生物浸出液(RBS)诱导急性髓系白血病细胞HL-60细胞凋亡的作用机制。应用MTT比色法检测不同时间与不同浓度雄黄微生物浸出液对HL-60细胞的抑制作用。应用Hoechst33258染色、流式细胞术、罗丹明123染色以及Western blot等多种方法,研究雄黄微生物浸出液对肿瘤细胞凋亡的作用机制。结果:雄黄微生物浸出液对HL-60细胞增殖有明显的抑制作用,且呈浓度梯度与时间梯度依赖性,阻滞HL-60细胞到G2/M期,促进细胞凋亡以及降低线粒体膜电位。下调cyclin B1、CDK1、c-myb、c-myc、Bcl-2的表达,上调cleaved-caspase3的表达。降低线粒体膜电位,通过线粒体途径激活caspase3,下调癌蛋白c-myc的表达以及降低抗凋亡蛋白Bcl-2可能是RBS对HL-60细胞G2/M阻滞以及诱导凋亡的潜在机制。
Realgar bioleaching solution( RBS) is made by using microbiological leaching method. Previous researches showed that RBS has a significant anti-proliferation effect on chronic myeloid leukemia cells,but the effect and mechanisms of acute myeloid leukemia cells are still not clear. To study anti-proliferation effect of RBS on acute myeloid leukemia cell lines HL-60 and to explore its apoptosis mechanism,HL-60 cells investigated the anti-proliferation effect by MTT assay by treating with RBS after 24,48 and72 h. By Hoechst 33258 dyeing HL-60 cells after treating with RBS in 24 h to explore the apoptotic body. After this,flow cytometry assay has been used to explore the cell cycle arrest and Annexin V/PI dye to explore apoptosis of HL-60. Then studying on the mitochondrial membrane potential level by rhodamine123 dye. At last,detect the protein expression of cyclin B1,CDK2,c-myb,c-myc and Bcl-2 by western blot. RBS has a significant inhibition ratio for HL-60 cells which is shown as concentration-dependent and timedependent,and IC50 are 3. 12,1. 27 and 0. 13 μg/m L in 24,48 and 72 h respectively. HL-60 was apoptosis with nuclear chromatin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies was observed by Hoechst 33258 dye.Apoptosis proportion is also be shown by the FCM results. In addition,RBS can also arrest the cell cycle within G2/M. Beyond this,rodamine123 dye demonstrated that RBS can reduce the mitochondrial membrane potential. According to the western blot,the protein expression of cyclin B1,CDK2,c-myc and Bcl-2 were decreased by treating with RBS for 24 h,while c-myb showed a little downtrend. In addition,the protein expression of cleaved-caspase3 was increased within concentration-dependent. RBS has a capacity of cell cycle arrest in G2/M and apoptosis. Reducing the mitochondrial membrane potential,activating caspase3,and down regulating the expression of protein c-myc and Bcl-2 which may be the potential mechanisms.
Realgar bioleaching solution( RBS) is made by using microbiological leaching method. Previous researches showed that RBS has a significant anti-proliferation effect on chronic myeloid leukemia cells,but the effect and mechanisms of acute myeloid leukemia cells are still not clear. To study anti-proliferation effect of RBS on acute myeloid leukemia cell lines HL-60 and to explore its apoptosis mechanism,HL-60 cells investigated the anti-proliferation effect by MTT assay by treating with RBS after 24,48 and72 h. By Hoechst 33258 dyeing HL-60 cells after treating with RBS in 24 h to explore the apoptotic body. After this,flow cytometry assay has been used to explore the cell cycle arrest and Annexin V/PI dye to explore apoptosis of HL-60. Then studying on the mitochondrial membrane potential level by rhodamine123 dye. At last,detect the protein expression of cyclin B1,CDK2,c-myb,c-myc and Bcl-2 by western blot. RBS has a significant inhibition ratio for HL-60 cells which is shown as concentration-dependent and timedependent,and IC50 are 3. 12,1. 27 and 0. 13 μg/m L in 24,48 and 72 h respectively. HL-60 was apoptosis with nuclear chromatin condensation and fragmentation as well as cell shrinkage and the formation of apoptotic bodies was observed by Hoechst 33258 dye.Apoptosis proportion is also be shown by the FCM results. In addition,RBS can also arrest the cell cycle within G2/M. Beyond this,rodamine123 dye demonstrated that RBS can reduce the mitochondrial membrane potential. According to the western blot,the protein expression of cyclin B1,CDK2,c-myc and Bcl-2 were decreased by treating with RBS for 24 h,while c-myb showed a little downtrend. In addition,the protein expression of cleaved-caspase3 was increased within concentration-dependent. RBS has a capacity of cell cycle arrest in G2/M and apoptosis. Reducing the mitochondrial membrane potential,activating caspase3,and down regulating the expression of protein c-myc and Bcl-2 which may be the potential mechanisms.
引文
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[14]Chen LB.Mitochondrial membrane potential in living cells[J].Annual Review Cell Biol,1988,4(4):151-181.
[15]陈长恒.Cdc2基因与细胞周期调控[J].中国实用医药,2007,2(13):105-107.
[16]Hartwell LH,MB Kastan.Cell cycle control and cancer[J].Science,1994,266(5192):1821-1828.
[17]Nakata Yuji,Susan Shetzline,Chizuko Sakashita,et al.c-Myb contributes to G2/M cell cycle transition in human hematopoietic cells by direct regulation of cyclin B1 expression[J].Mol Cell Biol,2007,27(6):2048-2058.
[18]蔡循,陈国强,陈竺,等.线粒体跨膜电位与细胞凋亡[J].生物化学与生物物理进展,2001,28(1):3-6.
[19]吴兰芳,杨爱珍,刘和,等.线粒体调控细胞凋亡的研究进展[J].中国农学通报,2010,26(8):63-68.
[20]Enari Masato,Hideki Sakahira,Hideki Yokoyama,et al.A caspase-activated DNase that degrades DNA during apoptosis,and its inhibitor ICAD[J].Nature,1998,391(6662):43-50.
[21]Kluck Ruth M,Wetzel E B,Green D R,et al.The release of cytochrome c from mitochondria:a primary site for Bcl-2 regulation of apoptosis[J].Science,1997,275(5303):1132-1136.
[22]何勤,秦苑,毕慧,等.急性白血病细胞bcl-2、c-myc和p53基因表达的研究[J].临床血液学杂志,2002,15(5):213-215.
[2]Lu DP,Wang Q.Current study of APL treatment in China[J].Int J Hematol,2002,76 Suppl 1:316-318.
[3]Lu DP,Qiu JY,Jiang B,et al.Tetra-arsenic tetra-sulfide for the treatment of acute promyelocytic leukemia:a pilot report[J].Blood,2002,99(9):3136-3143.
[4]Wang L,Zhou GB,Liu P,et al.Dissection of mechanisms of Chinese medicinal formula Realgar-Indigo naturalis as an effective treatment for promyelocytic leukemia[J].Proceed Nation Acad Sci USA,2008,105(12):4826-4831.
[5]Balá6 Peter,Martin Fabián,Michal Pastorek,et al.Mechanochemical preparation and anticancer effect of realgar As4S4 nanoparticles[J].Mater Lett,2009,63(17):1542-1544.
[6]Koch Iris,Steven Sylvester,Vivian W-M Lai,et al.Bioaccessibility and excretion of arsenic in Niu Huang Jie Du Pian pills[J].Toxicol Appl Pharmacol,2007,222(3):357-364.
[7]Zhang C,Huang SL,Xiang Y,et al.Study on realgar inducing apoptosis in T lymphocytic cell line CEM[J].Zhong Xi Yi Jie He Xue Bao,2003,1(1):42-43.
[8]黄士林.复方青黛片为主治疗急性早幼粒细胞白血病的临床研究[J].中华血液学杂志,1995,16(01):227-229.
[9]Zhang JH,Zhang X,Ni YQ,et al.Bioleaching of arsenic from medicinal realgar by pure and mixed cultures[J].Process Biochem,2007,42(9):1265-1271.
[10]Xie QJ,Cao XL,Bai L,et al.Anti-tumor effects and apoptosis induction by realgar bioleaching solution in Sarcoma-180 cells in vitro and transplanted tumors in mice in vivo[J].Asian Pac J Cancer Prev,2014,15(6):2883-2888.
[11]Wang X,Zhang X,Xu ZL,et al.Reversal effect of arsenic sensitivity in human leukemia cell line K562 and K562/ADM using realgar transforming solution[J].Biol Pharm Bull,2013,36(4):641-648.
[12]Liu DL,Zhi DJ,Zhou T,et al.Realgar bioleaching solution is a less toxic arsenic agent in suppressing the Ras/MAPK pathway in Caenorhabditis elegans[J].Environ Toxicol Pharmacol,2013,35(2):292-299.
[13]贾晋松,徐世荣,马劼,等.三氧化二砷对HL60细胞作用后细胞周期素相关基因的表达[J].中华内科杂志,2003,42(2):113-116.
[14]Chen LB.Mitochondrial membrane potential in living cells[J].Annual Review Cell Biol,1988,4(4):151-181.
[15]陈长恒.Cdc2基因与细胞周期调控[J].中国实用医药,2007,2(13):105-107.
[16]Hartwell LH,MB Kastan.Cell cycle control and cancer[J].Science,1994,266(5192):1821-1828.
[17]Nakata Yuji,Susan Shetzline,Chizuko Sakashita,et al.c-Myb contributes to G2/M cell cycle transition in human hematopoietic cells by direct regulation of cyclin B1 expression[J].Mol Cell Biol,2007,27(6):2048-2058.
[18]蔡循,陈国强,陈竺,等.线粒体跨膜电位与细胞凋亡[J].生物化学与生物物理进展,2001,28(1):3-6.
[19]吴兰芳,杨爱珍,刘和,等.线粒体调控细胞凋亡的研究进展[J].中国农学通报,2010,26(8):63-68.
[20]Enari Masato,Hideki Sakahira,Hideki Yokoyama,et al.A caspase-activated DNase that degrades DNA during apoptosis,and its inhibitor ICAD[J].Nature,1998,391(6662):43-50.
[21]Kluck Ruth M,Wetzel E B,Green D R,et al.The release of cytochrome c from mitochondria:a primary site for Bcl-2 regulation of apoptosis[J].Science,1997,275(5303):1132-1136.
[22]何勤,秦苑,毕慧,等.急性白血病细胞bcl-2、c-myc和p53基因表达的研究[J].临床血液学杂志,2002,15(5):213-215.