摘要
目的 研究对香豆酸组合物(total coumarins of Hedyotis diffusa,TCHD)对急性髓系白血病(AML)细胞的增殖抑制作用及其机制。方法 采用乙醇回流法提取TCHD,超高效液相色谱串联质谱系统(UPLC-MS/MS)检测其纯度。用不同浓度的TCHD(0.02、0.04、0.06、0.08、0.10 mg·mL-1),作用于对数生长期的Kasumi-1细胞、KG-1细胞、THP-1细胞、U937细胞和K562细胞,四甲基偶氮唑蓝(MTT)法检测各细胞增殖;作用于Kasumi-1细胞24 h后,采用AnnexinⅤ/PI流式细胞术检测细胞凋亡情况;运用流式细胞术检测药物作用后Kasumi-1细胞周期阻滞情况;Western blot法检测PARP、caspase-3、caspase-9、 caspase-8蛋白活性的变化;以及CDK4/6、cyclin D1、CDK2、p-CDK2、cyclin E、p21等细胞周期相关蛋白的变化。结果 TCHD在一定浓度范围内对AML细胞增殖有明显的抑制作用,对Kasumi-1、KG-1、THP-1、U937和K562细胞的IC50(24 h vs 48 h)分别为0.077 vs 0.059 (mg·mL-1)、0.083 vs 0.067(mg·mL-1)、0.096 vs 0.072(mg·mL-1)、0.087 vs 0.064(mg·mL-1)、0.096 vs 0.068(mg·mL-1),对Kasumi-1的抑制作用表现最明显,并且呈时间-浓度依赖性(r=0.357,P<0.05)。不同浓度 (0,0.02,0.04,0.06,0.08,0.10 mg·mL-1)的TCHD作用于Kasumi-1细胞株24 h后,凋亡率分别是(5.33±0.41)%、(7.99±0.45)%、(10.22±0.32)%、(20.10±1.99)%、(28.66±0.67)%和(33.24±2.12)%。0.02~0.06 mg·mL-1的TCHD处理Kasumi-1细胞24 h后用流式细胞术检测到该细胞G0/G1期的比例依次为(51.43±3.21)%、(62.91±2.35)%和(76.42±4.14) %,与空白对照组[(35.8±5.25)%]比较,差异均有统计学意义(P值均<0.05)。Western blot结果显示,不同浓度的TCHD能浓度依耐性激活caspase-8(P<0.01)、caspase-9、caspase-3和PARP(其余P<0.05),促进细胞色素C的表达;显著下调cyclin E、CDK6、CDK2、p-CDK2、cyclin D1,和上调p21(P<0.01)。结论 TCHD抑制白血病细胞株Kasumi-1的增殖呈时间浓度依赖性,其凋亡机制一方面与其激活caspase-3、caspase-9、PARP蛋白有关,另一方面则通过影响CDK2、p-CDK2、CDK4/6、cyclin E、cyclin D1和p21使Kasumi-1细胞被阻滞于G0/G1期,抑制细胞增殖。
Abstract
OBJECTIVE To explore the effect of total coumarins isolated from Hedyotis diffusa (total coumarins from Hedyotis diffusa, TCHD) on proliferation inhibition of leukemia cells, and to explore its related mechanism. METHODS The purity of TCHD prepared by ethanol reflux extraction was tested by ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system. The cells (KG-1 Kasumi-1, THP 1 cells, U937 cells and K562 cells) were treated with TCHD(0.02, 0.04, 0.06, 0.08, 0.10 mg·mL-1) for 24 or 48 h, the inhibitive effect of TCHD on cells growth were determined by MTT method. After Kasumi-1 cells were incubated with TCHD for 24 h,the apoptosis of cells were analyzed by flow cytometry stained with Annexin V/PI. The expression levels of caspase-3, caspase-8, caspase-9,PARP and Bcl-2 family protein were assayed by Western blot. RESULTS TCHD in certain concentration range could markedly inhibit the proliferation of AML cells, their IC50 on Kasumi-1, THP-1 KG-1, U937 and K562 cells were 0.077, 0.083, 0.096, 0.087, 0.096 mg·mL-1 for 24 h, and 0.059, 0.067, 0.072, 0.064, 0.068 mg·mL-1 for 48 h. TCHD has significant inhibitory effect on Kasumi-1, which was stronger than those on other cell lines, and showed a dose- and time-dependent manner(r=0.357,P<0.05). The apoptotic proportion of Kasumi-1cells in 0, 0.02, 0.04, 0.06, 0.08, 0.10 mg·mL-1 TCHD treatment groups for 24 h were (5.33±0.41)%, (7.99±0.45)%, (10.22±0.32)%, (20.10±1.99)%, (28.66±0.67)% and (33.24±2.12)%, respectively. After treated with TCHD(0.02-0.06 mg·mL-1) for 24 h, G0/G1 phase ratio of Kasumi-1 detected by flow cytometry were (51.43±3.21)%, (62.91±2.35)% and (76.42±4.14)%, respectively, which were significantly higher than that of the control group (35.8±5.25)% (P<0.05).Western blot results showed that different concentrations of TCHD could activate caspase-8, caspase-9, caspase-3 and PARP, promote the expression of cyto-C, down-regulate the cyclin E and CDK6, CDK2, p-CDK2 and cyclin D1 protein, and up-regulate the expression of p21 proteinin concentration- dependent manner(P<0.01). CONCLUSION TCHD can obviously inhibit the proliferation of Kasumi-1 in a dose- and time-dependent manner, which may relate to the apoptosis of Kasumi 1 induced by activating caspase-3, 9, PARP protein through the mitochondrial pathways and Kasumi-1 cell block in G0/G1 phase through the influence of CDK2, p-CDK2, CDK4/6, cyclin E, cyclin D1 and p21.
关键词
急性髓系白血病 /
对香豆酸组合物 /
细胞凋亡 /
细胞周期
{{custom_keyword}} /
Key words
acute myeloid leukemia /
total coumarins from Hedyotis diffusa /
apoptosis /
cell cycle
{{custom_keyword}} /
陈智,王博,裘玫,林圣云,熊昊.
对香豆酸组合物对白血病细胞的增殖抑制作用及机制[J]. 中国药学杂志, 2017, 52(17): 1503-1509 https://doi.org/10.11669/cpj.2017.17.006
CHEN Zhi, WANG Bo, QIU Mei,LIN Sheng-yun,XIONG Hao.
Proliferation Inhibition and Its Mechanism of Total Coumarins on Leukemia Cells[J]. Chinese Pharmaceutical Journal, 2017, 52(17): 1503-1509 https://doi.org/10.11669/cpj.2017.17.006
中图分类号：
R965
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] BYRD J C, DODGE R K, CARROLL A, et al. Patients with t (8; 21) (q22; q22) and acute myeloid leukemia have superior failure-free and overall survival when repetitive cycles of high-dose cytarabine are administered[J]. J Clin Oncol, 1999, 17(12): 3767-3775.
[2] GRIMWADE D, WALKER H, OLIVER F, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of1, 612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children′s Leukamia Working Parties[J]. Blood, 1998, 92(7):2322-2333.
[3] GILES F J, KEATING A, GOLDSTONE A H, et al. Acute myeloid leukemia[J]. Hematology, 2002, 1: 73.
[4] DE LA RUBIA J, REGADERA A, MARTIN G, et al. FLAG-IDA regimen (fludarabine, cytarabine, idarubicin and G-CSF) in the treatment of patients with high-risk myeloid malignancies.[J]. Leuk Res, 2002, 26(8):725-730.
[5] ZHAO C G, HANG L W, WANG H, et al. Study on anti-oxidation effects of cinnamic acid and its derivants[J]. Food Sci(食品科学),2005,26(1):218-222.
[6] ZHAO J, OU S Y. Simultaneous determination of p-coumaric and ferulic acid using ration spectra derivative spectrophotometry[J]. Food Sci(食品科学),2010,31(8):189-193.
[7] FEMIAA P, CANDERNI G, VAGNAIL F, et al. Effect of polyphenolic extracts from red wine and 4-OH-coumaric acid on 1,2-dimethylhydrazine-induced colon carcinogenesis in rats[J]. Eur J Nutr, 2005,44(2):79-84.
[8] JANICKE B, ONNING G, OREDSSON M S. Differential effects of ferulic acid and p-coumaric acid on S phase distribution and length of S phase in the human colonic cell line Caco-2[J]. J Agric Food Chem, 2005, 53(17):6658-6665.
[9] YAO T H,JIANG J P,LIN S Y, et al. Optimization of total flavonoids extraction from Hedyotis diffsua Willd.by means of response surface methodology[J]. J Zhejiang Univ Tradit Chin Med(浙江中医药大学学报), 2012, 36(4):425- 429.
[10] LU B Z,ZHOU L W,HOU G L, et al. Progress on antitumor activity of oldenlandiae herba[J]. Her Med(医药导报), 2009, 28(3):344-346.
[11] BEDI A, ZEHNBAUER B A, BARBER J P, et al. Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia[J]. Blood,1994,83(8):2038-2044.
[12] ERBA E. Cell cycle phase perturbations and apoptosis in tumour cells induced by aplidine[J]. Br J Cancer, 2002, 86(9):1510-1517.
[13] DI BACCO A, KEESHAN K, MCKENNA S L, et al. Molecular abnormalities in chronic myeloid leukemia: deregulation of cell growth and apoptosis[J]. Oncologist, 2000,5(5):405-415.
[14] JANICK B,ONNING G,OREDSSON M S. Differential effects of ferulic acid and p-coumaric acid on S phase distribution and length of S phase in the human colonic cell line Caco-2[J]. J Agric Food Chem, 2005,53(17):6658-6665.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
PDF(4103 KB)
