Effects of Artesunate on Proliferation and Apoptosis of Human Acute Myeloid Leukemia Stem Cell and Its K562 Cell Line

doi:10.12114/j.issn.1007-9572.2019.00.423

Abstract

Abstract: Background　At present，long-term treatment of acute myeloid leukemia （AML） still faces multiple challenges such as high recurrence rate，treatment-related death risks and chemotherapy-related toxic and side effects.As an new chemical compound （artesunate as its derivative） which has been extracted from compositae plants，artemisia annua for the first time by Chinese scholars，artemisinin has gradually attracted widespread attention in recent years in the treatment of leukemia and cancer.Objective　To explore the effects of artesunate on proliferation and apoptosis of human AML stem cells and K562 cell line.Methods　Density gradient method was performed to separate AML stem cells in bone marrow fluid of 24 children with newly diagnosed or relapsed AML n who met inclusion criteria and were treated in the Department of Hematology，Henan Children's Hospital，from October 2016 to October 2018.The AML K562 cell line was purchased for research.AML stem cells and K562 cell line were equally divided into control group and experimental group （control group 1 and experimental group 1，control group 2 and experimental group 2 in turn）．The control group 1 and control group 2 were not given artesunate for intervention.The experimental group 1 and experimental group 2 were given artesunate solution at a final concentration of 12.5，25.0 and 50.0 μg/ml，respectively （marked as experimental subgroup 1 and experimental subgroup 2 at a final concentration of 12.5，25.0 and 50.0 μg/ml）．The survival of AML stem cells and K562 cells was observed by cell counting method.The growth and inhibition condition were detected by MTT assay.The apoptosis was detected by flow cytometry.Results　At 48 h and 72 h after intervention，survival rate of AML stem cell in experimental subgroup 1 at a final concentration of 12.5，25.0 and 50.0 μg/ml was lower than that in control group 1 （P <0.05）．At 72 h after intervention，survival rate of AML stem cell in experimental subgroup 1 at a final concentration of 25.0 and 50.0 μg/ml was lower than that in experimental subgroup 1 at a final concentration of 12.5 μg/ml （P<0.05）．At 48 h and 72 h after intervention，survival rate of AML K562 cell line in experimental subgroup 2 at a final concentration of 12.5，25.0 and 50.0 μg/ml was lower than that in control group 2 （P<0.05）．At 72 h after intervention，survival rate of AML K562 cell line in experimental subgroup 2 at a final concentration of 25.0 and 50.0 μg/ml was lower than that in experimental subgroup 2 at a final concentration of 12.5 μg/ml （P<0.05）．At 24 h，48 h and 72 h after intervention，growth inhibition rate of AML stem cell in experimental subgroup 1 at a final concentration of 12.5，25.0 and 50.0 μg/ml was higher than that in control group 1 （P<0.05）．At 48 h and 72 h after intervention，growth inhibition rate of AML stem cell in experiment subgroup 1 at a final concentration of 25.0 and 50.0 μg/ml was higher than that in experimental subgroup 1 at a final concentration of 12.5 μg/ml （P<0.05）．At 24 h，48 h and 72 h after intervention，growth inhibition rate of AML stem cell in experimental subgroup 2 at a final concentration of 12.5，25.0 and 50.0 μg/ml was higher than that in control group 2 （P<0.05）．At 48 h and 72 h after intervention，growth inhibition rate of AML stem cell in experiment subgroup 2 at a final concentration of 25.0 and 50.0 μg/ml was higher than that in experimental subgroup 2 at a final concentration of 12.5 μg/ml （P<0.05）．At 48 h after intervention，apoptosis rate of AML stem cells in in experimental subgroup 1 at a final concentration of 12.5，25.0 and 50.0 μg/ml was higher than that in control group 1 （P<0.05）．At 48 h after intervention，apoptosis rate of AML stem cells in experimental subgroup 1 at a final concentration of 25.0 and 50.0 μg/ml was higher than that in experimental subgroup 1 at a final concentration of 12.5 μg/ml （P<0.05）．At 48 h after intervention，apoptosis rate of AML stem cells in experimental subgroup 2 at a final concentration of 12.5，25.0 and 50.0 μg/ml was higher than that in control group 2 （P<0.05）．At 48h after intervention，apoptosis rate of AML stem cells in experimental subgroup 2 at a final concentration of 25.0 and 50.0 μg/ml was higher than that in experimental subgroup 2 at a final concentration of 12.5 μg/ml （P<0.05）．Conclusion　Artesunate can effectively inhibit proliferation of AML stem cells and K562 cell line and induce their apoptosis，which can provide experimental evidence for artemisinin treating AML.

Key words: Leukemia, myeloid, acute；Artesunate；Primary cell；K562 cell line；Proliferation；Apoptosis

摘要： 背景　目前急性髓系白血病（AML）长期治疗仍面临着高复发率、治疗相关死亡风险及化疗相关毒副作用等多重挑战。而青蒿素作为我国学者首次从菊科植物黄花蒿中提取出的化合物（青蒿琥酯为其类衍生物），其近年来在白血病、肿瘤疾病治疗中的作用逐渐引起广泛关注。目的　探究青蒿虎酯对人AML原代细胞、细胞株K562增殖和凋亡的影响。方法　采用密度梯度法分离2016年10月—2018年10月于河南省儿童医院血液科就诊的符合纳入标准的24例初治或复发AML患儿的骨髓液中的AML原代细胞。并购买AML细胞株K562进行研究。将AML原代细胞及细胞株K562均分为对照组和实验组（依次为对照组1和实验组1，对照组2和实验组2），其中对照组1及对照组2不予青蒿琥酯干预，实验组1和实验组2分别添加终浓度为12.5、25.0、50.0 μg/ml的青蒿琥酯液（分别记为终浓度12.5、25.0、50.0 μg/ml实验亚组1及终浓度12.5、25.0、50.0 μg/ml实验亚组2）。采用细胞计数法观察AML原代细胞、细胞株K562存活情况；采用MTT法检测其生长抑制情况；采用流式细胞术检测其凋亡情况。结果　终浓度12.5、25.0、50.0 μg/ml实验亚组1干预48 h、72 h AML原代细胞存活率低于对照组1（P<0.05）；终浓度25.0、50.0 μg/ml实验亚组1干预72 h AML原代细胞存活率低于终浓度12.5 μg/ml实验亚组1（P<0.05）。终浓度12.5、25.0、50.0 μg/ml实验亚组2干预48 h、72 h AML细胞株K562存活率低于对照组2（P<0.05）；终浓度25.0、50.0 μg/ml实验亚组2干预72 h AML细胞株K562存活率低于终浓度12.5μg/ml实验亚组2（P<0.05）。终浓度12.5、25.0、50.0 μg/ml实验亚组1干预24 h、48 h、72 h AML原代细胞生长抑制率高于对照组1（P<0.05）；终浓度25.0、50.0 μg/ml实验亚组1干预48 h、72 h AML原代细胞生长抑制率高于终浓度12.5 μg/ml实验亚组1（P<0.05）。终浓度12.5、25.0、50.0 μg/ml实验亚组2干预24 h、48 h、72 h AML原代细胞生长抑制率高于对照组2（P<0.05）；终浓度25.0、50.0 μg/ml实验亚组2干预48 h、72 h AML原代细胞生长抑制率高于终浓度12.5 μg/ml实验亚组2（P<0.05）。终浓度12.5、25.0、50.0 μg/ml实验亚组1干预48 h AML原代细胞凋亡率高于对照组1（P<0.05）；终浓度25.0、50.0 μg/ml实验亚组1干预48 h AML原代细胞凋亡率高于终浓度12.5 μg/ml实验亚组1（P<0.05）。终浓度12.5、25.0、50.0 μg/ml实验亚组2干预48 h AML原代细胞凋亡率高于对照组2（P<0.05）；终浓度25.0、50.0 μg/ml实验亚组2干预48 h AML原代细胞凋亡率高于终浓度12.5 μg/ml实验亚组2（P<0.05）。结论　青蒿琥酯能够有效抑制AML原代细胞及细胞株K562的增殖并诱导其凋亡，可为青蒿素治疗AML提供实验依据。

关键词: 白血病, 髓样, 急性；青蒿琥酯；原代细胞；细胞株K562；增殖；凋亡

WANG Meng,JIA Guocun,CHENG Yibing, et al. Effects of Artesunate on Proliferation and Apoptosis of Human Acute Myeloid Leukemia Stem Cell and Its K562 Cell Line　[J]. Chinese General Practice, 2019, 22(30): 3694-3700. DOI: 10.12114/j.issn.1007-9572.2019.00.423.
王朦,贾国存,成怡冰等. 青蒿虎酯对人急性髓系白血病原代细胞及其细胞株K562增殖和凋亡的影响研究[J]. 中国全科医学, 2019, 22(30): 3694-3700. DOI: 10.12114/j.issn.1007-9572.2019.00.423.

References

［1］HASSERJIAN R P，CAMPIGOTTO F，KLEPEIS V，et al.De novo acute myeloid leukemia with 20% -29% blasts is less aggressive than acute myeloid leukemia with ≥30% blasts in older adults：a Bone Marrow Pathology Group study［J］．Am J Hematol，2014，89（11）：E193-199．DOI：10.1002/ajh.23808.
［2］张之南，沈悌.血液病诊断及疗效标准［M］．北京：科学出版社，2007：103-116.
［3］岳寒，刘丽英，王小倩.基质细胞共培养对急性髓系白血病细胞耐药性影响及机制研究［J］．中国实验血液学杂志，2017，25（2）：382-386．DOI：10.7534/j.issn.1009-2137.2017.02.013.
YUE H，LIU L Y，WANG X Q.Effect of stromal cell co-culture on drug resistance of acute myeloid leukemia cells and its mechanism［J］．Journal of Experimental Hematology，2017，25（2）：382-386．DOI：10.7534/j.issn.1009-2137.2017.02.013.
［4］SIVEEN K S，UDDIN S，MOHAMMAD R M.Targeting acute myeloid leukemia stem cell signaling by natural products［J］．Mol Cancer，2017，16（1）：13．DOI：10.1186/s12943-016-0571-x.
［5］王颖，牛志云，邢丽娜，等．青蒿琥酯对高危MDS细胞株SKM-1中DNMT1表达的影响［J］．河北医科大学学报，2016，37（12）：1374-1378．DOI：10.3969/j.issn.1007-3205.2016.12.003.
WANG Y，NIU Z Y，XING L N，et al.Effects of Artesunate on DNMT1 expression in high-risk MDS cell strain of SKM-1［J］．Journal of Hebei Medical University，2016，37（12）：1374-1378．DOI：10.3969/j.issn.1007-3205.2016.12.003.
［6］温桂海，王涛，朱涛.二氢青蒿素抑制胰腺癌PANC-1细胞GLUT1表达的实验研究［J］．西部医学，2016，28（2）：173-176．DOI：10.3969/j.issn.1672-3511.2016.02.008.
WEN G H，WANG T，ZHU T.Dihydroartemisinin suppresses expression of GLUT1 through PI3K/Akt signal pathway in pancreatic cancer PANC-1 cells［J］．Medical Journal of West China，2016，28（2）：173-176．DOI：10.3969/j.issn.1672-3511.2016.02.008.
［7］王玮琴，殷红.青蒿琥酯对人白血病 K562，K562/ADM细胞凋亡的作用及对NF-κB p65表达的影响［J］．中国药师，2014，17（5）：729-731.
［8］孔黛，张茵，马保根，等．雷公藤红素提高人白血病细胞化疗敏感性的实验研究［J］．中华实用诊断与治疗杂志，2017，31（10）：950-954．DOI：10.13507/j.issn.1674-3474.2017.10.005.
KONG D，ZHANG Y，MA B G，et al.Effect of celastrol on enhancing chemosensitivity of human leukemia cells［J］．Journal of Chinese Practical Diagnosis and Therapy，2017，31（10）：950-954．DOI：10.13507/j.issn.1674-3474.2017.10.005.
［9］魏万敏，高清平.大蒜素对人白血病细胞系K562细胞增殖和凋亡影响及其机制探讨［J］．辽宁中医药大学学报，2015，17（7）：49-51．DOI：10.13194/j.issn.1673-842x.2015.07.017.
WEI W M，GAO Q P.Effects of allicin on cell proliferation and apoptosis in K562 cells and its mechanisms［J］．Journal of Liaoning University of Traditional Chinese Medicine，2015，17（7）：49-51．DOI：10.13194/j.issn.1673-842x.2015.07.017.
［10］彭洪薇，张晓洁，李菲.黄芪多糖联合多柔比星抗白血病细胞HL-60/A耐药的机制研究［J］．中国实验血液学杂志，2017，25（3）：743-748．DOI：10.7534/j.issn.1009-2137.2017.03.019.
PENG H W，ZHANG X J，LI F.Mechanisms of astragalus polysaccharids synergized with doxorubicin against drug resistance in HL-60/A cells［J］．Journal of Experimental Hematology，2017，25（3）：743-748．DOI：10.7534/j.issn.1009-2137.2017.03.019.
［11］刘亮，左连富，王静.青蒿琥酯对食管癌Ec9706细胞线粒体膜电位及凋亡的影响［J］．解放军医学杂志，2014，39（1）：25-29．DOI：10.11855/j.issn.0577-7402.2014.01.06.
［12］李明，刘洪江，崔向军．青蒿素类药物在非疟疾疾病中的研究进展［J］．中国全科医学，2018，21（12）：1508-1512．DOI：10.3969/j.issn.1007-9572.2018.00.103.
LI M，LIU H J，CUI X J．Research progress in the use of artemisinin in the treatment of non-malarial diseases［J］．Chinese General Practice，2018，21（12）：1508-1512．DOI：10.3969/j.issn.1007-9572.2018.00.103．
［13］郭永泽，单铁英，任海霞，等．双氢青蒿素抑制四氯化碳诱导的小鼠肝纤维化的实验研究［J］．疑难病杂志，2016，15（5）：518-521．DOI：10.3969/j.issn.1671-6450.2016.05.021.
GUO Y Z，SHAN T Y，REN H X，et al．The inhibitor effect of dihydroartemisinin on liver fibrosis of mice induced by CCl4［J］．Chinese Journal of Difficult and Complicated Cases，2016，15（5）：518-521．DOI：10.3969/j.issn.1671-6450.2016.05.021．
［14］陈伟，王玲，杜苑苑，等．双氢青蒿素人对急性髓系白血病HL-60细胞凋亡的诱导作用［J］．广东医学，2011，32（13）：1641-1643．DOI：10.3969/j.issn.1001-9448.2011.13.001.
［15］彭宇，宋志群，赵玲玲，等．青蒿素衍生物SM1044对急性髓系白血病细胞株HL60作用的研究［J］．内科理论与实践，2013，8（3）：187-191.
［16］孙艳美，赵良中，李强，等．青蒿琥酯对白血病细胞增殖和凋亡的影响［J］．中国老年学杂志，2015，35（23）：6647-6648．DOI：10.3969/j.issn.1005-9202.2015.23.003.
［17］张萍，杨桂存，陈红霞，等．青蒿琥酯对白血病K562细胞自噬的影响及其相关机制的研究［J］．重庆医科大学学报，2017，42（11）：1457-1461．DOI：10.13406/j.cnki.cyxb.001097.
ZHANG P，YANG G C，CHEN H X，et al.Effect of artesunate on autophagy of leukemic K562 cells and its mechanism［J］．Journal of Chongqing Medical University，2017，42（11）：1457-1461．DOI：10.13406/j.cnki.cyxb.001097.
［18］李异兴，李曦雯，农晓琳.青蒿琥酯抗肿瘤作用机制与临床应用的研究进展［J］．天然产物研究与开发，2016，28（6）：986-989．DOI：10.16333/j.1001-6880.2016.6.028.
LI Y X，LI X W，NONG X L.Review on anti-tumor mechanism and clinical application of artesunate［J］．Natural Product Research and Development，2016，28（6）：986-989．DOI：10.16333/j.1001-6880.2016.6.028.
［19］欧瑞明，周长华，谭友平，等．青蒿琥酯对硼替佐米耐药的骨髓瘤细胞株增殖与凋亡的影响［J］．中国临床药理学杂志，2018，34（10）：1183-1186．DOI：10.13699/j.cnki.1001-6821.2018.10.014.
OU R M，ZHOU C H，TAN Y P，et al.Effects of artesunate on the proliferation and apoptosis of bortezomib-resistant multiple myeloma cells［J］．The Chinese Journal of Clinical Pharmacology，2018，34（10）：1183-1186．DOI：10.13699/j.cnki.1001-6821.2018.10.014.

[1]	ZHU Danmeng, HUANG Yuying, LUO Tong'an, HE Changyuan, CHEN Rilan. Effect of Pricking-bloodletting Therapy Combined with Zhuang-medicine-thread Moxibustion on TLRs/MyD88 Signal Pathway in a Rat Model of Acute Gouty Arthritis [J]. Chinese General Practice, 2023, 26(20): 2525-2531.
[2]	CHEN Xiaofeng, WANG Meng, LI Zhongyu, LI Jiajia. Clinical Study of Venetoclax with Chemotherapy for Relapsed/Refractory Acute Myeloid Leukemia [J]. Chinese General Practice, 2022, 25(08): 957-962.
[3]	MAO Xiaoyan, ZHOU Yan, LIU Li, YIN Runxiu, YANG Chunhui, CUI Tingting, FANG Chunlian, JIANG Hongchao, TIAN Xin. Ethnic-specific Clinical Features in Children with Non-M3 Acute Myeloid Leukaemia from Yunnan Province [J]. Chinese General Practice, 2022, 25(08): 979-983.
[4]	WANG Yina, DONG Bao, LI Xin, SHAO Chunying, ZUO Li, WANG Mei, YAN Yu. Clinicopathological Manifestations of Kidney Injury in Leukemia [J]. Chinese General Practice, 2022, 25(08): 952-956.
[5]	CHANG Quan，ZHANG Nana，MA Dong. Bioinformatics Analysis of Myelin KLF5 Deficiency Inhibiting Abdominal Aortic Aneurysm Formation in Mice　 [J]. Chinese General Practice, 2020, 23(6): 692-698.
[6]	GUO Zongfeng，WANG Xiang，CAO Su，XU Xingguo，ZHANG Changwei，YU Xiaoyan，XU Weidong. Effect of Dexmedetomidine with Ulinastatin on Perioperative Neurocognitive Disorders in Elderly Patients Undergoing Laparoscopic Surgery for Colorectal Cancer——a Multicenter，Randomized，Double-blind，Controlled Study　 [J]. Chinese General Practice, 2020, 23(36): 4578-4584.
[7]	LU Minqiu，BAO Li，CHU Bin，SHI Lei，GAO Shan，XIANG Qiuqing，FANG Lijun，WANG Yutong，LIU Xi，DING Yuehua. Efficacy of Decitabine Monotherapy in the Treatment of Moderate-to-high-Risk Myelodysplastic Syndrome and Acute Myeloid Leukemia　 [J]. Chinese General Practice, 2020, 23(35): 4443-4447.
[8]	CAI Zhi-mei，CHEN Ze，WANG Ying，WU Xiao-xie，XUE Lian-guo，WEI Ji-feng，ZHAO Li-dong. Clinical Study on the Timing for Changing Medications in the Treatment of Chronic Myelogenous Leukemia-chronic Phase with Imatinib　 [J]. Chinese General Practice, 2018, 21(23): 2814-2817.
[9]	PAN Yang-qiong1，CHEN Hao2，SONG Chun-lan1，CHENG Yi-bing1*. Expression of TAp63 Gene in Bone Marrow Mononuclear Cells Derived from Childhood Leukemia and Its Clinical Significance　 [J]. Chinese General Practice, 2018, 21(17): 2077-2081.