沉默DLL3基因增强白血病K562/ADM细胞对阿霉素的敏感性

王世广* 司旭艳 王丽君 仝雷 李阳杰

doi:10.16098/j.issn.0529-1356.2018.05.010

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解剖学报 ›› 2018, Vol. 49 ›› Issue (5) : 624-629. DOI: 10.16098/j.issn.0529-1356.2018.05.010

肿瘤生物学

沉默DLL3基因增强白血病K562/ADM细胞对阿霉素的敏感性

王世广^1* 司旭艳² 王丽君¹仝雷³ 李阳杰⁴

作者信息 +

Enhancing effect of DLL3 gene silencing on the sensibility in human leukemia K562/ADM cells to adriamycin

WANG Shi-guang ^1* SI Xu-yan² WANG Li-jun¹ TONG Lei³ LI Yang-jie⁴

Author information +

文章历史 +

摘要

目的探讨沉默Delta-like ligand 3（DLL3）基因对白血病K562/ADM细胞对阿霉素（ADM）耐药性的影响及其分子机制。方法将干扰人DLL3基因表达的shRNA质粒和无义对照质粒转染K562/ADM细胞，采用RT-PCR法检测DLL3的mRNA表达水平，采用CCK-8法检测ADM对K562和K562/ADM细胞的毒性作用，采用流式细胞术检测细胞凋亡和细胞内ADM浓度，采用Western blotting方法检测DLL3、谷胱甘肽S转移酶-π（GST-π）和P-糖蛋白（P-gp）的蛋白表达水平。结果 K562/ADM细胞DLL3的mRNA和蛋白表达水平均显著高于其亲代K562细胞（P<0.05）；ADM对K562和K562/ADM细胞的IC₅₀ 分别为1.08 mg/L和34.93 mg/L；沉默DLL3基因后，K562/ADM细胞的耐药倍数下降至13.12，反转倍数为2.47；尽管抑制DLL3基因表达未对K562/ADM细胞凋亡产生影响，但可下调P-gp和GST-π的蛋白表达水平（P<0.05），增加K562/ADM细胞内ADM的蓄积量（P<0.05），从而增强ADM诱导的K562/ADM细胞凋亡（P<0.05）。结论沉默DLL3基因可反转K562/ADM细胞对ADM的耐药性，这可能与下调P-gp和GST-π蛋白水平、从而减少K562/ADM细胞内阿霉素蓄积量有关。

Abstract

Objective To investigate whether Delta-like ligand 3 (DLL) gene silencing could influence the sensibility of human leukemia K562/ADM cells to adriamycin (ADM) in vitro . Methods K562/ADM cells were stably transfected with specific shRNA interference plasmid vector targeting for DLL3. The mRNA expression level of DLL3 were measured by RT-PCR. CCK-8 assay was employed to detect the cytotoxic effect of ADM in K562 and K562/ADM cells. The apoptosis and intracellular ADM concentration were analyzed by flow cytometry. Western blotting was performed to determine the protein expression levels of DLL3, glutathione S transferases-π, (GST-π) and P-glycoprotein (P-gp). Results K562/ADM cells had a significantly higher mRNA and protein expression level of DLL3 than K562 cells (P<0.05). The IC₅₀ value of K562 and K562/ADM cells to ADM were 1.08 and 34.93 mg/L respectively. After DLL3 gene silencing, the resistant factor of K562/ADM cells declined to 13.12 with a reversal fold of 2.47. DLL3 gene silencing had no effect on apoptosis of K562/ADM cells, but a significant effect on downregulating protein levels of P-gp and GST-π (P<0.05) and increasing intracellular ADM concentration (P<0.05), and resulting in an enhance of ADM-induced apoptosis (P<0.05). Conclusion DLL3 gene silencing may enhance the sensibility of K562/ADM cells to ADM by down-regulating P-gp and GST-π, and increasing intracellular ADM concentration.

导出引用

王世广司旭艳王丽君仝雷李阳杰. 沉默DLL3基因增强白血病K562/ADM细胞对阿霉素的敏感性[J]. 解剖学报. 2018, 49(5): 624-629 https://doi.org/10.16098/j.issn.0529-1356.2018.05.010

WANG Shi-guang SI Xu-yan WANG Li-jun TONG Lei LI Yang-jie. Enhancing effect of DLL3 gene silencing on the sensibility in human leukemia K562/ADM cells to adriamycin[J]. Acta Anatomica Sinica. 2018, 49(5): 624-629 https://doi.org/10.16098/j.issn.0529-1356.2018.05.010

参考文献

［1］Siebel C, Lendahl U. Notch signaling in development, tissue homeostasis, and disease［J］. Physiol Rev, 2017, 97(4):1235-1294.

［2］Chapman G, Dunwoodie SL. Role of Delta-like-3 in mammalian somitogenesis and vertebral column formation［J］. Adv Exp Med Biol, 2008, 638:95-112.

［3］Dunwoodie SL, Clements M, Sparrow DB, et al. Axial skeletal defects caused by mutation in the spondylocostal dysplasia/pudgy gene Dll3 are associated with disruption of the segmentation clock within the presomitic mesoderm［J］. Development, 2002, 129(7):1795-1806.

［4］Turnpenny PD, Whittock N, Duncan J, et al. Novel mutations in DLL3, a somitogenesis gene encoding a ligand for the Notch signalling pathway, cause a consistent pattern of abnormal vertebral segmentation in spondylocostal dysostosis［J］. J Med Genet, 2003, 40(5):333-339.

［5］Yan S, Ma D, Ji M, et al. Expression profile of Notch-related genes in multidrug resistant K562/ADM cells compared with parental K562 cells［J］. Int J Lab Hematol, 2010, 32(2):150-158.

［6］Maemura K, Yoshikawa H, Yokoyama K, et al. Delta-like 3 is silenced by methylation and induces apoptosis in human hepatocellular carcinoma［J］. Int J Oncol, 2013, 42(3):817-822.

［7］Deng SM, Yan XC, Liang L, et al. The Notch ligand delta-like 3 promotes tumor growth and inhibits Notch signaling in lung cancer cells in mice［J］. Biochem Biophys Res Commun, 2017, 483(1):488-494.

［8］Rudin CM, Pietanza MC, Bauer TM, et al. Rovalpituzumab tesirine, a DLL3-targeted antibody-drug conjugate, in recurrent small-cell lung cancer: a first-in-human, first-in-class, open-label, phase 1 study［J］. Lancet Oncol, 2017, 18(1):42-51.

［9］Gao FL, Liu ShM, Wu JL, et al. The reversal effect on mdr1 gene-mediated multidrug resistance in gastric cancer SGC7901/VCR cells by small interfering RNA ［J］. Acta Anatomica Sinica, 2006, 37(1):57-61. (in Chinese)

高福莲, 刘书漫, 吴景兰, 等. 小分子干扰RNA逆转胃癌SGC7901/VCR细胞mdr1介导的多药耐药［J］. 解剖学报, 2006, 37(1):57-61.

［10］Chufan EE, Sim HM, Ambudkar SV. Molecular basis of the polyspecificity of P-glycoprotein (ABCB1): recent biochemical and structural studies［J］. Adv Cancer Res, 2015, 125:71-96.

［11］Li D, Gale RP, Liu Y, et al. 5’-Triphosphate siRNA targeting MDR1 reverses multi-drug resistance and activates RIG-Ⅰ-induced immune-stimulatory and apoptotic effects against human myeloid leukaemia cells［J］. Leuk Res, 2017, 58:23-30.

［12］Kim HJ, Lee KY, Kim YW, et al. P-glycoprotein confers acquired resistance to 17-DMAG in lung cancers with an ALK rearrangement［J］. BMC Cancer, 2015, 15(1):553.

［13］Gu J, Fang X, Hao J, et al. Reversal of P-glycoprotein-mediated multidrug resistance by CD44 antibody-targeted nanocomplexes for short hairpin RNA-encoding plasmid DNA delivery［J］. Biomaterials, 2015, 45:99-114.

［14］Sheng L, Xiong M, Li C, et al. Reversing multidrug-resistant by RNA interference through silencing MDR1 gene in human hepatocellular carcinoma cells subline Bel-7402/ADM［J］. Pathol Oncol Res, 2014, 20(3):541-548.

［15］Zhu CY, Lv YP, Yan DF, et al. Knockdown of MDR1 increases the sensitivity to adriamycin in drug resistant gastric cancer cells［J］. Asian Pac J Cancer Prev, 2013, 14(11):6757-6760.

［16］Ramsay EE,Dilda PJ. Glutathione S-conjugates as prodrugs to target drug-resistant tumors［J］. Front Pharmacol, 2014, 5:181.

［17］Vasieva O. The many faces of glutathione transferase pi［J］. Curr Mol Med, 2011, 11(2):129-139.

［18］Zhao D, Jiang Y, Dong X, et al. Arsenic trioxide reduces drug resistance to adriamycin in leukemic K562/A02 cells via multiple mechanisms［J］. Biomed Pharmacother, 2011, 65(5):354-358.