Trivedi Rachana, Müller Gerhard A, Rathore Manohar S, Mishra Durga P, Dihazi Hassan
Clinic for Nephrology and Rheumatology, University Medical Centre, Georg-August University, Gx00F6;ttingen, Germany.
Cell Physiol Biochem. 2016;39(2):604-16. doi: 10.1159/000445652. Epub 2016 Jul 15.
BACKGROUND/AIMS: ER-Stress and activation of unfolded protein response belong to the major factors involved in chemoresistance in cancer cells. In this study we investigated the effect of shikonin on the survival of acute myeloid leukemia cells and the role of ER-stress protein ERP57, a protein disulfide isomerase, in improvement of chemotherapy.
Using MTT assay we studied cytotoxic effects of shikonin on HL-60 cells. The flow cytometry was adopted to examine the shikonin induced mode of cell death in HL-60 cells. The overall protein expression alteration resulting from shikonin treatment was investigated using proteomics methods. Western blotting was performed to quantify the alteration in protein expression in HL-60 after shikonin treatment. Silencing and overexpression studies were carried out to highlight the therapeutic role of ERP57 in shikonin effect on AML cells.
Shikonin induces apoptosis in HL-60 cells without significant effect on Primary cells from healthy volunteers. The apoptotic effect was dose and time dependent and was accompanied by strong alteration in cell proteome. Among the proteins targeted by shikonin, ERP57 was significantly downregulated in HL-60 after treatment. Compared to healthy control ERP57 was found to be highly expressed in AML cell line HL60 and was downregulated after shikonin treatment. Overexpression of ERP57 protected HL-60 from shikonin induced apoptosis, whereas knockdown of ERP57 expression resulted in increase in shikonin induced apoptosis.
Our results demonstrate that ERP57 plays a crucial role in resistance towards shikonin induced apoptosis in AML cells. Targeting of ERP57 might offer a new therapeutic option for the treatment of acute myeloid leukemia.
背景/目的：内质网应激和未折叠蛋白反应的激活是癌细胞化疗耐药的主要因素。在本研究中，我们研究了紫草素对急性髓系白血病细胞存活的影响以及内质网应激蛋白ERP57（一种蛋白质二硫键异构酶）在改善化疗中的作用。
使用MTT法研究紫草素对HL-60细胞的细胞毒性作用。采用流式细胞术检测紫草素诱导HL-60细胞的死亡方式。用蛋白质组学方法研究紫草素处理后导致的整体蛋白质表达变化。进行蛋白质印迹法以量化紫草素处理后HL-60中蛋白质表达的变化。进行沉默和过表达研究以突出ERP57在紫草素对AML细胞作用中的治疗作用。
紫草素诱导HL-60细胞凋亡，对健康志愿者的原代细胞无显著影响。凋亡作用呈剂量和时间依赖性，并伴有细胞蛋白质组的强烈变化。在紫草素靶向的蛋白质中，处理后HL-60中的ERP57显著下调。与健康对照相比，发现ERP57在AML细胞系HL60中高表达，紫草素处理后下调。ERP57的过表达保护HL-60免受紫草素诱导的凋亡，而ERP57表达的敲低导致紫草素诱导的凋亡增加。
我们的结果表明，ERP57在AML细胞对紫草素诱导的凋亡的抗性中起关键作用。靶向ERP57可能为急性髓系白血病的治疗提供一种新的治疗选择。
