Karami Hadi, Baradaran Behzad, Esfehani Ali, Sakhinia Masoud, Sakhinia Ebrahim
Immunology Research Center, Department of Medical Immunology, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, Iran E-mail :
Asian Pac J Cancer Prev. 2014;15(2):629-35. doi: 10.7314/apjcp.2014.15.2.629.
Acute myeloid leukemia (AML) is a fatal hematological malignancy which is resistant to a variety of chemotherapy drugs. Myeloid cell leukemia-1 (Mcl-1), a death-inhibiting protein that regulates apoptosis, has been shown to be overexpressed in numerous malignancies. In addition, it has been demonstrated that the expression level of the Mcl-1 gene increases at the time of leukemic relapse following chemotherapy. The aim of this study was to target Mcl-1 by small interference RNA (siRNA) and analyze its effects on survival and chemosensitivity of acute myeloid leukemia cell line HL-60.
siRNA transfection was performed with a liposome approach. The expression levels of mRNA and protein were measured by real-time quantitative PCR and Western blot analysis, respectively. Trypan blue assays were performed to evaluate tumor cell growth after siRNA transfection. The cytotoxic effects of Mcl-1 siRNA (siMcl-1) and etoposide were determined using MTT assay on their own and in combination. Apoptosis was quantified using a DNA-histone ELISA assay.
Transfection with siMcl-1 significantly suppressed the expression of Mcl-1 mRNA and protein in a time- dependent manner, resulting in strong growth inhibition and spontaneous apoptosis. Surprisingly, pretreatment with siMcl-1 synergistically enhanced the cytotoxic effect of etoposide. Furthermore, Mcl-1 down-regulation significantly increased apoptosis sensitivity to etoposide. No significant biological effects were observed with negative control siRNA treatment.
Our results suggest that specific suppression of Mcl-1 by siRNA can effectively induce apoptosis and overcome chemoresistance of leukemic cells. Therefore, siMcl-1 may be a potent adjuvant in leukemia chemotherapy.
急性髓系白血病（AML）是一种致命的血液系统恶性肿瘤，对多种化疗药物耐药。髓系细胞白血病-1（Mcl-1）是一种调节细胞凋亡的抗死亡蛋白，已证实在多种恶性肿瘤中过度表达。此外，还证实Mcl-1基因的表达水平在化疗后白血病复发时升高。本研究的目的是通过小分子干扰RNA（siRNA）靶向Mcl-1，并分析其对急性髓系白血病细胞系HL-60存活和化疗敏感性的影响。
采用脂质体法进行siRNA转染。分别通过实时定量PCR和蛋白质印迹分析测定mRNA和蛋白质的表达水平。进行台盼蓝试验以评估siRNA转染后肿瘤细胞的生长情况。单独及联合使用MTT试验测定Mcl-1 siRNA（siMcl-1）和依托泊苷的细胞毒性作用。使用DNA-组蛋白ELISA试验对细胞凋亡进行定量分析。
用siMcl-1转染以时间依赖性方式显著抑制Mcl-1 mRNA和蛋白质的表达，导致强烈的生长抑制和自发凋亡。令人惊讶的是，用siMcl-1预处理可协同增强依托泊苷的细胞毒性作用。此外，Mcl-1下调显著增加了对依托泊苷的凋亡敏感性。阴性对照siRNA处理未观察到明显的生物学效应。
我们的结果表明，通过siRNA特异性抑制Mcl-1可有效诱导白血病细胞凋亡并克服其化疗耐药性。因此，siMcl-1可能是白血病化疗中的一种有效佐剂。
