Down-Regulation of Mcl-1 by Small Interference RNA Induces Apoptosis and Sensitizes HL-60 Leukemia Cells to Etoposide

Hadi Karami,Ali Esfehani,Ebrahim Sakhinia,Masoud Sakhinia,Behzad Baradaran

doi:10.7314/apjcp.2014.15.2.629

Hadi Karami, Ali Esfehani + Show 3 more

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https://doi.org/10.7314/apjcp.2014.15.2.629

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Abstract

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.

Highlights

Acute myeloid leukemia (AML) is a genetic disorder characterized by accumulation of abnormal myeloid progenitor cells in the bone marrow, resulting in hematopoietic failure (Robak and Wierzbowska, 2009)
Acute myeloid leukemia (AML) is a fatal hematological malignancy which is resistant to a variety of chemotherapy drugs
It has been demonstrated that the expression level of the Myeloid cell leukemia-1 (Mcl-1) gene increases at the time of leukemic relapse following chemotherapy

Summary

Introduction

Acute myeloid leukemia (AML) is a genetic disorder characterized by accumulation of abnormal myeloid progenitor cells in the bone marrow, resulting in hematopoietic failure (Robak and Wierzbowska, 2009). The majority of patients with AML does not achieve complete remission (CR) or are expected to relapse even with intensive chemotherapy. This is mainly due to the development of drug resistance in tumor cells (Xiao et al, 2009; Kupsa et al, 2012; Szer, 2012). The cytotoxic effects of Mcl-1 siRNA (siMcl-1) and etoposide were determined using MTT assay on their own and in combination. Results: Transfection with siMcl-1 significantly suppressed the expression of Mcl-1 mRNA and protein in a timedependent manner, resulting in strong growth inhibition and spontaneous apoptosis. Conclusions: Our results suggest that specific suppression of Mcl-1 by siRNA can effectively induce apoptosis and overcome chemoresistance of leukemic cells. SiMcl-1 may be a potent adjuvant in leukemia chemotherapy

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