Dual Targeting of Anti-Apoptotic Proteins Enhances Chemosensitivity of the Acute Myeloid Leukemia Cells

Abstract

Background: Acute myeloid leukemia (AML) is a type of blood cancer characterized by fast cellular proliferation. Myeloid cell leukemia-1 (Mcl-1) and survivin, as anti-apoptotic proteins, are involved in cancer growth and resistance to chemotherapy. The aim of this study was to examine the combination effect of Mcl-1 and survivin specific siRNAs on chemosensitivity of the human HL-60 AML cells.

Methods: SiRNAs transfection was performed by using Lipofectamine™2000 reagent. The mRNA expression was analyzed by real-time quantitative PCR. The apoptosis analysis was measured by ELISA cell death assay.

Results: siRNAs markedly suppressed mRNA expression levels of Mcl-1 and survivin in a time-dependent manner, resulting in reduction of leukemic cell proliferation and enhanced spontaneous cell death. Surprisingly, Mcl-1 siRNA and survivin siRNA synergistically enhanced the cell toxic effects of etoposide. Furthermore, down-regulation of Mcl-1 and survivin significantly enhanced the apoptotic effect of etoposide.

Conclusions: Our investigation suggests that suppression of Mcl-1 and survivin by siRNA can effectually inhibit cell growth and overcome chemoresistance of AML cells. Therefore siRNAs may be an important adjuvant in chemotherapy for AML patients.

Keywords: Etoposide; Mcl-1; Survivin; acute myeloid leukemia; siRNA.

Figure 1

Mcl-1 and Survivin Expression Analysis in HL-60 Cells Treated with siRNA. The cells were transfected with NC siRNA, Mcl-1 siRNA and survivin siRNA for 24 and 48 h, and then relative Mcl-1 (A) and survivin (B) mRNA expression was measured by RT-qPCR. The results are expressed as mean±SD of three independent experiments. *p<0.05 versus blank control group or NC siRNA transfected cells

Figure 2

Effect of siRNAs in Combination with Etoposide on Cell Survival. The HL-60 cells were transfected with siRNA for 6 h and then exposed to etoposide at indicated concentrations. Twenty-four hours after transfection, the cell survival was measured using MTT assay. The cell survival curves were plotted by GraphPad software (A, B and C). Data are showed as mean ± SD of three experiments. The combination index (CI) values were calculated using the fractional affected (Fa) values of MTT assay and CalcuSyn software (D, E, F and G)

Figure 3

Proliferation Inhibition of HL-60 Leukemic Cells. The HL-60 cells were transfected with NC siRNA, Mcl-1 siRNA and survivin siRNA for 1-5 day, and the cell proliferation rate was measured using trypan blue assay at the end of each day. The data are represented as mean±SD of three experiments. *p<0.05 versus blank control or NC siRNA

Figure 4

The Effect of siRNAs and Etoposide on Apoptosis of HL-60 Cells. Cells were treated with negative control (NC) siRNA, Mcl-1 siRNA, survivin siRNA and etoposide (IC50 doses of 24 h), alone and in combination. Next, apoptosis was assessed using ELISA cell death assay. The data are presented as mean ± SD (n=3). *p<0.05 relative to the control; #p<0.05 versus single transfection; $ p<0.05 relative to siRNA in combination with etoposode