Dihydroartemisinin Enhances the Therapeutic Efficacy of BH3 Mimetic Inhibitor in Acute Lymphoblastic Leukemia Cells via Inhibition of Mcl-1

Abstract

Introduction: Up-regulation of the anti-apoptotic proteins such as Mcl-1 is associated with the primary and secondary resistance of tumor cells to ABT-737 Bcl-2 inhibitor. The combined treatment of Bcl-2 inhibitors with Mcl-1 inhibitors has been proposed as an attractive therapeutic strategy to overcome this drug resistance. Here, we investigated the effect of dihydroartemisinin on Mcl-1 expression and sensitization of T-ALL cells to ABT-737.

Methods: The cell growth and survival were tested by the cell proliferation and MTT assays, respectively. The mRNA levels of Bcl-2, Mcl-1, Bax and P21 were examined by qRT-PCR. Apoptosis were detected by Hoechst 33342 staining and caspase-3 activity assay.

Results: Our data showed that combination treatment with dihydroartemisinin and ABT-737 caused a significant decrease in the IC50 value and synergistically reduced the cell survival compared with dihydroartemisinin or ABT-737 alone. ABT-737 enhanced the Mcl-1 mRNA expression. Dihydroartemisinin also down-regulated the expression of Bcl-2 and Mcl-1 and enhanced the P21 and Bax expression. Moreover, dihydroartemisinin enhanced the apoptosis induced by ABT-737 in MOLT-4 and MOLT-17 cell lines.

Conclusion: In conclusion, dihydroartemisinin demonstrates anti-tumor activities in human ALL cells via inhibition of cell survival and growth. Dihydroartemisinin augments the apoptotic effect of ABT-737 by inhibiting the expression of Mcl-1.

Keywords: ABT-737; ALL; Apoptosis; Mcl-1; dihydroartemisinin.

Figure 1

Effect of Dihydroartemisinin and ABT-737 on Cell Survival. The MOLT-4 and MOLT-17 were treated with dihydroartemisinin (DHA) (A) and ABT-737 (B) at indicated concentrations. After 24 h, the cell survival rate was determined using MTT assay. The cell survival curves were plotted using GraphPad 6.1 software (C, D, F and G). Data are expressed as mean±SD of three experiments. The combination index (CI) values were determined using the fractional affected (Fa) values of MTT assay and CalcuSyn software (E and H)

Figure 2

Growth Inhibition of ALL Cells. The MOLT-4 and MOLT-17 cells were treated with ABT-737 and dihydroartemisinin (DHA) for 1-5 day, and the cell viability was determined using trypan blue exclusion assay at the end of each day. The data are expressed as mean±SD of three independent experiments. *p<0.05 versus blank control or solvent control

Figure 3

RT-qPCR Analysis of ALL Cells. The MOLT-4 and MOLT-17 cells were treated with ABT-737 and dihydroartemisinin (DHA) (IC50 doses). After 24 h, relative mRNA expression levels of Bcl-2 (A and E), Mcl-1 (B and F), Bax (C and G) and P21 (D and H) were measured by RT-qPCR using 2-(∆∆Ct) method. Data are presented as mean±SD (n=3). *p<0.05, relative to blank control

Figure 4

Combination Effects of ABT-737 and Dihydroartemisinin on Apoptosis of ALL Cells. MOLT-4 and MOLT-17 cells were treated with the IC50 doses ABT-737 and dihydroartemisinin (DHA) for 24 h. Next, the apoptosis was measured using Hoechst 33342 staining (A and B) and caspase-3 activity assay (C and D). The results are expressed as mean±SD (n=3) of three experiments. *p<0.05, compared with blank control. Arrows show the apoptotic cells