Int J Stem Cells.  2018 Jun;11(1):131-140. 10.15283/ijsc18021.

The Bromodomain Inhibitor JQ1 Enhances the Responses to All-trans Retinoic Acid in HL-60 and MV4-11 Leukemia Cells

• Kang C
• Kim CY
• Kim HS
• Park SP
• Chung HM

• KMID: 2413509
• DOI: http://doi.org/10.15283/ijsc18021

Abstract

All-trans retinoic acid (ATRA) is a highly effective treatment for acute promyelocytic leukemia (APL), a cytogenetically distinct subtype of acute myeloid leukemia (AML). However, ATRA-based treatment is not effective in other subtypes of AML. In non-APL AML, ATRA signaling pathway is impaired or downmodulated, and consequently fails to respond to pharmacological doses of ATRA. Therefore, complementary treatment strategies are needed to improve ATRA responsiveness in non-APL AML. In this study, we investigated the combined effect of ATRA and bromodomain inhibitor JQ1, proven to have potent anti-cancer activity mainly through inhibition of c-Myc. We showed that the combination of ATRA with JQ1 synergistically inhibited proliferation of AML cells. The synergistic growth inhibition was resulted from differentiation or apoptosis depending on the kind of AML cells. Concomitantly, the combined treatment of ATRA and JQ1 caused greater depletion of c-Myc and hTERT expression than each agent alone in AML cells. Taken together, these findings support the rationale for the use of the combination of ATRA and JQ1 as a therapeutic strategy for the treatment of AML.

Keyword

Acute myeloid leukemia; All-trans retinoic acid; JQ1; c-Myc; Differentiation; Apoptosis

MeSH Terms

Apoptosis
Leukemia*
Leukemia, Myeloid, Acute
Leukemia, Promyelocytic, Acute
Tretinoin*
Tretinoin

Figure

• 

  Fig. 1 JQ1 synergizes with ATRA to reduce proliferation of AML cells. (A) HL-60 cells and MV4-11 cells were treated with ATRA (20~100 nM) for the indicated days. Cell proliferation was measured using a CCK-8 assay. The data represent the means±SD of 3 independent experiments. *ATRA versus control (***p<0.001). (B) HL-60 cells and MV4-11 cells were treated with the indicated concentrations of JQ1 and/or 100 nM ATRA for 3 days. Cell proliferation was measured using a CCK-8 assay. The data represent the means±SD of 3 independent experiments. *combination versus ATRA (***p<0.001); †combination versus JQ1 (†††p<0.001). (C) CDI values at different concentrations of JQ1 and 100 nM ATRA were calculated as described in materials and methods.

• 

  Fig. 2 Effect of JQ1 on ATRA-induced HL-60 cell differentiation. HL-60 cells were treated with the indicated concentrations of JQ1 and/or 100 nM ATRA for 3 days. (A) Histogram (left) and bar graph (right) represented CD11b expression. Values were expressed as Mean fluorescence of intensity (MFI). Experiments were independently repeated three times and results were shown as mean±SD. (B) The mRNA levels of C/EBPɛ were determined by real-time PCR. The relative expression of C/EBPɛ mRNA was normalized to GAPDH and was reported as a percentage of untreated cells. (C) The protein levels of C/EBPɛ were measured by Western blot analysis using 20 μg protein. Similar results were obtained in three independent experiments. *combination versus ATRA (***p<0.001; **p<0.01).

• 

  Fig. 3 Effect of combined treatment with JQ1 and ATRA on apoptosis of AML cells. (A) HL-60 cells and (B) MV4-11 cells were treated with the indicated concentrations of JQ1 and/or 100 nM ATRA for 48 h. At the end of treatment, cells were washed with staining buffer and stained with Annexin V-FITC. Annexin-V positive, apoptotic cells was determined by flow cytometry. Numbers in each plot represented the percentage of Annexin-V positive cells. Bar graph showed means±SD of 3 independent experiments. *combination versus ATRA (**p<0.01; *p<0.05; ns, not significant).

• 

  Fig. 4 Effect of JQ1 and ATRA on the expression of c-Myc and hTERT mRNA. HL-60 cells (left) and MV4-11 cells (right) were treated with 100 nM ATRA and/or the indicated concentrations of JQ1 for 24 h. At the end of treatment, cells were harvest and analyzed in the following experiments. (A) The mRNA levels of c-Myc were determined by real-time PCR. The relative expression of c-Myc mRNA was normalized to GAPDH and was reported as a percentage of untreated cells. (B) The protein levels of c-Myc were measured by Western blot analysis using 20 μg protein. Similar results were obtained in three independent experiments. (C) The mRNA levels of hTERT were determined by real-time PCR. The relative expression of hTERT mRNA was normalized to GAPDH and was reported as a percentage of untreated cells. *combination versus ATRA (**p<0.01; *p<0.05); †combination versus JQ1 (††p<0.01; †p<0.05).

Reference

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