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Wnt signaling inhibitor FH535 selectively inhibits cell proliferation and potentiates imatinib-induced apoptosis in myeloid leukemia cell lines

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Abstract

Wnt signaling pathway plays a major role in leukemogenesis of myeloid leukemia. Aberrancy in its regulation results in hyperactivity of the pathway contributing to leukemia propagation and maintenance. To investigate effects of Wnt pathway inhibition in leukemia, we used human leukemia cell lines (i.e., K562, HL60, THP1, and Jurkat) and several Wnt inhibitors, including XAV939, IWP2 and FH535. Our results showed that leukemia cell lines (>95 % cells) had increased endogenous levels of β-catenin as compared to mononuclear cells from healthy donors (0 %). Among the tested inhibitors, FH535 demonstrated a markedly suppressive effect (IC50 = 358 nM) on mRNA levels of β-catenin target genes (LEF1, CCND1, and cMYC). In addition, FH535 significantly potentiated imatinib-induced apoptosis. Evaluation of erythrocyte and megakaryocyte lineage using flow cytometry demonstrated that the potentiation mechanism is independent of the developmental stage, and is more likely due to crosstalk between other pathways and β-catenin. FH535 also displayed antiproliferative properties in other cell lines used in this study. In summary, FH535 showed significantly high antiproliferative effects at submicromolar dosages, and additionally enhanced imatinib-induced apoptosis in human leukemia cell lines. Our results highlight its potential antileukemic promise when used in conjunction with other conventional therapeutic regimens.

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Acknowledgments

This work was supported by Mahidol University (A34/2557, Grant to KS), and The Central Instrument Facility (CIF), Faculty of Science, Mahidol University (58/023, Grant to KS).

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Authors and Affiliations

  1. Department of Pharmacology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand

    Kran Suknuntha, Thanyatip Thita & Piyanee Ratanachamnong

  2. Cell Therapy Core, Blood Systems Research Institute, San Francisco, CA, 94118, USA

    Padma Priya Togarrati

  3. Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand

    Patompon Wongtrakoongate

  4. Department of Pharmacology, Faculty of Dentistry, Mahidol University, Bangkok, 10400, Thailand

    Sirada Srihirun

  5. Department of Pathology and Laboratory Medicine, University of Wisconsin, Madison, WI, 53792, USA

    Igor Slukvin

  6. Wisconsin National Primate Research Center, University of Wisconsin, Madison, WI, 53715, USA

    Igor Slukvin

  7. Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, 10400, Thailand

    Suradej Hongeng

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  1. Kran Suknuntha
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Correspondence to Kran Suknuntha.

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Electronic supplementary material

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12185_2016_2116_MOESM1_ESM.tif

Supplement Fig. 1. Yellowish FH535 at high concentration interferes the absorbance at 450 nm. A) Stock solution of FH535 at 10 mM. B) Absorbance value reading at 450, 515, 562, 595, 630, and 750 nm wavelengths were shown. Red line indicates absorbance value reading at 450 nm. Error bars indicate mean ± SD (n = 3) (TIFF 872 kb)

12185_2016_2116_MOESM2_ESM.tif

Supplement Fig. 2. MTT assay at 24 h after treatment. K562 was treated with 1 μM of XAV939, IWP2, FH535, and IM for 21 h prior to MTT assay. Error bars indicate mean ± SD (n = 3). * indicates significance compared with DMSO, p < 0.05. XAV, XAV939; IWP, IWP2; FH, FH535; IM, imatinib (TIFF 62 kb)

12185_2016_2116_MOESM3_ESM.tif

Supplement Fig. 3. Total intracellular β-catenin could not monitor dynamic changes in Wnt signaling activity. A) Representative flow cytometry dot plots show total intracellular β-catenin in K562 after treatment with 1 μM of XAV939, IWP2, FH535, and IM for 48 h. B) Bar graphs show mean percentages of β-catenin+ cells ± SD from 3 independent experiments. * indicates significance, p < 0.05 (TIFF 451 kb)

12185_2016_2116_MOESM4_ESM.tif

Supplement Fig. 4. FH535 did not induce apoptosis in HL60, THP1, and Jurkat. Cells were treated with 1 μM of FH535 for 48 h prior to analysis using annexin V and 7AAD (TIFF 498 kb)

12185_2016_2116_MOESM5_ESM.tif

Supplement Fig. 5. CD41a expression in K562 cells. Cells were treated with 1 μM of XAV939, IWP2, FH535, IM, and FH + IM for 48 h prior to analysis. Representative flow cytometry histograms show CD41a vs. cell counts gated from live cells (7AAD-). XAV, XAV939; IWP, IWP2; FH, FH535; IM, imatinib (TIFF 427 kb)

12185_2016_2116_MOESM6_ESM.tif

Supplement Fig. 6. Dose–response curves of FH535 in HL60, THP1, Jurkat, and PBMCs. HL60, THP1, Jurkat, and normal PBMCs were treated with various concentrations of FH535 for 24 h in triplicate. Non-linear sigmoidal curve fit was generated from percentage of viable cells compared with no treatment control using trypan blue staining. Estimated IC50 was interpolated from the dose–response curve at half maximal effect. PBMCs were from 3 healthy donors. Error bars indicate mean ± SD from 2 independent experiments (TIFF 95 kb)

12185_2016_2116_MOESM7_ESM.tif

Supplement Fig. 7. Effect of FH535 + imatinib combination on apoptosis in primary CML mononuclear cells. Mononuclear cells from two CML patients were treated with 0.2 % DMSO, 1 μM of FH535, and FH + IM for 48 h prior to analysis using flow cytometry. Representative dot plots show 7AAD vs. annexin V and percentage of annexin V+ cell (TIFF 329 kb)

Supplement Table 1. Sequences of primers used in the study (DOCX 46 kb)

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Suknuntha, K., Thita, T., Togarrati, P.P. et al. Wnt signaling inhibitor FH535 selectively inhibits cell proliferation and potentiates imatinib-induced apoptosis in myeloid leukemia cell lines. Int J Hematol 105, 196–205 (2017). https://doi.org/10.1007/s12185-016-2116-x

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  • DOI: https://doi.org/10.1007/s12185-016-2116-x

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