International Journal of Hematology

, Volume 105, Issue 2, pp 196–205 | Cite as

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

  • Kran Suknuntha
  • Thanyatip Thita
  • Padma Priya Togarrati
  • Piyanee Ratanachamnong
  • Patompon Wongtrakoongate
  • Sirada Srihirun
  • Igor Slukvin
  • Suradej Hongeng
Original Article

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.

Keywords

Myeloid Leukemia Wnt inhibitor Apoptosis FH535 Imatinib Proliferation K562 HL60 THP1 Jurkat CML 

Supplementary material

12185_2016_2116_MOESM1_ESM.tif (873 kb)
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 (63 kb)
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 (451 kb)
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 (498 kb)
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 (428 kb)
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 (95 kb)
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 (329 kb)
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)
12185_2016_2116_MOESM8_ESM.docx (46 kb)
Supplement Table 1. Sequences of primers used in the study (DOCX 46 kb)

References

  1. 1.
    Logan CY, Nusse R. The Wnt signaling pathway in development and disease. Annu Rev Cell Dev Biol. 2004;20:781–810.CrossRefPubMedGoogle Scholar
  2. 2.
    Gough NR. Focus issue: Wnt and beta-catenin signaling in development and disease. Sci Signal 5(206):eg2.Google Scholar
  3. 3.
    Habas R, Dawid IB. Dishevelled and Wnt signaling: is the nucleus the final frontier? J Biol. 2005;4(1):2.CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Luis TC, Naber BA, Roozen PP, Brugman MH, de Haas EF, Ghazvini M, et al. Canonical wnt signaling regulates hematopoiesis in a dosage-dependent fashion. Cell Stem Cell. 2011;9(4):345–56.CrossRefPubMedGoogle Scholar
  5. 5.
    Zhao C, Blum J, Chen A, Kwon HY, Jung SH, Cook JM, et al. Loss of beta-catenin impairs the renewal of normal and CML stem cells in vivo. Cancer Cell. 2007;12(6):528–41.CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Wang Y, Krivtsov AV, Sinha AU, North TE, Goessling W, Feng Z, et al. The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML. Science. 2010;327(5973):1650–3.CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Barker N, Clevers H. Mining the Wnt pathway for cancer therapeutics. Nat Rev Drug Discov. 2006;5(12):997–1014.CrossRefPubMedGoogle Scholar
  8. 8.
    Behrens J, von Kries JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R, et al. Functional interaction of beta-catenin with the transcription factor LEF-1. Nature. 1996;382(6592):638–42.CrossRefPubMedGoogle Scholar
  9. 9.
    Chen B, Dodge ME, Tang W, Lu J, Ma Z, Fan CW, et al. Small molecule-mediated disruption of Wnt-dependent signaling in tissue regeneration and cancer. Nat Chem Biol. 2009;5(2):100–7.CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Huang SM, Mishina YM, Liu S, Cheung A, Stegmeier F, Michaud GA, et al. Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling. Nature. 2009;461(7264):614–20.CrossRefPubMedGoogle Scholar
  11. 11.
    Handeli S, Simon JA. A small-molecule inhibitor of Tcf/beta-catenin signaling down-regulates PPARgamma and PPARdelta activities. Mol Cancer Ther. 2008;7(3):521–9.CrossRefPubMedGoogle Scholar
  12. 12.
    Gedaly R, Galuppo R, Daily MF, Shah M, Maynard E, Chen C, et al. Targeting the Wnt/beta-catenin signaling pathway in liver cancer stem cells and hepatocellular carcinoma cell lines with FH535. PLoS One. 2014;9(6):e99272.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Sebaugh JL. Guidelines for accurate EC50/IC50 estimation. Pharm Stat. 2011;10(2):128–34.CrossRefPubMedGoogle Scholar
  14. 14.
    Reya T, Duncan AW, Ailles L, Domen J, Scherer DC, Willert K, et al. A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature. 2003;423(6938):409–14.CrossRefPubMedGoogle Scholar
  15. 15.
    Wang P, Henning SM, Heber D. Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols. PLoS One. 2010;5(4):e10202.CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Luis TC, Ichii M, Brugman MH, Kincade P, Staal FJ. Wnt signaling strength regulates normal hematopoiesis and its deregulation is involved in leukemia development. Leukemia. 2012;26(3):414–21.CrossRefPubMedGoogle Scholar
  17. 17.
    Minke KS, Staib P, Puetter A, Gehrke I, Gandhirajan RK, Schlosser A, et al. Small molecule inhibitors of WNT signaling effectively induce apoptosis in acute myeloid leukemia cells. Eur J Haematol. 2009;82(3):165–75.CrossRefPubMedGoogle Scholar
  18. 18.
    Albanell J, Rojo F, Averbuch S, Feyereislova A, Mascaro JM, Herbst R, et al. Pharmacodynamic studies of the epidermal growth factor receptor inhibitor ZD1839 in skin from cancer patients: histopathologic and molecular consequences of receptor inhibition. J Clin Oncol. 2002;20(1):110–24.CrossRefPubMedGoogle Scholar
  19. 19.
    Jacquel A, Herrant M, Legros L, Belhacene N, Luciano F, Pages G, et al. Imatinib induces mitochondria-dependent apoptosis of the Bcr-Abl-positive K562 cell line and its differentiation toward the erythroid lineage. FASEB J. 2003;17(14):2160–2.CrossRefPubMedGoogle Scholar
  20. 20.
    Corbin AS, Agarwal A, Loriaux M, Cortes J, Deininger MW, Druker BJ. Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity. J Clin Invest. 2011;121(1):396–409.CrossRefPubMedGoogle Scholar
  21. 21.
    Jiang X, Zhao Y, Smith C, Gasparetto M, Turhan A, Eaves A, et al. Chronic myeloid leukemia stem cells possess multiple unique features of resistance to BCR-ABL targeted therapies. Leukemia. 2007;21(5):926–35.PubMedGoogle Scholar
  22. 22.
    Holtz MS, Forman SJ, Bhatia R. Nonproliferating CML CD34+ progenitors are resistant to apoptosis induced by a wide range of proapoptotic stimuli. Leukemia. 2005;19(6):1034–41.CrossRefPubMedGoogle Scholar
  23. 23.
    Hu Y, Chen Y, Douglas L, Li S. beta-Catenin is essential for survival of leukemic stem cells insensitive to kinase inhibition in mice with BCR-ABL-induced chronic myeloid leukemia. Leukemia. 2009;23(1):109–16.CrossRefPubMedGoogle Scholar
  24. 24.
    Muller-Tidow C, Steffen B, Cauvet T, Tickenbrock L, Ji P, Diederichs S, et al. Translocation products in acute myeloid leukemia activate the Wnt signaling pathway in hematopoietic cells. Mol Cell Biol. 2004;24(7):2890–904.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Wachter J, Neureiter D, Alinger B, Pichler M, Fuereder J, Oberdanner C, et al. Influence of five potential anticancer drugs on wnt pathway and cell survival in human biliary tract cancer cells. Int J Biol Sci. 2012;8(1):15–29.CrossRefPubMedGoogle Scholar
  26. 26.
    Iida J, Dorchak J, Lehman JR, Clancy R, Luo C, Chen Y, et al. FH535 inhibited migration and growth of breast cancer cells. PLoS One. 2012;7(9):e44418.CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Graham SM, Jorgensen HG, Allan E, Pearson C, Alcorn MJ, Richmond L, et al. Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro. Blood. 2002;99(1):319–25.CrossRefPubMedGoogle Scholar
  28. 28.
    Saito Y, Uchida N, Tanaka S, Suzuki N, Tomizawa-Murasawa M, Sone A, et al. Induction of cell cycle entry eliminates human leukemia stem cells in a mouse model of AML. Nat Biotechnol. 2010;28(3):275–80.PubMedGoogle Scholar
  29. 29.
    Suknuntha K, Ishii Y, Tao L, Hu K, McIntosh BE, Yang D, et al. Discovery of survival factor for primitive chronic myeloid leukemia cells using induced pluripotent stem cells. Stem Cell Res. 2015;15(3):678–93.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Li H, Kim JH, Koh SS, Stallcup MR. Synergistic effects of coactivators GRIP1 and beta-catenin on gene activation: cross-talk between androgen receptor and Wnt signaling pathways. J Biol Chem. 2004;279(6):4212–20.CrossRefPubMedGoogle Scholar
  31. 31.
    Jiang X, Lopez A, Holyoake T, Eaves A, Eaves C. Autocrine production and action of IL-3 and granulocyte colony-stimulating factor in chronic myeloid leukemia. Proc Natl Acad Sci USA. 1999;96(22):12804–9.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Sadras T, Perugini M, Kok CH, Iarossi DG, Heatley SL, Brumatti G, et al. Interleukin-3-mediated regulation of beta-catenin in myeloid transformation and acute myeloid leukemia. J Leukoc Biol. 2014;96(1):83–91.CrossRefPubMedGoogle Scholar

Copyright information

© The Japanese Society of Hematology 2016

Authors and Affiliations

  • Kran Suknuntha
    • 1
  • Thanyatip Thita
    • 1
  • Padma Priya Togarrati
    • 2
  • Piyanee Ratanachamnong
    • 1
  • Patompon Wongtrakoongate
    • 3
  • Sirada Srihirun
    • 4
  • Igor Slukvin
    • 5
    • 6
  • Suradej Hongeng
    • 7
  1. 1.Department of Pharmacology, Faculty of ScienceMahidol UniversityBangkokThailand
  2. 2.Cell Therapy CoreBlood Systems Research InstituteSan FranciscoUSA
  3. 3.Department of Biochemistry, Faculty of ScienceMahidol UniversityBangkokThailand
  4. 4.Department of Pharmacology, Faculty of DentistryMahidol UniversityBangkokThailand
  5. 5.Department of Pathology and Laboratory MedicineUniversity of WisconsinMadisonUSA
  6. 6.Wisconsin National Primate Research CenterUniversity of WisconsinMadisonUSA
  7. 7.Department of Pediatrics, Faculty of Medicine, Ramathibodi HospitalMahidol UniversityBangkokThailand

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