International Journal of Hematology

, Volume 78, Issue 3, pp 219–225 | Cite as

N-(4-Hydroxyphenyl)retinamide (4-HPR) Induces Leukemia Cell Death via Generation of Reactive Oxygen Species

  • Hiroaki Goto
  • Hiroyuki Takahashi
  • Hisaki Fujii
  • Koichiro Ikuta
  • Shumpei Yokota
Progress in Hematology


The role of reactive oxygen species (ROS) in the cytotoxicity of N-(4-hydroxyphenyl)retinamide (4-HPR) was studied with use of the B-precursor lymphoblastic leukemia cell line YCUB-2. The increase in intracellular ROS measured with 2′-7′-dichlorodihydrofluorescein diacetate after 3 hours’ incubation was 3.7-fold with 1 μM 4-HPR and 5.8-fold with 5 μM 4-HPR. The rate of apoptosis after 48 hours’ incubation was 9.8% and 56.4% in comparison with untreated cells. Hydroethidine, which is a more specific indicator of superoxide anion radical level, did not effectively detect 4-HPR-induced ROS. The antioxidant 3-methyl-1-phenyl-2-pyrazolin-5-one suppressed 4-HPR-induced ROS production and apoptosis. The cytotoxicity of 4-HPR was analyzed in 4 other leukemia/lymphoma lines (CCRF-HSB2, Molt-4, KG-1, HL-60). We found that the cytotoxicity of 4-HPR correlated with the amount of ROS produced in cell lines, except in HL-60 cells. The intracellular glutathione level varied among the 5 cell lines, the highest levels occurring in Molt-4 and KG-1, which were less sensitive to 4-HPR. Suppression of glutathione by buthionine sulfoximine enhanced the level of 4-HPR-induced ROS production and apoptosis in Molt-4. Our findings suggest that ROS play a significant role in the antileukemia effect of 4-HPR and that the glutathione level in leukemias may be associated the sensitivity of the cells to 4-HPR.

Key words

Fenretinide 4-HPR Leukemia ROS Glutathione 


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  1. 1.
    Simon HU, Haj-Yehia A, Levi-Schaffer F. Role of reactive oxygen species (ROS) in apoptosis induction.Apoptosis. 2000;5:415–418.CrossRefPubMedGoogle Scholar
  2. 2.
    Groninger E, Meeuwsen-De Boer GJ, De Graaf SS, Kamps WA, De BontES. Vincristine induced apoptosis in acute lymphoblastic leukaemia cells: a mitochondrial controlled pathway regulated by reactive oxygen species?Int J Oncol. 2002;21:1339–1345.PubMedGoogle Scholar
  3. 3.
    Sugimoto K, Tamayose K, Sasaki M, Hayashi K, Oshimi K. Low- dose doxorubicin-induced necrosis in Jurkat cells and its acceleration and conversion to apoptosis by antioxidants.Br J Haematol. 2002;1 18:229–238.CrossRefGoogle Scholar
  4. 4.
    Iacobini M, Menichelli A, Palumbo G, Multari G, Werner B, Del Principe D. Involvement of oxygen radicals in cytarabine-induced apoptosis in human polymorphonuclear cells.Biochem Pharmacol. 2001;61:1033–1040.CrossRefPubMedGoogle Scholar
  5. 5.
    Mansat-de Mas V, Bezombes C, Quillet-Mary A, et al. Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin.Mol Pharmacol. 1999;56:867–874.CrossRefGoogle Scholar
  6. 6.
    Reynolds CP, Lemons RS. Retinoid therapy of childhood cancer.Hematol Oncol Clin North Am. 2001;15:867–910.CrossRefPubMedGoogle Scholar
  7. 7.
    Garewal HS, List A, Meyskens F, Buzaid A, Greenberg B, Katakkar S. Phase II trial of fenretinide [N-(4-hydroxyphenyl) retinamide] in myelodysplasia: possible retinoid-induced disease acceleration.Leuk Res. 1989;13:339–343.CrossRefPubMedGoogle Scholar
  8. 8.
    O’Donnell PH, Guo WX, Reynolds CP, Maurer BJ. N-(4- Hydroxyphenyl)retinamide increases ceramide and is cytotoxic to acute lymphoblastic leukemia cell lines, but not to non-malignant lymphocytes.Leukemia. 2002;16:902–910.CrossRefPubMedGoogle Scholar
  9. 9.
    Wu JM, DiPietrantonio AM, Hsieh TC. Mechanism of fenretinide (4-HPR)-induced cell death.Apoptosis. 2001;6:377–388.CrossRefPubMedGoogle Scholar
  10. 10.
    Takahashi H, Goto H, Eunabiki T, et al. Expression of two types of E2A-HLF fusion proteins in YCUB-2, a novel cell line established from B-lineage leukemia with t(17;19).Leukemia. 2001;15:995–997.CrossRefPubMedGoogle Scholar
  11. 11.
    Anderson CP, Tsai JM, Meek WE, et al. Depletion of glutathione by buthionine sulfoxine is cytotoxic for human neuroblastoma cell lines via apoptosis.Exp Cell Res. 1999;246:183–192.CrossRefPubMedGoogle Scholar
  12. 12.
    Bestwick CS, Milne L. Quercetin modifies reactive oxygen levels but exerts only partial protection against oxidative stress within HL-60 cells.Biochim Biophys Acta. 2001;1528:49–59.CrossRefPubMedGoogle Scholar
  13. 13.
    Benov L, Sztejnberg L, Fridovich I. Critical evaluation of the use of hydroethidine as a measure of superoxide anion radical.Free Radic Biol Med. 1998;25:826–831.CrossRefPubMedGoogle Scholar
  14. 14.
    Darzynkiewicz Z, Bruno S, Del Bino G, et al. Features of apoptotic cells measured by flow cytometry.Cytometry. 1992;13:795–808.CrossRefPubMedGoogle Scholar
  15. 15.
    Ikeda T, Xia YX, Kaneko M, Sameshima H, Ikenoue T. Effect of the free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), on hypoxia-ischemia-induced brain injury in neonatal rats.Neurosci Lett. 2002;329:33–36.CrossRefPubMedGoogle Scholar
  16. 16.
    Stull ND, Polan DP, Iacovitti L. Antioxidant compounds protect dopamine neurons from death due to oxidative stress in vitro.Brain Res. 2002;931:181–185.CrossRefPubMedGoogle Scholar
  17. 17.
    Asumendi A, Morales MC, Alvarez A, Arechaga J, Perez-Yarza G. Implication of mitochondria-derived ROS and cardiolipin peroxidation in N-(4-hydroxyphenyl)retinamide-induced apoptosis.Br J Cancer. 2002;86:1951–1956.CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Hursting SD, Shen JC, Sun XY, Wang TT, Phang JM, Perkins SN. Modulation of cyclophilin gene expression by N-4- (hydroxyphenyl)retinamide: association with reactive oxygen species generation and apoptosis.Mol Carcinog. 2002;33:16–24.CrossRefPubMedGoogle Scholar
  19. 19.
    Suzuki S, Higuchi M, Proske RJ, Oridate N, Hong WK, Lotan R. Implication of mitochondria-derived reactive oxygen species, cytochrome C and caspase-3 in N-(4-hydroxyphenyl)retinamide- induced apoptosis in cervical carcinoma cells.Oncogene. 1999;18:6380–63877.CrossRefPubMedGoogle Scholar
  20. 20.
    You KR, Wen J, Lee ST, Kim DG. Cytochrome c oxidase subunit III: a molecular marker for N-(4-hydroxyphenyl)retinamise-induced oxidative stress in hepatoma cells.J Biol Chem. 2002;277:3870–3877.CrossRefPubMedGoogle Scholar
  21. 21.
    Hail N Jr, Lotan R. Mitochondrial respiration is uniquely associated with the prooxidant and apoptotic effects of N-(4-hydroxyphenyl)retinamide.J Biol Chem. 2001;276:45614–45621.CrossRefPubMedGoogle Scholar
  22. 22.
    Sun SY, Li W, Yue P, Lippman SM, Hong WK, Lotan R. Mediation of N-(4-hydroxyphenyl)retinamide-induced apoptosis in human cancer cells by different mechanisms.Cancer Res. 1999;59:2493–2498.PubMedGoogle Scholar
  23. 23.
    Wagner BA, Buettner GR, Oberley LW, Darby CJ, Burns CP. Myeloperoxidase is involved in H2O2-induced apoptosis of HL-60 human leukemia cells.J Biol Chem. 2000;275:22461–22469.CrossRefPubMedGoogle Scholar
  24. 24.
    Maurer BJ, Metelitsa LS, Seeger RC, Cabot MC, Reynolds CP. Increase of ceramide and induction of mixed apoptosis/necrosis by N-(4-hydroxyphenyl)-retinamide in neuroblastoma cell lines.J Natl Cancer Inst. 1999;91:1138–1146.CrossRefPubMedGoogle Scholar
  25. 25.
    Wolf D, Rotter V. Major deletions in the gene encoding the p53 tumor antigen cause lack of p53 expression in HL-60 cells.Proc Natl Acad Sci USA. 1985;82:790–794.CrossRefPubMedGoogle Scholar
  26. 26.
    Shiohara M, Akashi M, Gombart AF, Yang R, Koeffler HP. Tumor necrosis factor alpha: posttranscriptional stabilization of WAF1 mRNA in p53-deficient human leukemic cells.J Cell Physiol. 1996; 166:568–576.CrossRefPubMedGoogle Scholar
  27. 27.
    Andrieu-Abadie N, Gouaze V, Salvayre R, Levade T. Ceramide in apoptosis signaling: relationship with oxidative stress.Free Radic BiolMed. 2001;31:717–728.CrossRefGoogle Scholar
  28. 28.
    Corda S, Laplace C, Vicaut E, Duranteau J. Rapid reactive oxygen species production by mitochondria in endothelial cells exposed to tumor necrosis factor-alpha is mediated by ceramide.Am J Respir Cell Mol Biol. 2001;24:762–768.CrossRefPubMedGoogle Scholar
  29. 29.
    Phillips DC, Allen K, Griffiths HR. Synthetic ceramides induce growth arrest or apoptosis by altering cellular redox status.Arch Biochem Biophys. 2002;407:15–24.CrossRefPubMedGoogle Scholar
  30. 30.
    Lavrentiadou SN, Chan C, Kawcak T, et al. Ceramide-mediated apoptosis in lung epithelial cells is regulated by glutathione.Am J Respir Cell Mol Biol. 2001;25:676–684.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Lewandowicz GM, Britt P, Elgie AW, et al. Cellular glutathione content, in vitro chemoresponse, and the effect of BSO modulation in samples derived from patients with advanced ovarian cancer.Gynecol Oncol. 2002;85:298–304.CrossRefPubMedGoogle Scholar
  32. 32.
    Gamcsik MP, Dubay GR, Cox BR. Increased rate of glutathione synthesis from cystine in drug-resistant MCF-7 cells.Biochem Pharmacol. 2002;63:843–851.CrossRefPubMedGoogle Scholar
  33. 33.
    Tsukamoto N, Chen J, Yoshida A. Enhanced expressions of glucose- 6-phosphate dehydrogenase and cytosolic aldehyde dehydrogenase and elevation of reduced glutathione level in cyclophosphamide- resistant human leukemia cells.Blood Cells Mol Dis. 1998;24:231–2388.CrossRefPubMedGoogle Scholar
  34. 34.
    Tanner B, Hengstler JG, Dietrich B, et al. Glutathione, glutathione S-transferase alpha and pi, and aldehyde dehydrogenase content in relationship to drug resistance in ovarian cancer.Gynecol Oncol. 1997;65:54–62.CrossRefPubMedGoogle Scholar
  35. 35.
    Richardson ME, Siemann DW. DNA damage in cyclophosphamide- resistant tumor cells: the role of glutathione.Cancer Res. 1995;55:1691–16955.PubMedGoogle Scholar
  36. 36.
    Anderson CP, Reynolds CP. Synergistic cytotoxicity of buthionine sulfoximine (BSO) and intensive melphalan (L-PAM) for neuroblastoma cell lines established at relapse after myeloablative therapy.Bone Marrow Transplant. 2002;30:135–140.CrossRefPubMedGoogle Scholar
  37. 37.
    Gartenhaus RB, Prachand SN, Paniaqua M, Li Y, Gordon LI. Arsenic trioxide cytotoxicity in steroid and chemotherapy-resistant myeloma cell lines: enhancement of apoptosis by manipulation of cellular redox state.Clin Cancer Res. 2002;8:566–572.PubMedGoogle Scholar
  38. 38.
    Sipos EP, Witham TF, Ratan R, et al. L-Buthionine sulfoximine potentiates the antitumor effect of 4-hydroperoxycyclophosphamide when administered locally in a rat glioma model.Neurosurgery. 2001;48:392–400.PubMedGoogle Scholar

Copyright information

© The Japanese Society of Hematology 2003

Authors and Affiliations

  • Hiroaki Goto
    • 1
  • Hiroyuki Takahashi
    • 1
  • Hisaki Fujii
    • 1
  • Koichiro Ikuta
    • 1
  • Shumpei Yokota
    • 1
  1. 1.Department of PediatricsYokohama City University School of MedicineYokohamaJapan

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