Transcriptional Profiling Identifies Altered Intracellular Labile Iron Homeostasis as a Contributing Factor to the Toxicity of Adaphostin: Decreased Vascular Endothelial Growth Factor Secretion Is Independent of Hypoxia-Inducible Factor-1 Regulation

Purpose: Adaphostin was developed as an inhibitor of the p210bcr-abl tyrosine kinase, but as its activity is not limited to tumor cell lines containing this translocation, transcriptional profiling was used as a tool to elucidate additional mechanisms responsible for adaphostin cytotoxicity. Experimental design: Profiles of drug-induced transcriptional changes were measured in three hematopoietic cell lines following 1 and 10 μmol/L adaphostin for 2 to 6 hours and then confirmed with real-time reverse transcription-PCR (2-24 hours). These data indicated altered iron homeostasis, and this was confirmed experimentally. Alteration of vascular endothelial growth factor (VEGF) secretion through hypoxia-inducible factor-1 (HIF-1) regulation was also investigated. Results: Drug-induced genes included heat shock proteins and ubiquitins, but an intriguing response was the induction of ferritins. Measurement of the labile iron pool showed release of chelatable iron immediately after treatment with adaphostin and was quenched with the addition of an iron chelator. Pretreatment of cells with desferrioxamine and N-acetyl-cysteine reduced but did not ablate the sensitivity of the cells to adaphostin, and desferrioxamine was able to modulate adaphostin-induced activation of p38 and inactivation of AKT. VEGF secretion was shown to be reduced in cell lines after the addition of adaphostin but was not dependent on HIF-1. Conclusions: Adaphostin-induced cytotoxicity is caused in part by a rapid release of free iron, leading to redox perturbations and cell death. Despite this, reduced VEGF secretion was found to be independent of regulation by the redox responsive transcription factor HIF-1. Thus, adaphostin remains an interesting agent with the ability to kill tumor cells directly and modulate angiogenesis.

[1]  G. Minotti,et al.  Doxorubicin Paradoxically Protects Cardiomyocytes against Iron-mediated Toxicity , 2004, Journal of Biological Chemistry.

[2]  K. Jungermann,et al.  A Fenton reaction at the endoplasmic reticulum is involved in the redox control of hypoxia-inducible gene expression , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[3]  K. Pantopoulos.  Iron Metabolism and the IRE/IRP Regulatory System: An Update , 2004, Annals of the New York Academy of Sciences.

[4]  E. Sausville,et al.  Induction of apoptosis in human leukemia cells by the tyrosine kinase inhibitor adaphostin proceeds through a RAF-1/MEK/ERK- and AKT-dependent process , 2004, Oncogene.

[5]  Y. Pommier,et al.  Topoisomerase I-Mediated Inhibition of Hypoxia-Inducible Factor 1 , 2004, Cancer Research.

[6]  E. Sausville,et al.  Involvement of reactive oxygen species in adaphostin-induced cytotoxicity in human leukemia cells. , 2003, Blood.

[7]  E. Sausville,et al.  In vitro and in vivo evaluations of the tyrosine kinase inhibitor NSC 680410 against human leukemia and glioblastoma cell lines , 2002, Cancer Chemotherapy and Pharmacology.

[8]  D. Scudiero,et al.  Identification of small molecule inhibitors of hypoxia-inducible factor 1 transcriptional activation pathway. , 2002, Cancer research.

[9]  J. Joseph,et al.  Transferrin Receptor-dependent Iron Uptake Is Responsible for Doxorubicin-mediated Apoptosis in Endothelial Cells , 2002, The Journal of Biological Chemistry.

[10]  A. Urbano,et al.  Mechanisms of apoptosis by the tyrphostin AG957 in hematopoietic cells. , 2002, Biochemical pharmacology.

[11]  E. Sausville,et al.  Effects of the Bcr/abl kinase inhibitors STI571 and adaphostin (NSC 680410) on chronic myelogenous leukemia cells in vitro. , 2002, Blood.

[12]  P. Sharma,et al.  Release of iron from ferritin by metabolites of benzene and superoxide radical generating agents. , 2001, Toxicology.

[13]  A. Harris,et al.  Relation of vascular endothelial growth factor production to expression and regulation of hypoxia-inducible factor-1 alpha and hypoxia-inducible factor-2 alpha in human bladder tumors and cell lines. , 2001, Clinical cancer research : an official journal of the American Association for Cancer Research.

[14]  G. Semenza.  HIF-1 and mechanisms of hypoxia sensing. , 2001, Current opinion in cell biology.

[15]  Elizabeth C. Theil,et al.  Combinatorial mRNA Regulation: Iron Regulatory Proteins and Iso-iron-responsive Elements (Iso-IREs)* , 2000, The Journal of Biological Chemistry.

[16]  A. Harris,et al.  Relationship of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha expression to vascular endothelial growth factor induction and hypoxia survival in human breast cancer cell lines. , 2000, Cancer research.

[17]  F. Petrat,et al.  Determination of the chelatable iron pool of single intact cells by laser scanning microscopy. , 2000, Archives of biochemistry and biophysics.

[18]  R. Eisenstein.  Iron regulatory proteins and the molecular control of mammalian iron metabolism. , 2000, Annual review of nutrition.

[19]  E. Sausville,et al.  Effects of the bcr/abl kinase inhibitors AG957 and NSC 680410 on chronic myelogenous leukemia cells in vitro. , 2000, Clinical cancer research : an official journal of the American Association for Cancer Research.

[20]  C. Beaumont,et al.  H-ferritin subunit overexpression in erythroid cells reduces the oxidative stress response and induces multidrug resistance properties. , 1999, Blood.

[21]  M. Buchwald,et al.  Loss of FancC Function Results in Decreased Hematopoietic Stem Cell Repopulating Ability , 1999, Blood.

[22]  E. Sausville,et al.  Different early effets of tyrphostin AG957 and geldanamycins on mitogen-activated protein kinase and p120cbl phosphorylation in anti CD-3-stimulated T-lymphoblasts. , 1999, Biochemical pharmacology.

[23]  R. Bhatia,et al.  Tyrphostin AG957, a tyrosine kinase inhibitor with anti-BCR/ABL tyrosine kinase activity restores β1 integrin-mediated adhesion and inhibitory signaling in chronic myelogenous leukemia hematopoietic progenitors , 1998, Leukemia.

[24]  C. Beaumont,et al.  Role of Ferritin in the Control of the Labile Iron Pool in Murine Erythroleukemia Cells* , 1998, The Journal of Biological Chemistry.

[25]  E. Sausville,et al.  Altered physical state of p21bcr—abl in tyrphostin AG957-treated K562 cells , 1996, Anti-cancer drugs.

[26]  J. Folkman,et al.  Fighting cancer by attacking its blood supply. , 1996, Scientific American.

[27]  D. Hanahan,et al.  Patterns and Emerging Mechanisms of the Angiogenic Switch during Tumorigenesis , 1996, Cell.

[28]  M. Hentze,et al.  Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[29]  G. Semenza,et al.  Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[30]  E. Sausville,et al.  Tyrphostin induced growth inhibition: correlation with effect on p210bcr-abl autokinase activity in K562 chronic myelogenous leukemia. , 1994, Anti-cancer drugs.

[31]  Georg Breier,et al.  Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo , 1992, Nature.

[32]  Elizabeth C. Theil.  Regulation of ferritin and transferrin receptor mRNAs. , 1990, The Journal of biological chemistry.

[33]  D A Scudiero,et al.  Display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and COMPARE algorithm. , 1989, Journal of the National Cancer Institute.

[34]  D. Hanahan,et al.  Induction of angiogenesis during the transition from hyperplasia to neoplasia , 1989, Nature.