ABSTRACT
Acute Lymphoblastic Leukemia (ALL) is the most frequent childhood malignancy. In the effort to find new anti-leukemic agents, we evaluated the small drug SB225002 (N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea). Although initially described as a selective antagonist of CXCR2, later studies have identified other cellular targets for SB225002, with potential medicinal use in cancer. We found that SB225002 has a significant pro-apoptotic effect against both B- and T-ALL cell lines. Cell cycle analysis demonstrated that treatment with SB225002 induces G2-M cell cycle arrest. Transcriptional profiling revealed that SB225002-mediated apoptosis triggered a transcriptional program typical of tubulin binding agents. Network analysis revealed the activation of genes linked to the JUN and p53 pathways and inhibition of genes linked to the TNF pathway. Early cellular effects activated by SB225002 included the up-regulation of GLIPR1, a p53-target gene shown to have pro-apoptotic activities in prostate and bladder cancer. Silencing of GLIPR1 in B- and T-ALL cell lines resulted in increased resistance to SB225002. Although SB225002 promoted ROS increase in ALL cells, antioxidant N-Acetyl Cysteine pre-treatment only modestly attenuated cell death, implying that the pro-apoptotic effects of SB225002 are not exclusively mediated by ROS. Moreover, GLIPR1 silencing resulted in increased ROS levels both in untreated and SB225002-treated cells. In conclusion, SB225002 induces cell cycle arrest and apoptosis in different B- and T-ALL cell lines. Inhibition of tubulin function with concurrent activation of the p53 pathway, in particular, its downstream target GLIPR1, seems to underlie the anti-leukemic effect of SB225002.
Subject(s)
Antineoplastic Agents/pharmacology , Cell Cycle/drug effects , Cell Death/drug effects , Neoplasm Proteins/drug effects , Nerve Tissue Proteins/drug effects , Phenylurea Compounds/pharmacology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Animals , Apoptosis/drug effects , Blotting, Western , Cell Line, Tumor , Gene Expression Regulation, Neoplastic/drug effects , Gene Silencing , Humans , Jurkat Cells , Membrane Proteins , Mice, Inbred NOD , Mice, SCID , Neoplasm Transplantation , Oligonucleotide Array Sequence Analysis , Reactive Oxygen Species/metabolism , Real-Time Polymerase Chain ReactionABSTRACT
[This corrects the article DOI: 10.1371/journal.pone.0129298.].
ABSTRACT
Current monitoring of acute lymphoblastic leukemia (ALL) in living mice is based on FACS analysis of blood hCD45+ cells. In this work, we evaluated the use of human IGFBP2, B2M or Hsp90 as soluble markers of leukemia. ELISA for B2M and IGFBP2 resulted in high background levels in healthy animals, precluding its use. Conversely, plasma levels of Hsp90 showed low background and linear correlation to FACS results. In another experiment, we compared Hsp90 levels with percentage of hCD45+ cells in blood, bone marrow, liver and spleen of animals weekly sacrificed. Hsp90 levels proved to be a superior method for the earlier detection of ALL engraftment and correlated linearly to ALL burden and progression in all compartments, even at minimal residual disease levels. Importantly, the Hsp90/hCD45+ ratio was not altered when animals were treated with dexamethasone or a PI3K inhibitor, indicating that chemotherapy does not directly interfere with leukemia production of Hsp90. In conclusion, plasma Hsp90 was validated as a soluble biomarker of ALL, useful for earlier detection of leukemia engraftment, monitoring leukemia kinetics at residual disease levels, and pre-clinical or mouse avatar evaluations of anti-leukemic drugs.
Subject(s)
HSP90 Heat-Shock Proteins/blood , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Animals , Antineoplastic Agents, Hormonal/therapeutic use , Biomarkers, Tumor/blood , Dexamethasone/therapeutic use , Disease Progression , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Insulin-Like Growth Factor Binding Protein 2/blood , Leukocyte Common Antigens/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Phosphatidylinositol 3-Kinases/metabolism , Phosphoinositide-3 Kinase Inhibitors , Precursor Cell Lymphoblastic Leukemia-Lymphoma/blood , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
The AP-1 transcription factor complex has been implicated in a variety of biological processes including cell differentiation, proliferation, apoptosis and oncogenic transformation. We previously established that activation of the AP-1 family member JunD contributes to deregulated expression of the anti-apoptotic IL-6 gene in prostate cancer cells. We now show that inhibition of JunD in prostate cancer cells results in GADD45α- and γ-dependent induction of cell death and inhibition of tumor growth that is mediated at least partially via c-Jun N-terminal kinase (JNK) and p38 kinase activation. Apoptosis induction by dominant negative JunD and JNK and p38 kinase activation are impeded upon knock down of GADD45α and γ expression by small interfering RNA, most vividly demonstrating the central role of GADD45α and γ in JunD-mediated escape of prostate cancer cells from programmed cell death.
Subject(s)
Apoptosis , Cell Cycle Proteins/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Nuclear Proteins/metabolism , Prostatic Neoplasms/metabolism , Proto-Oncogene Proteins c-jun/metabolism , Cell Cycle Proteins/antagonists & inhibitors , Cell Cycle Proteins/genetics , Cell Line, Tumor , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , Intracellular Signaling Peptides and Proteins/antagonists & inhibitors , Intracellular Signaling Peptides and Proteins/genetics , Male , Mitogen-Activated Protein Kinase 8/metabolism , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins c-jun/antagonists & inhibitors , RNA Interference , RNA, Small Interfering/metabolism , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
BACKGROUND: The interactions of acute lymphoblastic leukemia (ALL) blasts with bone marrow (BM) stromal cells have a positive impact on leukemia cell survival. In the present study, we proposed to identify and investigate the role of molecules critically involved in leukemia--microenvironment crosstalk. PROCEDURE: Gene expression profiling analyses of BM mesenchymal stem cells (BMMSC) were performed following stimulation by ALL cells. CCL2 and IL-8 plasma levels were evaluated from ALL patients and controls. Expression of the CCL2 and IL-8 receptors in ALL was determined by RT-PCR. The biological effects of CCL2, IL-8 or its neutralizing antibodies in primary precursor-B ALL and BMMSC cells were evaluated using in vitro assays. RESULTS: Leukemia stimulation of BMMSC upregulated the expression of several inflammatory chemokines, including CCL2 and IL-8. The BM plasma levels of CCL2 and IL-8 in children at diagnosis were significantly higher than in healthy controls (P < 0.001). Functional studies revealed that CCL2 and IL-8 enhanced the capacity of BMMSC to support adhesion of ALL cells. CCL2 and IL-8 were also found to enhance BMMSC survival and to increase their proliferation. ALL cells were not directly affected by CCL2 or IL-8. CONCLUSIONS: The leukemic BM microenvironment had increased levels of CCL2 and IL-8. These chemokines are known to have suppressive effects in normal hematopoiesis. Our data indicate that CCL2 and IL-8 have a positive impact on BMMSC survival, proliferation, and adhesiveness to ALL cells. Leukemia-associated CCL2 and IL-8 upregulation may represent one possible mechanism of microenvironment perversion in favor of ALL cells.
