Multiwalled carbon nanotube buckypaper induces cell cycle arrest and apoptosis in human leukemia cell lines through modulation of AKT and MAPK signaling pathways

Abstract JAZ (just another zinc finger protein) was previously identified in our laboratory as a unique ZFP that preferentially binds to double-stranded (ds) RNA rather than dsDNA. We found that interleukin-3 growth factor withdrawal upregulates JAZ expression in factor-dependent hematopoietic cells in association with p53 activation and induction of apoptotic cell death. We recently discovered JAZ as a novel direct, positive regulator of p53 transcriptional activity. The mechanism involves direct binding to p53’s C-terminal (negative) regulatory domain to activate “latent” p53 in response to non-genotoxic stress signals. Our preliminary data indicate that JAZ is differentially expressed in murine and human bone marrow cells and in normal and malignant hematopoietic tissues and cell lines. Thus, we have explored JAZ as a potentially novel molecular target in human leukemia by identifying small molecules that bind and activate JAZ. Using a high-throughput, “molecular docking” strategy, we have screened approximately 240,000 small molecules for their ability to interact with JAZ. Based on the Lipinski Rules for Drug Likeness (molecular characteristics favorable for absorption and permeability), we identified ~70 putative “drug-like” binding molecules with high scores and obtained ~40 of them from the NCI Developmental Therapeutics Program. We first tested their cytotoxic effect on various human leukemia cell lines including wt p53 expressing Reh pre-B lymphoblastic leukemia and Molt-3 T-cell lymphoblastic leukemia cells, and p53-deficient U937 leukemic monocyte lymphoma and KU812 and K562 chronic myelogenous leukemia cells. We have selected four “candidate” JAZ-targeting (J1-J4) compounds for further investigation because they are potent (IC50 = <1 to ~50 μM) in killing leukemia cells in association with upregulation of JAZ protein expression and p53 activation. Since we previously demonstrated that JAZ can induce G1 cell cycle arrest prior to apoptosis in NIH3T3 mouse fibrablast cells in association with upregulation of p21, dephosphorylation of Rb and repression of cyclin A, we have tested these J-compounds for their potential effect on cell cycle progression. Drug treatment followed by flow cytometry analysis was carried out in human leukemia cell lines. Results reveal that the J2, J3 and J4 but not J1 compounds induce significant G1 cell cycle arrest followed by cell death in a dose- and time-dependent manner (e.g. an increase in the G1 population by up to 35 % at 24 hr following the treatment at doses of 0.1 to 50 μM). These data indicate that the J2-J4 compounds can not only induce leukemia cell killing but also mediate growth arrest. Interestingly, J3 and J4 are FDA-approved drugs (for the treatment of non-cancer diseases), suggesting a potentially novel role for these clinically available drugs as therapy for hematologic malignancies. Therefore, while further in vitro and in vivo characterization remains to be carried out, the JAZ-“targeting” compound(s) points the way to develop a potentially novel therapeutic strategy targeting JAZ to treat human leukemia.

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Growth inhibition and apoptosis in human Philadelphia chromosome-positive lymphoblastic leukemia cell lines by treatment with the dual PPARα/γ ligand TZD18

Blood ◽

10.1182/blood-2005-05-2103 ◽

2006 ◽

Vol 107 (9) ◽

pp. 3683-3692 ◽

Cited By ~ 36

Author(s):

Hongyu Liu ◽

Chuanbing Zang ◽

Martin H. Fenner ◽

Dachuan Liu ◽

Kurt Possinger ◽

...

Keyword(s):

Cell Cycle ◽

Growth Inhibition ◽

Cell Cycle Arrest ◽

Kinase Inhibitor ◽

Philadelphia Chromosome ◽

Leukemia Cell ◽

Lymphocytic Leukemia ◽

Cycle Arrest ◽

Leukemia Cell Lines ◽

G1 Cell Cycle Arrest

Treatment of adult Philadelphia chromosome-positive lymphocytic leukemia is rarely successful. We report here the effects of TZD18, a novel dual ligand specific for peroxisome proliferator-activated receptor α and γ (PPARα/γ) on Ph+ lymphocytic leukemia cell lines BV173, SD1, and SupB-15. Exposure of these cells to TZD18 resulted in growth inhibition in a dose- and time-dependent manner that was associated with G1 cell cycle arrest. This effect was much stronger than that mediated by the PPARγ ligand pioglitazone (PGZ), which also belongs to the thiazolidinediones (TZD) class of ligands. However, it may not be mediated through PPARγ or PPARα activation because antagonists of PPARγ and PPARα cannot reverse it. Study of the key regulators of cell cycle progression by Western blot analysis showed that the expression of the cyclin-dependent kinase inhibitor (CDKI) p27kip1, but not that of p21cip1, was enhanced, whereas that of c-Myc, cyclin E, cyclin D2, and cyclin-dependent kinases 2 and 4 (CDK-2 and CDK-4) was decreased when these cells were treated with TZD18 (10 or 20 μM). Therefore, the up-regulation of p27kip1 and the down-regulation of CDK-2 and CDK-4 may, at least in part, account for the G1 cell cycle arrest. Furthermore, a remarkable induction of apoptosis was observed in the cells treated with this dual ligand. No obvious alteration of bcl-2 protein level occurred, but bax was up-regulated in these TZD18-treated cells. Activation of caspase 8 and caspase 9 by TZD18 was also observed. Importantly, NF-κB DNA-binding activity was markedly decreased by the TZD18 treatment. In addition, TZD18 enhanced the growth inhibitory effect of imatinib, a specific tyrosine kinase inhibitor therapeutically used in the treatment of Ph+ leukemia. Overall, our findings strongly suggest that TZD18 may offer a new therapeutic approach to aid in the treatment of Ph+ lymphocytic leukemia.

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