Leukemia stem cells (LSCs) are thought to play a central role in the pathogenesis of acute leukemia and likely contribute to both disease initiation and relapse. Therefore, identification of agents that target LSCs is an important consideration for the development of new therapies. To this end, we have previously demonstrated that the naturally occurring compound parthenolide (PTL) can induce death of human LSCs in vitro while sparing normal hematopoietic cells. However, PTL has relatively poor pharmacologic properties that limit its potential clinical use.Consequently, we generated a family of PTL analogs designed to improve solubility and bioavailability. These studies identified an analog, dimethylaminoparthenolide (DMAPT), which induces rapid death of primary human LSCs from both myeloid and lymphoid leukemias, and is also highly cytotoxic to bulk leukemic cell populations. Molecular studies indicate the prevalent activities of DMAPT include induction of oxidative stress responses, inhibition of NF-B, and activation of p53. The compound has approximately 70% oral bioavailability, and pharmacologic studies using both mouse xenograft models and spontaneous acute canine leukemias demonstrate in vivo bioactivity as determined by functional assays and multiple biomarkers. Therefore, based on the collective preclinical data, we propose that the novel compound DMAPT has the potential to target human LSCs in vivo. IntroductionRecent studies have demonstrated that myeloid leukemia and certain forms of lymphoid leukemia arise from malignant stem cells (called leukemia stem cells [LSCs]). [1][2][3] LSCs are typically found in a quiescent state and are thus unlikely to respond to standard chemotherapeutic agents that preferentially eradicate actively cycling cells. [4][5][6][7] Indeed, the persistence of LSCs following chemotherapy may be a major factor contributing to clinical relapse. 8,9 In addition, conventional leukemia therapy is also substantially toxic to normal hematopoietic cells and frequently results in severe myelosuppression. Therefore, given the drugrefractory nature of LSCs, and the importance of normal hematopoiesis, identification of less toxic and more specific forms of therapy are important priorities for the development of better therapeutic regimens.As a foundation for developing more selective leukemia treatments, our previous experiments have investigated basic properties of primitive acute myelogenous leukemia (AML) cells. These studies showed that LSCs from different AML subtypes share characteristics 10 that are unique to AML and thus represent potential therapeutic targets for the selective ablation of LSCs relative to their normal counterparts. 11,12 Specifically, we reported that NF-B, a known regulator of growth and survival, is constitutively active in LSCs but not in normal hematopoietic stem cells (HSCs). 13 Notably, many traditional cancer therapies induce activation of NF-B, a potentially undesirable characteristic likely to facilitate survival of malignant cells. 14,15 Given the abi...

No abstract

No abstract

Background: Eradication of primary human leukemia cells represents a major challenge. Therapies have not substantially changed in over 30 years. Results: Using normal versus leukemia specimens enriched for primitive cells, we document aberrant regulation of glutathione metabolism. Conclusion: Aberrant glutathione metabolism is an intrinsic property of human leukemia cells. Significance: Interventions based on modulation of glutathione metabolism represent a powerful means to improve therapy.

No abstract

In this study, we characterized nuclear factor B (NF- B IntroductionB-cell chronic lymphocytic leukemia (CLL) is a malignancy characterized by the accumulation of CD5, CD19, and CD23 positive lymphocytes. Diagnosis is aided by the CLL immunophenotyping score which includes assessment of CD5 and CD23, FMC7, CD79b, and surface IgM. 1 Although CLL is the commonest leukemia in the Western world, it manifests a very heterogeneous clinical course, with some patients having normal age-adjusted survival, whereas the median survival for those patients with advanced stage disease is only 3 years. 2 The factors that contribute to the pathogenesis and progression of this disease are poorly understood, but decreased susceptibility to apoptosis 3 and dysregulated proliferation have been implicated. 4 Clinical studies have shown that high ZAP-70 expression, high CD38 expression, unmutated V H genes, and cytogenetic abnormalities (especially deletions of 11q and 17p) are all associated with a poor prognosis. [5][6][7][8][9] Nuclear factor B (NF-B) is a collective name for a group of inducible homodimeric and heterodimeric transcription factors made up of members of the Rel family of DNA-binding proteins. In humans, this family is composed of c-Rel, Rel B, p50, p52, and Rel A (p65) which, when bound in the cytoplasm to inhibitor of NF-B (IB) proteins, are inactive. 10,11 Various factors, including ligation of CD40 or the B-cell receptor (BCR), result in proteosomal degradation of IB releasing NF-B, which then translocates to the nucleus. 10,11 Once in the nucleus, NF-B can enhance survival by inducing apoptosis inhibitory proteins, including inhibitor of apoptosis proteins (IAPs), Fas-associated death domain (FADD)-like interleukin (IL)-1␤-converting enzyme (FLICE), and FADD-like IL-1␤-converting enzyme-inhibitory protein (FLIP). 12-14 CLL cells have been reported to exhibit high constitutive NF-B activation compared with normal B lymphocytes. [15][16][17] Although the exact factors responsible for the constitutive expression of NF-B are not fully resolved, many factors, including Akt activation, BCR signaling, CD40 ligation, IL-4, and B-cell activating factor (BAFF), have been shown to increase NF-B activity and enhance CLL cell survival, with members of the Bcl-2 family being principal transcriptional targets. [18][19][20][21][22] Several recent studies have demonstrated the proof of concept of the effectiveness of targeting NF-B in hematologic malignancies, including CLL 23,24 and acute myeloid leukemia. 25,26 In this study, we first set out to determine the range of constitutive DNA binding of NF-B within our patient cohort and to characterize the specific subunits of NF-B in these primary CLL cells. We then went on to investigate the ability of freshly isolated CLL cells to induce NF-B expression in response to BCR Submitted November 20, 2007; accepted January 25, 2008. Prepublished online as Blood First Edition paper, January 28, 2008; DOI 10.1182 DOI 10. /blood-2007 An Inside Blood analysis of this article appears at ...

Thymoquinone (TQ), the most abundant constituent in black seed, was shown to possess potent chemopreventive activities against DMBA-initiated TPA-promoted skin tumors in mice. Despite the potential interest in TQ as a skin antineoplastic agent, its mechanism of action has not been examined yet. Using primary mouse keratinocytes, papilloma (SP-1) and spindle (I7) carcinoma cells, we studied the cellular and molecular events involved in TQ's antineoplastic activity. We show that non-cytotoxic concentrations of TQ reduce the proliferation of neoplastic keratinocytes by 50%. The sensitivity of cells to TQ treatment appears to be stage dependent such that papilloma cells are twice as sensitive to the growth inhibitory effects of TQ as the spindle cancer cells. TQ treatment of SP-1 cells induced G0/G1 cell-cycle arrest, which correlated with sharp increases in the expression of the cyclin-dependent kinase inhibitor p16 and a decrease in cyclin D1 protein expression. TQ-induced growth inhibition in I7 cells by inducing G2/M cell-cycle arrest, which was associated with an increase in the expression of the tumor suppressor protein p53 and a decrease in cyclin B1 protein. At longer times of incubation, TQ induced apoptosis in both cell lines by remarkably increasing the ratio of Bax/Bcl-2 protein expression and decreasing Bcl-xL protein. The apoptotic effects of TQ were more pronounced in SP-1 than in I7 cells. Collectively, these findings support a potential role for TQ as a chemopreventive agent, particularly at the early stages of skin tumorigenesis.

Lobeline attenuates the behavioral effects of psychostimulants in rodents and inhibits the function of nicotinic receptors (nAChRs), dopamine transporters (DATs), and vesicular monoamine transporters (VMAT2s). Monoamine transporters are considered valid targets for drug development for the treatment of methamphetamine abuse. In the current study, a series of lobeline analogs were evaluated for affinity and selectivity at these targets. None of the analogs was more potent than nicotine at the [ 3 H]methyllycaconitine binding site ‫ء7␣(‬ nAChR subtype). Lobeline tosylate was equipotent with lobeline in inhibiting [3 H]nicotine binding but 70-fold more potent in inhibiting nicotine-evoked 86 Rb ϩ efflux, demonstrating antagonism of ‫ء2␤4␣‬ nAChRs. Compared with lobeline, the defunctionalized analogs lobelane, mesotransdiene, and (Ϫ)-trans-transdiene showed dramatically reduced affinity at ‫ء2␤4␣‬ nAChRs and a 15-to 100-fold higher affinity (K i ϭ 1.95, 0.58, and 0.26 M, respectively) at DATs. Mesotransdiene and (Ϫ)-transtransdiene competitively inhibited DAT function, whereas lobelane and lobeline acted noncompetitively. 10S/10R-MEPP [N-methyl-2R-(2R/2S-hydroxy-2-phenylethyl)6S-(2-phenylethyl)piperidine] and 10R-MESP [N-methyl-2R-(2R-hydroxy-2-phenylethyl)6S-(2-phenylethen-1-yl)piperidine] were 2 to 3 orders of magnitude more potent (K i ϭ 0.01 and 0.04 M, respectively) than lobeline in inhibiting [3 H]serotonin uptake; 10S/10R-MEPP showed a 600-fold selectivity for this transporter. Uptake results using hDATs and human serotonin transporters expressed in human embryonic kidney-293 cells were consistent with native transporter assays. Lobelane and ketoalkene were 5-fold more potent (K i ϭ 0.92 and 1.35 M, respectively) than lobeline (K i ϭ 5.46 M) in inhibiting [ 3 H]methoxytetrabenazine binding to VMAT2 in vesicle preparations. Thus, structural modification (defunctionalization) of the lobeline molecule markedly decreases affinity for ‫ء2␤4␣‬ and ‫ء7␣‬ nAChRs while increasing affinity for neurotransmitter transporters, affording analogs with enhanced selectivity for these transporters and providing new leads for the treatment of psychostimulant abuse.Lobeline, an alkaloid from Indian tobacco, inhibits the behavioral and neurochemical effects of psychostimulant drugs of abuse. For example, lobeline attenuates d-amphetamine-, methamphetamine-and nicotine-induced hyperactivity (Green et al., 2001;Miller et al., 2001Miller et al., , 2002 and inhibits the discriminative stimulus effects of methamphetamine ). Although lobeline is not selfadministered, it decreases methamphetamine self-administration in rats, which is not surmounted by increasing methamphetamine unit doses (Harrod et al., , 2003. These results suggest that lobeline lacks abuse liability while decreasing the stimulant and rewarding effects of methamphetamine via a noncompetitive mechanism of action.Psychostimulant-induced behavioral activation and reinforcement are at least partly mediated via interaction with neurotransmitter transporters that r...