[Show abstract][Hide abstract] ABSTRACT: Triptolide, isolated from the herb Tripterygium wilfordii, has been shown to potently induce apoptosis in various malignant cells by inhibiting RNA synthesis and nuclear factor-κB activity. Previously, we showed that triptolide promotes apoptosis in acute myeloid leukemia (AML) cells via the mitochondria-mediated pathway, in part, by decreasing levels of the anti-apoptotic proteins XIAP and Mcl-1. MRx102 is a triptolide derivative, currently in preclinical development. Here we show that MRx102 potently promoted apoptosis in AML cell lines, with EC(50) values of 14.5±0.6 nM and 37.0±0.9 nM at 48 h for OCI-AML3 and MV4-11 cells, respectively. MRx102, at low nanomolar concentrations, also induced apoptosis in bulk, CD34(+) progenitor, and more importantly, CD34(+)CD38(-) stem/progenitor cells from AML patients, even when they were protected by coculture with bone marrow derived mesenchymal stromal cells. MRx102 decreased XIAP and Mcl-1 protein levels and inhibited RNA synthesis in OCI-AML3 cells. In vivo, MRx102 greatly decreased leukemia burden and increased survival time in non-obese diabetic/severe combined immunodeficiency mice harboring Ba/F3-ITD cells. Collectively, we demonstrated that MRx102 has potent antileukemic activity both in vitro and in vivo, has the potential to eliminate AML stem/progenitor cells and overcome microenvironmental protection of leukemic cells, and warrants clinical investigation.
Leukemia: official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 09/2011; 26(3):443-50. · 10.16 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nucleoside analogs are structurally similar antimetabolites that have a broad range of action and are clinically active in both solid tumors and hematological malignancies. Many of these agents are incorporated into DNA by polymerases during normal DNA synthesis, an action that blocks further extension of the nascent strand and causes stalling of replication forks. The molecular mechanisms that sense stalled replication forks activate cell cycle checkpoints and DNA repair processes, which may contribute to drug resistance. When replication forks are not stabilized by these molecules or when subsequent DNA repair processes are overwhelmed, apoptosis is initiated either by these same DNA damage sensors or by alternative mechanisms. Recently, strategies aimed at targeting DNA damage checkpoints or DNA repair processes have demonstrated effectiveness in sensitizing cells to nucleoside analogs, thus offering a means to elude drug resistance. In addition to their DNA synthesis-directed actions many nucleoside analogs trigger apoptosis by unique mechanisms, such as causing epigenetic modifications or by direct activation of the apoptosome. A review of the cellular and molecular responses to clinically relevant agents provides an understanding of the mechanisms that cause apoptosis and may provide rationale for the development of novel therapeutic strategies.
[Show abstract][Hide abstract] ABSTRACT: Not Available Bibtex entry for this abstract Preferred format for this abstract (see Preferences) Find Similar Abstracts: Use: Authors Title Return: Query Results Return items starting with number Query Form Database: Astronomy Physics arXiv e-prints
Annals of the New York Academy of Sciences 12/2006; 284(1):91 - 102. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 17-Allylamino-17-demethoxygeldanamycin (17-AAG) is a new anticancer agent currently in clinical trials. The ability of 17-AAG to abrogate the function of heat-shock protein Hsp90 and modulate cellular sensitivity to anticancer agents has prompted recent research to use this compound in drug combination therapy. Here we report that 17-AAG has striking opposite effects on the activity of arsenic trioxide (ATO) and ara-C. Combination of 17-AAG with ATO exhibited a synergistic effect in leukemia cells, whereas coincubation of 17-AAG and ara-C showed antagonistic activity. Mechanistic studies revealed that ATO exerted cytotoxic action by reactive oxygen species generation, and activated Akt survival pathway. 17-AAG abrogated Akt activation and enhanced the activity of ATO. In contrast, treatment of leukemia cells with 17-AAG caused a G1 arrest, a decrease in DNA synthesis and reduced ara-C incorporation into DNA, leading to antagonism. The ability of 17-AAG to enhance the antileukemia activity of ATO was further demonstrated in primary leukemia cells isolated from patients with acute myeloid leukemia and chronic lymphocytic leukemia, including cells from refractory patients. Our data suggest that combination of 17-AAG and ATO may be an effective therapeutic regimen. Caution should be exercised in using 17-AAG together with ara-C, as their combination effects are schedule dependent.
[Show abstract][Hide abstract] ABSTRACT: Resistance to purine analogs is emerging as a major problem in the management of patients with chronic lymphocytic leukemia (CLL). Most of these patients have already been exposed to and have become refractory to alkylating agents. To define the natural history of fludarabine (Fludara) refractory patients with CLL, we reviewed the response to first salvage therapy of 147 patients who were refractory to Fludara or had a remission less than six months in duration after a Fludara-containing regimen. Thirty-three (22%) patients responded to their first salvage attempt. However, the median survival was only 10 months. Responders survived significantly longer than non-responders. The most effective salvage regimens were combinations of purine analogs and cyclophosphamide. Patients still possibly sensitive to alkylating agents had a superior response than alkylating agent resistant or naive patients. Subsequent salvage therapy was administered to 61 patients. The most promising results noted in the group were transplantation and the use of Campath-1H antibody. The major morbidity and cause of death were associated with infections. The probability of infection was most strongly associated with the response to salvage therapy. Gram-positive organisms were most commonly associated with infection. However, gram-negative bacilli or opportunistic infection such as fungi, Pneumocystis carinii, acid-fast bacilli and legionella were prominent causes of infection. Fludara-refractory patients are a poor prognosis group and need more effective therapeutic regimens and well-designed infection prophylactic regimens.
Leukemia and Lymphoma 10/2002; 43(9):1755-62. · 2.61 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Therapeutic nucleoside analogues such as ara-C, gemcitabine, and fludarabine exert their cytotoxic activity against cancer cells mainly by incorporation into DNA and disruption of further DNA synthesis, resulting in the triggering of apoptosis. However, the molecules that recognize the incorporated analogues in DNA and subsequently initiate the downstream cellular responses remain to be identified. Here, we report that the DNA-dependent protein kinase (DNA-PK) and p53 are able to form a protein complex that interacts with the gemcitabine-containing DNA and plays a role in signaling to apoptotic pathways. DNA-PK/Ku and p53 were copurified in a protein fraction that binds to gemcitabine-containing DNA in preference to normal DNA. Immunoprecipitation experiments revealed that the two proteins physically associate in a complex. Treatment with gemcitabine resulted in an increase of DNA-PK and p53 protein and an increase in the phosphorylation of p53 at Ser15. Furthermore, confocal microscopy demonstrated a colocalization of DNA-PK and p53 to the nucleus in cells treated with gemcitabine. The nuclear localization of the DNA-PK/p53 complex was coincident with the induction of apoptosis in these cells. Although the wild-type p53 present in the protein complex exhibited 3'-5' exonuclease activity, it was incapable of excising the incorporated gemcitabine from DNA. The binding of the p53/DNA-PK complex to DNA substantially blocked further DNA synthesis by DNA polymerases alpha and epsilon in vitro, indicating a stalling of this complex at the site of drug incorporation. These data suggest that DNA-PK and p53 may form a sensor complex that detects the disruption of DNA replication caused by nucleoside analogue incorporation and may subsequently signal for apoptosis.
Cancer Research 01/2002; 61(24):8723-9. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chronic lymphocytic leukemia (CLL) lymphocytes respond to DNA alkylation by excision repair, with the extent of repair increasing as the cells acquire resistance to alkylating agents. Because incorporation of nucleotide analogues into the repair patches elicits death signals in quiescent cells, the increased capacity for excision repair in alkylator-resistant cells could facilitate incorporation of nucleotide analogues. We hypothesized that the mechanism-based interaction of nucleoside analogues with alkylating agents could elicit greater than additive killing of CLL cells.
Lymphocytes from 50 patients with CLL that were not refractory to alkylators were treated in vitro with 4-hydroperoxycyclophosphamide (4-HC) with or without prior incubation with fludarabine nucleoside (F-ara-A) or with clofarabine (Cl-F-ara-A). DNA damage repair kinetics were determined by the single-cell gel electrophoresis (comet) assay. Cytotoxicity was assessed by staining with annexin V.
CLL lymphocytes promptly initiated and completed excision repair in response to 4-HC. A 2-h preincubation with 10 microM F-ara-A or 10 microM Cl-F-ara-A inhibited the repair initiated by 4-HC, with inhibition peaking at the intracellular concentrations of 50 microM F-ara-ATP or 5 microM Cl-F-ara-ATP. Combining 4-HC with either F-ara-A or Cl-F-ara-A produced more than additive apoptotic cell death than the sum of each alone. The increase in cytotoxicity was proportional to the initial magnitude of the DNA incision and to the extent of repair inhibition by the nucleoside analogues, suggesting close correlation between the repair inhibition and induction of cell death.
DNA repair, which is active in CLL lymphocytes, may be a biological target for facilitating the incorporation of nucleoside analogues and increasing their cytotoxicity. Thus, the increased repair capacity associated with resistant disease may be manipulated to therapeutic advantage.
Clinical Cancer Research 12/2001; 7(11):3580-9. · 7.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mammalian cell cycle is exquisitely controlled by the cyclin-dependent kinases, which regulate cell cycle progression. Cell cycle transitions are, in turn, controlled by checkpoints that monitor the integrity and replication status of the genetic material before cells commit to either replicate or segregate their DNA. On activation, checkpoints interface with cyclin-Cdk complexes to block the cell cycle. Pharmacologic compounds that exploit our current knowledge of cell cycle and checkpoint pathway regulation offer insights into the development of novel therapeutic strategies.
Current Opinion in Oncology 12/2001; 13(6):484-90. · 4.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To evaluate the efficacy, toxicity, and optimal dose rate of gemcitabine in adult patients with advanced soft tissue sarcomas (STS) by comparing levels of gemcitabine triphosphate (GTP) in peripheral-blood mononuclear cells (PBMCs) of patients receiving two different dose rates.
Fifty-six assessable patients with STS (17 gastrointestinal [GI] leiomyosarcomas and 39 other histologies) were treated on a two-arm phase II study. Gemcitabine was given at 1 g/m2 as a 30-minute infusion weekly for up to 7 weeks followed by 1 week of rest and reassessment of tumor. Subsequent cycles were given at 1 g/m2 weekly for 3 weeks followed by 1 week of rest. Nine patients underwent cellular pharmacologic studies at two different dose rates (1 g/m2 over a standard 30-minute infusion on week 1 and over pharmacologically based infusion of 150 minutes on week 2) to evaluate GTP levels in PBMCs.
Seven partial responses were noted among 39 patients, for an overall response rate of 18% (95% confidence interval, 7% to 29%). Median duration of response was 3.5 months (range, 2 to 13 months). Four of 10 patients with non-GI leiomyosarcomas achieved a partial response. No objective responses were noted in 17 patients with GI leiomyosarcomas. One patient had a mixed response. Median time to progression for all patients (both arms) was 3 months; median survival was 13.9 months. Treatment was generally well tolerated. Comparison of cellular pharmacology demonstrated a significant 1.4-fold increase in the concentration of GTP with the 150-minute infusion.
Given the limited therapeutic armamentarium for STS, the activity of gemcitabine is encouraging. Its potential for combination therapy in the salvage setting should be studied with pharmacologically guided fixed dose-rate infusion.
Journal of Clinical Oncology 09/2001; 19(15):3483-9. · 18.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanism of 2'-C-cyano-2'-deoxy-1-beta-D-arabino-pentofuranosylcytosine (CNDAC) action was investigated in human lymphoblastoid CEM cells and myeloblastic leukemia ML-1 cells. CNDAC was metabolized to its 5'-triphosphate and incorporated into DNA, which was associated with inhibition of DNA synthesis. After incubation of cells with [(3)H]CNDAC, metabolites were detected in 3'-->5' phosphodiester linkage and at the 3' terminus of cellular DNA. Specific enzymatic hydrolysis of DNA demonstrated that the parent nucleoside and its 2'-epimer 2'-C-cyano-2'-deoxy-2-ribo-pentofuranosylcytosine accounted for approximately 65% of the total analogs incorporated into DNA and essentially all of the drug in the 3'-->5' phosphodiester linkage. In contrast, all detectable radioactivity at 3' termini was associated with 2'-C-cyano-2',3'-didehydro-2',3'-dideoxycytidine. This de facto DNA chain-terminating nucleotide arises from an electronic characteristic and cleavage of the 3'-phosphodiester bond subsequent to the addition of a nucleotide to the incorporated CNDAC moiety by beta-elimination, a process that generates a single strand break in DNA. Investigation of the biological consequences of these actions indicated that, after incubation with cytostatic concentrations of CNDAC, cell cycle progression was delayed during S phase, but that cells arrested predominantly in the G(2) phase. This differed from the S phase-arresting actions of ara-C and gemcitabine, other deoxycytidine analogs that inhibit DNA replication but do not cause strand breaks. Thus, once incorporated into DNA, the CNDAC molecule appears to act by a dual mechanism that 1) delays the progress of further DNA replication, but 2) upon addition of a deoxynucleotide results in the conversion of the incorporated analog to a de facto DNA chain terminator at the 3' terminus of a single strand break. It is likely that DNA strand breaks trigger cell cycle arrest in G(2).
[Show abstract][Hide abstract] ABSTRACT: A pilot protocol was designed to evaluate the efficacy of fludarabine with nelarabine (the prodrug of arabinosylguanine [ara-G]) in patients with hematologic malignancies. The cellular pharmacokinetics was investigated to seek a relationship between response and accumulation of ara-G triphosphate (ara-GTP) in circulating leukemia cells and to evaluate biochemical modulation of cellular ara-GTP metabolism by fludarabine triphosphate.
Nine of the 13 total patients had indolent leukemias, including six whose disease failed prior fludarabine therapy. Two patients had T-acute lymphoblastic leukemia, one had chronic myelogenous leukemia, and one had mycosis fungoides. Nelarabine (1.2 g/m(2)) was infused on days 1, 3, and 5. On days 3 and 5, fludarabine (30 mg/m(2)) was administered 4 hours before the nelarabine infusion. Plasma and cellular pharmacokinetic measurements were conducted during the first 5 days.
Seven patients had a partial or complete response, six of whom had indolent leukemias. The disease in four responders had failed prior fludarabine therapy. The median peak intracellular concentrations of ara-GTP were significantly different (P =.001) in responders (890 micromol/L, n = 6) and nonresponders (30 micromol/L, n = 6). Also, there was a direct relationship between the peak fludarabine triphosphate and ara-GTP in each patient (r = 0.85). The cellular elimination of ara-GTP was slow (median, 35 hours; range, 18 to > 48 hours). The ratio of ara-GTP to its normal counterpart, deoxyguanosine triphosphate, was higher in each patient (median, 42; range, 14 to 1,092) than that of fludarabine triphosphate to its normal counterpart, deoxyadenosine triphosphate (median, 2.2; range, 0.2 to 27).
Fludarabine plus nelarabine is an effective, well-tolerated regimen against leukemias. Clinical responses suggest the need for further exploration of nelarabine against fludarabine-refractory diseases. Determination of ara-GTP levels in the target tumor population may provide a prognostic test for the activity of nelarabine.
Journal of Clinical Oncology 04/2001; 19(8):2142-52. · 18.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The mechanisms of resistance to nucleoside analogues established in preclinical models are rarely found in primary tumors resistant to therapy with these agents. We tested the hypothesis that cells sense sublethal incorporation of analogues into DNA during replication and react by arresting further DNA synthesis and cell cycle progression. After removal of drug, cells may be able to repair damaged DNA and continue proliferation, thus escaping nucleoside analogue toxicity. As a corollary, we evaluated whether dysregulation of this mechanism causes cell death. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, we found that DNA synthesis decreased, cells arrested in S-phase transit, and 60-70% of the population accumulated in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium. S-phase-arrested cells were then treated with otherwise nontoxic concentrations of UCN-01, which caused rapid onset of apoptosis without cell cycle progression specifically in cells with an S-phase DNA content. Thus, S-phase arrest by nucleoside analogues sensitizes cells to UCN-01, which appears to activate signaling for death mechanisms and/or inhibit survival pathways. These results differ from those in cells arrested at the G2 checkpoint, in which UCN-01 abrogates cell cycle arrest, permitting cells to progress in the cell cycle before apoptosis.
Cancer Research 03/2001; 61(3):1065-72. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation of the c-Jun NH2-terminal kinase type 1 (JNK1) signaling pathway is often associated with apoptosis. In this report, we elucidated the role of this kinase in the programmed cell death induced by the nucleoside analogue 9-beta-D-arabinosyl-2-fluoroadenine (F-ara-A). Treatment of ML-1 cells with 3 or 10 microM F-ara-A specifically killed cells in the S-phase of the population. Incorporation of F-ara-ATP, the nucleoside triphosphate of F-ara-A, into DNA resulted in the activation of JNK1 in a time- and dose-dependent fashion. Activation of JNK1 temporally preceded DNA fragmentation. When incorporation of F-ara-A into DNA was blocked by pretreatment of the cells with aphidicolin to inhibit DNA synthesis, neither JNK1 signaling nor apoptosis was evident. Furthermore, inhibition of JNK1 by treatment of the cells with forskolin or by pretreatment with an antisense oligonucleotide directed against JNK1 mRNA resulted in a decrease in F-ara-A-induced apoptosis. Finally, the JNK1 signaling pathway appeared to be upstream to that of the effector caspases in nucleoside analogue-induced apoptosis. Thus, our data strongly suggest that JNK1 is involved in transduction of F-ara-A-induced distress signals into an apoptotic response.
Cancer Research 12/2000; 60(22):6408-15. · 8.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Gemcitabine (dFdC) is a prodrug that undergoes metabolism by cytidine deaminase to form an inactive metabolite, 2',2'-difluorodeoxyuridine (dFdU). The pharmacokinetics of dFdC and dFdU have been studied; however, their disposition has never been evaluated in a patient with ascites. A patient with pancreatic cancer and malignant ascites was treated with dFdC 1,500 mg/m2 over 150 minutes weekly for 3 weeks, repeated every 4 weeks. Serial plasma and ascites samples were obtained on weeks 1 and 2 of cycle 2. High-pressure liquid chromatography was used to quantify dFdC and dFdU in plasma and ascites. The systemic dispositions of dFdC and dFdU were similar to those reported in patients without ascites. The concentration of dFdC in ascites approached 1 mg/ml. Ascitic fluid did not serve as a depot for dFdC, and the agent's concentration in ascites approached that at which its phosphorylation is saturated.
[Show abstract][Hide abstract] ABSTRACT: Superoxide dismutases (SOD) are essential enzymes that eliminate superoxide radical (O2-) and thus protect cells from damage induced by free radicals. The active O2- production and low SOD activity in cancer cells may render the malignant cells highly dependent on SOD for survival and sensitive to inhibition of SOD. Here we report that certain oestrogen derivatives selectively kill human leukaemia cells but not normal lymphocytes. Using complementary DNA microarray and biochemical approaches, we identify SOD as a target of this drug action and show that chemical modifications at the 2-carbon (2-OH, 2-OCH3) of the derivatives are essential for SOD inhibition and for apoptosis induction. Inhibition of SOD causes accumulation of cellular O2- and leads to free-radical-mediated damage to mitochondrial membranes, the release of cytochrome c from mitochondria and apoptosis of the cancer cells. Our results indicate that targeting SOD may be a promising approach to the selective killing of cancer cells, and that mechanism-based combinations of SOD inhibitors with free-radical-producing agents may have clinical applications.
[Show abstract][Hide abstract] ABSTRACT: Fludarabine is a nucleotide analog effective in the clinical treatment of chronic lymphocytic leukemia (CLL) and other indolent lymphocytic malignancies. Although the incorporation of fludarabine into DNA is a key event in causing cytotoxicity in proliferating leukemia cells, the precise mechanisms by which fludarabine kills CLL cells remain unclear because of the quiescent nature of this malignancy. The present study demonstrated that inhibition of RNA transcription correlated significantly with the cytotoxic action of fludarabine in CLL cells. In contrast, suppression of the low level of DNA synthesis did not affect the survival of the leukemia cells. In addition, inhibition of fludarabine incorporation into cellular DNA through repair synthesis in CLL cells did not alter the cytotoxicity of this drug. Rather, inhibition of RNA synthesis by fludarabine led to a specific diminishment of certain cellular proteins from CLL cells. The combination of fludarabine with another RNA synthesis inhibitor, actinomycin D, or with the protein synthesis inhibitor, puromycin, substantially enhanced the cytotoxic activity against CLL cells. These results suggest that termination of mRNA transcription and the consequent depletion of proteins required for cell survival may be a novel biochemical mechanism of action of fludarabine in CLL cells. Thus, inhibition of RNA/protein synthesis may provide a new therapeutic strategy for the treatment of CLL patients.