Development of a new isogenic cell-xenograft system for evaluation of NAD(P)H:quinone oxidoreductase-directed antitumor quinones: evaluation of the activity of RH1. (49/274)

PURPOSE: The purpose of our study was to develop and validate an isogenic cell line pair that differs only in the expression of NAD(P)H:quinone oxidoreductase (NQO1) that can be used to examine the in vitro and in vivo role of NQO1 in the bioactivation of the antitumor quinone RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), a compound currently in Phase I clinical trials. EXPERIMENTAL DESIGN: MDA-MB-468 (MDA468) human breast adenocarcinoma cells, homozygous for a polymorphism in NQO1 (NQO1*2/*2) and with low levels of NQO1 activity, were stably transfected with human NQO1 to generate a clone (NQ16) expressing very high NQO1 activity. We examined levels of other reductases and looked at biochemical systems that might influence response to antitumor quinones to validate that the isogenic cell line pair differed only in the expression of NQO1. The 3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium (MTT) assay was used to determine the differential toxicity of various quinones, including the most recent NQO1-directed antitumor quinone, RH1, between the two cell lines. Human tumor xenografts were established from both MDA468 and NQ16 cells, and the antitumor activity of RH1 was evaluated. RESULTS: Levels of cytochrome P450 reductase, cytochrome b(5) reductase, soluble thiols, and superoxide dismutase in the NQ16 line were unchanged from the parental line. The functional significance of wild-type NQO1 expression was confirmed by measurement of the differential toxicity of compounds activated or deactivated by NQO1 in the two cell lines. The toxicity of the NQO1-directed antitumor quinones RH1 and streptonigrin were markedly greater and the toxicity of menadione, which is detoxified by NQO1, was ameliorated in the NQ16 line. High levels of NQO1 expression were observed throughout xenograft tumors established from the NQ16 cell line. RH1 treatment was effective at statistically reducing tumor volume in NQ16 xenografts at all of the doses tested (0.1, 0.2, 0.4 mg/kg every day for 5 days), whereas only the highest dose of RH1 resulted in a significant reduction in tumor volume in MDA468 xenografts. CONCLUSIONS: The MDA468/NQ16 isogenic cell line pair is a useful model system for evaluating the role of NQO1 in the bioactivation of antitumor quinones in both cell lines and xenografts. In addition, our data demonstrate that the novel antitumor quinone RH1, is effectively activated by NQO1 both in vitro and in vivo.  (+info)

Thiol oxidation coupled to DT-diaphorase-catalysed reduction of diaziquone. Reductive and oxidative pathways of diaziquone semiquinone modulated by glutathione and superoxide dismutase. (50/274)

DT-diaphorase [NAD(P)H:quinone oxidoreductase; EC] catalysed the two-electron reduction of the anti-tumour quinone 2,5-bis-(1-aziridinyl)-3,6-bis(ethoxycarbonylamino)-1,4-benzoquino ne (AZQ) to the hydroquinone form (AZQH2). Although DT-diaphorase catalysis of AZQ was not significantly affected by pH, the hydroquinone product was effectively stabilized by protonation at pH values below 7, whereas, above that pH, hyroquinone autoxidation, evaluated in terms of H2O2 production, increased exponentially. The autoxidation of AZQH2 entailed the formation of diverse radicals, such as O2-.,HO., and the semiquinone form of AZQ (AZQ-.), which contributed to different extents to the e.p.r. spectrum. Superoxide dismutase enhanced the autoxidation of AZQH2 and suppressed the e.p.r. signal ascribed to AZQ-., in agreement with a displacement of the equilibrium of the semiquinone autoxidation reaction (AZQ-.+O2 in equilibrium with AZQ+O2-.) upon enzymic withdrawal of O2-.. GSH increased the steady-state concentration of AZQH2 formed during DT-diaphorase catalysis and inhibited temporarily its autoxidation. This effect was accompanied by oxidation of the thiol to the disulphide within a process involving glutathionyl radical (GS.) formation, the relative contribution of which to the e.p.r. spectrum was enhanced by increasing GSH concentrations. GS. formation in this experimental model can be rationalized as originating from the reaction of GSH with AZQ-., rather than with O2-. or HO., for thiol oxidation was not affected significantly by superoxide dismutase, and GS. formation was insensitive to catalase. In addition, GSH suppressed the e.p.r. signal attributed to AZQ-.. No glutathionyl-quinone conjugate was detected during the DT-diaphorase-catalysed reduction of AZQ; although the chemical requirements for alkylation were partly fulfilled (quinone ring aromatization and acid-assisted aziridinyl ring opening), the negligible dissociation of GSH (GS(-)+H+ in equilibrium with GSH) at low pH prevented any nucleophilic addition to occur. Therefore the redox transitions of AZQ during DT-diaphorase catalysis seemed to be centred on the semiquinone species, the fate of which was inversely affected by catalytic amounts of superoxide dismutase and large amounts of GSH: the former enhanced AZQ-. autoxidation and the latter favoured AZQ-. reduction. Accordingly, superoxide dismutase and GSH suppressed the semiquinone e.p.r. signal. These results are discussed in terms of three interdependent redox transitions (comprising one-electron transfer reactions involving the quinone, oxygen and the thiol) and the thermodynamic and kinetic properties of the reactions involved.  (+info)

Outcome of induction and postremission therapy in younger adults with acute myeloid leukemia with normal karyotype: a cancer and leukemia group B study. (51/274)

PURPOSE: Evaluate the outcome of induction and postremission therapy in adults younger than 60 years with normal cytogenetics acute myeloid leukemia (AML). PATIENTS AND METHODS: In 490 patients, induction included cytarabine and daunorubicin (AD) or cytarabine and escalated doses of daunorubicin and etoposide +/- PSC-833 (ADE/ADEP). Intensification included one cycle of high-dose cytarabine (HDAC) followed by etoposide/cyclophosphamide and mitoxantrone/diaziquone (group I), three HDAC cycles (group II), four intermediate-dose cytarabine (IDAC) or HDAC cycles (group III), or one HDAC/etoposide cycle and autologous stem-cell transplantation (ASCT; group IV). RESULTS: Of 350 patients receiving AD, 73% achieved complete remission (CR), compared with 82% of 140 receiving ADE/ADEP (P = .04). Splenomegaly was associated with a lower CR rate (P < .001), and ADE/ADEP, with a higher CR rate in younger patients (P = .005). The 5-year disease-free survival (DFS) rate was 28% each for intensification groups I and II, compared with 41% and 45% for groups III and IV, respectively (P = .02). The 5-year cumulative incidence of relapse (CIR) was 62% and 67% for groups I and II, respectively, compared with 54% and 44% for groups III and IV, respectively (P = .049). The type of postremission intensification remained significant for DFS and CIR in multivariable analysis. CONCLUSION: In younger adults with normal cytogenetics AML, splenomegaly predicts a lower CR rate, and the postremission strategies of either four cycles of I/HDAC or one cycle of HDAC/etoposide followed by ASCT are associated with improved DFS and reduced relapse compared with therapies that include fewer cycles of cytarabine or no transplantation.  (+info)

In vitro activity of the novel indoloquinone EO-9 and the influence of pH on cytotoxicity. (52/274)

The novel indoloquinone compound EO-9 is shortly to undergo phase I clinical evaluation as a potential bioreductive drug. Preclinical studies have shown that EO-9 has greater activity against cells derived from human solid tumours than leukaemias in vitro. The results of this study extend the preclinical data available on EO-9 by demonstrating that EO-9 induces a broad spectrum of activity (IC50 values range from 8 to 590 ng ml-1) against a panel of human and murine tumour cell lines. Some evidence exists of selectivity towards leukaemia and human colon cell lines as opposed to murine colon cells. The response of cells to Mitomycin C were not comparable to EO-9 suggesting that the mechanism of action of these compounds is different. The cytotoxic properties of EO-9 under aerobic conditions are significantly influenced by extracellular pH. Reduction of pH from 7.4 to 5.8 increases cell kill from 40% to 95% in DLD-1 cells. In addition, EO-9 is unstable at acidic pH (T1/2 = 37 min at pH 5.5) compared to neutral pH T1/2 = 6.3 h). The major breakdown product in vitro was identified as EO-5A which proved relatively inactive compared to EO-9 (IC50 = 50 and 0.6 ug ml-1 respectively). These studies suggest that if EO-9 can be delivered to regions of low pH within solid tumours, a therapeutic advantage may be obtained.  (+info)

The p53-dependent apoptotic pathway of breast cancer cells (BC-M1) induced by the bis-type bioreductive compound aziridinylnaphthoquinone. (53/274)

INTRODUCTION: Several aziridinylbenzoquinone drugs have undergone clinical trials as potential antitumor drugs. These bioreductive compounds are designed to kill cells preferentially within the hypoxia tumor microenvironment. The bioreductive compound of bis-type naphthoquinone synthesized in our laboratory, 2-aziridin-1-yl-3-[(2-{2-[(3-aziridin-1-yl-1,4-dioxo-1,4-dihydronaphthalen-2-yl)t hio]ethoxy}ethyl)thio]naphthoquinone (AZ-1), had the most potent death effect on the breast cancer cells BC-M1 in our previous screening. In the present study, we determined that the mechanism of the death effect of BC-M1 cells induced by AZ-1 was mediated by the apoptosis pathway. METHODS: We evaluated the cytotoxicity of AZ-1 and the anti-breast cancer drugs tamoxifen and paclitaxel to BC-M1 cells and MCF-7 cells by the MTT assay and measured the apoptosis phenomena by Hoechst 33258 staining for apoptotic bodies. We also quantified the sub-G1 peak area and the ratio of the CH2/CH3 peak area of the cell membrane in BC-M1 cells by flow cytometry and 1H-NMR spectra, respectively. The apoptosis-related protein expressions, including p53, p21, the RNA-relating protein T-cell restricted intracellular antigen-related protein, cyclin-dependent kinase 2 (cell cycle regulating kinase) and pro-caspase 3, were detected by western blot, and the caspase-3 enzyme activity was also quantified by an assay kit. RESULTS: AZ-1 induced two of the breast cancer cell lines, with IC50 = 0.51 microM in BC-M1 cells and with IC50= 0.57 microM in MCF-7 cells, and showed less cytotoxicity to normal fibroblast cells (skin fibroblasts) with IC50= 5.6 microM. There was a 10-fold difference between two breast cancer cell lines and normal fibroblasts. Of the two anti-breast cancer drugs, tamoxifen showed IC50= 0.12 microM to BC-M1 cells and paclitaxel had much less sensitivity than AZ-1. The expression of p53 protein increased from 0.5 to 1.0 microM AZ-1 and decreased at 2.0 microM AZ-1. The p21 protein increased from 0.5 microM AZ-1, with the highest at 2 microM AZ-1. Regarding the AZ-1 compound-induced BC-M1 cells mediating the apoptosis pathway, the apoptotic body formation, the sub-G1 peak area, the ratio of CH2/CH3 of phospholipids in the cell membrane and the enzyme activity of caspase-3 were all in direct proportion with the dose-dependent increase of the concentration of AZ-1. The death effect-related proteins, including T-cell restricted intracellular antigen-related protein, cyclin-dependent kinase 2, and pro-caspase-3, all dose-dependently decreased with AZ-1 concentration. CONCLUSIONS: The AZ-1-induced cell death of BC-M1 cells mediating the apoptosis pathway might be associated with p53 protein expression, and AZ-1 could have the chance to be a candidate drug for anti-breast cancer following more experimental evidence, such as animal models.  (+info)

Gating-enhanced accessibility of hydrophobic sites within the transmembrane region of the nicotinic acetylcholine receptor's {delta}-subunit. A time-resolved photolabeling study. (54/274)

General anesthetics often interact more strongly with sites on open than on closed states of ligand-gated ion channels. To seek such sites, Torpedo membranes enriched in nicotinic acetylcholine receptors (nAChRs) were preincubated with the hydrophobic probe 3-(trifluoromethyl)-3-(m-iodophenyl) diazirine ([125I]TID) and exposed to agonist for either 0 ms (closed state), 1.5 and 10 ms (activated states), 1 s (fast desensitized state), or > or =1 h (equilibrium or slow desensitized state) and then rapidly frozen (<1 ms) and photolabeled. Within 1.5 ms, the fractional change in photoincorporation relative to the closed state decreased to 0.7 in the beta- and gamma-subunits, whereas in the alpha-subunit, it changed little. The most dramatic change occurred in the delta-subunit, where it increased to 1.6 within 10 ms but fell to 0.7 during fast desensitization. Four residues in the delta-subunit's transmembrane domain accounted for the enhanced photoincorporation induced by a 10-ms agonist exposure both when TID was added simultaneously with agonist and when it was preincubated with membranes. In the published closed state structure, two residues (deltaThr274 and deltaLeu278) are situated toward the extracellular end of helix M2, both contralateral to the ion channel and adjacent to the third residue (deltaPhe232) on M1. The fourth labeled residue (deltaIle288) is toward the end of the M2-M3 loop. Contact with these residues occurs on the time scale of a rapid phase of TID inhibition in Torpedo nAChRs, suggesting the formation of a transient hydrophobic pocket between M1, M2, and M3 in the delta-subunit during gating.  (+info)

RH1 induces cellular damage in an NAD(P)H:quinone oxidoreductase 1-dependent manner: relationship between DNA cross-linking, cell cycle perturbations, and apoptosis. (55/274)

Structure-based development of NAD(P)H:quinone oxidoreductase (NQO1)-directed antitumor quinones resulted in development of RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone], a methyl-substituted diaziridinyl quinone. We conducted experiments to evaluate the mechanism of RH1-induced cytotoxicity and the inter-relationship between DNA cross-linking, cell cycle changes, and apoptosis using an isogenic cell line pair developed from the human breast cancer cell line MDA-MB-468 differing only in expression of wtNQO1 (NQ16 cells). Statistically significant DNA cross-linking was detected using a modified comet assay in cells with wtNQO1 within 1 h of dosing, whereas in parental cells, only marginal DNA cross-linking was observed and required a concentration up to 50 times higher. Cross-linking in NQ16 cells could be abrogated with 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione, a mechanism-based inhibitor of NQO1. RH1 prolonged S phase and caused a G(2)/M block. Cell cycle changes were observed up to 10-fold lower in RH1 concentrations in NQ16 cells relative to parental cells. Apoptosis was similarly observed morphologically in both cell lines after RH1 treatment but was induced preferentially in NQ16 cells at lower concentrations and earlier time points. Marked cleavage of caspase-3 was observed in NQ16 cells relative to parental cells using lower concentrations of RH1. Temporally, low doses of RH1-induced rapid DNA cross-linking in NQ16 cells followed by induction of apoptosis at times when a G(2)/M block was not observed. This suggests that cell cycle arrest is not required for RH1-induced apoptosis and that DNA damage may directly initiate apoptotic events. In summary, RH1-induced preferential DNA cross-linking, cell cycle changes, and apoptosis in an NQO1-dependent manner.  (+info)

Conversion of DNA methyltransferases into azidonucleosidyl transferases via synthetic cofactors. (56/274)

Aziridine-based cofactor mimics have been synthesized and are shown to undergo methyltransferase-dependent DNA alkylation. Notably, each cofactor mimic possesses an azide functionality, to which can be attached an assortment of unnatural groups following methyltransferase-dependent DNA delivery. DNA duplexes modified with these cofactor mimics are capable of undergoing the Staudinger ligation with phosphines tethered to biological functionalities following enzymatic modification. This methodology provides a new tool by which to selectively modify DNA in a methyltransferase-dependent way. The conversion of biological methyltransferases into azidonucleosidyl transferases demonstrated here also holds tremendous promise as a means of identifying, as yet, unknown substrates of methylation.  (+info)