Inhibition of microtubule assembly in tumor cells by 3-bromoacetylamino benzoylurea, a new cancericidal compound

We have synthesized a new compound, 3-bromoacetylamino benzoylurea (3-BAABU), which showed strong cancericidal activity by inducing irreversible mitotic arrest and subsequently apoptosis in human T cell leukemic cells (CEM), human biphenotypic leukemic cells (SP), a human prostate cancer cell line (PC-3), murine melanoma cells (B-16), and murine lymphoma/leukemia cells (EL4) in vitro with an ID50 in the range of 0.013-0.07 microg/ml (0.04-0.22 microM). Treatment of tumor cells for 12-24 h with 3-BAABU resulted in mitotic arrest at prometaphase/metaphase/anaphase, with separation and dispersion of chromosomes and with the absence of mitotic spindle apparatus in cytoplasm. Treatment with 3-BAABU had no cytotoxic and mitotic blocking effect in normal human lymphocytes, proliferating fibroblast cells (3T3), or proliferating myocardial cells (MOT). Cell cycle analyses showed that most treated leukemic cells accumulated at M phase 12 h after treatment. By the end of 48 h of treatment, the cells underwent apoptosis with DNA fragmentation. 3-BAABU inhibited the assembly of microtubules from tubulin but did not interfere with the disassembly of microtubules. The presence and the position of bromine and urea groups on the benzoic ring are the determining factors for its inhibition of microtubule assembly. Replacing bromine with chlorine yielded much less mitotic blocking activity and increased the ID50 40-fold. Substitution of the urea group with ethyl ester abrogated the activity of blocking mitosis but induced apoptosis. Moving the bromoacetylamino group from the 3-position to the 4-position removed blocking activity for mitosis but induced necrosis. These results suggest that 3-BAABU possesses a unique and functional structure and is a potential agent for cancer chemotherapy.     

Desoxyepothilone B is curative against human tumor xenografts that are refractory to paclitaxel

The epothilones are naturally occurring, cytotoxic macrolides that function through a paclitaxel (Taxol)-like mechanism. Although structurally dissimilar, both classes of molecules lead to the arrest of cell division and eventual cell death by stabilizing cellular microtubule assemblies. The epothilones differ in their ability to retain activity against multidrug-resistant (MDR) cell lines and tumors where paclitaxel fails. In the current account, we focus on the relationship between epothilone and paclitaxel in the context of tumors with multiple drug resistance. The epothilone analogue Z-12,13-desoxyepothilone B (dEpoB) is >35,000-fold more potent than paclitaxel in inhibiting cell growth in the MDR DC-3F/ADX cell line. Various formulations, routes, and schedules of i.v. administration of dEpoB have been tested in nude mice. Slow infusion with a Cremophor-ethanol vehicle proved to be the most beneficial in increasing efficacy and decreasing toxicity. Although dEpoB performed similarly to paclitaxel in sensitive tumors xenografts (MX-1 human mammary and HT-29 colon tumor), its effects were clearly superior against MDR tumors. When dEpoB was administered to nude mice bearing our MDR human lymphoblastic T cell leukemia (CCRF-CEM/paclitaxel), dEpoB demonstrated a full curative effect. For human mammary adenocarcinoma MCF-7/Adr cells refractory to paclitaxel, dEpoB reduced the established tumors, markedly suppressed tumor growth, and surpassed other commonly used chemotherapy drugs such as adriamycin, vinblastine, and etoposide in beneficial effects.     

Desoxyepothilone B: an efficacious microtubule-targeted antitumor agent with a promising in vivo profile relative to epothilone B

A new class of 16-membered macrolides, the epothilones (Epos), has been synthesized and evaluated for antitumor potential in vitro and in vivo. Recent studies in these and other laboratories showed that epothilones and paclitaxel (paclitaxel) share similar mechanisms of action in stabilizing microtubule arrays as indicated by binding-displacement studies, substitution for paclitaxel in paclitaxel-dependent cell growth, and electron microscopic examinations. The present study examined cell growth-inhibitory effects in two rodent and three human tumor cell lines and their drug-resistant sublines. Although paclitaxel showed as much as 1, 970-fold cross-resistance to the sublines resistant to paclitaxel, adriamycin, vinblastine, or actinomycin D, most epothilones exhibit little or no cross-resistance. In multidrug-resistant CCRF-CEM/VBL100 cells, IC50 values for EpoA (1), EpoB (2), desoxyEpoA (3) (dEpoA), desoxyEpoB (4) (dEpoB), and paclitaxel were 0.02, 0.002, 0.012, 0.017, and 4.14 microM, respectively. In vivo studies, using i.p. administration, indicated that the parent, EpoB, was highly toxic to mice and showed little therapeutic effect when compared with a lead compound, dEpoB. More significantly, dEpoB (25-40 mg/kg, Q2Dx5, i.p.) showed far superior therapeutic effects and lower toxicity than paclitaxel, doxorubicin, camptothecin, or vinblastine (at maximal tolerated doses) in parallel experiments. For mammary adenocarcinoma xenografts resistant to adriamycin, MCF-7/Adr, superior therapeutic effects were obtained with dEpoB compared with paclitaxel when i.p. regimens were used. For ovarian adenocarcinoma xenografts, SK-OV-3, dEpoB (i.p.), and paclitaxel (i. v.) gave similar therapeutic effects. In nude mice bearing a human mammary carcinoma xenograft (MX-1), marked tumor regression and cures were obtained with dEpoB.     

Paclitaxel and nocodazole differentially alter endocytosis in cultured cells

PURPOSE: Microtubule-based transport facilitates the endocytosis of exogenous macromolecules. We have determined how microtubule accumulation and disassembly alter endocytosis. METHODS: The effects of paclitaxel, which promotes microtubule assembly, and nocodazole, which promotes microtubule disassembly, on fluid-phase and receptor-mediated endocytosis were measured using uptake of horseradish peroxidase and 125I-transferrin, respectively. Changes in membrane and microtubule organization were examined by fluorescence microscopy. RESULTS: Neither paclitaxel (4 microM, 60 min pretreatment) nor nocodazole (1 microgram/ml, 60 min pretreatment) significantly inhibited fluid-phase endocytosis. However, paclitaxel caused a redistribution of fluorescent fluid-phase marker to the periphery. Both paclitaxel and nocodazole treatment significantly (p < or = 0.05) reduced the initial uptake of 125I-transferrin at 5 min to approximately 50% of control. Despite the similarity of the effects on initial endocytic uptake, the effects on steady state accumulation of 125I-transferrin were quite distinct. Exposure of CV-1 cells to paclitaxel for an additional 30, 60 or 90 min also showed reduced accumulation of 125I-transferrin up to a maximum significant (p < or = 0.05) inhibition of 48% +/- 10% of control at 90 min. In contrast, nocodazole caused an initial significant (p < or = 0.05) increase in 125I-transferrin accumulation after 30 min (159% +/- 13% of control), while by 90 min 125I-transferrin accumulation had returned to control levels. Microtubule content, particularly of stable microtubules, was increased in CV-1 cells by paclitaxel, but abolished by nocodazole treatment. CONCLUSIONS: Our data show that changes in the microtubule array can alter the dynamics of receptor movement through the endosomal pathway. However, microtubule assembly versus disassembly have different effects.     

Novel inhibitors for multidrug resistance: 1,3,5-triazacycloheptanes

1,3,5-Triazacycloheptanes were synthesized and examined for reversal of the multidrug resistance dependent on P-glycoprotein. Most of these compounds increased the intracellular uptake of vinblastine in multidrug-resistant mouse leukemia P388/ADR cells without influence upon the vinblastine accumulation in P388/S cells. The efficacy of 1,5-dibenzyl-1,3,5-triazacycloheptanes in increasing the vinblastine accumulation was in the order of 2,4-dithioxo (5) > 2-oxo-4-thioxo (4) approximately 4-(methylthio)-2-oxo (6) > 2,4-dioxo (2). The efficacy was further increased when the benzyl group was converted to a chlorobenzyl group. Among these compounds, 6c [1,5-bis(4-chlorobenzyl)-1,5,6,7-terahydro-4-(methylthio)-2H-1,3,5 - triazepin-2-one] potentiated the in vitro cell growth-inhibitory effect of vinblastine, adriamycin, and mitomycin C on P388/ADR cells and prolonged the life span of P388/ADR-bearing mice in combined therapy with vinblastine more than vinblastine alone.     

Protein kinase Cdelta-dependent induction of manganese superoxide dismutase gene expression by microtubule-active anticancer drugs

Bacterial lipopolysaccharide can induce manganese superoxide dismutase (MnSOD) gene expression in a variety of cells. Paclitaxel (taxol) shares many properties of lipopolysaccharide. Here we report that paclitaxel can induce MnSOD gene expression in human lung adenocarcinoma cell line A549 in a time- and dose-dependent manner. Additional anticancer drugs, vinblastine and vincristine, also induced MnSOD gene expression. We have shown previously (Das, K. C., and White, C. W. (1997) J. Biol. Chem. 272, 14914-14920) that these drugs can activate protein kinase C (PKC). The PKC agonists thymeleatoxin (0.5 microM) and 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA; 10 nM) potently induced MnSOD gene expression. Calphostin C and GF109203X, both specific inhibitors of PKC, each inhibited MnSOD gene expression by anticancer agents. Down-regulation of PKC by prolonged treatment with phorbol 12-myristate 13-acetate (PMA) also inhibited induction of MnSOD by anticancer drugs, indicating an important role of PKC in MnSOD signaling by these agents. Of 11 PKC isoenzymes, only PKCdelta translocated to the cell membrane after stimulation with anticancer drugs. By contrast, dPPA, PMA, and thymeleatoxin caused translocation of PKCalpha, betaI, delta, and mu isotypes. Anticancer drug-stimulated cells also had increased total PKC activity in membrane and cytosolic fractions. Thus, paclitaxel, vinblastine, and vincristine each specifically activate PKCdelta, whereas PMA, thymeleatoxin, and dPPA activate multiple isoenzymes. PKCdelta was the only isoform activated by each agent in both groups of compounds effective in MnSOD induction.     

The moderate affinity of clozapine at H3 receptors is not shared by its two major metabolites and by structurally related and unrelated atypical neuroleptics

We determined the affinity and/or potency of two metabolites of clozapine (clozapine-N-oxide and N-desmethylclozapine) and of five atypical neuroleptics, chemically related (olanzapine) or unrelated to clozapine (remoxipride, risperidone, thioridazine, zotepine), at H3 receptors. The specific binding of 3H-N alpha-methylhistamine to rat brain cortex homogenates was inhibited by the seven compounds; the pKi values were: N-desmethylclozapine (5.33); clozapine-N-oxide (4.18); olanzapine (5.45); thioridazine (4.91); zotepine (4.75); remoxipride (4.51) and risperidone (4.43). Three compounds were examined in a functional H3 receptor model as well. The electrically evoked tritium overflow from superfused mouse brain cortex slices, which represents quasi-physiological noradrenaline release, was not affected by N-desmethylclozapine (3.2 and 10 microM), clozapine-N-oxide (3.2-100 microM) and olanzapine (3.2-32 microM). On the other hand, the three compounds shifted to the right the concentration-response curve of histamine for its inhibitory effect on the evoked overflow; the apparent pA2 values were 5.84, 4.21 and 5.80, respectively. The present study shows that five atypical neuroleptics of different chemical classes and the two major metabolites of clozapine possess a lower affinity and/or antagonistic potency at H3 receptors than clozapine itself (pKi 6.15, pA2 6.33; Kathmann M, Schlicker E, G?thert M (1994). Psychopharmacology 116: 464-468).     

Modulation of P-glycoprotein activity by estramustine is limited by binding to plasma proteins

BACKGROUND: Estramustine previously has been shown to interact with P-glycoprotein and to restore intracellular accumulation of vinblastine and paclitaxel in cells overexpressing this drug transporter. However, the ability of estramustine to potentiate the cytotoxicities of several drugs was less than that expected. To resolve this apparent discordance, the authors examined the effects of serum on the actions of estramustine. METHODS: The cytotoxicities of anticancer drugs with or without estramustine or verapamil toward MCF-7 breast carcinoma cells and a P-glycoprotein-overexpressing subline MCF-7/ADR were determined using the sulforhodamine-binding assay. The extent of intracellular accumulation of [3H]vinblastine and [3H]paclitaxel was determined for each using standard methods, and the binding of radiolabeled drugs to plasma proteins was characterized by equilibrium dialysis. RESULTS: Without serum, the sensitivities of MCF-7/ADR cells to several P-glycoprotein-transported drugs were increased by estramustine and verapamil. Conversely, when the cells were treated with a 10% serum, the cytotoxicities of these drugs were increased by verapamil, but not by estramustine. Without serum, intracellular accumulation of [3H]vinblastine and [3H]paclitaxel by MCF-7/ADR cells was increased markedly by verapamil and estramustine; however, serum suppressed the effects of estramustine much more strongly than those of verapamil. Equilibrium dialysis experiments demonstrated that [3H]estramustine binds to plasma proteins, predominantly albumin, whereas [3H]paclitaxel binds to albumin and alpha 1-acid-glycoprotein, and [3H]vinblastine binds predominantly to alpha 1-acid-glycoprotein. CONCLUSION: Although estramustine can bind to P-glycoprotein, its effectiveness as a reversing agent in vivo likely is limited by binding to plasma proteins.     

Functionality of protective colloids affecting the formation, size uniformity and morphology of drug-free polylactic acid microspheres

Colchicine is a microtubule depolymerizing agent used extensively in the study of cytoskeleton-dependent cell functions. In studying the possible functional interaction between the GABA(A) receptor and the cytoskeleton, we found that colchicine inhibits GABA(A) receptor function by mechanisms independent of microtubule depolymerization. Human GABA(A) receptor alpha1beta2gamma2L subunits were co-expressed in Xenopus oocytes and the effects of colchicine on GABA(A) receptor function was assessed using the two-electrode voltage-clamp technique. Co-application of GABA (10 microM) with colchicine (100 microM) resulted in a 59.9% inhibition of GABA-gated chloride currents. This effect was instantaneous in onset with no pre-incubation required and reversed within seconds. Other depolymerizing agents, such as nocodazole (20 microM) and vinblastine (200 microM), did not affect GABA(A) receptor function using the same co-application protocol used with colchicine. The polymerizing agent taxol (10-50 microM) did not affect colchicine inhibition of the GABA responses and did not itself alter GABA-gated chloride currents. The inhibitory effect of colchicine was present under conditions in which the oocyte microtubules had been depolymerized by cold temperature. These results indicate that colchicine inhibits the GABA(A) receptor via mechanisms unrelated to microtubule depolymerization. To further examine the inhibitory effect of colchicine on the GABA response, GABA (10-3000 microM) concentration-response curves were performed in the absence or presence of various concentrations of colchicine (30-300 microM). In the presence of colchicine, the GABA concentration-response curve was shifted to the right in a parallel fashion. A Schild plot of this data yielded a linear slope indicating that colchicine acts as a competitive antagonist at the GABA binding site. We conclude that colchicine is a competitive antagonist at the GABA(A) receptor and that studies using colchicine to examine the functional interaction between GABA(A) receptors and microtubules should be interpreted with caution.     

Functional role of M2 and M3 muscarinic receptors in the urinary bladder of rats in vitro and in vivo

1. Urinary bladder smooth muscle is enriched with muscarinic receptors, the majority of which are of the M2 subtype whereas the remaining minority belong to the M3 subtype. The objective of the present study was to assess the functional role of M2 and M3 receptors in the urinary bladder of rat in vitro and in vivo by use of key discriminatory antagonists. 2. In the isolated bladder of rat, (+)-cis-dioxolane produced concentration-dependent contractions (pEC50 = 6.3) which were unaffected by tetrodotoxin (0.1 microM). These contractions were antagonized by muscarinic antagonists with the following rank order of affinity (pA2) estimates: atropine (9.1) > 4-diphenyl acetoxy-methyl piperidine methiodide (4-DAMP) (8.9) > darifenacin (8.5) > para fluoro hexahydrosiladifenidol (p-F-HHSiD) (7.4) > pirenzepine (6.8) > methoctramine (5.9). These pA2 estimates correlated most favourably (r = 0.99, P < 0.001) with the binding affinity (pKi) estimates of these compounds at human recombinant muscarinic m3 receptors expressed in Chinese hamster ovary cells, suggesting that the receptor mediating the direct contractile responses to (+)-cis-dioxolane equates with the pharmacologically defined M3 receptor. 3. As M2 receptors in smooth muscle are negatively coupled to adenylyl cyclase, we sought to determine whether a functional role of M2 receptors could be unmasked under conditions of elevated adenylyl cyclase activity (i.e., isoprenaline-induced relaxation of KCl pre-contracted tissues). Muscarinic M3 receptors were preferentially alkylated by exposing tissues to 4-DAMP mustard (40 nM, 1 h) in the presence of methoctramine (0.3 microM) to protect M2 receptors. Under these conditions, (+)-cis-dioxolane produced concentration-dependent reversal (re-contraction) of isoprenaline-induced relaxation (pEC50 = 5.8) but had marginal effects on pinacidil-induced, adenosine 3':5'-cyclic monophosphate (cyclic AMP)-independent, relaxation. The re-contractions were antagonized by methoctramine and darifenacin, yielding pA2 estimates of 6.8 and 7.6, respectively. These values are intermediate between those expected for these compounds at M2 and M3 receptors and were consistent with the involvement of both of these subtypes. 4. In urethane-anaesthetized rats, the cholinergic component (approximately 55%) of volume-induced bladder contractions was inhibited by muscarinic antagonists with the following rank order of potency (ID35%inh, nmol kg-1, i.v.): 4-DAMP (8.1) > atropine (20.7) > methoctramine (119.9) > darifenacin (283.3) > pirenzepine (369.1) > p-F-HHSiD (1053.8). These potency estimates correlated most favourably (r = 0.89, P = 0.04) with the pKi estimates of these compounds at human recombinant muscarinic m2 receptors. This is consistent with a major contribution of M2 receptors in the generation of volume-induced bladder contractions, although the modest potency of darifenacin does not exclude a role of M3 receptors. Pretreatment with propranolol (1 mg kg-1, i.v.) increased the ID35%inh of methoctramine significantly from 95.9 to 404.5 nmol kg-1 but had no significant effects on the inhibitory responses to darifenacin. These data suggest an obligatory role of beta-adrenoceptors in M2 receptor-mediated bladder contractions in vivo. 5. The findings of the present study suggest that both M2 and M3 receptors can cause contraction of the rat bladder in vitro and may also mediate reflex bladder contractions in vivo. It is proposed that muscarinic M3 receptor activation primarily causes direct contraction of the detrusor whereas M2 receptor activation can contract the bladder indirectly by reversing sympathetically (i.e. beta-adrenoceptor)-mediated relaxation. This dual mechanism may allow the parasympathetic nervous system, which is activated during voiding, to cause more efficient and complete emptying of the bladder.     

Novel systemically active antagonists of the glycine site of the N-methyl-D-aspartate receptor: electrophysiological, biochemical and behavioral characterization

A series of novel tricyclic pyrido-phthalazine-dione derivatives was tested for antagonistic effects at the strychnine-insensitive modulatory site of the N-methyl-D-aspartate (NMDA) receptor (glycineB). All compounds displaced [3H]MDL-105,519 binding to rat cortical membranes with IC50 values of between 90 nM and 3.6 microM. In patch-clamp experiments, steady-state inward current responses of cultured hippocampal neurons to NMDA (200 microM, glycine 1 microM) were antagonized by these same compounds with IC50 values of 0.14 to 13.8 microM. The antagonism observed was typical for glycineB antagonists, i.e., they induced desensitization and their effects were not use or voltage dependent. Moreover, increasing concentrations of glycine were able to decrease their apparent potency. Much higher concentrations (>100 microM) were required to antagonize alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced currents. They were potent, systemically active NMDA receptor antagonists in vivo against responses of single neurons in the rat spinal cord to microelectrophoretic application of NMDA with ID50 values in the low milligram per kilogram i.v. range. They also inhibited pentylenetetrazol-, NMDA- and maximal electroshock-induced convulsions in mice with ED50 values ranging from 8 to 100 mg/kg i.p. The duration of anticonvulsive action was rather short but was prolonged by the organic acid transport inhibitor probenecid (200 mg/kg). The agents tested represent a novel class of systemically active glycineB antagonists with greatly improved bioavailability.     

Topology and function of the Na+/proline transporter of Escherichia coli, a member of the Na+/solute cotransporter family

Penidiamide, a new tripetide containing dehydrotryptamine, glycine and anthranilic acid linked together by two amide bonds, and oxindole were isolated from submerged cultures of Penicillium sp. 62-92. Both compounds preferentially inhibited human synovial phospholipase A2, penidiamide with an IC50 of 30 microM and oxindole of 380 microM. With the exception of U 937 cells (leukemia, human), no cytotoxic activities were detected against HL-60- (leukemia, human), HeLa S3- (epitheloid carcinoma, human), BHK 21- (kidney fibroblasts, hamster), and L1210-cells (leukemia, mouse). No antimicrobial activity was detected for oxindole, and only weak antibacterial activity for penidiamide. The structure of penidiamide was elucidated by spectroscopic methods.     

Characterization of the interaction of cryptophycin 1 with tubulin: binding in the Vinca domain, competitive inhibition of dolastatin 10 binding, and an unusual aggregation reaction

The antimitotic depsipeptide cryptophycin 1 (CP1) was compared to the antimitotic peptide dolastatin 10 (D10) as an antiproliferative agent and in its interactions with purified tubulin. The potent activity of CP1 as an inhibitor of cell growth was confirmed. The agent had an IC50 of 20 pM against L1210 murine leukemia cells versus 0.5 nM for D10. Both drugs were comparable as inhibitors of the glutamate-induced assembly of purified tubulin, with D10 being slightly more potent. CP1, like D10, was a noncompetitive inhibitor of the binding of [3H]vinblastine to tubulin (apparent Ki, 3.9 microM); and the depsipeptide was a competitive inhibitor of the binding of [3H]D10 to tubulin (apparent Ki, 2.1 microM). CP1 was less potent than D10 as an inhibitor of nucleotide exchange on tubulin, but the two drugs were equivalent in stabilizing the colchicine binding activity of tubulin. CP1, like D10, caused the formation of extensive structured aggregates of tubulin when present in stoichiometric amounts relative to the protein. Whereas at lower concentrations the drugs were equivalent in causing formation of small oligomers detected by gel permeation high-performance liquid chromatography, there were notable differences in the aggregation reactions induced by the two drugs. The electron micrographic appearance of the D10-induced aggregate differed substantially from that of the CP1-induced aggregate. With D10, but not CP1, aggregate morphology was greatly altered in the presence of microtubule-associated proteins. Finally, although CP1 caused the formation of massive aggregates, as did D10, there was little turbidity change with the depsipeptide as opposed to the peptide.     

Inhibition of murine hepatic cytochrome P450 activities by natural and synthetic phenolic compounds

1. The effect of the phenolic compounds protocatechuic acid, chlorogenic acid, tannic acid, gallates and silybin on ethoxyresorufin O-dealkylase (CYP1A1), methoxyresorufin O-dealkylase (CYP1A2) and pentoxy-O-dealkylase (CYP2B) was examined in mouse liver microsomes from induced animals. 2. All compounds tested could inhibit cytochrome P450-mediated enzyme activities, but to different extents. Tannic acid was the most potent inhibitor, especially toward EROD activity with an IC50=2.6 microM. Synthetic dodecyl gallate was also relatively selective toward this enzyme activity with an IC50=120 microM. 3. Protocatechuic acid, chlorogenic and silybin were more selective towards PROD and MROD activities. Their relative inhibitory potency for PROD activity was as follows: chlorogenic acid > protocatechuic acid > silybin > dodecyl gallate > propyl gallate. Protocatechuic acid was a more effective inhibitor of MROD activity than chlorogenic acid, and propyl gallate more effective than dodecyl gallate. Thus, no clear structure-activity or selectivity relationship was observed. 4. Analysis of the kinetics of inhibition revealed that the inhibition in most cases was non-competitive in nature.     

Phase II trial of 96-hour paclitaxel plus oral estramustine phosphate in metastatic hormone-refractory prostate cancer

PURPOSE: To evaluate the antitumor activity of 96-hour paclitaxel and daily oral estramustine phosphate (EMP) in patients with metastatic hormone-refractory prostate cancer (HRPC). PATIENTS AND METHODS: Thirty-four patients with adenocarcinoma of the prostate that progressed after one or more hormonal therapies and a trial of antiandrogen withdrawal were enrolled onto this phase II trial. Patients received paclitaxel 120 mg/m2 by 96-hour intravenous (i.v.) infusion on days 1 through 4 of each 21-day cycle, together with daily oral EMP 600 mg/m2/d, continuously. RESULTS: Four of nine patients with measurable disease had objective responses (one complete response [CR] and three partial responses [PRs]) in liver (two patients) or nodes (two patients) of 2, 6, 8, and 20 months' duration. Of 25 assessable patients with metastases limited to bone, 14 had a > or = 50% decline in pretreatment prostate-specific antigen (PSA) level sustained for at least 6 weeks and seven had a > or = 80% decline. Overall, 17 of 32 patients (53.1%) with elevated pretreatment PSA levels had a > or = 50% decline of PSA and nine (28.1%) had a > or = 80% decrease. The main toxicities (> or = grade 2) were nausea, fluid retention, and fatigue, which occurred in 33%, 33%, and 24.2% of patients. Median time to progression, based on increasing PSA level and other clinical criteria, was 22.5 weeks. The estimated median overall survival time is 69 weeks. CONCLUSION: The combination of EMP and 96-hour paclitaxel is an active regimen for patients with HRPC. These results further support the therapeutic strategy of combining agents that impair microtubule function by complementary mechanisms.     

Biochemical characterization of glycyrrhizin as an effective inhibitor for hyaluronidases from bovine testis

The inhibitory effects of several anti-inflammatory agents, including glycyrrhizin (GL), on the activities of hyaluronidases (HAses) purified from bovine testes and Streptomyces were investigated in vitro. It was found that (i) GL inhibits the activity of HAse (p55) from bovine testes in a dose-dependent manner, but does not affect HAse from Streptomyces; (ii) GL was the most effective of the compounds tested on bovine testis HAse activity (50% inhibition with approx. 3 microM GL); and (iii) glycyrrhetinic acid (GA), a derivative (oGA) of GA and diglucuronic acid had no detectable effects on HAse activity at 9.0 microM. The GL-induced inhibition of HAse activity is uncompetitive for its substrates. Data are provided to support the contentions that (i) bovine testis HAse (p55) is a GL-binding protein; and (ii) GL acts as a potent inhibitor of HAse in vitro.     

Cytotoxic 2,3-secoaromadendrane-type sesquiterpenoids from the liverwort Plagiochila ovalifolia

The ether extract of the Japanese liverwort, Plagiochila ovalifolia Mitt. (Plagiochilaceae) yielded three 2,3-secoaromadendrane-type sesquiterpenoids, plagiochiline-A-15-yl octanoate, 14-hydroxyplagiochiline-A-15-yl 2E,4E-dodecadienoate and 14-hydroxyplagiochiline-A-15-yl 2E,4E,8Z-tetradecatrienoate. The two latter compounds were first isolated from the Plagiochila species. The structures of the new compounds were elucidated by spectroscopy. Plagiochiline-A-15-yl octanoate and 14-hydroxyplagiochiline-A-15-yl 2E,4E-dodecadienoate showed significant cytotoxicity against P-388 murine leukemia tumor cells, both compounds exhibiting an ID50 of 0.05 microgram/ml. Plagiochiline A also showed cytotoxic activity. The ID50 was 3.0 micrograms/ml.     

Effects of veratridine on the action potentials and contractility of right and left ventricles from normo- and hypertensive rats

Induction of nitric oxide synthase and generation of nitric oxide in pancreatic islet beta-cells may mediate cytokine-induced dysfunction leading to insulin-dependent diabetes mellitus. Nitric oxide generation can be regulated by availability of arginine substrate which, in turn, may be affected by substrate utilization in competing pathways such as the arginase-catalysed formation of ornithine and urea. In this study we have investigated the activity of arginase in the rat insulinoma-derived cell line RINm5F and the effect on this of interleukin 1beta, the nitric oxide synthase reaction intermediate NG-hydroxy-l-arginine and the nitric oxide-generating compounds 3-morpholinosydnonimine and S-nitrosoglutathione. Cytosols from RINm5F cells treated with or without interleukin 1beta (0.1nM, 18h) were incubated (45min, 37 degrees C) with [U-14C]arginine. Radiolabelled products ([14C]citrulline from nitric oxide synthase, [14C]ornithine and [14C]urea from arginase) were separated by high-performance liquid chromatography or ion-exchange chromatography. Interleukin 1beta increased citrulline production (from 0.01+/-0.002 to 0.58+/-0.03 pmol/microg cell protein), indicating induction of nitric oxide synthase, and significantly decreased production of both ornithine (from 4.60+/-0.20 to 3.40+/-0.20 pmol/microg) and urea (0.93+/-0.05 to 0.69+/-0.04 pmol/microg) (P<0.001), indicating decreased activity of arginase. Arginase was significantly inhibited by NG-hydroxy-l-arginine (IC50=50 microM), S-nitrosoglutathione (500 microM: 69+/-7% of control) and 3-morpholinosydnonimine (1 mM: 57+/-7% of control) (P<0.05). We conclude that during cytokine-directed beta-cell assault nitric oxide synthase-catalysed production of NG-hydroxy-l-arginine and nitric oxide may inhibit arginase thereby increasing the availability of arginine for nitric oxide production.     

Mitochondrial electron transport chain activity, but not ATP synthesis, is required for drug-induced apoptosis in human leukaemic cells: a possible novel mechanism of regulating drug resistance

There is increasing evidence for an association between mitochondrial function and susceptibility to apoptosis. It has been shown that the vinblastine-resistant leukaemic cell line CEM/VLB100 has a more active mitochondrial electron transport chain (ETC) than the parental CCRF-CEM cell line. Inhibition of mitochondrial DNA replication by ethidium bromide (EB) depleted the activity of the ETC and reduced cellular respiratory rate. Depletion of mitochondrial DNA was associated with increased resistance to vinblastine-induced apoptosis in both cell lines. In contrast, the highly specific inhibitor of the energy producing mitochondrial enzyme F1Fzero-ATPase, oligomycin, rendered CEM/VLB100 cells more sensitive to vinblastine by inhibiting the energy-dependent P-glycoprotein (Pgp) pump, suggesting that the effect of EB is independent of energy generation and ATPase activity. Both mitochondrial ETC depletion and ATPase inhibition decreased vinblastine-induced cell cycle changes in the CCRF-CEM cell line, suggesting that cell cycle changes are dependent on ATP generation. However, EB-induced ETC depletion in CEM/VLB100 cells inhibited apoptosis in response to high concentration of vinblastine, but not G2M arrest. We suggest that: (1) over-expression of Pgp by drug-resistant cells may up-regulate mitochondrial energy production; (2) mitochondrial ETC activity is required for DNA fragmentation in response to vinblastine, but the mechanism is independent of Pgp activity and ATP generation; (3) down-regulation of mitochondrial ETC activity may confer resistance to vinblastine-induced apoptosis; (4) the mitochondrial ETC is involved in vinblastine-induced apoptosis downstream of microtubule disruption and cell cycle changes.