Dual inhibition of topoisomerase I and tubulin polymerization by BPR0Y007, a novel cytotoxic agent

Through the screening of DNA topoisomerase I (Top I) inhibitors, a new cytotoxic agent, BPR0Y007 [2,5-bis(4-hydroxy-3-methoxybenzylidene)cyclopentanone], was identified. BPR0Y007 was less potent than camptothecin (CPT) in the inhibition of Top I in vitro. Also, in vitro data showed that BPR0Y007 induces DNA cleavage in the presence of Top I at micromolar concentrations, with a cleavage specificity similar to that of CPT. High concentrations of BPR0Y007 did not produce detectable DNA unwinding, suggesting that BPR0Y007 is not a DNA intercalator. However, BPR0Y007 displaced Hoechst 33342 dye, suggesting that BPR0Y007 binds to DNA at the Hoechst 33342 binding site. Furthermore, BPR0Y007 generated protein-linked DNA breaks in a cell-based study. Cell cycle analysis demonstrated that the cell cycle effect of BPR0Y007 differs from that of CPT. Cells accumulated in the S-phase when treated with high concentrations of CPT, whereas cells accumulated gradually in the G(2)/M phase when treated with increasing concentrations of BPR0Y007. Further studies showed that BPR0Y007 inhibits tubulin polymerization in vivo and in vitro, and induces apoptosis in a concentration-dependent manner. No cross-resistance with BPR0Y007 was observed in CPT-, VP-16-, or vincristine-resistant cell lines. The IC(50) of BPR0Y007 for various human cancer cell lines ranged from 1 to 8 microM. Taken together, these results suggest that BPR0Y007 acts on both Top I and tubulin. Given its unique biochemical mechanisms of action, BPR0Y007 warrants exploration as an antitumor compound.     

DNA structural alterations induced by bis-netropsins modulate human DNA topoisomerase I cleavage activity and poisoning by camptothecin

Bis-netropsins (bis-Nts) are efficient catalytic inhibitors of human DNA topoisomerase I (top I). These DNA minor groove binders are considered to serve as suppressors of top I-linked DNA breaks, which is generally believed to be related to their affinity to DNA. In this study, it was found that bis-Nts exhibit sequence-specificity of suppression of the strong top I-specific DNA cleavage sites and that this sequence-specificity is determined by differential ligand-induced structural alterations of DNA. Raman scattering analysis of bis-Nts interactions with double-stranded oligonucleotides, each containing the site of specific affinity to one of bis-Nts and a distinctly located top I degenerate consensus, demonstrated that bis-Nts induce not only structural changes in duplex DNA at their loading position, but also conformational changes in a distant top I-specific DNA cleavage site. The ability to alter the DNA structure correlates with the anti-top I inhibitory activities of the ligands. In addition, DNA structural alterations induced by bis-Nts were shown to be responsible for modulation of the camptothecin (CPT)-mediated DNA cleavage by top I. This effect is expressed in the bis-Nts-induced enhancement of some of the CPT-dependent DNA cleavage sites as well as in the CPT-induced enhancement of some of the top I-specific DNA cleavage sites suppressed by bis-Nts in the absence of CPT.     

Circadian regulation of mouse topoisomerase I gene expression by glucocorticoid hormones

Because glucocorticoid hormones modulate various biological processes, the endogenous rhythm of their secretion is thought to be an important factor affecting the efficacy and/or toxicity of many drugs. Topoisomerase I (Topo I) is a nuclear target of the anticancer drug camptothecin (CPT). In this study, we demonstrate that Topo I expression in tumor-bearing mice and the efficacy of CPT on the tumor are affected by the 24-h variation in circulating glucocorticoid levels. A single administration of corticosterone (CORT) to the tumor-bearing mice resulted in a significant increase in Topo I mRNA levels not only in the tumor masses but also in other healthy tissues such as liver and skeletal muscle. The CORT-induced increase in Topo I mRNA was suppressed by pretreating the mice with RU486, a glucocorticoid receptor antagonist. Significant 24-h oscillations in the Topo I mRNA levels were observed in the tumor and healthy liver without exogenous CORT, and were eliminated by adrenalectomy of the mice. This result suggests that endogenous glucocorticoid hormones are involved in the circadian regulation of Topo I gene expression. Furthermore, the anti-tumor efficacy of the Topo I inhibitor CPT-11 on the tumor-bearing mice was enhanced by administering the drug at the time when the Topo I activity was increased. Our present results demonstrate that glucocorticoid is involved in the 24-h oscillation mechanism of Topo I gene expression and suggest that monitoring the circadian rhythm in Topo I activity is useful for choosing the most appropriate time of day to administer of Topo I inhibitors.     

Secalonic acid D as a novel DNA topoisomerase I inhibitor from marine lichen-derived fungus Gliocladium sp. T31

Context: DNA topoisomerase I (topo I) is an essential enzyme which regulates the conformational changes in DNA topology by cleaving and rejoining DNA strands during normal cell growth. The inhibitors of topo I represent a major class of anticancer drugs. In our projects to isolate new anticancer agents from marine-derived fungi, secalonic acid D (SAD) with inhibitory activity on topo I was isolated from the fermentation broth of marine lichen-derived fungus Gliocladium sp. T31, which was collected from marine sediments in South Pole.

Objective: The inhibitory activity of SAD on topo I was investigated for the first time.

Materials and methods: The inhibitory effect of SAD on topo I was determined via in vitro supercoil relaxation assays and electrophoretic mobility shift assay (EMSA) using plasmid substrate, pBR322.

Results: SAD displays a considerable inhibition on topo I in a dose-dependent manner with the minimum inhibitory concentration (MIC) of 0.4 µM. Unlike the prototypic DNA topo I poison camptothecin (CPT), SAD inhibits the binding of topo I to DNA but does not induce the formation of topo I-DNA covalent complexes.

Discussion and conclusion: SAD is an excellent topo I inhibitor and thus a significantly potential anticancer candidate.     

Inhibition of DNA topoisomerases I and II,and growth inhibition of human cancer cell lines by a marine microalgal polysaccharide

We have previously reported purification of an extracellular polysaccharide GA3P, D-galactan sulfate associated with L-(+)-lactic acid, produced by a toxic marine microalga Dinoflagellate Gymnodinium sp. A(3) (GA3), and induction thereby of apoptosis on human myeloid leukemia K562 cells. In the present report, we show that the GA3P is a potent inhibitor of DNA topoisomerase (topo) I and topo II, irrespective of the presence or absence of the lactate group. Dextran sulfate also showed similar level of inhibition of topo I and topo II. We also demonstrated that, unlike camptothecin (CPT) or teniposide (VM-26), the inhibition of topo I or topo II by the polysaccharide does not involve accumulation of DNA-topo I/II cleavable complexes, clearly showing that they are not topo poisons but catalytic inhibitors with dual activity. Furthermore, the polysaccharide, when added to the reaction mixture with CPT or VM-26, inhibited stabilization of cleavable complex induced by the latter compounds. In addition, when added to the reaction mixture after the formation of the cleavable complexes by topo poisons, CPT for topo I and VM-26 for topo II, either GA3P or dextran sulfate diminished the amount of the complexes already accumulated, i.e. reversal of the reaction. These results suggest that the polysaccharides bind to the enzymes with high affinities, and that, as for topo I/II inhibition, the GA3P shares a common mechanism with dextran sulfate. As examined in vitro with a human cancer cell line panel, GA3P exhibited significant cytotoxicity against a variety of cancer cells. These findings show that the polysaccharide GA3P would prove to be a potential anticancer chemotherapeutic agent with dual activity of topo I and topo II catalytic inhibition.     

Increased cytotoxicity of an unusual DNA topoisomerase II inhibitor compound C-1305 toward HeLa cells with downregulated PARP-1 activity results from re-activation of the p53 pathway and modulation of mitotic checkpoints

Our previous studies have shown that murine fibroblast cells, in which PARP-1 gene was inactivated by gene disruption, are extremely sensitive to triazoloacridone compound C-1305, an inhibitor of DNA topoisomerase II with unusual properties. Here, we show that pharmacological inhibition of PARP-1 activity by its inhibitor compound NU1025, sensitizes human cervical carcinoma HeLa cells to compound C-1305 compared to treatment with drug alone. Cytotoxic effect of drug/NU1025 of other topoisomerase II inhibitors varied depending on the dose of PARP-1 inhibitor. Increased cytotoxicity of topoisomerase II inhibitor/NU1025 combinations was attributable to the re-activation of the p53 pathway in drug-treated HeLa cells. This lead to a more stringent cell cycle checkpoint control during G2 and M and enhanced cell death by mitotic catastrophe induced by drug/NU1025 combinations. Interestingly, treatment of HeLa cells with NU1025 alone also increased p53 expression. This effect is, at least in part, related to the inhibition of proteasome activity by drug treatments. Together, our results show that concomitant inhibition of topoisomerase II and PARP-1 leads to the synergistic cytotoxic effect toward tumor cells that may be important for combination therapies with NU1025 and topoisomerase II inhibitors. We also confirmed our earlier work and show the important role of PARP-1 activity in the maintenance of the G2 arrest induced by DNA damaging drugs. Finally, based on our studies we propose that NU1025 and possibly other inhibitors of PARP-1 may be used as non-genotoxic agents to activate p53 in tumor cells with non-functional p53 pathways.     

Inhibition of topoisomerase II by 8-chloro-adenosine triphosphate induces DNA double-stranded breaks in 8-chloro-adenosine-exposed human myelocytic leukemia K562 cells

8-Chloro-cAMP and 8-chloro-adenosine (8-Cl-Ado) are known to inhibit proliferation of cancer cells by converting 8-Cl-Ado into an ATP analog, 8-chloro-ATP (8-Cl-ATP). Because type II topoisomerases (Topo II) are ATP-dependent, we infer that 8-Cl-Ado exposure might interfere with Topo II activities and DNA metabolism in cells. We found that 8-Cl-Ado exposure inhibited Topo II-catalytic activities in K562 cells, as revealed by decreased relaxation of the supercoiled pUC19 DNA and inhibited decatenation of the kinetoplast DNA (kDNA). In vitro assays showed that 8-Cl-ATP, but not 8-Cl-Ado, could directly inhibit Topo IIα-catalyzed relaxation and decatenation of substrate DNA. Furthermore, 8-Cl-ATP inhibited Topo II-catalyzed ATP hydrolysis and increased salt-stabilized closed clamp. In addition, 8-Cl-Ado exposure decreased bromo-deoxyuridine (BrdU) incorporation into DNA and led to enhanced DNA double-stranded breaks (DSBs) and to increased formation of γ-H2AX nuclear foci in exposed K562 cells. Together, 8-Cl-Ado/8-Cl-ATP can inhibit Topo II activities in cells, thereby inhibiting DNA synthesis and inducing DNA DSBs, which may contribute to 8-Cl-Ado-inhibited proliferation of cancers.     

Enhanced clearance of topoisomerase I inhibitors from human colon cancer cells by glucuronidation

As part of a program to identify novel mechanisms of resistance to topoisomerase I (topo I) inhibitors, the cellular pharmacology of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of clinically used irinotecan (CPT-11) and NU/ICRF 505, an anthraquinone-tyrosine conjugate, has been investigated in two human colorectal cancer (CRC) cell lines. Two novel metabolites of NU/ICRF 505 (M1 and M2) and a single metabolite of SN-38 (M1) were detected by high performance liquid chromatography in the culture medium of HT29 cells but were absent in HCT116 cells. Identities of all three metabolites were established by a combination of biochemical and physicochemical techniques. M1 of SN-38 was the C10-(beta)-glucuronide of the parent lactone while M1 of NU/ICRF 505 was the C4-O-glucuronide and M2 the tyrosine-O-glucuronide, both of the parent compound. Drug transport studies revealed that by 24hr HT29 cells had effectively cleared 82.5% of NU/ICRF 505 (10 microM) into the culture medium as the two glucuronides. In contrast, intracellular concentrations of NU/ICRF 505 were maintained in HCT116 cells in the absence of glucuronidation at a level 550 times greater than in HT29 cells. HT29 cells cleared 40.9% of SN-38 (1 microM) as the glucuronide to the culture medium, while the parent drug was maintained at a level 2-fold greater in HCT116 cells. Enhanced drug clearance due to glucuronidation may contribute to intrinsic drug resistance of human CRC.     

Novel amidine analogue of melphalan as a specific multifunctional inhibitor of growth and metabolism of human breast cancer cells

A novel amidine analogue of melphalan (AB4) was compared to its parent drug, melphalan in respect to cytotoxicity, DNA and collagen biosynthesis in MDA-MB-231 and MCF-7 human breast cancer cells. It was found that AB4 was more active inhibitor of DNA and collagen synthesis as well more cytotoxic agent than melphalan. The topoisomerase I/II inhibition assay indicated that AB4 is a potent catalytic inhibitor of topoisomerase II. Data from the ethidium displacement assay showed that AB4 intercalated into the minor-groove at AT sequences of DNA. The greater potency of AB4 to suppress collagen synthesis was found to be accompanied by a stronger inhibition of prolidase activity and expression compared to melphalan. The phenomenon was related to the inhibition of beta(1)-integrin and IGF-I receptor mediated signaling caused by AB4. The expression of beta(1)-integrin receptor, as well as Sos-1 and phosphorylated MAPK, ERK(1) and ERK(2) but not FAK, Shc, and Grb-2 was significantly decreased in cells incubated for 24h with 20 microM AB4 compared to the control, not treated cells, whereas in the same conditions melphalan did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. These results indicate the amidine analogue of melphalan, AB4 represent multifunctional inhibitor of breast cancer cells growth and metabolism.     

1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methoxyphenyl)-4-piperidinol,a novel subtype selective inhibitor of the mouse type II GABA-transporter

The selectivity of new derivatives of the γ-aminobutyric acid (GABA)-uptake inhibitor, tiagabine was characterized at the four cloned mouse GABA transporters (mGAT1 through mGAT4) by measuring [³H]-GABA uptake into stably transfected baby hamster kidney cells. While tiagabine is a highly selective inhibitor of mGAT1 (K_i=0.11±0.02 μM), these derivatives exhibited low potencies at mGAT1 but differential activities at mGAT2, mGAT3 and mGAT4. In particular, 1-(3-(9H-carbazol-9-yl)-1-propyl)-4-(2-methoxyphenyl)-4-piperidinol (NNC 05-2090) was a potent inhibitor of mGAT2 (K_i=1.4±0.3 μM) showing at least 10 fold selectivity over mGAT1, mGAT3 and mGAT4. NNC 05-2090 is the first subtype selective inhibitor of mGAT2 and may represent a novel useful tool for investigating the physiological roles of GAT2 in the brain and periphery.     

A novel cyclo-oxygenase-2 inhibitor modulates catabolic and antiinflammatory mediators in osteoarthritis

ITB (6-(p-bromophenyl)amino-7-(p-chlorophenyl)indazolo[2',3':1,5]-1,2,4-triazolo[4,3-a]-1,3,5-benzotriazepine) is a novel inhibitor of cyclo-oxygenase-2 (COX-2) with antiinflammatory activity in animal models. In the present study, we investigated the effect of this compound on the production of catabolic or antiinflammatory mediators in osteoarthritis (OA) cartilage. In OA cartilage explants, ITB inhibited the production of prostaglandin E(2) (PGE(2)), tumour necrosis factor-alpha (TNF-alpha) and matrix metalloproteinase-13 (MMP-13) in a concentration-dependent manner, whereas nitrite was partially reduced. On the contrary, ITB increased the production of interleukin (IL)-10 and the expression of heme oxygenase-1 (HO-1). ITB inhibited the production of catabolic mediators at concentrations able to increase IL-10 and HO-1 in OA cartilage, suggesting that this compound may be useful in the prevention of cartilage degradation.     

Actions of derivatives of dehydroaltenusin, a new mammalian DNA polymerase alpha-specific inhibitor

Dehydroaltenusin was found to be an inhibitor of mammalian DNA polymerase alpha (pol. alpha) in vitro, but did not influence the activities of the other replicative DNA polymerases including even other vertebrate pol. alpha. In this study, we purified or synthesized various slightly modified derivatives of dehydroaltenusin, and using them, investigated the relationship between the chemical structure and the inhibitory effects, and the in vitro and in vivo effects of dehydroaltenusin to determine to what extent the pol. alpha activity inhibition influences cell proliferation. Most of the derivatives lost the enzyme species-specific inhibitory effect, suggesting that dehydroaltenusin is three-dimensionally inserted into a pocket present only in mammalian pol. alpha. Dehydroaltenusin inhibited the cell proliferation of the human gastric cancer cell line NUGC-3 by arresting the cells at G1/S-phase, and prevented the incorporation of thymidine into the cells, indicating that it blocks the primary step of in vivo DNA replication by inhibiting pol. alpha. This compound also induced apoptosis of the cells. Dehydroaltenusin is a mammalian pol. alpha-specific inhibitor useful in both of in vivo and in vitro experiments.     

Discovery of Trp-Nle-Tyr-Met as a novel agonist for human formyl peptide receptor-like 1

Formyl peptide receptor-like 1 (FPRL1) is a structural homologue of FPR, which binds chemotactic peptides as small as three amino acids (e.g., fMet-Leu-Phe, fMLF) and activates potent bactericidal functions in neutrophils. In comparison, FPRL1 ligands include peptides of 6-104 amino acids, such as Trp-Lys-Tyr-Met-Val-[d]Met (WKYMVm) and other synthetic peptides. To determine the core peptide sequence required for FPRL1 activation, we prepared various analogues based on WKYMVm and evaluated their bioactivities in an FPRL1-transfected cell line. Although substitution of d-Met(6) resulted in loss of activity, removal of Val(5) together with d-Met(6) produced a peptide that retained most of the bioactivities of the parent peptide. The resulting peptide, WKYM, represents a core structure for an FPRL1 ligand. Further substitution of Lys(2) with Nle slightly improved the potency of the tetrapeptide, which selectively activates FPRL1 over FPR. Based on these structure-activity relationship studies, we propose a model in which the modified tetrapeptide Trp-Nle-Tyr-Met (WNleYM) binds to FPRL1 through aromatic interactions involving the side chains of Trp(1) and Tyr(3), hydrophobic interaction of Nle(2), and the thio-based hydrogen bonding of Met(4), with the respective residues in FPRL1 which have not been identified. The identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for FPRL1-related disorders.     

Preclinical pharmacology profile of CS-706, a novel cyclooxygenase-2 selective inhibitor, with potent antinociceptive and anti-inflammatory effects

We report here the preclinical anti-inflammatory profile of CS-706 [2-(4-ethoxyphenyl)-4-methyl-1-(4-sulfamoylphenyl)-1H-pyrrole], a novel cyclooxygenase-2 (COX-2) selective inhibitor. CS-706 selectively inhibited COX-2 in a human whole blood assay with an IC(50) of 0.31 microM, compared with an IC(50) of 2.2 microM for COX-1. The selectivity ratio of CS-706 was higher than those of the conventional non-steroidal anti-inflammatory drugs naproxen, indomethacin, and Diclofenac-Na, whereas it was lower than those of rofecoxib, valdecoxib and etoricoxib. It was similar to that of celecoxib. The pharmacokinetic profile of CS-706 showed rapid absorption and dose-proportional exposure after oral administration to rats. CS-706 inhibited prostaglandin E(2) production in inflamed tissue induced by yeast-injection in rats with potency similar to that of indomethacin. However, it inhibited gastric mucosal prostaglandin E(2) production in normal rats weakly compared with indomethacin. CS-706 ameliorated both yeast-induced inflammatory acute pain (ED(50)=0.0090 mg/kg) and adjuvant-induced chronic arthritic pain (ED(50)=0.30 mg/kg) in rats. CS-706 showed more potent antinociceptive activity than celecoxib and rofecoxib in these models. In an adjuvant-induced arthritic model in rats, CS-706 suppressed foot swelling prophylactically with an ID(50) of 0.10 mg/kg/day, and decreased foot swelling in the established arthritis therapeutically in a dose range of 0.040 to 1.0 mg/kg/day. Single administration of up to 100 mg/kg of CS-706 induced no significant gastric lesions in rats. In conclusion, CS-706 is a COX-2-selective inhibitor with a potent antinociceptive and anti-inflammatory activity and a gastric safety profile.     

Metabolic activation of zebularine, a novel DNA methylation inhibitor, in human bladder carcinoma cells

Zebularine (2(1H)-pyrimidinone riboside, Zeb), a synthetic analogue of cytidine that is a potent inhibitor of cytidine deaminase, has been recently identified as a general inhibitor of DNA methylation. This inhibition of DNA methyltransferase (DNMT) is hypothesized to be mechanism-based and result from formation of a covalent complex between the enzyme and zebularine-substituted DNA. Metabolic activation of Zeb thus requires that it be phosphorylated and incorporated into DNA. We have quantitatively assessed the phosphorylation and DNA incorporation of Zeb in T24 cells using 2-[(14)C]-Zeb in conjunction with gradient anion-exchange HPLC and selected enzymatic and spectroscopic analyses. The corresponding 5'-mono-, di- and triphosphates of Zeb were readily formed in a dose- and time-dependent manner. Two additional Zeb-containing metabolites were tentatively identified as diphosphocholine (Zeb-DP-Chol) and diphosphoethanolamine adducts. Intracellular concentrations of Zeb-TP and Zeb-DP-Chol were similar and greatly exceeded those of other metabolites. DNA incorporation occurred but was surpassed by that of RNA by at least seven-fold. Equivalent levels and similar intracellular metabolic patterns were also observed in the Molt-4 (human T-lymphoblasts) and MC38 (murine colon carcinoma) cell lines. For male BALB/c nu/nu mice implanted s.c. with the EJ6 variant of T24 bladder carcinoma and treated i.p. with 500mg/kg 2-[(14)C]-Zeb, the in vivo phosphorylation pattern of Zeb in tumor tissue examined 24h after drug administration was similar to that observed in vitro. The complex metabolism of Zeb and its limited DNA incorporation suggest that these are the reasons why it is less potent than either 5-azacytidine or 5-aza-2'-deoxycytidine and requires higher doses for equivalent inhibition of DNMT.     

An amperometric biosensor with human CYP3A4 as a novel drug screening tool

We developed a biosensor based on the redox properties of human CYP3A4 to directly monitor electron transfer to the heme protein. Enzyme films were assembled on gold electrodes by alternate adsorption of a CYP3A4 layer on top of a polycation layer. Direct, reversible electron transfer between the electrode and CYP3A4 was observed with voltammetry under anaerobic conditions. In the presence of oxygen, the oxidation peak of the hemoprotein disappeared, and the reduction peak increased 2- to 3-fold. Addition of CYP3A4 substrates (verapamil, midazolam, quinidine, and progesterone) to the oxygenated solution caused a concentration-dependent increase in the reduction current in cyclic voltammetric and amperometric experiments. Product analyses after electrolysis with the enzyme film showed catalytic activity of the biosensor depending on substrate concentration, its inhibition by ketoconazole, and a minor contribution of H(2)O(2) to the catalytic cycle. These results suggest that electron exchange between the electrode and the immobilized CYP3A4 occurred, and that metabolic activity of the enzyme was maintained. Thus, important requirements for the application of human CYP biosensors in order to identify drugs or drug candidates as substrates or inhibitors to the attached enzyme are fulfilled.     

Protective effects of hesperidin against oxidative stress of tert-butyl hydroperoxide in human hepatocytes

Increasing evidence regarding free radical generating agents and the inflammatory process suggest that accumulation of reactive oxygen species (ROS) could involve hepatotoxicity. Hesperidin, a naturally occurring flavonoid presents in fruits and vegetables, has been reported to exert a wide range of pharmacological effects that include antioxidant, anti-inflammatory, antihypercholesterolemic, and anticarcinogenic actions. However, the cytoprotection and mechanism of hesperidin to neutralize oxidative stress in human hepatic L02 cells remain unclear. In this work, we assessed the capability of hesperidin to prevent tert-butyl hydroperoxide (t-BuOOH)-induced cell damage by augmenting cellular antioxidant defense. Hesperidin significantly protected hepatocytes against t-BuOOH-induced cell cytotoxicity, such as mitochondrial membrane potential (MMP) deplete and lactate dehydrogenase (LDH) release. Hesperidin also remarkably prevented indicators of oxidative stress, such as the ROS and lipid peroxidation level in a dose-dependent manner. Western blot showed that hesperidin facilitated ERK/MAPK phosphorylation which appeared to be responsible for nuclear translocation of Nrf2, thereby inducing cytoprotective heme oxygenase-1 (HO-1) expression. Based on the results described above, it suggested that hesperidin has potential as a therapeutic agent in the treatment of oxidative stress-related hepatocytes injury and liver dysfunctions.     

Inhibition of dipeptidyl peptidase I in the human mast cell line HMC-1: blocked activation of tryptase, but not of the predominant chymotryptic activity

The mast cell proteases tryptase and chymase are synthesised as inactive precursors, but are stored and secreted as active enzymes. The cysteinyl protease dipeptidyl peptidase I (DPPI, cathepsin C) can activate the corresponding proenzymes in cell-free systems, but it is unknown whether it fulfils this role within the intact cell. We, therefore, tested the effect the DPPI-selective inhibitor Gly-Phe diazomethyl ketone (Gly-Phe-CHN(2)) on the tryptic and chymotryptic activity of the human mast cell-like cell line, HMC-1, and monitored any changes in the amount of immunodetectable enzymes by flow cytometry. Culture in Gly-Phe-CHN(2) produced a significant decrease in tryptase activity in cell lysates within 24hr and further decreases during continued culturing to 216 hr with periodic replenishment of Gly-Phe-CHN(2)-containing media. Flow cytometry showed no significant change in the levels of immunoreactive tryptase. In contrast, chymotryptic activity in treated cells did not differ significantly from untreated cells at any time point. Treatment of 216 hr cell lysates with DPPI revealed significant amounts of activatable protryptase in Gly-Phe-CHN(2)-treated cells, but not in controls, whereas activatable prochymotryptic activity was found in both treated and control cells. Chymase was detected immunologically, though small differences in substrate specificity and molecular mass were observed. These results strongly suggest that DPPI plays a role in the activation of tryptase, but not of the predominant chymotryptic activity of HMC-1 cells. As inhibitors of tryptase have proven efficacious in models of allergic disease, these results also indicate that inhibitors of DPPI might provide an additional point of therapeutic control.     

The anti-neoplastic and novel topoisomerase II-mediated cytotoxicity of neoamphimedine,a marine pyridoacridine

Topoisomerase IIalpha (top2) is a target of some of the most useful anticancer drugs. All clinically approved top2 drugs act to stabilize a drug-enzyme-DNA cleavable complex. Here we report the novel top2 activity of neoamphimedine, an isomer of the marine pyridoacridine amphimedine. Neoamphimedine was cytotoxic in yeast and mammalian cell lines. Neoamphimedine exhibited enhanced toxicity in top2 over-expressing yeast cells and was toxic in every mammalian cell line tested. However, neoamphimedine did not possess enhanced toxicity in a mammalian cell line sensitive to stabilized cleavable complexes. Therefore, we hypothesized that neoamphimedine is a top2-dependent drug, whose primary mechanism of action is not the stabilization of cleavable complexes. Top2-directed activity was determined in purified enzyme systems. Neoamphimedine-induced catenation of plasmid DNA only in the presence of active top2. This catenation correlated with the ability of neoamphimedine to aggregate DNA. Catenation was also observed using a filter-binding assay and transmission electron microscopy. Catenation was confirmed when only restriction enzyme digestion could resolve the catenated plasmid complex to monomer length plasmid DNA. Neoamphimedine also showed potent anti-neoplastic activity in human xenograft tumors in athymic mice. Neoamphimedine was as effective as etoposide in mice bearing KB tumors and as effective as 9-aminocamptothecin in mice bearing HCT-116 tumors. Amphimedine did not induce DNA aggregation or catenation in vitro, nor did it display any significant anti-neoplastic activity. These results suggest that neoamphimedine has a novel top2-mediated mechanism of cytotoxicity and anticancer potential.