Green Tea (Camellia sinensis) Extract Increased Topoisomerase IIß, Improved Antioxidant Defense, and Attenuated Cardiac Remodeling in an Acute Doxorubicin Toxicity Model.

Experimental studies have shown the action of green tea in modulating cardiac remodeling. However, the effects of green tea on the cardiac remodeling process induced by doxorubicin (DOX) are not known. Therefore, this study is aimed at evaluating whether green tea extract could attenuate DOX-induced cardiac remodeling, assessed by cardiac morphological and functional changes and associated with the evaluation of different modulators of cardiac remodeling. The animals were divided into four groups: the control group (C), the green tea group (GT), the DOX group (D), and the DOX and green tea group (DGT). Groups C and GT received intraperitoneal sterile saline injections, D and DGT received intraperitoneal injections of DOX, and GT and DGT were fed chow supplemented with green tea extract for 35 days prior to DOX injection. After forty-eight hours, we performed an echocardiogram and euthanasia and collected the materials for analysis. Green tea attenuated DOX-induced cardiotoxicity by increasing cardiac function and decreasing the concentric remodeling. Treatment with DOX increased oxidative stress in the heart, marked by a higher level of lipid hydroperoxide (LH) and lower levels of antioxidant enzymes. Treatment with green tea increased the antioxidant enzymes' activity and decreased the production of LH. Green tea extract increased the expression of Top2-ß independent of DOX treatment. The activity of ATP synthase, citrate synthase, and complexes I and II decreased with DOX, without the effects of green tea. Both groups that received DOX presented with a lower ratio of P-akt/T-akt and a higher expression of CD45, TNFα, and intermediate MMP-2, without the effects of green tea. In conclusion, green tea attenuated cardiac remodeling induced by DOX and was associated with increasing the expression of Top2-ß and lowering oxidative stress. However, energy metabolism and inflammation probably do not receive the benefits induced by green tea in this model.

New series of fused pyrazolopyridines: Synthesis, molecular modeling, antimicrobial, antiquorum-sensing and antitumor activities.

New series of fused pyrazolopyridines were prepared and assessed for antimicrobial, antiquorum-sensing and antitumor activities. Antimicrobial evaluation toward selected Gram-positive bacteria, Gram-negative bacteria and fungi indicated that 5-phenylpyrazolopyridotriazinone 4a has good and broad-spectrum antimicrobial activity. In addition, 5-(4-chlorophenyl)pyrazolopyridotriazinone 4b and 5-(4-(dimethylamino)phenyl)pyrazolopyridotriazinone 4c exhibited good activity against the selected Gram-positive bacteria and A. fumigatus, whereas 5-amino-4-phenylpyrazolopyridopyrimidine 6a demonstrated good activity against B. cereus and P. aeruginosa. Furthermore, 6-amino-5-imino-4-phenylpyrazolopyridopyrimidine 7a and 6-amino-4-(4-chlorophenyl)-5-iminopyrazolopyridopyrimidine 7b demonstrated promising activity against the tested Gram-negative bacteria and fungi, and moderate activity against Gram-positive bacteria. Antiquorum-sensing screening over C. violaceum illustrated that 4a, 6a and 7a-c have strong activity. In vitro antiproliferative assessment of the new derivatives against HepG2, HCT-116 and MCF-7 cancer cells revealed that 7a is the most active analog against all tested cell lines. Likewise, 3,7-dimethyl-4-phenylpyrazolopyridopyrimidinone 2a and 6-amino-4-(4-chlorophenyl)-5-iminopyrazolopyridopyrimidine 7b manifested strong activity against all examined cell lines. In vivo antitumor testing of 2a, 7a and 7b against EAC cells in mice indicated that 7a has the highest activity. Cytotoxicity toward WI38 and WISH normal cells was also assessed and results assured that all of the investigated analogs have lower cytotoxicity than doxorubicin. DNA-binding affinity and topoisomerase IIß inhibitory activity were evaluated, and results revealed that 5b, 7a and 7b bind strongly to DNA; in addition, 2a, 4a, 7a and 7b manifested higher topoisomerase IIß inhibitory activity than that of doxorubicin. Analogs 5b, 7a and 7b were docked into topoisomerase IIß, and results indicated that 7a and 7b have the highest binding affinity toward topoisomerase IIß. In silico simulation studies referred that most of the new analogs comply with the optimum needs for good oral absorption. Also, computational carcinogenicity evaluation was predicted.

6,6'-Dihydroxythiobinupharidine as a poison of human type II topoisomerases.

A number of natural products with medicinal properties increase DNA cleavage mediated by type II topoisomerases. In an effort to identify additional natural compounds that affect the activity of human type II topoisomerases, a blind screen of a library of 341 Mediterranean plant extracts was conducted. Extracts from Nuphar lutea, the yellow water lily, were identified in this screen. N. lutea has been used in traditional medicine by a variety of indigenous populations. The active compound in N. lutea, 6,6'-dihydroxythiobinupharidine, was found to enhance DNA cleavage mediated by human topoisomerase IIα and IIß âˆ¼8-fold and ∼3-fold, respectively. Mechanistic studies with topoisomerase IIα indicate that 6,6'-dihydroxythiobinupharidine is a "covalent poison" that acts by adducting the enzyme outside of the DNA cleavage-ligation active site and requires the N-terminal domain of the protein for its activity. Results suggest that some of the medicinal properties of N. lutea may result from the interactions between 6,6'-dihydroxythiobinupharidine and the human type II enzymes.

Discovery of a novel anticancer agent with both anti-topoisomerase I and II activities in hepatocellular carcinoma SK-Hep-1 cells in vitro and in vivo: Cinnamomum verum component 2-methoxycinnamaldehyde.

Cinnamomum verum is used to make the spice cinnamon and has been used as a traditional Chinese herbal medicine for various applications. We evaluated the anticancer effect of 2-methoxycinnamaldehyde (2-MCA), a constituent of the bark of the plant, and its underlying molecular biomarkers associated with carcinogenesis in human hepatocellular carcinoma SK-Hep-1 cell line. The results show that 2-MCA suppressed proliferation and induced apoptosis as indicated by mitochondrial membrane potential loss, activation of caspase-3 and caspase-9, increase in the DNA content in sub-G1, and morphological characteristics of apoptosis, including blebbing of plasma membrane, nuclear condensation, fragmentation, apoptotic body formation, and long comet tail. In addition, 2-MCA also induced lysosomal vacuolation with increased volume of acidic compartments, suppressions of nuclear transcription factors NF-κB, cyclooxygenase-2, prostaglandin E2 (PGE2), and both topoisomerase I and II activities in a dose-dependent manner. Further study reveals the growth-inhibitory effect of 2-MCA was also evident in a nude mice model. Taken together, the data suggest that the growth-inhibitory effect of 2-MCA against SK-Hep-1 cells is accompanied by downregulations of NF-κB-binding activity, inflammatory responses involving cyclooxygenase-2 and PGE2, and proliferative control involving apoptosis, both topoisomerase I and II activities, together with an upregulation of lysosomal vacuolation and volume of acidic compartments. Similar effects (including all of the above-mentioned effects) were found in other tested cell lines, including human hepatocellular carcinoma Hep 3B, lung adenocarcinoma A549, squamous cell carcinoma NCI-H520, colorectal adenocarcinoma COLO 205, and T-lymphoblastic MOLT-3 (results not shown). Our data suggest that 2-MCA could be a potential agent for anticancer therapy.

Inhibition of cellular proliferation by diterpenes, topoisomerase II inhibitor.

We examined the effects of 12 terpene compounds derived from the roots of Euphorbia kansui on the proliferative activity of Xenopus embryo cells. Eight of these compounds showed significant inhibition of cellular proliferation even at low concentrations, while four of them needed to be present at higher concentrations to inhibit cellular proliferation. In order to define the mechanism of inhibition of cellular proliferation by these compounds, the effects of diterpene compounds on the activity of topoisomerase II were measured. Most of the diterpene compounds that inhibited cellular proliferation also inhibited topoisomerase II activity.

Effects of albaconol from the basidiomycete Albatrellus confluens on DNA topoisomerase II-mediated DNA cleavage and relaxation.

The effect of albaconol on the growth of human tumor cell, DNA topoisomerase (topo)-mediated DNA cleavage and direct DNA breakage was investigated. Albaconol inhibited significantly the growth of the human tumor cell lines K562, A549, BGC-823 and Bcap-37, the IC5s values were 7.99 +/- 0.4, 3.17 +/- 0.89, 4.18 +/- 0.14 and 7.45 +/- 2.5 microM, respectively. Albaconol stabilized and increased the topo 11-mediated DNA cleavable complex and inhibited the religation activity of topo II in a dose-dependent manner, but it failed to affect the activity of topo I. Albaconol directly broke pBR322 DNA at high concentrations, but there was no effect on the macromolecule of K562 cells. These results strongly suggest that albaconol targeted specifically to DNA topo II and that this is one of the mechanisms of its antitumor action; the direct action of albaconol on DNA may partly contribute to its anti-tumor activity at high concentrations.

Circadian variation of topoisomerase II-alpha in human rectal crypt epithelium: implications for reduction of toxicity of chemotherapy.

Topoisomerase II-alpha is a target of common chemotherapeutic agents such as doxorubicin and etoposide, which induce DNA damage by altering the activity of this enzyme. We took rectal biopsies at 4-hour intervals over a 24-hour period (seven total) from each of 10 healthy volunteers and examined immunoperoxidase-stained coded anti-topoisomerase II-alpha-stained sections. A significant circadian periodicity was seen in the number of rectal crypt epithelial cell nuclei that were stained (P =.01). Mean peak staining was at 7:23 a.m. +/- 45 minutes, and the mean rate of change (difference between peak and trough expression) was 40%. Topoisomerase II-alpha expression in rectal epithelium has a significant circadian variation similar to that of tritiated thymidine incorporation. Although direct confirmation is needed, giving topoisomerase II-targeted chemotherapeutic agents at the proper time of day might reduce their mucositis side effects.

Three new triterpenoids from Peganum nigellastrum.

Three new triterpenoids, 3alpha,27-dihydroxylup-20(29)-en-28-oic acid methyl ester, 3alpha-acetoxy-27-hydroxylup-20(29)-en-28-oic acid methyl ester, and 3alpha-acetoxyolean-12-ene-27,28-dioic acid 28-methyl ester, were isolated from the roots of Peganum nigellastrum along with four known lupene-type triterpenoids. The structures of the new triterpenoids were determined by NMR spectroscopic means. The new triterpene, 3alpha, 27-dihydroxylup-20(29)-en-28-oic acid methyl ester is a DNA topoisomerase II inhibitor (IC(50) = 8.9 microM/mL).

Resistance issues and treatment implications: pneumococcus, Staphylococcus aureus, and gram-negative rods.

During the last decade there has been an unexpectedly rapid evolution of antimicrobial resistance in the respiratory pathogens for community- and hospital-acquired pneumonia. In order to choose the most optimal therapy for their patients, it is essential that physicians be aware of the prevalence and mechanisms of resistance and their implications on the effectiveness of the various antimicrobials.

A simple, inexpensive apparatus for performance of preparative scale solution phase multiple parallel synthesis of drug analogs. II. Biological evaluation of a retrospective library of quinolone antiinfective agents.

A series of pure fluoroquinolone antiinfective agents was prepared by multiple parallel synthesis using a simple new apparatus. These compounds were evaluated biologically against Gram-positive and Gram-negative microorganisms and against a BCG strain transfected with luciferase in a fluorescence-based antitubercular assay. Activity against relatively fast growing, acid-fast Mycobacterium smegmatis was determined in part by agar-dilution streak assays. Data obtained against Escherichia coli-derived DNA gyrase does not correlate well with whole cell assays against E. coli. These compounds were assayed by a convenient glass-fiber filter binding method modified for high throughput screening. In these analogs, the results with a N-1 cyclopropyl substituent were often inferior to those obtained with a N-1 2',4'-difluorophenyl substituent. None of the new compounds prepared was superior in its antimycobacterial potency to ciprofloxacin or temafloxacin.

Preclinical studies of water-insoluble camptothecin congeners: cytotoxicity, development of resistance, and combination treatments.

Water-insoluble camptothecin (CPT) congeners are rapidly establishing themselves as promising anticancer drugs. In vitro, they have exhibited: (a) insensitivity to elevated levels of P-glycoprotein that confers multidrug resistance; (b) selective killing of malignant cells traversing the S-phase of the cell cycle, while leaving viable normal cells, which either are arrested at the S-G2 boundary or continue to divide; (c) no cross-resistance with several other anticancer drugs; and (d) potentiation or enhancement of cytotoxicity when appropriately used in combination with tumor necrosis factor, ionizing radiation, and hyperthermia. In addition, development of cell resistance to water-insoluble CPT congeners in vitro is accompanied by increased sensitivity to other anticancer drugs. Furthermore, water-insoluble CPT congeners have exhibited an unprecedented activity against a wide variety of human tumors xenografted in nude mice by inhibiting growth and inducing regression of carcinomas of the lung, breast, ovary, colon, stomach, pancreas, and prostate, as well as malignant melanoma, lymphoma, and leukemia. More importantly, oral administration of the water-insoluble CPT congeners in clinical studies with cancer patients makes other route(s) of administration unnecessary.

Hyperthermia can reduce cytotoxicity from etoposide without a corresponding reduction in the number of topoisomerase II-DNA cleavable complexes.

The chemotherapeutic drug etoposide (VP-16) causes the equilibrium reaction between noncleavable and cleavable topoisomerase II-DNA complexes to shift in favor of the cleavabel complex [H. Zang, P. D'Arpa, and L.F. Liu, Cancer Cells (Cold Spring Harbor), 2:23-27, 1990]. Pulsed-field gel electrophoresis was used to study induction and removal of cleavable complexes in cells heated before, during, or after VP-16 treatment. Pulsed-field gel electrophoresis results were evaluated both as the fraction of activity (DNA) released from the plug and as the number of double-strand breaks (DSBs) calculated from molecular weight distributions; both end points led to the same conclusions. When cells were heated at 42 degrees C during treatment with VP-16 (12 micrograms/ml up to 60 min), a slight decrease in cleavable complexes (from 30 to 20 DSBs/100 megabase pairs) was detected immediately after treatment when compared with cells treated with the drug at 37 degree C. Furthermore, heating at 42 degrees C caused a slight decrease in drug cytotoxicity as measured by less than a 2-fold increase in clonogenic survival. When cells were heated for 10 min at 45.5 degrees C prior to or after treatment with the drug, there was a reduction (approximately 50%) immediately after treatment in the number of DSBs/100 megabase pairs compared with unheated cells. The rate of removal of cleavable complexes was decreased slightly by heat. After 120 min at 37 degrees C, the number of DSB/100 megabase pairs decreased to approximately 6 for both unheated cells and those heated prior to drug treatment and to approximately 8 for cells heated after drug treatment. In agreement with a low effect of heat on the number of cleavable complexes after drug treatment, there was no significant effect of this heating protocol on drug cytotoxicity. However, heating at 45.5 degrees C prior to drug treatment at 37 degrees C protected cells from drug cytotoxicity (e.g., increased survival after 12 micrograms/ml for 60 min by approximately 100-fold) despite the similarity in the induction and rate of removal of cleavable complexes when compared with nonheated cells. Thus, when cells are heated prior to administration of VP-16, drug cytotoxicity does not correlate with the number of cleavable complexes measured either immediately after treatment or 180 min later when approximately 75% of the initial number have been removed. Finally, since hyperthermia can actually decrease drug cytotoxicity, the use of hyperthermia as an adjuvant to chemotherapy involving topoisomerase II poisons, such as VP-16, should be approached with caution.

Partial purification and characterization of DNA topoisomerase II from Plasmodium falciparum.

DNA topoisomerase II from Plasmodium falciparum was partially purified by FPLC using three columns: Econo-Pac Q, heparin-agarose and Mono Q. The enzyme showed ATP- and Mg2 +/- dependent activities in a decatenation assay, with optimum concentrations of 0.5 and 10 mM, respectively. Furthermore, highest activity was detected in the presence of 100 mM KCI. Enzyme decatenation activity was not inhibited by the DNA topoisomerase I inhibitor, camptothecin, but was sensitive to both prokaryotic and eukaryotic DNA topoisomerase II inhibitors.

In vitro and in vivo antibacterial activities of AM-1155, a new 6-fluoro-8-methoxy quinolone.

AM-1155 is a new quinolone with a wide spectrum of antibacterial activity against various bacteria including anaerobes and Mycoplasma pneumoniae. AM-1155 was 2- to 16-fold more active than ciprofloxacin and ofloxacin against Staphylococcus aureus including methicillin-resistant strains, Staphylococcus epidermidis, Streptococcus pneumoniae, and Enterococcus faecalis; its MICs for 90% of strains tested were 0.10 to 0.78 micrograms/ml. The activity of AM-1155 was comparable to that of ciprofloxacin against members of the family Enterobacteriaceae, Branhamella catarrhalis, Haemophilus influenzae, and Neisseria gonorrhoeae, but was fourfold less than that of ciprofloxacin against Pseudomonas aeruginosa. Against Xanthomonas maltophilia, Acinetobacter calcoaceticus, and Campylobacter jejuni, AM-1155 was two- to fourfold more active than ciprofloxacin. At a concentration of 1.56 micrograms/ml, AM-1155 inhibited 90% of Bacteroides fragilis strains tested; its activity was 8- to 10-fold higher than those of ofloxacin and ciprofloxacin. Development of resistance to AM-1155 in S. aureus and S. epidermidis occurred at a lower frequency than did that to ciprofloxacin after eight transfers in the presence of drug. In the oral treatment of mouse systemic infections, AM-1155 was four- to eightfold more effective than ciprofloxacin against gram-positive cocci and was as active as ciprofloxacin against gram-negative rods. The efficacy of an oral or a subcutaneous dose of AM-1155 was two- to fivefold greater than that of ofloxacin. Against experimental pneumonia with Klebsiella pneumoniae and P. aeruginosa, AM-1155 was two- to fourfold more active than ciprofloxacin and ofloxacin. AM-1155 also had good efficacy against mouse ascending urinary tract infections with Escherichia coli and P. aeruginosa. These results suggest that AM-1155 may be a potent antibacterial agent applicable to various infections.