ABSTRACT
IMPORTANCE: African swine fever virus (ASFV) is a highly fatal swine disease that severely affects the pig industry. Although ASFV has been prevalent for more than 100 years, effective vaccines or antiviral strategies are still lacking. In this study, we identified four Bacillus subtilis strains that inhibited ASFV proliferation in vitro. Pigs fed with liquid biologics or powders derived from four B. subtilis strains mixed with pellet feed showed reduced morbidity and mortality when challenged with ASFV. Further analysis showed that the antiviral activity of B. subtilis was based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II. Our findings offer a promising new strategy for the prevention and control of ASFV that may significantly alleviate the economic losses in the pig industry.
Subject(s)
African Swine Fever Virus , African Swine Fever , Bacillus subtilis , Animals , African Swine Fever/prevention & control , Antiviral Agents/pharmacology , DNA Topoisomerases, Type II/pharmacology , Genistein/pharmacology , SwineABSTRACT
BACKGROUND: Castration-resistant prostate cancer (CRPC) is refractory to hormone treatment and the therapeutic options are continuously advancing. This study aims to discover the anti-CRPC effects and underlying mechanisms of small-molecule compounds targeting topoisomerase (TOP) II and cellular components of DNA damage repair. METHODS: Cell proliferation was determined in CRPC PC-3 and DU-145 cells using anchorage-dependent colony formation, sulforhodamine B assay and flow cytometric analysis of CFSE staining. Flow cytometric analyses of propidium iodide staining and JC-1 staining were used to examine the population of cell-cycle phases and mitochondrial membrane potential, respectively. Nuclear extraction was performed to detect the nuclear localization of cellular components in DNA repair pathways. Protein expressions were determined using Western blot analysis. RESULTS: A series of azathioxanthone-based derivatives were synthesized and examined for bioactivities in which WC-A13, WC-A14, WC-A15, and WC-A16 displayed potent anti-CRPC activities in both PC-3 and DU-145 cell models. These WC-A compounds selectively downregulated both TOP IIα and TOP IIß but not TOP I protein expression. WC-A13, WC-A14, and WC-A15 were more potent than WC-A16 on TOP II inhibition, mitochondrial dysfunction, and induction of caspase cascades indicating the key role of amine-containing side chain of the compounds in determining anti-CRPC activities. Furthermore, WC-A compounds induced an increase of γH2AX and activated ATR-Chk1 and ATM-Chk2 signaling pathways. P21 protein expression was also upregulated by WC-A compounds in which WC-A16 showed the least activity. Notably, WC-A compounds exhibited different regulation on Rad51, a major protein in homologous recombination of DNA in double-stranded break repair. WC-A13, WC-A14, and WC-A15 inhibited, whereas WC-A16 induced, the nuclear translocation of Rad51. CONCLUSION: The data suggest that WC-A compounds exhibit anti-CRPC effects through the inhibition of TOP II activities, leading to mitochondrial stress-involved caspase activation and apoptosis. Moreover, WC-A13, WC-A14, and WC-A15 but not WC-A16 display inhibitory activities of Rad51-mediated DNA repair pathway which may increase apoptotic effect of CRPC cells.
Subject(s)
Antineoplastic Agents , Prostatic Neoplasms, Castration-Resistant , Male , Humans , Antineoplastic Agents/therapeutic use , Prostatic Neoplasms, Castration-Resistant/drug therapy , Prostatic Neoplasms, Castration-Resistant/metabolism , Cell Line, Tumor , Apoptosis , Cell Proliferation , Caspases/metabolism , Caspases/pharmacology , Caspases/therapeutic use , DNA Repair , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/pharmacology , DNA Topoisomerases, Type II/therapeutic useABSTRACT
Aim: The previously reported dual histone deacetylase type II (HDAC II) / topoisomerase type I (Topo I) inhibitors suffer pharmacokinetic limitations because of their huge molecular weights. Materials & methods: We report the design and synthesis of a smarter novel set of uracil-linked Schiff bases (19-30) as dual HDAC II/Topo I inhibitors keeping the essential pharmacophoric features. Cytotoxicity of all compounds was assessed against three cancer cell lines. Studies of their effects on the apoptotic BAX and antiapoptotic BCL2 genes, molecular docking studies, and absorption, distribution, metabolism and excretion studies were conducted. Results: Compounds 22, 25 and 30 exhibited significant activities. The bromophenyl derivative 22 displayed the best selectivity index, with IC50 values against HDAC II and Topo I of 1.12 and 13.44 µM, respectively. Conclusion: Compound 22 could be considered a lead HDAC II/Topo I inhibitor.
Subject(s)
Antineoplastic Agents , Histone Deacetylase Inhibitors , Histone Deacetylase Inhibitors/pharmacology , Topoisomerase I Inhibitors/pharmacology , Histone Deacetylases/metabolism , Cell Line, Tumor , Molecular Docking Simulation , Schiff Bases/pharmacology , Structure-Activity Relationship , Antineoplastic Agents/pharmacology , Cell Proliferation , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/pharmacologyABSTRACT
Two series of the ferrocenyl and ruthenocenyl analogues of etoposide bearing 1,2,3-triazolyl or aminoalkyl linker were synthesized and evaluated for their cytotoxic properties, influence on the cell cycle, ability to induce tubulin polymerization, and inhibition of topoisomerase II activity. We found that the replacement of the etoposide carbohydrate moiety with a metallocenyl group led to organometallic conjugates exhibiting differentiated antiproliferative activity. Biological studies demonstrated that two ferrocenylalkylamino conjugates were notably more active than etoposide, with submicromolar or low-micromolar IC50 values towards SW620, etoposide-resistant SW620E, and methotrexate-resistant SW620M cancer cell lines. Moreover, the simplest ferrocenylmethylamino conjugate exerted dual inhibitory action against tubulin polymerization and topoisomerase II activity while other studied compounds affected only topoisomerase II activity.
Subject(s)
Antineoplastic Agents , Tubulin , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/pharmacology , Drug Screening Assays, Antitumor , Etoposide/pharmacology , Polymerization , Structure-Activity Relationship , Topoisomerase II Inhibitors/pharmacology , Tubulin/metabolismABSTRACT
Merbarone is a derivative of thiobarbituric acid, possessing catalytic inhibitory potential against human topoisomerase IIα (hTopoIIα). Merbarone was reported to inhibit DNA cleavage by hTopoIIα. It is important to understand the molecular mechanism of hTopoIIα inhibition by merbarone, as these details guide the rational design of new ligands. In this work, a systematic pharmacoinformatics analysis has been reported to analyze the merbarone-hTopoIIα interactions and to identify merbarone analogs as potential hTopoIIα inhibitors. The reported crystal structure of hTopoIIα-DNA complex (PDB ID: 4FM9) is not suitable for analyzing the merbarone-binding domain, because it is a biological assembly of hTopoIIα in C-gate open conformation. Therefore, 3D structure of hTopoIIα-DNA complex suitable for molecular modeling analysis at merbarone binding site was first generated. Using this generated complex, molecular docking analysis and molecular dynamics simulations were performed to explore the effect of merbarone on hTopoIIα-DNA complex. The binding energy for the enol form of merbarone with hTopoIIα-DNA was estimated to be -51.28â¯kcal/mol. The explored binding site and identified molecular recognition interactions were in accordance with the previously reported interference in the DNA-cleavage by merbarone. Virtual screening was performed using drug likeness filters, toxicity filters and ADMET descriptor based filters followed by molecular docking (ZINC database). Sixteen compounds were identified as merbarone-functional analogs suitable for hTopoIIα inhibition. These identified molecules can be considered for further evaluation of their anti-hTopoIIα activity.
Subject(s)
Binding Sites , Computational Biology/methods , DNA Topoisomerases, Type II/chemistry , Thiobarbiturates/chemistry , Topoisomerase II Inhibitors/chemistry , DNA Topoisomerases, Type II/pharmacology , Humans , Hydrogen Bonding , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Protein Binding , Reproducibility of Results , Structure-Activity Relationship , Thiobarbiturates/pharmacology , Topoisomerase II Inhibitors/pharmacologyABSTRACT
Three anthraquinones (1, 2 and 4), three stilbenes (5, 6 and 7) and 3,5-dihydroxybenzyl alcohol (3) were isolated from Reynoutria japonica. Their structures were identified as emodin (1), emodin-8-O-ß-D-glucoside (2), 3,5-dihydroxybenzyl alcohol (3), citreorosein (4), cis-resveratrol (5), trans-resveratrol (6) and trans-resveratrol-5-O-ß-D-glucopyranoside (7) by comparing their physicochemical and spectral data with published data. Compound 3 was isolated for the first time from the Polygonaceae family. Among the purified compounds, 3 showed more potent inhibitory activity against topoisomerase I (IC50: 4 µM) than camptothecin, as the positive control (IC50: 18 µM). Compounds 3, 4, 5, 6 and 7 showed stronger inhibitory activities toward DNA topoisomerase II (IC50: 0.54, 14, 15, 0.77 and 3 µM, respectively) than the positive control, etoposide (IC50: 44 µM). Compounds 1 and 4 displayed weak cytotoxicities against human lung cancer (A549), ovarian cancer (SK-OV-3), human liver hepatoblastoma (HepG2) and colon adenocarcinoma (HT-29) cell lines.
Subject(s)
Antigens, Neoplasm/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , DNA Topoisomerases, Type II/pharmacology , DNA-Binding Proteins/pharmacology , Fallopia japonica/chemistry , Neoplasms/drug therapy , Plant Roots/chemistry , Topoisomerase I Inhibitors/pharmacology , Animals , Anthraquinones/chemistry , Anthraquinones/isolation & purification , Anthraquinones/pharmacology , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/isolation & purification , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/isolation & purification , Benzyl Alcohols/chemistry , Benzyl Alcohols/isolation & purification , Benzyl Alcohols/pharmacology , Cattle , Cell Line, Tumor , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/isolation & purification , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/isolation & purification , Drug Discovery , Glucosides/chemistry , Glucosides/isolation & purification , Glucosides/pharmacology , Humans , Inhibitory Concentration 50 , Medicine, Korean Traditional , Plant Extracts/chemistry , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Republic of Korea , Resorcinols/chemistry , Resorcinols/isolation & purification , Resorcinols/pharmacology , Stilbenes/chemistry , Stilbenes/isolation & purification , Stilbenes/pharmacology , Topoisomerase I Inhibitors/chemistry , Topoisomerase I Inhibitors/isolation & purification , Transition TemperatureABSTRACT
PURPOSE: Solid tumors encounter a growth-limiting hypoxic microenvironment as they develop. Hypoxia-inducible factors (HIF) play important roles in hypoxia-associated tumor development and therapeutic resistance. Targeting the HIF pathway (especially HIF-1α) represents a promising cancer treatment strategy. Here, we report a novel class of HIF-1α inhibitors and the possible molecular basis of inhibition. EXPERIMENTAL DESIGN: We analyzed the inhibitory effects of clinically used topoisomerase II (TOP2)-targeting drugs on HIF-1α expression with a primary focus on mitoxantrone. The potential role of TOP2 in mitoxantrone-inhibited HIF-1α expression was studied using pharmacologic inhibition, a knockdown approach, and TOP2 mutant cells. Moreover, involvement of mitoxantrone in proteasome-mediated degradation, transcription, and translation of HIF-1α was examined. RESULTS: The TOP2-targeting mitoxantrone, but neither doxorubicin nor etoposide (VP-16), strongly inhibited HIF-1α expression under hypoxic conditions in a dose- and time-dependent manner. Surprisingly, the mitoxantrone-mediated inhibition of HIF-1α expression was largely independent of two TOP2 isozymes, proteasomal degradation, and transcription. Furthermore, mitoxantrone inhibited HIF-1α expression and function in a similar fashion as cycloheximide, suggesting that mitoxantrone might inhibit HIF-1α via a blockage at its translation step. In vitro translation experiments using HIF-1α mRNA further confirmed inhibition of HIF-1α translation by mitoxantrone. Interestingly, levels of the polysome-bound HIF-1α and VEGF-A mRNA were elevated and decreased after mitoxantrone treatment, respectively. CONCLUSIONS: We have identified the TOP2-targeting compound, mitoxantrone, as an HIF-1α inhibitor possibly through a translation inhibition mechanism, suggesting the possibility of an additional anticancer activity for mitoxantrone.
Subject(s)
Colorectal Neoplasms/metabolism , DNA Topoisomerases, Type II/pharmacology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Kidney Neoplasms/metabolism , Mitoxantrone/pharmacology , Antineoplastic Agents/pharmacology , Cell Line, Tumor , DNA Topoisomerases, Type II/genetics , Gene Knockout Techniques , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Mutation , Proteasome Endopeptidase Complex/metabolism , Protein Biosynthesis/drug effects , RNA, Messenger/metabolism , Signal TransductionABSTRACT
The tumor suppressor BRCA1 functions in DNA homologous recombination, and mutations in BRCA1 increase the risk of breast and ovarian cancers. RAP80 is a component of BRCA1-containing complexes that is required for recruitment of BRCA1 to sites of DNA damage. To evaluate the role of RAP80 in DNA damage repair, we genetically disrupted both RAP80 alleles in the recombinogenic avian DT40 cell line. The resulting RAP80(-/-) cells were proficient at homologous recombination and nonhomologous end-joining (NHEJ), but were specifically sensitized to the topoisomerase II inhibitor etoposide. Notably, doubly mutant RAP80(-/-)BRCA1(-/-) cells were more sensitive to etoposide than were BRCA1(-/-) cells, revealing that RAP80 performs a BRCA1-independent repair function. Moreover, jointly impairing the function of CtIP, a distinct BRCA1 effector protein, rendered RAP80(-/-) cells more sensitive to etoposide compared with singly mutant cells, again illustrating a BRCA1-independent role of RAP80. Based on our findings, we propose that RAP80 exerts a specific function in repair of the topoisomerase-cleavage complex, such as the removal of covalently bound polypeptides from double-strand break ends independently of BRCA1.
Subject(s)
BRCA1 Protein/physiology , Carrier Proteins/physiology , DNA Damage/drug effects , DNA Repair/drug effects , DNA Topoisomerases, Type II/pharmacology , Nuclear Proteins/physiology , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Carrier Proteins/metabolism , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Cell Nucleus/radiation effects , Chickens , Cisplatin/pharmacology , DNA Damage/radiation effects , DNA Repair/radiation effects , DNA-Binding Proteins , Endodeoxyribonucleases , Etoposide/pharmacology , Female , Fluorescent Antibody Technique , Gamma Rays , Histone Chaperones , Humans , Hydrogen Peroxide/pharmacology , Nuclear Proteins/metabolism , Oxidants/pharmacology , Recombination, Genetic , Subcellular Fractions , Ultraviolet RaysABSTRACT
Cytotoxicity of the topoisomerase II (topoII) poison etoposide has been ascribed to the persistent covalent trapping of topoII in DNA cleavage complexes that become lethal as cells replicate their DNA. However, short term etoposide treatment also leads to subsequent cell death, suggesting that the lesions that lead to cytotoxicity arise rapidly and prior to the onset DNA replication. In the present study 1h treatment with 25muM etoposide was highly toxic and initiated a double-stranded DNA damage response as reflected by the recruitment of ATM, MDC1 and DNA-PKcs to gammaH2AX foci. While most DNA breaks were rapidly repaired upon withdrawal of the etoposide treatment, the repair machinery remained engaged in foci for at least 24h following withdrawal. TopoII siRNA ablation showed the etoposide toxicity and gammaH2AX response to correlate with the inability of the cell to correct topoIIalpha-initiated DNA damage. gammaH2AX induction was resistant to the inhibition of DNA replication and transcription, but was increased by pre-treatment with the histone deacetylase inhibitor trichostatin A. These results link the lethality of etoposide to the generation of persistent topoIIalpha-dependent DNA defects within topologically open chromatin domains.
Subject(s)
Antigens, Neoplasm/immunology , Antineoplastic Agents, Phytogenic/therapeutic use , DNA Damage/drug effects , DNA Repair/genetics , DNA Topoisomerases, Type II/pharmacology , DNA-Binding Proteins/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Antigens, Neoplasm/metabolism , Antigens, Neoplasm/pharmacology , Apoptosis/drug effects , Cell Cycle Proteins/drug effects , Cell Cycle Proteins/metabolism , Cell Survival/drug effects , Cell Survival/physiology , DNA/drug effects , DNA Breaks, Double-Stranded/drug effects , DNA Damage/genetics , DNA Repair/drug effects , DNA Replication/drug effects , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Drug Resistance, Neoplasm/drug effects , Drug Resistance, Neoplasm/genetics , Etoposide , Flow Cytometry , G2 Phase/drug effects , G2 Phase/genetics , Humans , K562 Cells , Promoter Regions, Genetic/drug effects , Promoter Regions, Genetic/genetics , Tumor Suppressor ProteinsABSTRACT
Collisions with DNA tracking systems are critical for the conversion of transient topoisomerase-DNA cleavage complexes to permanent strand breaks. Since DNA is overwound ahead of tracking systems, cleavage complexes most likely to produce permanent strand breaks should be formed between topoisomerases and positively supercoiled molecules. Therefore, the ability of human topoisomerase IIalpha and IIbeta and topoisomerase I to cleave positively supercoiled DNA was assessed in the absence or presence of anticancer drugs. Topoisomerase IIalpha and IIbeta maintained approximately 4-fold lower levels of cleavage complexes with positively rather than negatively supercoiled DNA. Topoisomerase IIalpha also displayed lower levels of cleavage with overwound substrates in the presence of nonintercalative drugs. Decreased drug efficacy was due primarily to a drop in baseline (i.e., nondrug) cleavage, rather than an altered interaction with the enzyme-DNA complex. Similar results were seen for topoisomerase IIbeta, but the effects of DNA geometry on drug-induced scission were somewhat less pronounced. With both topoisomerase IIalpha and IIbeta, intercalative drugs displayed greater relative cleavage enhancement with positively supercoiled DNA. This appeared to result from negative effects of high concentrations of intercalative agents on underwound DNA. In contrast to the type II enzymes, topoisomerase I maintained approximately 3-fold higher levels of cleavage complexes with positively supercoiled substrates and displayed an even more dramatic increase in the presence of camptothecin. These findings suggest that the geometry of DNA supercoils has a profound influence on topoisomerase-mediated DNA scission and that topoisomerase I may be an intrinsically more lethal target for anticancer drugs than either topoisomerase IIalpha or IIbeta.
Subject(s)
Antineoplastic Agents/pharmacology , DNA Topoisomerases, Type II/pharmacology , DNA, Superhelical/chemistry , Antigens, Neoplasm/metabolism , Antigens, Neoplasm/pharmacology , Antineoplastic Agents/metabolism , DNA Topoisomerases, Type II/metabolism , DNA, Superhelical/drug effects , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/pharmacology , Dose-Response Relationship, Drug , Etoposide/metabolism , Etoposide/pharmacology , Humans , Intercalating Agents/metabolism , Intercalating Agents/pharmacology , Plasmids/chemistry , Plasmids/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae , Time FactorsABSTRACT
Helsinn Healthcare SA is developing becatecarin, a topoisomerase II inhibitor for the potential intravenous treatment of cancer.
Subject(s)
Carbazoles/pharmacology , Carbazoles/therapeutic use , Glucosides/pharmacology , Glucosides/therapeutic use , Indoles/pharmacology , Indoles/therapeutic use , Animals , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carbazoles/chemical synthesis , Clinical Trials as Topic , DNA Topoisomerases, Type II/pharmacology , DNA Topoisomerases, Type II/therapeutic use , Fluorouracil/therapeutic use , Glucosides/chemical synthesis , Humans , Indoles/chemical synthesis , Leucovorin/therapeutic use , Randomized Controlled Trials as Topic , Survival , Topoisomerase II InhibitorsABSTRACT
Etoposide is a DNA topoisomerase II inhibitor widely used in the treatment of a variety of malignancies that is also associated with therapy-related leukemia. The cytochrome P450 (P450)-derived catechol and quinone metabolites of etoposide may be important in the damage to the MLL (mixed lineage leukemia) gene and other genes resulting in leukemia-associated chromosomal translocations. Kinetic analysis of catechol formation by recombinant P450s was determined using liquid chromatography/selected reaction monitoring/mass spectrometry. CYP3A4 was found to play a major role in etoposide metabolism (K(m) = 77.7 +/- 27.8 microM; V(max) = 314 +/- 84 pmol of catechol/min/nmol of P450). However, CYP3A5 (K(m) = 13. 9 +/- 3.1 microM; V(max) = 19.4 +/- 0.4 pmol of catechol/min/nmol of P450) may be involved in etoposide metabolism at therapeutic concentrations of free drug. Other P450s do not appear to be involved in etoposide catechol formation. Real-time polymerase chain reaction and Western blot analysis revealed significantly increased CYP3A4 mRNA and protein levels in hepatocytes treated with 10 microM rifampicin compared with untreated cells, but only modest effects of rifampicin on CYP3A5 induction. Etoposide (40, 5, 1, and 0.25 microM) caused a slight increase in CYP3A4 mRNA in three of five batches of hepatocytes but did not result in proportionately increased CYP3A4 protein levels. At high concentrations, etoposide induced only a modest increase in CYP3A5 mRNA and protein levels in four of five batches of hepatocytes. Alternatively, coadministration of other drugs with etoposide may account for the increase in etoposide catechol formation during therapy with etoposide.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Etoposide/metabolism , Adult , Blotting, Western/methods , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/drug effects , Cytochrome P-450 Enzyme System/genetics , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/pharmacology , Etoposide/analogs & derivatives , Etoposide/chemical synthesis , Female , Hepatocytes/chemistry , Hepatocytes/drug effects , Hepatocytes/physiology , Humans , Kinetics , Male , Middle Aged , Polymerase Chain Reaction/drug effects , Polymerase Chain Reaction/methods , RNA, Messenger , Rifampin/pharmacology , Spectrometry, Mass, Electrospray Ionization/methods , Subcellular Fractions , Teniposide/metabolism , Topoisomerase II InhibitorsABSTRACT
DA-125, a novel derivative of adriamycin, is known for its anti-cancer activity. In this study, the inhibitory mechanism of DA-125 on topoisomerase was investigated in the simian virus 40 (SV40) replicating CV-1 cell by studying the SV40 DNA replication intermediates and DNA-topoisomerase complexes. DNA-protein complexes that were formed in the drug-treated cells were quantitated by using a glass filter assay. SV40 DNA replication intermediates that were accumulated in the drug-treated CV-1 cell were analyzed in a high resolution gel. DA-125 did not accumulate B-dimers of SV40 DNA replication intermediates which were found in the adriamycin-treated CV-1 cells. DA-125 induced a dose-dependent formation of the DNA-protein complexes, while adriamycin did not. When adriamycin and etoposide (VP16) were added to the SV40-infected cells at the same time, adriamycin blocked the formation of the DNA-protein complexes induced by VP16 in a dose-dependent manner. However, DA-125 blocked the formation of the DNA-protein complexes induced by VP16 up to the maximum level of the DNA-protein complexes that were induced by DA-125 alone. Adriamycin and DA-125 did not inhibit the formation of the DNA-protein complexes that were caused by camptothecin, a known topoisomerase I poison. DA-125 is bifunctional in inhibiting topoisomerase II because it simultaneously has the properties of the topoisomerase II poison and the DNA intercalator. As a topoisomerase II poison, DA-125 alone induced dose-dependent formation of the DNA-protein complexes. However, as a DNA intercalator, it quantitatively inhibited the formation of the DNA-protein complexes induced by a strong topoisomerase II poison VP16. Furthermore considering that the levels of the DNA-protein complex induced by VP16 were decreased by DA-125 in terms of the topoisomerase II poison, we suggest that DA-125 has a higher affinity to the drug-binding sites of DNA than VP16 has.
Subject(s)
Antineoplastic Agents/pharmacology , DNA Topoisomerases, Type II/pharmacology , DNA/drug effects , Doxorubicin/analogs & derivatives , Doxorubicin/pharmacology , Intercalating Agents/pharmacology , Topoisomerase II Inhibitors , Animals , Camptothecin/pharmacology , Chlorocebus aethiops , DNA/chemistry , DNA Replication/drug effects , DNA Replication/physiology , DNA Topoisomerases, Type II/biosynthesis , Dimerization , Dose-Response Relationship, Drug , Etoposide/antagonists & inhibitors , Etoposide/pharmacology , Haplorhini , Methods , Protein Biosynthesis , Proteins/chemistry , Simian virus 40/drug effects , Simian virus 40/growth & development , Vero CellsSubject(s)
Clusia/chemistry , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , DNA Topoisomerases, Type I/drug effects , DNA Topoisomerases, Type I/pharmacology , DNA Topoisomerases, Type II/drug effects , DNA Topoisomerases, Type II/pharmacology , Drug Screening Assays, Antitumor , Humans , Telomerase/drug effects , Telomerase/pharmacology , Tumor Cells, CulturedABSTRACT
A wide variety of cellular responses that may afford tumor cells drug-tolerance characteristics. Overexpression of plasma membrane efflux pumps, up-regulation of anti-apoptosis factors, down-regulation of proapoptosis factors, subcellular redistribution of drug targets, and up-regulation of detoxifying enzymes are just a few known mechanisms of cancer cell resistance. In addition to these individual cell adaptations, cellular drug resistance also appears to be mediated by the binding of tumor cells to extracellular matrix (ECM) proteins. Cell adhesion-mediated drug resistance (CAM-DR) is particularly relevant in hematologic malignancies such as multiple myeloma, where myeloma cells localize in the bone marrow and interact with stroma and stromal cells, initiating the production of proteins that stimulate or support tumor survival. Thus, CAM-DR provides a plausible explanation for the protective mechanisms associated with myeloma cell adhesion and demonstrates that the tumor microenvironment may hold the key to elucidating how tumor cells resist chemotherapy.
Subject(s)
Apoptosis/physiology , Cell Adhesion/physiology , Drug Resistance, Neoplasm/physiology , Multiple Myeloma/physiopathology , Bone Marrow/pathology , Bone Marrow/physiology , Cell Survival , Cysteine Endopeptidases/pharmacology , DNA Topoisomerases, Type II/pharmacology , Humans , Multienzyme Complexes/antagonists & inhibitors , Multienzyme Complexes/pharmacology , Proteasome Endopeptidase Complex , Signal Transduction , Up-Regulation , fas ReceptorABSTRACT
Several observations highlight the importance of the carbohydrate moiety for the biological activity of antitumoural anthracyclines. Here is reported the synthesis, cytotoxicity and topoisomerase II-mediated DNA cleavage intensity of the new oligosaccharide anthracyclines 1--4 modified in the sugar residue. Evaluation of cytotoxic potency on different cell lines, resulted in quite similar values among the different analogues. On the other hand, topoisomerase II-mediated DNA breaks level was different for the various compounds, and was not related to cytotoxicity, thus supporting previous observations reported for some monosaccharide anthracyclines modified in the carbohydrate portion.
Subject(s)
Antibiotics, Antineoplastic/chemical synthesis , Oligosaccharides/pharmacology , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacology , Carbohydrates/chemistry , Cell Survival/drug effects , DNA/metabolism , DNA Topoisomerases, Type II/drug effects , DNA Topoisomerases, Type II/pharmacology , Drug Screening Assays, Antitumor , Humans , Oligosaccharides/chemical synthesis , Oligosaccharides/chemistry , Structure-Activity Relationship , Tumor Cells, Cultured/drug effectsABSTRACT
Collisions between replication forks and topoisomerase-drug-DNA ternary complexes result in the inhibition of DNA replication and the conversion of the normally reversible ternary complex to a nonreversible form. Ultimately, this can lead to the double strand break formation and subsequent cell death. To understand the molecular mechanisms of replication fork arrest by the ternary complexes, we have investigated molecular events during collisions between DNA helicases and topoisomerase-DNA complexes. A strand displacement assay was employed to assess the effect of topoisomerase IV (Topo IV)-norfloxacin-DNA ternary complexes on the DnaB, T7 gene 4 protein, SV40 T-antigen, and UvrD DNA helicases. The ternary complexes inhibited the strand displacement activities of these DNA helicases. Unlike replication fork arrest, however, this general inhibition of DNA helicases by Topo IV-norfloxacin-DNA ternary complexes did not require the cleavage and reunion activity of Topo IV. We also examined the reversibility of the ternary complexes after collisions with these DNA helicases. UvrD converted the ternary complex to a nonreversible form, whereas DnaB, T7 gene 4 protein, and SV40 T-antigen did not. These results suggest that the inhibition of DnaB translocation may be sufficient to arrest the replication fork progression but it is not sufficient to generate cytotoxic DNA lesion.
Subject(s)
Anti-Infective Agents/pharmacology , DNA Helicases/metabolism , DNA Replication/drug effects , DNA Topoisomerases, Type II/pharmacology , Norfloxacin/pharmacology , Base Sequence , DNA Damage , DNA Helicases/antagonists & inhibitors , DNA Topoisomerase IV , Molecular Sequence Data , Nucleic Acid ConformationABSTRACT
Recent studies have shown that the anticancer drugs VM-26 and mitoxantrone stabilize preferentially the binding of topoisomerase IIalpha to replicating compared to nonreplicating DNA. To further understand the mechanisms by which cleavable complex-forming topoisomerase II inhibitors interfere with DNA replication, we examined the effects of VM-26 on this process in human leukemia CEM cells. Both the inhibition of DNA synthesis and cell survival were directly related to the total amount of drug-stabilized cleavable complexes formed in VM-26-treated cells. DNA chain elongation was also inhibited in a concentration-dependent fashion in these cells, which suggested that VM-26-stabilized cleavable complexes interfered with the movement of DNA replication forks. To test this hypothesis directly, we monitored replication fork progression at a specific site of VM-26-induced DNA cleavage. A topoisomerase II-mediated cleavage site was detected in the first exon of the c-myc gene in VM-26-treated cells. This cleavage site was downstream of a putative replication origin located in the 5' flanking region of the gene. Replication forks, which moved through this region of the c-myc gene in the 5' to 3' direction, were specifically arrested at this site in VM-26-treated cells, but not in untreated or aphidicolin-treated cells. These studies provide the first direct evidence that a VM-26-stabilized topoisomerase II-DNA cleavable complex acts as a replication fork barrier at a specific genomic site in mammalian cells. Furthermore, the data support the hypothesis that the replication fork arrest induced by cleavable complex-forming topoisomerase II inhibitors leads to the generation of irreversible DNA damage and cytotoxicity in proliferating cells.
Subject(s)
DNA Damage , DNA Replication/drug effects , DNA Topoisomerases, Type II/pharmacology , Leukemia, T-Cell/drug therapy , Nucleic Acid Synthesis Inhibitors/pharmacology , Teniposide/pharmacology , DNA Topoisomerases, Type II/metabolism , Enzyme Stability/drug effects , Genes, myc/drug effects , Humans , Leukemia, T-Cell/genetics , Macromolecular Substances , Topoisomerase II Inhibitors , Tumor Cells, CulturedABSTRACT
BACKGROUND: The combination of VP-16 and cisplatin is one of the most active regimens available for the treatment of small cell lung cancer (SCLC), however, most tumors eventually become resistant to these drugs. METHODS: To investigate the problem of resistance to VP-16 and cisplatin in patients with SCLC, we established two resistant sublines from the drug sensitive human SCLC line, NCI-H209, by in vitro selection in VP-16 and cisplatin. RESULTS: The VP-16-selected cell line, H209/VP, was more than 100-fold resistant to VP-16, and displayed cross-resistance to VM-26 and other topoisomerase II interactive drugs, but not to vinca alkaloids. There was no difference in accumulation of VP-16 in H209/VP compared with its parent cell line. The level of topoisomerase II-alpha was reduced to 8% of that in the parent cell line, and there was an altered form of this enzyme with a molecular weight of 160 kilodaltons (kDa), in addition to the normal 170 kDa protein. The cisplatin-selected cell line, H209/CP, was 11.5-fold resistant to cisplatin, with only a low level of cross-resistance to other platinum compounds including carboplatin, tetraplatin, iproplatin, and lobaplatin. This line was highly cross-resistant to vinca alkaloids, but not to anthracyclines or epipodophyllotoxins. The H209/CP cell line was not resistant to cadium chloride, suggesting that alterations in metallothionein are unlikely to be a cause of resistance. Although glutathione (GSH) levels were increased nearly 2-fold in H209/CP, there was no difference in levels of the GSH-related enzymes glutathione-S-transferase, glutathione peroxidase, and glutathione reductase, compared with the parent line. The H209/CP line had a 1.4-fold elevation of topoisomerase II-alpha. The accumulation of cisplatin was reduced in this cell line, and there were fewer DNA-interstrand cross links formed in the presence of cisplatin in H209/CP, compared with the parent line. Neither H209/VP nor H209/CP expressed MDR1, the gene for P-glycoprotein. The MRP gene was expressed at a slightly higher level in the H209/VP cell line, but there was no significant increase in expression of this gene in the H209/CP cell line. CONCLUSIONS: The resistance of the H209/VP cell line is associated with an alteration of topoisomerase II-alpha, whereas the resistance in the H209/CP line is associated with reduced drug accumulation.
Subject(s)
Antineoplastic Agents, Phytogenic/therapeutic use , Antineoplastic Agents/therapeutic use , Carcinoma, Small Cell/drug therapy , Cisplatin/therapeutic use , Etoposide/therapeutic use , Lung Neoplasms/drug therapy , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Antibiotics, Antineoplastic/pharmacology , Antibiotics, Antineoplastic/therapeutic use , Antineoplastic Agents/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Cadmium/pharmacology , Cadmium/therapeutic use , Cadmium Chloride , Carcinoma, Small Cell/enzymology , Carcinoma, Small Cell/physiopathology , Cell Line , Chlorides/pharmacology , Chlorides/therapeutic use , Cisplatin/pharmacology , Cross Reactions , DNA/drug effects , DNA Topoisomerases, Type II/analysis , DNA Topoisomerases, Type II/pharmacology , DNA Topoisomerases, Type II/therapeutic use , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/physiology , Etoposide/pharmacology , Gene Expression Regulation, Neoplastic , Glutathione/analysis , Glutathione Peroxidase/analysis , Glutathione Reductase/analysis , Glutathione Transferase/analysis , Humans , Lung Neoplasms/enzymology , Lung Neoplasms/physiopathology , Metallothionein/metabolism , Platinum Compounds/pharmacology , Platinum Compounds/therapeutic use , Podophyllotoxin/pharmacology , Podophyllotoxin/therapeutic use , Teniposide/pharmacology , Teniposide/therapeutic use , Tumor Cells, Cultured , Vinca Alkaloids/pharmacology , Vinca Alkaloids/therapeutic useABSTRACT
Comparison between five human leukemic lines (BV173, HL60, U937, K562, KCL22) suggest that the main determinant of their sensitivity to topoisomerase I (camptothecin) and II (VP-16) inhibitors is their ability to regulate cell cycle progression in response to specific DNA damage, then to die through apoptosis: the more the cells inhibit cell cycle progression, the less sensitive they are. The final pathway of apoptosis induction involves a cytoplasmic signal, active at neutral pH, needing magnesium, sensitive to various protease inhibitors and activated directly by staurosporine. Modulators of intracellular signaling (calcium chelators, calmodulin inhibitors, PKC modulators, kinase and phosphatase inhibitors) have no significant influence upon apoptosis induction. Conversely, apoptosis induction pathway is modified during monocytic differentiation of HL60 cells induced by phorbol esters. Lastly, poly(ADP-ribosyl)ation and chromatine structure should regulate apoptotic DNA fragmentation that is prevented by 3-aminobenzamide and spermine, respectively.