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Biomedical subjects

B W Day

Publications and source records attributed to B W Day.

At least 19 recordsLinked to original sources
PubMed2001

Identification of an inhibitor of hsc70-mediated protein translocation and ATP hydrolysis.

Members of the hsc70 family of molecular chaperones are critical players in the folding and quality control of cellular proteins. Because several human diseases arise from defects in protein folding, the activity of hsc70 chaperones is a potential therapeutic target for these disorders. By using a known hsc70 modulator, 15-deoxyspergualin, as a seed, we identified a novel inhibitor of hsc70 activity. This compound, R/1, inhibits the endogenous and DnaJ-stimulated ATPase activity of hsc70 by 48 and 51%, respectively, and blocks the hsc70-mediated translocation of a preprotein into yeast endoplasmic reticulum-derived microsomal vesicles. Biochemical studies demonstrate that R/1 most likely exerts these effects by altering the oligomeric state of hsc70.

Antibiotics, Antineoplastic↗
PubMed2001

A mathematical model of in vitro cancer cell growth and treatment with the antimitotic agent curacin A.

A mathematical model of cancer cell growth and response to treatment with the experimental antimitotic agent curacin A is presented. Rate parameters for the untreated growth of MCF-7/LY2 breast cancer and A2780 ovarian cell lines are determined from in vitro growth studies. Subsequent growth studies following treatments with 2.5, 25 and 50 nanomolar (nM), concentrations of curacin A are used to determine effects on the cell cycle and cell viability. The model's system of ordinary differential equations yields an approximate analytical solution which predicts the minimum concentration necessary to prevent growth. The model shows that cell growth is arrested when the apoptotic rate is greater than the mitotic rate and that the S-phase transition rate acts to amplify this effect. Analysis of the data suggests that curacin A is rapidly absorbed into both cell lines causing an increase in the S-phase transition and a decrease in the M-phase transition. The model also indicates that the rate of apoptosis remains virtually constant for MCF-7/LY2 while that of A2780 increases 38% at 2.5 nM and 59% at 50 nM as compared to the untreated apoptotic rate.

Antineoplastic Agents↗
PubMed2001

Synthesis and biological activity of a photoaffinity etoposide probe.

The epipodophyllotoxin etoposide is a potent and widely used anticancer drug that targets DNA topoisomerase II. The synthesis, photochemical, and biological testing of a photoactivatable aromatic azido analogue of etoposide also containing an iodo group is described. This azido analogue should prove useful for identifying the etoposide interaction site on topoisomerase II. Irradiation of the azido analogue and an aldehyde-containing azido precursor with UV light produced changes in their UV--visible spectra that were consistent with photoactivation. The azido analogue strongly inhibited topoisomerase II and inhibited the growth of Chinese Hamster Ovary cells. Azido analogue-induced topoisomerase II--DNA covalent complexes were significantly increased subsequent to UV irradiation of drug-treated human leukemia K562 cells as compared to etoposide-treated cells. These results suggest that the photoactivated form of etoposide is a more effective topoisomerase II poison either by interacting directly with the enzyme or with DNA subsequent to topoisomerase II-mediated strand cleavage.

Animals↗
PubMed2001

Structure-function analysis of D9N and N291S mutations in human lipoprotein lipase using molecular modelling.

Lipoprotein lipase (LPL) plays a central role in lipid metabolism. The D9N and N291S mutations in the LPL gene are associated with elevated triglyceride and decreased HDL-cholesterol levels. Published in vitro expression studies suggest that these two mutations are associated with reduced LPL enzymatic activity. We sought to gain further insight on the impact of these two mutations on the LPL structure and function by molecular modelling techniques. Homology modelling was used to develop a three-dimensional (3D) structure of LPL from human pancreatic lipase. Two separate LPL models for the D9N and N291S substitutions were constructed and compared with the wild type LPL for differences in hydrophobicity, atomic burial, hydrogen bond pattern, and atomic mobility. In comparison to the wild type model, the 9N model was associated with significantly increased atomic mobility of its neighboring residues, but the catalytic site was not affected. The region near residue 9 in the upper part of the N-domain was considered a candidate site for protein-protein interaction. In the N291S model, alterations in H-bonds and constrained atomic mobility were among conformational changes in the region where the substitution had occurred. These are hypothesized to cause an increase in the rate of dissociation in LPL dimerization, subsequently affecting the LPL enzymatic activity. We also modelled the C-domain of apoCII, the obligatory cofactor of LPL, from 2D NMR data and docked the model with LPL to explore their interaction site. These docking experiments suggest that the C-domain of apoCII interacts with the interface of N- and C-domains of LPL and part of the lid structure that covers the catalytic site. In summary, we provide molecular modelling data on two well-known mutations in the LPL gene to help explain the published in vitro expression findings and propose a possible LPL-apoCII interaction site. Our data indicate that molecular modelling of LPL mutations could provide a valuable tool to understand the effects of a mutation on the structure-function of this important enzyme.

Amino Acid Sequence↗
PubMed2001

Antimitotic actions of a novel analog of the fungal metabolite palmarumycin CP1.

The pentacyclic palmarumycins are structurally unique natural products with both antifungal and antibacterial activities but their antineoplastic effects are not well established. We have examined their antiproliferative actions against tumor cells using a temperature-sensitive tsFT210 mouse mammary carcinoma cell line and found that a novel palmarumycin analog, [8-(furan-3-ylmethoxy)-1-oxo-1,4-dihydronaphthalene-4-spiro-2'-naphtho[1",8"-de][1',3'][dioxin] or SR-7, prominently blocked mammalian cell cycle transition in G2/M but not in G1 phase. We found no evidence for inhibition of the critical mitosis-controlling cyclin-dependent kinase Cdk1, or its regulator, the dual specificity phosphatase Cdc25. Moreover, Cdk1 was hypophosphorylated and not directly inhibited by SR-7. SR-7 also failed in vitro to hypernucleate bovine tubulin, did not compete with colchicine for tubulin binding, and only modestly blocked GTP-induced assembly. In addition, SR-7 caused almost equal inhibition of paclitaxel-sensitive and -resistant cell growth. Moreover, unlike benchmark tubulin-disrupting agents, SR-7 did not cause hyperphosphorylation of the antiapoptotic protein Bcl-2. Thus, SR-7 represents a novel chemical structure that can inhibit G2/M transition by a mechanism that appears to be independent of marked tubulin disruption.

Animals↗
PubMed2000

Z-1,1-Dichloro-2,3-diphenylcyclopropanes block human prostate carcinoma cell proliferation, inhibit prostate-specific antigen expression, and initiate apoptosis.

BACKGROUND: Z-1,1-Dichloro-2,3-diphenylcyclopropane (A(II)) has long been known to be active against models of breast carcinoma. Microtubule perturbation and interaction at type II estrogen binding sites mediate its actions. METHODS: Since these targets are potentially useful for treatment of prostate tumors, we studied the drug's effects on androgen-sensitive (LNCaP) and -independent (PC-3) human prostatic carcinoma lines. Effects on cell growth and morphology, prostate-specific antigen (PSA) expression, and cell cycle kinetics were determined by microscopy, antibody-based methods, flow cytometry, and electrophoresis. RESULTS: At 100 microM, A(II) reduced survival of both lines by 50% in 12-24 hr, whereas 10 microM A(II) caused a prolonged block of proliferation in both lines, and parallel and complete block of PSA in LNCaP cells. At 10 microM, A(II) caused no major changes in chromatin, morphology or cell cycle distributions, whereas 100 microM drug caused rapid, large-scale cell detachment, nuclear and internucleosomal DNA fragmentation, and hypodiploidy. These effects were also accompanied by dissolution of cellular microtubule arrays. A more potent tubulin assembly-inhibiting congener of A(II), Z-1, 1-dichloro-2-(4-methoxy-phenyl)-3-phenylcyclopropane, slightly more effectively inhibited cell growth, caused little hypodiploidy, but potently and dose-dependently caused G(2)/M accumulation. CONCLUSIONS: These and previous data suggest that the Z-1, 1-dichloro-2,3-diarylcyclo-propanes may be useful in the treatment of human prostate disease.

Androgens↗
PubMed2000

Quantitation of benzo[a]pyrene-DNA adducts by postlabeling with 14C-acetic anhydride and accelerator mass spectrometry.

Quantitation of carcinogen-DNA adducts provides an estimate of the biologically effective dose of a chemical carcinogen reaching the target tissue. In order to improve exposure-assessment and cancer risk estimates, we are developing an ultrasensitive procedure for the detection of carcinogen-DNA adducts. The method is based upon postlabeling of carcinogen-DNA adducts by acetylation with 14C-acetic anhydride combined with quantitation of 14C by accelerator mass spectrometry (AMS). For this purpose, adducts of benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide (BPDE) with DNA and deoxyguanosine (dG) were synthesized. The most promutagenic adduct of BPDE, 7R,8S,9R-trihydroxy-10S-(N(2)-deoxyguanosyl)-7,8,9, 10-tetrahydrobenzo[a]pyrene (BPdG), was HPLC purified and structurally characterized. Postlabeling of the BPdG adduct with acetic anhydride yielded a major product with a greater than 60% yield. The postlabeled adduct was identified by liquid chromatography-mass spectrometry as pentakis(acetyl) BPdG (AcBPdG). Postlabeling of the BPdG adduct with 14C-acetic anhydride yielded a major product coeluting with an AcBPdG standard. Quantitation of the 14C-postlabeled adduct by AMS promises to allow detection of attomolar amounts of adducts. The method is now being optimized and validated for use in human samples.

Acetic Anhydrides↗
PubMed2000

Activation of the nitric oxide synthase 2 pathway in the response of bone marrow stromal cells to high doses of ionizing radiation.

Reverse transcription-polymerase chain reaction and immunofluorescence analysis of D2XRII murine bone marrow stromal cells showed that gamma irradiation with doses of 2-50 Gy from (137)Cs stimulated expression of nitric oxide synthase 2 (Nos2, also known as iNos). The activation of Nos2 was accompanied by an increase in the fluorescence of 4,5-diaminofluorescein diacetate, a nitric oxide trap, and accumulation of 3-nitrotyrosine within cellular proteins in a dose-dependent manner. These effects were inhibited by actinomycin D and by N-[3-(aminomethyl)benzyl]acetamidine dihydrochloride, a specific inhibitor of Nos2. The induction of Nos2 expression and Nos2-dependent release of nitric oxide in D2XRII cells was observed within 24 h after irradiation and was similar in magnitude to that observed in cultures incubated with Il1b and Tnf. We conducted (1) confocal fluorescence imaging of 3-nitrotyrosine in bone marrow cells of irradiated C57BL/6J mice and (2) 3-nitrotyrosine fluorescence imaging of FDC-P1JL26 hematopoietic cells that were cocultured with previously irradiated D2XRII bone marrow stromal cells. Exposure to ionizing radiation increased the production of 3-nitrotyrosine in irradiated bone marrow cells in vivo and in nonirradiated FDC-P1JL26 cells cocultured with irradiated D2XRII cells for 1 or 4 h. We suggest that nitrative/oxidative stress to the transplanted multilineage hematopoietic cells due to exposure to nitric oxide released by host bone marrow stromal cells may contribute to the genotoxic events associated with malignant alterations in bone marrow tissue of transplant recipients who are prepared for engraftment by total-body irradiation.

Animals↗
PubMed1999

Induction of human breast cancer cell apoptosis from G2/M preceded by stimulation into the cell cycle by Z-1,1-dichloro-2,3-diphenylcyclopropane.

We have shown previously that Z-1,1-dichloro-2,3-diphenylcyclopropane (a.k.a. Analog II, A(II)) inhibits human breast cancer cell proliferation regardless of estrogen receptor status or estrogen sensitivity, and that its cellular targets include microtubules. In the present study, we investigated the apoptosis-inducing effects of A(II). MCF-7, MCF-7/LY2, and MDA-MB-231 cells all showed nuclear fragmentation in response to 100 microM A(II) when stained with Hoechst 33342 and examined by fluorescence microscopy. Pulsed field gel electrophoretic analysis showed that each of the cell lines also developed specific high molecular weight DNA fragments: a low level of 1-2 Mb fragments appeared after 6 hr, while 30-50 kb fragments accumulated subsequently. At 24 hr of drug exposure, the majority of cells became nonadherent, and the 30-50 kb fragments were restricted to detached MCF-7 and MDA-MB-231 cells. Both adherent and detached MCF-7/LY2 cells exhibited these fragments. A previous study by single-color (propidium) flow cytometry demonstrated that A(II) blocks MDA-MB-231 cells in G2/M of the cell cycle. More refined analyses in the present study showed this same result for MDA-MB-231 cells, but MCF-7 and MCF-7/LY2 cells did not reveal apparent drug-induced cell cycle block. A(II) demonstrated growth inhibitory, cell cycle-perturbing, and hypodiploidy-inducing activity against other human breast carcinoma lines, i.e. BT-20, CAMA-1, and SKBR-3, but no such actions in the non-tumorigenic, "normal" human breast epithelial line MCF-10A. Bromodeoxyuridine labeling and two-color flow cytometric analysis, however, suggested that A(II) caused stimulation into S phase, and that G2/M was the phase of the cell cycle from which cells apoptosed. A(II) caused cell rounding, detachment from the growth matrix, and nuclear shrinkage and fragmentation in parallel with biochemical changes. Cycloheximide inhibited A(II)-induced cell death, indicating that its toxicity requires de novo protein synthesis.

Antineoplastic Agents↗
PubMed1999

Peroxidase-catalyzed pro- versus antioxidant effects of 4-hydroxytamoxifen: enzyme specificity and biochemical sequelae.

Some studies have shown the potential relevance of the oxidation products of 4-hydroxytamoxifen (4OHTAM) in carcinogenesis. Other studies show 4OHTAM has antioxidant properties. We characterized the one-electron oxidative activation reactions of 4OHTAM and three other phenolics, 3-hydroxytamoxifen (3OHTAM), 1-(4-hydroxyphenyl)-1, 2-diphenylethene, and phenol (PhOH), catalyzed by myeloperoxidase (MPx), horseradish peroxidase (HRP), lactoperoxidase, mushroom tyrosinase, and nonenzymatic initiators in vitro under a variety of conditions and in cells. Differences in activation of the phenolics by the enzymes were directly compared using cis-parinaric acid (PnA)-loaded human serum albumin. All phenolics were substrates for the enzymes, but MPx only weakly activated 4OHTAM to its phenoxyl radical. In HL60 cells loaded metabolically with PnA so that effects on phospholipids could be monitored by HPLC with fluorescence detection, PhOH plus H2O2 caused massive oxidation across all phospholipid classes. 4OHTAM dose-dependently protected phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine against both H2O2-induced and normal metabolic oxidation. This suggested 4OHTAM is a poor substrate for intracellular MPx. In rat aorta smooth muscle cells loaded with PnA, 4OHTAM also protected against AMVN-induced peroxidation of those three phospholipids and sphingomyelin, whereas 3OHTAM did not. Spin trapping of glutathionyl radicals (GS*) with DMPO and quantifying the ESR-silent nitrone form of the GS-DMPO adduct by HPLC showed that neither 3OHTAM plus H2O2 nor 4OHTAM plus H2O2 caused a significant level of GSH oxidation with isolated MPx, nor did the latter in HL60 cells, whereas PhOH plus H2O2 was a potent source of GS* in both systems. Both 4OHTAM and 3OHTAM formed the nitrone adduct under cell-free conditions when activated with HRP. The data show that the substrate specificity of a given (myelo)peroxidase determines if a phenolic exerts pro- (through generation of reactive phenoxyl radicals) or antioxidant (through radical scavenging) properties in intracellular environments.

Animals↗
PubMed1999

Intracellular S-glutathionyl adducts in murine lung and human bronchoepithelial cells after exposure to diisocyanatotoluene.

Diisocyanatotoluene (toluene diisocyanate, TDI), a 4:1 mixture of 2, 4- and 2,6-isomers used in the preparation of polyurethanes, causes occupational asthma by an as yet unknown mechanism. We previously showed that it forms adducts with the apical surface of the bronchoepithelium in vivo, and with ciliary microtubules in cultured human bronchoepithelial (HBE) cells. These results suggested that TDI may not enter HBE cells. In vitro studies, however, showed that TDI avidly forms bis adducts with glutathione (GSH) and that these adducts transfer monoisocyanato-monoglutathionyl-TDI to a sulfhydryl-containing peptide. This study sought to elucidate intracellular reactions of TDI. Using an electron paramagnetic resonance spectrometric (EPR) method, we established that the level of thiol-dependent quenching of phenoxyl radicals of etoposide was decreased >40% in pulmonary tissue of mice that received TDI intrabronchially. Similarly, HBE cells exposed to 100 ppb TDI vapor experienced a >30% reduction in thiol levels as determined with a thiol-specific fluorescent probe (ThioGlo 1). HPLC/UV analysis of lysates from HBE cells exposed to 200 and 500 ppb TDI vapor suggested a dose-related formation of S-glutathionyl adducts. Data from the 500 ppb TDI-treated HBE cells verified the identity of the 2-monoglutathionyl-4-monoisocyanato adduct. The results provide firm evidence that TDI enters pulmonary cells and reacts with GSH. This rapid reaction leading to formation of S-glutathionyl adducts of TDI suggests the importance of cellular thiols in TDI-induced pulmonary disease.

Animals↗
PubMed1998

Glutamate-induced cytotoxicity in PC12 pheochromocytoma cells: role of oxidation of phospholipids, glutathione and protein sulfhydryls revealed by bcl-2 transfection.

Incubation of mock-transfected PC12 rat pheochromocytoma cells (PC12) for 2 h with increasing concentrations of glutamate caused progressive loss of viability (e.g., 67% with 15 mM glutamate). In contrast, the viability of bcl-2-transfected cells (PC12/bcl-2) was unaffected by glutamate. Neither PC12 nor PC12/bcl-2 cells showed a significant incidence of apoptosis in response to glutamate. Conventional phospholipid analysis by high-performance TLC and phosphorous determination showed no significant changes in the phospholipid composition of either cell line incubated with </=15 mM glutamate. Phospholipid peroxidation was quantified in the cells using our newly developed method based on fluorescence-HPLC analysis of metabolically incorporated oxidation-sensitive and fluorescent fatty acid, cis-parinaric acid. Unlike previous studies that measured total phospholipid oxidation, this novel technology permitted quantitation of oxidative stress in different classes of labeled phospholipids (the amount of labeled phospholipids in the cells did not exceed 1% of total phospholipids). Significant peroxidation of phosphatidylcholine and phosphatidylethanolamine occurred in PC12 cells treated with >5 mM glutamate. The peroxyl radical initiator 2,2'-azobis(2,4-dimethylvaleronitrile) caused a pronounced loss of all major phospholipid classes in PC12 cells, but no loss of cell viability. No phospholipid peroxidation was detected in PC12/bcl-2 cells incubated with </=15 mM glutamate or with 2, 2'-azobis(2,4-dimethylvaleronitrile). These results directly demonstrate that peroxidation of membrane phospholipids is not responsible for the cytotoxicity of glutamate in PC12 cells. Total cellular thiol, protein thiol and GSH reserves were quantified by a previously described electron paramagnetic resonance spectrometric method. Total thiols were ca. 1.5-fold greater in PC12/bcl-2 than in PC12 cells. Glutamate (</=5 mM) caused a progressive and equally significant decrease in total thiols and GSH in both PC12 and PC12/bcl-2 cells. High glutamate concentrations caused oxidation of protein sulfhydryls in PC12 cells, but not in PC12/bcl-2 cells. The results suggest that the changes in cellular milieu caused by bcl-2 gene transfection protect PC12 cells from the toxic effects of glutamate in a manner consistent with prevention of protein sulfhydryl oxidation.

Adrenal Gland Neoplasms↗
PubMed1998

Nitric oxide protects cardiomyocytes against tert-butyl hydroperoxide-induced formation of alkoxyl and peroxyl radicals and peroxidation of phosphatidylserine.

We studied protective effects of nitric oxide against tert-butyl hydroperoxide-induced oxidative damage to cardiac myocytes. Two distinct free radicals species--alkoxyl radicals associated with non-heme iron catalytic sites and myoglobin protein-centered peroxyl radicals--were found in low-temperature EPR spectra of cardiac myocytes exposed to t-BuOOH. The t-BuOOH-induced radical formation was accompanied by site-specific oxidative stress in membrane phospholipids (peroxidation of phosphatidylserine) assayed by fluorescence HPLC after metabolic labeling of cell phospholipids with oxidation-sensitive cis-parinaric acid. An NO-donor, (Z)-1-[N-(3-ammonio-propyl)-N-(n-propyl) amino]-diazen-1-ium-1,2-diolate], protected cardiac myocytes against tert-butyl hydroperoxide-induced: (i) formation of non-protein- and protein-centered free radical species and (ii) concomitant peroxidation of phosphatidylserine. Thus nitric oxide can act as an effective antioxidant in live cardiomyocytes.

Alcohols↗
PubMed1998

Prospective association between lipid soluble antioxidants and coronary heart disease in men. The Multiple Risk Factor Intervention Trial.

A nested case-control study was performed using participants enrolled in the Multiple Risk Factor Intervention Trial (MRFIT). The cases involved nonfatal myocardial infarction or death from coronary heart disease. Serum samples (n = 734) obtained at baseline and frozen for approximately 20 years were analyzed for the antioxidants, carotenoids, retinol, and alpha-, gamma-, and total tocopherol. The concentrations of antioxidants were in the expected range and their association with low density lipoprotein (LDL) cholesterol reflected their absorption and transport mechanisms. Among nonsmokers, the odds ratios (95% confidence intervals (CI)) for quartile IV versus quartile I were 1.40 (0.40-4.89), 0.77 (0.20-2.96), 1.45 (0.38-5.56), 2.34 (0.56-9.81), and 2.40 (0.52-11.07) for retinol, total carotenoids, and alpha-, gamma-, and total tocopherol, respectively. The equivalent odds ratios (95% CI) for smokers were 0.90 (0.34-2.41), 0.66 (0.23-1.84), 0.67 (0.21-2.13), 2.04 (0.88-4.73), and 0.52 (0.16-1.67), respectively. This analysis of antioxidant concentrations by quartiles indicated no significant association of antioxidant levels with the risk of coronary disease death or nonfatal myocardial infarction.

Adult↗
PubMed1998

Effects of bulky polycyclic aromatic hydrocarbon adducts on DNA replication by exonuclease-deficient T7 and T4 DNA polymerases.

In vitro DNA replication by exonuclease-deficient T7 DNA polymerase (Sequenase) and an exonuclease deficient T4 DNA polymerase was examined on a 244-nucleotide DNA template treated with three electrophilic polycyclic aromatic hydrocarbon (PAH) metabolites: racemic trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaPDE), trans-2,3-dihydroxy-anti-1,10b-epoxy-10b,1,2,3-tetrahydrofluoranthene (FADE), or 3,4-epoxy-3,4-dihydrocyclopenta[cd]pyrene (CPPE). The DNA replication terminated opposite template guanines and, to a lesser extent, at template adenines, as expected, as purines were modified preferentially by the chemical treatments. Analysis of the products synthesized on the damaged templates indicated that bypass replication by Sequenase proceeded in three steps: (1) replication first terminated one base 3' to each adduct; (2) a nucleotide was then incorporated opposite the PAH-modified base; and (3) replication continued at some sites to give full bypass of the lesions. The rate of lesion bypass was affected by the type of chemical adduct, the sequence context of the adduct, and the concentration of deoxynucleoside triphosphates. Short DNA repeats appeared to facilitate translesion replication.

Bacteriophage T4↗
PubMed1998

The potent microtubule-stabilizing agent (+)-discodermolide induces apoptosis in human breast carcinoma cells--preliminary comparisons to paclitaxel.

(+)-Discodermolide, a sponge-derived natural product, stabilizes microtubules more potently than paclitaxel despite the lack of any obvious structural similarities between the drugs. It competitively inhibits the binding of paclitaxel to tubulin polymers, hypernucleates microtubule assembly more potently than paclitaxel, and inhibits the growth of paclitaxel-resistant ovarian and colon carcinoma cells. Because paclitaxel shows clinical promise for breast cancer treatment, its effects in a series of human breast cancer cells were compared to those of (+)-discodermolide. Growth inhibition, cell and nuclear morphological, and electrophoretic and flow cytometric analyses were performed on (+)-discodermolide-treated MCF-7 and MDA-MB231 cells. (+)-Discodermolide potently inhibited the growth of both cell types (IC50 < 2.5 nM) at concentrations similar to those observed with paclitaxel. Complete inhibition of growth occurred with 10 nM or greater of each drug and was not reversed by removal. (+)-Discodermolide-treated cells exhibited condensed and highly fragmented nuclei. Flow cytometric comparison of cells treated with either drug at 10 nM, a concentration well below that achieved clinically with paclitaxel, showed both caused cell cycle perturbation and induction of a hypodiploid cell population. (+)-Discodermolide caused these effects more extensively and at earlier time points. The timing and type of high molecular weight DNA fragmentation induced by the two agents was consistent with induction of apoptosis. The results suggest that (+)-discodermolide has promise as a new chemotherapeutic agent against breast and other cancers.

Alkanes↗
PubMed1998

Structure-activity analysis of the interaction of curacin A, the potent colchicine site antimitotic agent, with tubulin and effects of analogs on the growth of MCF-7 breast cancer cells.

Originally purified as a major lipid component of a strain of the cyanobacterium Lyngbya majuscula isolated in Curaçao, curacin A is a potent inhibitor of cell growth and mitosis, binding rapidly and tightly at the colchicine site of tubulin. Because its molecular structure differs so greatly from that of colchicine and other colchicine site inhibitors, we prepared a series of curacin A analogs to determine the important structural features of the molecule. These modifications include reduction and E-to-Z transitions of the olefinic bonds in the 14-carbon side chain of the molecule; disruption of and configurational changes in the cyclopropyl moiety; disruption, oxidation, and configurational reversal in the thiazoline moiety; configurational reversal and substituent modifications at C13; and demethylation at C10. Inhibitory effects on tubulin assembly, the binding of colchicine to tubulin, and the growth of MCF-7 human breast carcinoma cells were examined. The most important portions of curacin A required for its interaction with tubulin seem to be the thiazoline ring and the side chain at least through C4, the portion of the side chain including the C9-C10 olefinic bond, and the C10 methyl group. Only two modifications totally eliminated the tubulin-drug interaction. The inactive compounds were a segment containing most of the side chain, including its two substituents, and analogs in which the methyl group at the C13 oxygen atom was replaced by a benzoate residue. Antiproliferative activity comparable with that observed with curacin A was only reproduced in compounds that were potent inhibitors of the binding of colchicine to tubulin. Molecular modeling and quantitative structure-activity relationship studies demonstrated that most active analogs overlapped extensively with curacin A but failed to provide an explanation for the apparent structural analogy between curacin A and colchicine.

Animals↗