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Biomedical subjects

E A Sausville

Publications and source records attributed to E A Sausville.

At least 19 recordsLinked to original sources
PubMed2006

Adaphostin and other anticancer drugs quench the fluorescence of mitochondrial potential probes.

Fluorescent dyes are widely used to monitor changes in mitochondrial transmembrane potential (DeltaPsim). When MitoTracker Red CMXRos, tetramethylrhodamine methyl ester (TMRM), and 3,3'dihexyloxacarbocyanine iodide (DiOC6(3)) were utilized to examine the effects of the experimental anticancer drug adaphostin on intact cells or isolated mitochondria, decreased fluorescence was observed. In contrast, measurement of tetraphenylphosphonium uptake by the mitochondria using an ion-selective microelectrode failed to show any effect of adaphostin on DeltaPsim. Instead, further experiments demonstrated that adaphostin quenches the fluorescence of the mitochondrial dyes. Structure-activity analysis revealed that the adamantyl and p-aminobenzoic acid moieties of adaphostin are critical for this quenching. Anticancer drugs containing comparable structural motifs, including mitoxantrone, aminoflavone, and amsacrine, also quenched the mitochondrial probes. These results indicate the need for caution when mitochondrial dyes are utilized to examine the effects of xenobiotics on DeltaPsim and suggest that some previously reported direct effects of anticancer drugs on mitochondria might need re-evaluation.

Adamantane↗
PubMed2004

The hollow fibre model in cancer drug screening: the NCI experience.

The in vivo hollow fibre model was developed by the National Cancer Institute (NCI) in the United States of America (USA) at a time when the number of potential anti-cancer drugs arising from in vitro screening efforts exceeded the available capacity for testing in traditional xenograft models. Updated analysis of the predictive value of the hollow fibre model continues to indicate that the greater the response in the hollow fibre assay, the more likely it is that activity will be seen in subsequent xenograft models. The original 12 cell line hollow fibre panel has been supplemented with histology-specific panels, and we begin here to analyse their utility in predicting activity in subsequent in vivo models. The key goal of using the hollow fibre model as a way to decrease the cost, both financial and in the number of animals used, to evaluate initial evidence of a compound's capacity to act across physiological barriers continues to be reinforced with our enlarging experience.

Animals↗
PubMed2004

A potential role for imaging technology in anticancer efficacy evaluations.

The introduction of imaging methods suitable for rodents offers opportunities for new anticancer efficacy models. Traditional models do not provide the level of sensitivity afforded by these precise and quantitative techniques. Bioluminescent endpoints, now feasible because of sensitive charge-coupled device cameras, can be non-invasively detected in live animals. Currently, the most common luminescence endpoint is firefly luciferase, which, in the presence of O(2) and ATP, catalyses the cleavage of the substrate luciferin and results in the emission of a photon of light. In vivo implantation of tumour cells transfected with the luciferase gene allows sequential monitoring of tumour growth within the viscera by measuring these photon signals. Furthermore, tumour cell lines containing the luciferase gene transcribed from an inducible promoter offer opportunities to study molecular-target modulation without the need for ex vivo evaluations of serial tumour samples. In conjunction with this, transgenic mice bearing a luciferase reporter mechanism can be used to monitor the tumour microenvironment as well as to signal when transforming events occur. This technology has the potential to reshape the efficacy evaluations and drug-testing algorithms of the future.

Animals↗
PubMed2004

Sensitivity of renal cell carcinoma to aminoflavone: role of CYP1A1.

PURPOSE: The aminoflavone analogue (AF) exhibits antitumor activity in vitro, particularly against neoplastic cells of renal origin. We identified cellular correlates of responsiveness to AF in continuous human tumor renal cell carcinoma lines and in tumor cell isolates, termed renal carcinoma cell strains, from patients with clear cell and papillary renal neoplasms. MATERIALS AND METHODS: In vitro antiproliferative activity of AF was evaluated using the sulforhodamine B protein dye assay. In vivo antitumor activity of the drug was determined in mice bearing xenografts. Covalent binding of AF/metabolite(s) was assessed following exposure of cells to AF for 16 hours. CYP1A1 and CYP1B1 mRNA and apoptosis were quantitated by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: AF produced total growth inhibition in vitro in 3 of 6 human tumor renal cell lines at concentrations of 90 to 400 nM. In vivo treatment of mice bearing xenografts of the Caki-1 renal cell carcinoma, sensitive to AF in vitro, resulted in significant antitumor activity, including tumor-free survivors. Studies in 13 renal cell strains isolated from patients with clear cell (9) or papillary (4) renal cell carcinoma indicated that 3 of 4 papillary strains were sensitive to AF compared with 2 of 9 clear cell strains. AF sensitive renal cell lines and strains exhibited induction of CYP1A1 and CYP1B1 gene expression, increased covalent binding of AF metabolite(s) and apoptosis. CONCLUSIONS: AF has noteworthy antitumor activity against certain human tumor renal cell lines in vitro and in vivo, which correlates with drug metabolism to covalently binding metabolites after CYP1A1 and CYP1B1 gene expression. We hypothesize that it leads to apoptosis induction. AF sensitive renal cell strains are predominantly of the papillary histological type. These results are limited by the small numbers of cell lines and cell strains but they are suggestive of the need for further testing in larger collections of cell strains.

Animals↗
PubMed2004

Target selection issues in drug discovery and development.

Targets for cancer treatments have been proposed which address oncogenes mutated in tumors (pathogenic targets). Yet additional opportunities may come from molecules expressed in the tissue of origin (ontogenic); allow drug handling (pharmacological) or address the tumor microenvironment. The key is to define an assay that defines a therapeutic index which can be achieved in modulated target function.

Animals↗
PubMed2004

Geldanamycin analogs.

Benzoquinoid ansamycins can downregulate molecules which are chaperoned by heat shock protein (Hsp90), including numerous tyrosine kinases, steroid receptors, and cell cycle regulatory kinases. 17allylaminol7demethoxygeldanamycin (17AAG) has entered the clinic, but 17dimethyl-aminoethylaminogeldanamycin (17DMA) has emerged as an analog with potential preferential pharmaceutical features.

Antineoplastic Agents↗
PubMed2003

DNA damage and cell cycle arrest induced by 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is attenuated in aryl hydrocarbon receptor deficient MCF-7 cells.

The fluorinated benzothiazole analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is a novel agent with potent and selective antitumour properties and, in the form of its L-lysylamide prodrug Phortress (NSC 710305), is a current candidate for early phase clinical studies. Previous findings have indicated that cytochrome P450 1A1 (CYP1A1) may play a role in the antitumour activity of molecules in the benzothiazole series including the nonfluorinated parent compound 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495) (Kashiyama et al, 1999; Chua et al, 2000; Loaiza-Pérez et al, 2002). In this study, we assessed and verified that a fully functional aryl hydrocarbon receptor (AhR) signalling pathway is a necessary requisite for the induction of efficient cytotoxicity by 5F 203 in MCF-7 wild-type sensitive cells. Drug exposure caused MCF-7 sensitive cells to arrest in G(1) and S phase, and induced DNA adduct formation, in contrast to AhR-deficient AH(R100) variant MCF-7 cells. In sensitive MCF-7 cells, induction of CYP1A1 and CYP1B1 transcription (measured by luciferase reporter assay and real-time reverse transcriptase-polymerase chain reaction (RT-PCR)), and 7-ethoxyresorufin-O-deethylase (EROD) activity was demonstrated, following treatment with 5F 203. In contrast, in resistant AH(R100) cells, drug treatment did not affect CYP1A1 and CYP1B1 transcription and EROD activity. Furthermore, AH(R100) cells failed to produce either protein/DNA complexes on the xenobiotic responsive element (XRE) sequence of CYP1A1 promoter (measured by electrophoretic mobility shift assay) or DNA adducts. The data confirm that activation of the AhR signalling pathway is an important feature of the antitumour activity of 5F 203.

Aryl Hydrocarbon Hydroxylases↗
PubMed2002

In vitro evaluation of amino acid prodrugs of novel antitumour 2-(4-amino-3-methylphenyl)benzothiazoles.

Novel 2-(4-aminophenyl)benzothiazoles possess highly selective, potent antitumour properties in vitro and in vivo. They induce and are biotransformed by cytochrome P450 (CYP) 1A1 to putative active as well as inactive metabolites. Metabolic inactivation of the molecule has been thwarted by isosteric replacement of hydrogen with fluorine atoms at positions around the benzothiazole nucleus. The lipophilicity of these compounds presents limitations for drug formulation and bioavailability. To overcome this problem, water soluble prodrugs have been synthesised by conjugation of alanyl- and lysyl-amide hydrochloride salts to the exocyclic primary amine function of 2-(4-aminophenyl)benzothiazoles. The prodrugs retain selectivity with significant in vitro growth inhibitory potency against the same sensitive cell lines as their parent amine, but are inactive against cell lines inherently resistant to 2-(4-aminophenyl)benzothiazoles. Alanyl and lysyl prodrugs rapidly and quantitatively revert to their parent amine in sensitive and insensitive cell lines in vitro. Liberated parent compounds are sequestered and metabolised by sensitive cells only; similarly, CYP1A1 activity and protein expression are selectively induced in sensitive carcinoma cells. Amino acid prodrugs meet the criteria of aqueous solubility, chemical stability and quantitative reversion to parent molecule, and thus are suitable for in vivo preclinical evaluation.

Amines↗
PubMed2002

Developmental therapeutics program at the NCI: molecular target and drug discovery process.

As the drug discovery and developmental arm of the National Cancer Institute (NCI), the Developmental Therapeutics Program (DTP) plans, conducts and facilitates development of therapeutic agents for cancer and AIDS. DTP's goal is to turn 'molecules into medicine for the public health'. Areas of support by DTP are discovery, development and pathways to development for the intramural and the extramural community. The Developmental Therapeutics Program (DTP) operates a repository of synthetic and pure natural products, which are evaluated as potential anticancer agents. The repository derives from a historical database of greater than 600 000 compounds, which have been supplied to DTP from a variety of sources worldwide. The in vitro anti-cancer drug cell line screen established at DTP is unique in several respects. It has changed the NCI emphasis from a compound-oriented drug discovery effort to a disease-panel oriented exercise, emphasized human tumor cells derived from solid tumors, developed a high volume screening method that can adapt to processing of numerous chemical agents or natural source-derived extracts, that has minimized the use of animals, and saved on the amount of material required for the initial screening. The hollow fiber assay created at the DTP has demonstrated the ability to provide quantitative initial indices of in vivo drug efficacy, with minimum expenditures of time and materials and is currently being utilized as the initial in vivo experience for agents found to have reproducible activity in the in vitroanticancer drug screen. Drugs showing activity with unique mechanisms of actions are being further developed for treatment of hematopoietic neoplasms, prominent examples being flavopiridol, UCN-01 and depsipeptide among others.

Animals↗
PubMed2001

Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol.

BACKGROUND: Flavopiridol, a flavonoid currently in cancer clinical trials, inhibits cyclin-dependent kinases (CDKs) by competitively blocking their ATP-binding pocket. However, the mechanism of action of flavopiridol as an anti-cancer agent has not been fully elucidated. RESULTS: Using DNA microarrays, we found that flavopiridol inhibited gene expression broadly, in contrast to two other CDK inhibitors, roscovitine and 9-nitropaullone. The gene expression profile of flavopiridol closely resembled the profiles of two transcription inhibitors, actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), suggesting that flavopiridol inhibits transcription globally. We were therefore able to use flavopiridol to measure mRNA turnover rates comprehensively and we found that different functional classes of genes had distinct distributions of mRNA turnover rates. In particular, genes encoding apoptosis regulators frequently had very short half-lives, as did several genes encoding key cell-cycle regulators. Strikingly, genes that were transcriptionally inducible were disproportionately represented in the class of genes with rapid mRNA turnover. CONCLUSIONS: The present genomic-scale measurement of mRNA turnover uncovered a regulatory logic that links gene function with mRNA half-life. The observation that transcriptionally inducible genes often have short mRNA half-lives demonstrates that cells have a coordinated strategy to rapidly modulate the mRNA levels of these genes. In addition, the present results suggest that flavopiridol may be more effective against types of cancer that are highly dependent on genes with unstable mRNAs.

Antineoplastic Agents↗
PubMed2001

Quantitative determination of perifosine, a novel alkylphosphocholine anticancer agent, in human plasma by reversed-phase liquid chromatography-electrospray mass spectrometry.

A sensitive and selective reversed-phase LC-ESI-MS method to quantitate perifosine in human plasma was developed and validated. Sample preparation utilized simple acetonitrile precipitation without an evaporation step. With a Develosil UG-30 column (10 x 4 mm I.D.), perifosine and the internal standard hexadecylphosphocholine were baseline separated at retention times of 2.2 and 1.1 min, respectively. The mobile phase consisted of eluent A, 95% 9 mM ammonium formate (pH 8) in acetonitrile-eluent B, 95% acetonitrile in 9 mM ammonium formate (pH 8) (A-B, 40:60, v/v), and the flow-rate was 0.5 ml/min. The detection utilized selected ion monitoring in the positive-mode at m/z 462.4 and 408.4 for the protonated molecular ions of perifosine and the internal standard, respectively. The lower limit of quantitation of perifosine was 4 ng/ml in human plasma, and good linearity was observed in the 4-2,000 ng/ml range fitted by linear regression with 1/x weight. The total LC-MS run time was 5 min. The validated LC-MS assay was applied to measure perifosine plasma concentrations from patients enrolled on a phase I clinical trial for pharmacokinetic/pharmacodynamic analyses.

Antineoplastic Agents↗
PubMed2001

Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials.

An analysis of the activity of compounds tested in pre-clinical in vivo and in vitro assays by the National Cancer Institute's Developmental Therapeutics Program was performed. For 39 agents with both xenograft data and Phase II clinical trials results available, in vivo activity in a particular histology in a tumour model did not closely correlate with activity in the same human cancer histology, casting doubt on the correspondence of the pre-clinical models to clinical results. However, for compounds with in vivo activity in at least one-third of tested xenograft models, there was correlation with ultimate activity in at least some Phase II trials. Thus, an efficient means of predicting activity in vivo models remains desirable for compounds with anti-proliferative activity in vitro. For 564 compounds tested in the hollow fibre assay which were also tested against in vivo tumour models, the likelihood of finding xenograft activity in at least one-third of the in vivo models tested rose with increasing intraperitoneal hollow fibre activity, from 8% for all compounds tested to 20% in agents with evidence of response in more than 6 intraperitoneal fibres (P< 0.0001). Intraperitoneal hollow fibre activity was also found to be a better predictor of xenograft activity than either subcutaneous hollow fibre activity or intraperitoneal plus subcutaneous activity combined. Since hollow fibre activity was a useful indicator of potential in vivo response, correlates with hollow fibre activity were examined for 2304 compounds tested in both the NCI 60 cell line in vitro cancer drug screen and hollow fibre assay. A positive correlation was found for histologic selectivity between in vitro and hollow fibre responses. The most striking correlation was between potency in the 60 cell line screen and hollow fibre activity; 56% of compounds with mean 50% growth inhibition below 10(-7.5) M were active in more than 6 intraperitoneal fibres whereas only 4% of compounds with a potency of 10(-4) M achieved the same level of hollow fibre activity (P< 0.0001). Structural parameters of the drugs analysed included compound molecular weight and hydrogen-bonding factors, both of which were found to be predictive of hollow fibre activity.

Animals↗
PubMed2001

Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms.

PURPOSE: To define the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of the novel protein kinase inhibitor, UCN-01 (7-hydroxystaurosporine), administered as a 72-hour continuous intravenous infusion (CIV). PATIENTS AND METHODS: Forty-seven patients with refractory neoplasms received UCN-01 during this phase I trial. Total, free plasma, and salivary concentrations were determined; the latter were used to address the influence of plasma protein binding on peripheral tissue distribution. The phosphorylation state of the protein kinase C (PKC) substrate alpha-adducin and the abrogation of DNA damage checkpoint also were assessed. RESULTS: The recommended phase II dose of UCN-01 as a 72-hour CIV is 42.5 mg/m(2)/d for 3 days. Avid plasma protein binding of UCN-01, as measured during the trial, dictated a change in dose escalation and administration schedules. Therefore, nine patients received drug on the initial 2-week schedule, and 38 received drug on the recommended 4-week schedule. DLTs at 53 mg/m(2)/d for 3 days included hyperglycemia with resultant metabolic acidosis, pulmonary dysfunction, nausea, vomiting, and hypotension. Pharmacokinetic determinations at the recommended dose of 42.5 mg/m(2)/d for 3 days included mean total plasma concentration of 36.4 microM (terminal elimination half-life range, 447 to 1176 hours), steady-state volume of distribution of 9.3 to 14.2 L, and clearances of 0.005 to 0.033 L/h. The mean total salivary concentration was 111 nmol/L of UCN-01. One partial response was observed in a patient with melanoma, and one protracted period ( > 2.5 years) of disease stability was observed in a patient with alk-positive anaplastic large-cell lymphoma. Preliminary evidence suggests UCN-01 modulation of both PKC substrate phosphorylation and the DNA damage-related G(2) checkpoint. CONCLUSION: UCN-01 can be administered safely as an initial 72-hour CIV with subsequent monthly doses administered as 36-hour infusions.

Adult↗
PubMed2001

MS-275, a histone deacetylase inhibitor, selectively induces transforming growth factor beta type II receptor expression in human breast cancer cells.

Transcriptional repression of the transforming growth factor (TGF)-1P type II receptor (TPRII) gene appears to be a major mechanism to inactivate TGF-beta responsiveness in many human cancers. Because histone acetylation/deacetylation plays a role in transcriptional regulation, we have examined the effect of MS-275, a synthetic inhibitor of histone deacetylase, in human breast cancer cell lines. MS-275 showed antiproliferative activity against all human breast cancer cell lines examined and induced TbetaRII mRNA, but not TGF-beta type I receptor mRNA. MS-275 caused an accumulation of acetylated histones H3 and H4 in total cellular chromatin. An increase in the accumulation of acetylated histones H3 and H4 was detected in the TbetaRII promoter after treatment with MS-275. However, the level of histone acetylation did not change in chromatin associated with the TGF-beta type I receptor gene. MS-275 treatment enhanced TGF-beta1-induced plasminogen activator inhibitor 1 expression. Thus, antitumor activity of MS-275 may be mediated in part through the induction of TbetaRII expression and consequent potentiation of TGF-beta signaling.

Acetylation↗
PubMed2001

The challenge of pathway and environment-mediated drug resistance.

New insights into cancer biology have allowed the definition of pathways critical to the success of the cancer cell. These include enhanced activity of positive regulators of cell proliferation, including oncogenes, and loss of tumor suppressor gene function; inactivation of cell death or enhancement of srvival functions; activation of telomerase, and enhanced ability of tumor cells to invade and reorganize host stroma. It follows then that these pathways may also mediate drug resistance, in a way that is fundamentally different from classically defined mechanisms for drug resistance which focus on altered handling of the drug or the drug's target by the resistant tumor cell. It is expected that resistance will occur that is predicated on altered pathway predominance in a tumor and altered tumor microenvironment. Cases which exemplify this possibility are presented, including up-regulation of angiogenesis-related regulators in relation to inhibition of EGF-related proliferative and angiogenesis-promoting functions. The implications of this thinking in relation to development of combination regimens targeting distinct pathways are considered.

Antineoplastic Agents↗
PubMed2001

Dolastatin 11, a marine depsipeptide, arrests cells at cytokinesis and induces hyperpolymerization of purified actin.

The successful synthesis of dolastatin 11, a depsipeptide originally isolated from the mollusk Dolabella auricularia, permitted us to study its effects on cells. The compound arrested cells at cytokinesis by causing a rapid and massive rearrangement of the cellular actin filament network. In a dose-and time-dependent manner, F-actin was rearranged into aggregates, and subsequently the cells displayed dramatic cytoplasmic retraction. The effects of dolastatin 11 were most similar to those of the sponge-derived depsipeptide jasplakinolide, but dolastatin 11 was about 3-fold more cytotoxic than jasplakinolide in the cells studied. Like jasplakinolide, dolastatin 11 induced the hyperassembly of purified actin into filaments of apparently normal morphology. Dolastatin 11 was qualitatively more active than jasplakinolide and, in a quantitative assay we developed, dolastatin 11 was twice as active as jasplakinolide and 4-fold more active than phalloidin. However, in contrast to jasplakinolide and phalloidin, dolastatin 11 did not inhibit the binding of a fluorescent phalloidin derivative to actin polymer nor was it able to displace the phalloidin derivative from polymer. Thus, despite its structural similarity to other agents that induce actin assembly (all are peptides or depsipeptides), dolastatin 11 may interact with actin polymers at a distinct drug binding site.

Actin Cytoskeleton↗