ognizant Communication Corporation



Oncology Research, Volume 13, pp. 455-461
0965-0407/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Novel Sigma Binding Site Ligands as Inhibitors of Cell Proliferation in Breast Cancer

Federica Barbieri,1 Anna Sparatore,2 Angela Alama,1 Federica Novelli,3 Cristina Bruzzo,1 and Fabio Sparatore3

1Laboratory of Pharmacology and Neuroscience, National Institute for Cancer Research, Largo R. Benzi 10, 16132, Genoa, Italy
2Institute of Pharmaceutical Chemistry, University of Milan, Viale Abruzzi 42, 20131, Milan, Italy
3Department of Pharmaceutical Sciences, University of Genoa, Viale Benedetto XV, 3, 16132, Genoa, Italy

Sigma receptors, namely j1 and j2, have been shown to be expressed in a variety of human cell lines playing a role in cell growth. In the human breast, they are absent in normal mammary tissue but expressed in tumors, particularly in the proliferating stage. The study presented here concerns nine newly synthesized ligands for sigma receptors. The compounds are of general structure consisting of: five (1a/1b-arylalkyl)quinolizidines including two thioisosteres and four spiro-[3,4-dihydro-1,2,4-benzotriazino-3,4´-(1´-substituted) piperidines]. These compounds exhibited varying degrees of affinity for sigma receptors and were able to inhibit the growth of MCF-7 and MDA-MB 231 human breast cancer cell lines, in vitro. Good to moderate binding to j1 receptors occurred with all tested ligands. However, affinity for j2 appeared more evident with compounds FN/C-2 and FN/C-4 (spiro-[3,4-dihydro-1,2,4-benzotriazino-3,4´-(1´-substituted) piperidines] derivatives). In addition, higher cytotoxic activity of FN/C-2 and FN/C-4 with IC50 values below 100 mM in MCF-7 and lower than 40 mM in MDA-MB 231 was revealed. The data from the current study show that these novel sigma receptor ligands exhibit interesting cytotoxic activity and suggest their potential for development as antitumor agents.

Key words: Sigma receptor ligands; MCF-7; MDA-MB 231; Cytotoxic activity

Address correspondence to Angela Alama, Laboratory of Pharmacology and Neuroscience, National Institute for Cancer Research, Largo R. Benzi 10, 16132, Genoa, Italy. Tel: +39-010-5600934; Fax: +39-010-5600937; E-mail: angela.alama@istge.it

Oncology Research, Volume 13, pp. 463-469
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Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Flavonoids as Inhibitors of MRP1-Like Efflux Activity in Human Erythrocytes. A Structure-Activity Relationship Study

Malgorzata Bobrowska-Hägerstrand,1 Anna Wróbel,2 Lucyna Mrówczynska,3 Thomas Söderström,1,4 Yoshiaki Shirataki,5 Noboru Motohashi,6 Joseph Molnár,7 Krystyna Michalak,8 and Henry Hägerstrand1

1Department of Biology, Åbo Akademi University, FIN-20520 Åbo/Turku, Finland
2Institute of Physics, Wroclaw University of Technology, PL-50370 Wroclaw, Poland
3Department of Cytology and Histology, A. Mickiewicz University, PL-61701, Poznan, Poland
4Turku Center for Biotechnology, University of Turku and Åbo Akademi University, FIN-20520 Åbo/Turku, Finland
5Faculty of Pharmaceutical Sciences, Josai University, Sakado, Saitama, 350-0295 Japan
6Meiji Pharmaceutical University, Tokyo, 204-8588, Japan
7Department of Microbiology, Albert Szent-Györgyi Medical University, H-6720 Szeged, Hungary
8Department of Biophysics, Wroclaw Medical University, Poland

The potency of flavonoids (isoflavones, flavones, and flavanones) to inhibit efflux of 2´,7´-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes was investigated. Structure-activity relationship analysis showed that the strongest inhibitors were found among flavanones bearing a hydrophobic prenyl, geranyl, or lavandulyl group at position 8 (and hydroxyl groups at 5 and 7) in ring A. A prenyl group at position 5´ or stilbene at positions 4´-5´ in ring B further seemed to increase inhibitor potency. The most efficient flavanones, euchrestaflavanone A and sophoraflavanone H, were ~20 times more efficient than genistein, and induced 50% inhibition of BCPCF efflux (IC50) at 3 mM (60 min, 37°C). This is comparable to IC50 of benzbromarone (4 mM) and lower than IC50 of indomethacin (10 mM), both known MRP1 (ABCC1) inhibitors. It is suggested that BCPCF efflux is mainly due to MRP1 activity. Our results indicate that flavonoid molecular structure provides a promising base for development of potent MRP1 inhibitors.

Key words: Multidrug resistance; Red blood cell; BCPCF; Flavanone; Structure-activity analysis; Benzbromarone; Indomethacin

Address correspondence to Henry Hägerstrand, Department of Biology, Åbo Akademi University, Biocity, FIN-20520, Åbo/Turku, Finland. Tel: +358-2-2154089; Fax: +358-2-2154748; E-mail: henry.hagerstrand@abo.fi

Oncology Research, Volume 13, pp. 471-478
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Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Three-Dimensional Pharmacophore Mapping of Certain Anticancer a-Methylene-g-Butyrolactones

Kuan-Han Lee1 and Bor-Ruey Huang2

1Department of Pharmacy, Tajen Institute of Technology, Pingtung 907, Taiwan
2School of Chemistry, Kaohsiung Medical University, Kaohsiung 807, Taiwan

A definition of pharmacophore hypotheses for certain anticancer a-methylene-g-butyrolactones was carried out by consideration of a three-dimensional model that correlates the chemical structures of a series of a-methylene-g-butyrolactones with their cytostatic activity. Training sets consisting of 20 a-methylene-g-butyrolactones were selected in each case on the basis of the information content of the structures and the activity data as required by the HypoGen program in the Catalyst software. In this case, the best pharmacophore in terms of statistics and predictive value consisted of four features: two hydrogen bond acceptors (HBA), one hydrophobic (HP) feature, and one aromatic ring (AR) feature. The selected pharmacophore hypothesis yielded a correlation coefficient of 0.942 with a cost differece (null cost minus total cost) of 40.342. The pharmacophore was validated on a set of test compounds. This validation study provided confidence for the usefulness of the selected pharmacophore model to identify compounds with diverse structures from a database search. This investigation has demonstrated a practical approach toward the development of anticancer lead compounds through a three-dimensional pharmacophore mapping.

Key words: Pharmacophore mapping; Anticancer drugs; a-Methylene-g-butyrolactone

Address correspondence to K. H. Lee. No 20, Wei-Shin Rd, Shin-Ell Tsun, Yan-Puu Hsiang, Pingtung 907, Taiwan. Tel: 886-8-7624002; Fax: 886-8-7622899; E-mail: khlee@ccsun.tajen.edu.tw

Oncology Research, Volume 13, pp. 479-489
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Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Detection of Labile Anthracycline-DNA Adducts By Real-Time PCR

Damian M. S. Spencer,1 Suzanne M. Cutts,1 Ken-ichi Kimura,2 Peter J. Gray,3 and Don R. Phillips1

1Department of Biochemistry, La Trobe University, Victoria, 3086, Australia
2Department of Agro-bioscience, Iwate University, Ueda, Morioka, Iwate, 020-8550, Japan
3Platforms Sciences Laboratory, Defence Science and Technology Organisation, P.O. Box 4331, Melbourne, Victoria, 3001, Australia

Barminomycin was employed as a model anthracycline that yields thermally stable drug-DNA adducts. Real-time PCR was utilized for the detection of these barminomycin-DNA adducts at drug levels as low as 100 nM in a cell-free assay system, with the lowest level of detection at approximately 20 nM. By contrast, doxorubicin-DNA adducts are heat labile and their levels were underestimated by conventional real-time PCR unless the DNA denaturation temperature was lowered by the addition of glycerol. Doxorubicin-DNA adduct levels of 5.5 per 10 kb were detected by real-time PCR (in the presence of 24% glycerol) following treatment with 0.5 mM doxorubicin (and 2 mM formaldehyde), considerably more sensitive than that detected by a gene-specific Southern-based procedure. Both the absolute fluorescence intensity in the linear PCR amplification range and the crossing point method provided useful dose-dependent estimates of adduct levels. The time required for a complete real-time PCR analysis of drug-induced adduct levels was approximately 40 min, and this may ultimately provide oncologists with a rapid means with which to monitor drug-DNA adduct levels in patients under treatment with anthracyclines. Responses to these drugs could be quickly and efficiently monitored in patients, thereby facilitating optimization of drug dosages as well as early detection of resistance to these agents.

Key words: Real-time PCR; Doxorubicin; DNA adducts; Barminomycin; Anthracyclines

Address correspondence to D. R. Phillips, Department of Biochemistry, La Trobe University, Victoria, 3086, Australia. Tel: +61-3-9479-2182; Fax: +61-3-9479-2467; E-mail: d.phillips@latrobe.edu.au

Oncology Research, Volume 13, pp. 491-502
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Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Mode of Cell Death Induced in Chinese Hamster Cells by Sequence-Selective DNA Minor Groove Binding Nitrogen Mustards: Comparison With Untargeted Mustards and With Hoechst 33342

Glenn Whiteside,1 Lynnette R. Ferguson,1,2 William A. Denny,1 and Iona E. Weir3*

1Auckland Cancer Society Research Centre, The University of Auckland, PB92019, Auckland, New Zealand
2Discipline of Nutrition, The University of Auckland, Auckland, New Zealand
3Horticulture and Food Research Institute of New Zealand Ltd, PB 92169, Auckland, New Zealand

The clinical antitumor efficacy of nitrogen mustards such as chlorambucil may relate to their ability to cause programmed cell death (apoptosis), probably through their DNA cross-linking properties. In contrast, bisbenzimidazoles such as Hoechst 33342 interact noncovalently with the minor groove of DNA, and appear to cause apoptosis in a fundamentally different way, which may involve the inhibition of topoisomerase (topo) I enzymes. A series of DNA minor groove binding nitrogen mustards with selective DNA affinity and in vivo antitumor activity in animal models was studied. Although two examples of such compounds proved to inhibit topo I enzymes in vitro, they were equally toxic towards topo I-proficient and- deficient strains of yeast, suggesting that topo I inhibition was not involved in cell killing. Flow cytometric analysis of Chinese hamster cells highlighted the differences in the propensity to cause apoptosis by chlorambucil compared with Hoechst 33342, revealing two distinct apoptotic populations in cells treated with the latter drug. Unexpectedly, the bisbenzimidazole mustards showed a novel peak of apoptotic activity, distinct from that shown by either parent drug. Exploring these different mechanisms of apoptosis may provide new directions for the development of antitumor drugs.

Key words: Nitrogen mustard; Bisbenzimidazole; Topoisomerase I; Cell cycle; Apoptosis

Address correspondence to Lynnette R. Ferguson, Nutrition/Auckland Cancer Society Research Centre, Private Bag 92019, Auckland 1000, New Zealand. Tel: 64 9 373 7599, ext. 6372; Fax: 64 9 373 7502; E-mail: l.ferguson@auckland.ac.nz

*Formerly known as Iona O'Brien.

Oncology Research, Volume 13, pp. 503-512
0965-0407/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Synthesis and Antimelanoma Activity of Tertiary Amide Analogues of N-Acetyl-4-S-cysteaminylphenol

Vikki C. Pearson,1 Jillian Ferguson,1 Paul M. Rogers,2 Lloyd R. Kelland,3 and David J. Robins1

1Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK
2Cancer Research UK Centre for Cancer Therapeutics, Institute of Cancer Research, Haddow Laboratories, 15 Cotswold Road, Belmont, Sutton, Surrey SM2 5NG, UK
3Antisoma plc, St Georges Hospital Medical School, Cranmer Terrace, London SW17 0QS, UK

The biosynthetic pathway to melanin is a realistic target for therapeutic intervention in the development of new drugs to combat malignant melanoma. N-Acetyl-4-S-cysteaminylphenol (1) is an analogue of a biosynthetic intermediate in the pathway to melanin. It probably acts as a prodrug and is oxidized selectively in melanocytes to an o-quinone, which can alkylate cellular nucleophiles resulting in interference with cell growth and proliferation. We previously synthesized a range of more lipophilic analogues of 1 by varying the acyl portion and introducing substitution a to the nitrogen. Most of the new compounds displayed greater cytotoxicity than the original lead compound 1. We have now prepared 12 new compounds with varying acyl portions and three different substituents on the nitrogen of the amide in order to increase lipophilicity and to reduce the possibility of hydrolysis of the amides. Most of the tertiary amides showed greater cytotoxicity towards five representative melanoma cell lines than the parent secondary amide. The highest cytotoxicity, comparable to cisplatin, was observed for the benzyl substituted compounds 4, 8, 12, and 16 and the cyclohexylacetamide derivatives 13-15 against these five cell lines. The moderate activity of 13-16 against SK-Mel-24 (non-tyrosinase containing) and an ovarian cell line suggests that interference with the melanin pathway may not be the only mode of action of these new compounds.

Key words: Antimelanoma; Cysteaminylphenol; Tyrosinase; Amide

Address correspondence to Professor David J. Robins, Department of Chemistry, University of Glasgow, Glasgow, G12 8QQ UK. Tel: 0141-330-4378; Fax: 0141-330-4888; E-mail: D.Robins@chem.gla.ac.uk