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Oncology Research/Anti-Cancer Drug Design, Volume 13, pp. 123-135
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Copyright © 2002 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Development of Polyamine Analogs as Cancer Therapeutic Agents

Thresia Thomas,1,3,4 Srivani Balabhadrapathruni,2 Michael A. Gallo,1,4 and T. J. Thomas2,3,4

Departments of 1Environmental & Community Medicine, 2Medicine, 3Environmental and Occupational Health Sciences Institute, and 4The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ 08903

Natural polyamines (putrescine, spermidine, and spermine) are aliphatic cations with multiple functions in cell growth and differentiation. Alterations in the polyamine structure provide a strategy to synthesize analogs that can interfere with the cellular functions of natural polyamines. Analogs of spermine are particularly effective in modifying the synthesis, catabolism, and uptake of natural polyamines. The increased requirement of natural polyamines in cancer cell growth makes it possible to utilize the polyamine pathway as a therapeutic target in cancer cells. Because polyamine functions extend from membrane phospholipid structure and signal transduction to DNA structure and conformational transitions, it is likely that the action of polyamine analogs also permeates to these sites of polyamine action. For the same reason, toxicity of polyamine analogs might be considerable. However, it is possible to design polyamine analogs that target a specific function of polyamines in cancer cells, thereby enhancing selectivity for inducing apoptosis of cancer cells. Alternatively, polyamine analogs may potentiate the action of other anticancer agents and become an effective tool in cancer chemotherapy. In either case, further research into the action of polyamine analogs will open up new opportunities in the fight against cancer.

Key words: Polyamines; Polyamine analogs; bis(ethyl)Polyamines; Anticancer drugs

Address correspondence to T. J. Thomas, 125 Paterson Street, Clinical Academic Building, Room 7090, New Brunswick, NJ 08903. Tel: (732) 235-8460; E-mail: thomastj@UMDNJ.edu

Oncology Research/Anti-Cancer Drug Design, Volume 13, pp. 137-146
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Copyright © 2002 Cognizant Comm. Corp.
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Constitutive Production of Parathyroid Hormone-Related Protein (PTHrP) by Fibroblasts Derived From Normal and Pathological Human Breast Tissue

Magali Cros,1 Christophe Cataisson,1 Yong Mee Cho,4 Yolande Berthois,2 Oana Bernard-Poenaru,3 Maryannick Denne,1 Anne-Marie Graulet,3 Marie-Christine De Vernejoul,1 John Foley,4 and Zhor Bouizar1

1INSERM U349, Laboratoire de Biologie Cellulaire et Moléculaire de l'Os et du Cartilage, Hôpital Lariboisière, 75475 Paris Cedex 10, France
2Laboratoire de Cancérologie Expérimentale, Faculté de Médecine de Marseille II, 13916 Marseille Cedex 20, France
3Laboratoire de Biologie Endocrinienne, Hôpital Lariboisière, 75475 Paris Cedex 10, France
4Medical Sciences, Indiana University School of Medicine, Bloomington, IN 47405

Parathyroid hormone-related protein (PTHrP) is expressed in the mammary gland and appears to be critical to the morphogenesis of this structure. PTHrP production in the breast is generally attributed to epithelial cells. Because the stromal component of the breast produces factors implicated in proliferation and differentiation of mammary epithelial tissue and tumors, the aim of this study was to investigate the PTHrP expression by mammary fibroblasts from breast cancer tumors and normal breast. PTHrP antibodies labeled intralobular fibroblasts in normal breast and stromal fibroblasts that surround tumor cells. PTHrP was constitutively produced by the cultured mammary fibroblasts, independent of serum stimulation. Normal (15.83 ± 1.72 fmol/106 cells) and pathological breast fibroblasts (19.87 ± 5.76) secreted similar amounts of PTHrP. PTH/PTHrP receptor mRNA was detected by RT-PCR in all the samples tested. Fibroblasts from normal breast were both PTH and PTHrP-cAMP responsive (453 ± 133% and 513 ± 133%, respectively, from basal stimulation), whereas pathological breast fibroblasts were minimally PTHrP-cAMP responsive (183 ± 36%). The production of other fibroblastic factors implicated in tumor growth and invasiveness was also examined. Interleukin-6 (IL-6), tumor necrosis factor-a (TNF-a), and pro-matrix metalloproteinase (MMP)-1 were not affected by the status of the tissue. In contrast, increased levels of pro-MMP-2 were produced in fibroblasts that originated from pathological (290 ± 62 ng/106 cells) samples compared with those from normal donors (125 ± 41 ng/106 cells). PTHrP production was correlated with TNF-a and pro-MMP-2 production. However, inhibition with specific neutralizing antibodies against TNF-a or PTHrP, or with a PTHrP antagonist, showed that these factors did not regulate each other. In conclusion, breast fibroblasts are constitutive PTHrP-producing cells with the potential for autocrine signaling through the PTH/PTHrP receptor.

Key words: Parathyroid hormone-related protein (PTHrP); Breast cancer; Fibroblasts; Cytokines; Metalloproteinases

Address correspondence to Dr. Z. Bouizar, Laboratoire de Biologie Cellulaire et Moléculaire de l'Os et du Cartilage, INSERM U349, Hôpital Lariboisière, 2 rue Ambroise Par,, 75475 Paris Cedex 10, France. Tel: 01 49 95 63 58; Fax: 01 49 95 84 52; E-mail: zhor.bouizar@inserm.larb.ap-hop-Paris.fR

Oncology Research/Anti-Cancer Drug Design, Volume 13, pp. 147-160
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Copyright © 2002 Cognizant Comm. Corp.
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Embryonal Mass and Hormone-Associated Effects of Pregnancy Inducing a Differential Growth of Four Murine Tumors

Oscar D. Bustuoabad,* Pedro D. di Gianni, Marcela Franco, Edith C. Kordon,* Silvia I. Vanzulli, Roberto P. Meiss, Lorena C. Grion, Graciela S. Díaz, Sergio H. Nosetto, Pablo Hockl, M. Gabriela Lombardi, Christiane Dosne Pasqualini,* and Raúl A. Ruggiero*

División Medicina Experimental, Instituto de Investigaciones Hematológicas y Centro de Estudios Oncológicos, Academia Nacional de Medicina. Las Heras 3092, 1425 Buenos Aires, Argentina

A differential effect of pregnancy on the growth of subcutaneous implants of four murine tumors has been observed. Two tumors lacking receptors for progesterone and estrogen [methylcholanthrene-induced fibrosarcoma (MC-C) and spontaneous lymphoid leukemia (LB)] exhibited slow kinetics throughout the course of pregnancy, although inhibition was stronger beyond day 10. On the other hand, one of two tumors bearing receptors for progesterone and estrogen [medroxyprogesterone (MPA)-induced mammary adenocarcinoma (C7HI)] exhibited three phases: up to days 8-10 of gestation the tumor grew faster than in virgins, between days 8-10 and 15 it reached a plateau, and beyond day 15 a sharp reduction in tumor mass was observed. The other tumor [mouse mammary tumor virus (MMTV)-induced mammary carcinoma (T2280)] behaved as a typical pregnancy-dependent tumor (i.e., it grew in pregnant but not in virgin mice, regressed soon after delivery, and reassumed its growth at the middle of a second round of pregnancy). Neither MPA nor estrogen affected MC-C and LB tumor growth. On the other hand, MPA-treated mice enhanced C7HI tumor and reciprocally C7HI tumor-bearing mice treated with estrogen strongly inhibited tumor growth. As for T2280, neither MPA nor estrogen alone could promote tumor growth and, in consequence, no tumor developed. However, when MPA plus estrogen was administered in a schedule simulating the successive appearance of these hormones in pregnancy, T2280 grew even faster than in pregnant mice. When the four tumors were implanted in mice bearing grafts of embryonal tissues (teratomas), all of them were inhibited. This antitumor effect was similar to that observed in pregnancy when tumors unresponsive to progesterone and estrogen were tested. On the other hand, with tumors bearing progesterone and estrogen receptors, differences in tumor growth were detected in pregnant and teratoma-bearing mice. This suggested the existence during pregnancy of two factors potentially acting on tumor growth. First, a progesterone and estrogen-mediated hormonal component, which would exert either inhibitory or stimulatory effects only evidenced with tumors bearing hormonal receptors. Secondly, an antitumor effect proportional to the growing embryonal mass, inhibiting all tumors independently of their origin or hormone responsiveness. This antitumor effect could be attributed to a heat-resistant serum factor (1000-1200 Da molecular weight) presumably associated with the pathway of the arachidonic acid metabolism. The interplay between the hormonal component and the serum factor associated with embryonal mass could account for some of the largely heterogeneous and otherwise unexplained effects of pregnancy on tumor growth reported in the literature and illustrated by the four tumors studied here.

Key words: Murine tumors; Pregnancy; Tumor dormancy; Embryonal mass; Hormone regulation

Address correspondence to Dr. Christiane Pasqualini, División Medicina Experimental, Instituto de Investigaciones Hematológicas, Academia Nacional de Medicina, Las Heras 3092, 1425 Buenos Aires, Argentina. Tel/Fax: 54-11-4805-8176; E-mail: chdosne@hotmail.com

*Member of Research Career, CONICET.

Oncology Research/Anti-Cancer Drug Design, Volume 13, pp. 161-167
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Copyright © 2002 Cognizant Comm. Corp.
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Analysis of Differentially Expressed Genes in Hepatocellular Carcinoma With Hepatitis C Virus by Suppression Subtractive Hybridization

Masayo Kotaka,1 George G. Chen,1,2 Paul B. S. Lai,1 Wan Y. Lau,1 Paul K. S. Chan,3 Thomas W. T. Leung,4 and Arthur K. C. Li1

1Department of Surgery, 2Sir Y.K. Pao Center for Cancer, 3Department of Microbiology, and 4Department of Clinical Oncology, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong

Hepatitis C virus (HCV) infection is associated with pathogenesis of hepatocellular carcinoma (HCC). We carried out suppression subtractive hybridization to identify variable expression of genes linked to HCC with HCV infection. RNA from both tumorous (tester) and nontumorous (driver) liver tissues was isolated. The cDNA clones were subjected to MegaBACE PCR sequencing to identify those that hybridized to the subtracted library with preference. Nucleic acid sequences generated were searched against the human UniGene database. Among 576 clones screened in the tumorous liver tissue, we identified 30 genes and 28 Expressed Sequence Tags (ESTs). Among 30 genes detected, 23 were with known functions and 7 with unknown functions. The known genes identified had diversified functions and could be divided into 10 functional categories. Twenty percent of these genes were previously known to be tumor related and those most frequently appearing were haptoglobin alpha(2FS)-beta precursor, haptoglobin related protein, and alpha-2-macroglobulin. Four out of 30 known genes (immunoglobulin lambda light chain, kappa immunoglobulin, spliceosomal protein, and X-ray repair cross-complementing protein) were related to chromosome translocation and nucleotide repair. These four genes may contribute to carcinogenesis caused by DNA-damaged agents and to the efficiency of anticancer therapy. The genes with unknown function, which were most frequently detected, were PRO2760 and PRO2955; both encode proteins that express in fetal liver. Twenty-one known and six novel genes were discovered in the nontumorous liver tissue. Apparently, these 27 genes were lost in the tumorous liver tissues. Therefore, using suppression subtractive hybridization, we have identified a number of genes associated with HCC with HCV infection. Most of these genes have not been reported in HCC. Further characterization of these differentially expressed known and unknown genes will provide useful information in understanding the genes responsible for the development of HCC.

Key words: Hepatocellular carcinoma; Hepatitis C virus; Suppression subtractive hybridization

Address correspondence to George G. Chen, Department of Surgery, Prince of Wales Hospital, The Chinese University of Hong Kong, Shatin, N.T. Hong Kong. Tel: 852 2632 3934; Fax: 852 2645 0605; E-mail:gchen@cuhk.edu.hk

Oncology Research/Anti-Cancer Drug Design, Volume 13, pp. 169-174
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Copyright © 2002 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

A Sensitive Bioassay for Measuring Blood Levels of 12-O-Tetradecanoylphorbol-13-acetate (TPA) in Patients: Preliminary Pharmacokinetic Studies

Xiao Xing Cui,1 Richard L. Chang,1 Xi Zheng,1 Donald Woodward,2 Roger Strair,3 and Allan H. Conney1,3

1Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020
2Department of Pharmacy Practice and Administration, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020
3The Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ 08901

12-O-Tetradecanoylphorbol-13-acetate (TPA) is a potent stimulator of differentiation in myelocytic leukemia cells, and it has been shown to have activity in patients with acute myelocytic leukemia. Because attempts to develop a suitable mass spectrometry assay for TPA were unsuccessful (because of the lack of sufficient sensitivity), we developed a novel and highly sensitive blood level bioassay for TPA that measures ethyl acetate-extractable differentiating activity in blood. Differentiating activity in ethyl acetate extracts of blood was measured in HL-60 cells by measuring the formation of adherent cells. The sensitivity of the assay was ~0.1 ng TPA/ml blood. The assay for TPA has a high degree of specificity and does not measure deesterifed potential metabolites (phorbol, phorbol-13-acetate, or phorbol-12-myristate), and the presence of GM-CSF, G-CSF, interferon-a, or interferon-g does not interfere with the assay. Blood levels of TPA as measured by the bioassay immediately after an IV infusion of TPA (0.125 mg/m2; ~0.25 mg per patient) and 1 and 3 h later were 1.75 ± 0.55, 0.93 ± 0.54, and 0.69 ± 0.42 ng/ml, respectively (mean ± SD from eight infusions in five patients). Terminal half-lives were determined in a few patients where TPA blood levels were measured at multiple time intervals after the TPA infusion. In these patients, the terminal half-life was 11.1 ± 3.9 h (from five infusions in four patients). To the best of our knowledge, this is the first analytical method for the measurement of TPA.

Key words: 12-O-Tetradecanoylphorbol-13-acetate (TPA); Myelocytic leukemia; Bioassay; Blood levels

Address correspondence to Dr. Allan H. Conney, Susan Lehman Cullman Laboratory for Cancer Research, Department of Chemical Biology, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 164 Frelinghuysen Road, Piscataway, NJ 08854-8020. Tel: (732) 445-4940; Fax: (732) 445-0687; E-mail: aconney@rci.rutgers.edu

Oncology Research/Anti-Cancer Drug Design, Volume 13, pp. 175-189
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Copyright © 2002 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Antitumor Polycyclic Acridines. Part 12. Physical and Biological Properties of 8,13-Diethyl-6-methylquino[4,3,2-kl]acridinium Iodide: A Lead Compound in Anticancer Drug Design

Sotiris Missailidis,1 Johnson Stanslas,1* Chetna Modi,1 Michael J. Ellis,1 R. Adrian Robins,2 Charles A. Laughton,1 and Malcolm F. G. Stevens1

1Cancer Research Laboratories, School of Pharmaceutical Sciences and 2Division of Immunology, School of Clinical Laboratory Sciences, University of Nottingham, Nottingham, NG7 2RD, UK

The biophysical and biological characterization of 8,13-diethyl-6-methylquino[4,3,2-kl]acridinium iodide (6) is reported. The compound binds to DNA, as measured by UV, fluorescence, and circular dichroism studies, and stabilizes the double helix and higher order DNA structures (DNA triplexes and quadruplexes) against thermal denaturation. Unlike many DNA ligands, (6) shows no specificity for binding to specific base pair combinations and does not inhibit topoisomerase I (topo I) or topo II activity. Furthermore, the biological fingerprint elicited by (6) in in vitro evaluations does not compare with clinical agents of the topo II inhibition class. The compound provokes cell cycle arrest in response to DNA damage and the biological sequelae are dependent on the p53 status of the cell line. DNA damage by (6) upregulates p53 and p21CIP/WAF1 proteins. The unusual structure of (6) and its ease of synthesis in a "one-pot" reaction are features that are being exploited in the design and development of a new series of G-quadruplex stabilizing telomerase inhibitors. However, although the second-generation compounds that resulted from (6) present strong telomerase inhibition, (6) in itself presents yet a different mode of action, with a strong preference for triplex DNA, sequences often found in a number of genes.

Key words: Polycyclic acridine derivatives; DNA binding; Spectrophotometric analysis; UV; Fluorescence; Circular dichroism

Address correspondence to Dr. S. Missailidis, Chemistry Department, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK. E-mail: s.missailidis@open.ac.uk

*Present address: Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, University Putra Malaysia, 43400 Serdang, Selangor, Malaysia