ognizant Communication Corporation



Oncology Research, Volume 13, pp. 245-252
0965-0407/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Differential Ability of Cytostatics From Anthraquinone Group to Generate Free Radicals in Three Enzymatic Systems: NADH Dehydrogenase, NADPH Cytochrome P450 Reductase, and Xanthine Oxidase

Jolanta Pawlowska,1 Jolanta Tarasiuk,1 C. Roland Wolf,2 Mark J. I. Paine,2 and Edward Borowski1

1Department of Pharmaceutical Technology and Biochemistry, Technical University of Gdansk, Narutowicza St 11/12, 80-952 Gdansk, Poland
2Imperial Cancer Research Fund Molecular Pharmacology Unit, Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee, UK

The antitumor drugs of the anthraquinone group are widely used agents in the treatment of a variety of human neoplasms. However, their clinical effectiveness is limited by several factors, among which dose-dependent cardiotoxicity is of great importance. Numerous data indicate that the cardiac effects of these drugs are the consequence of one-electron transfer from reduced nucleotides to atmospheric oxygen. This process is catalyzed primarily by NADH dehydrogenase, NADPH cytochrome P450 reductase, and xanthine oxidase, and leads to the formation of reactive oxygen species. In our previous studies we have shown that the NADH dehydrogenase catalyzed electron transfer phenomenon is correlated with the affinity of anthraquinone drugs to the enzyme. In this work data are presented on the ability of compounds belonging to several structural types of anthraquinone cytostatics (sugar-and quinone-modified derivatives of DR and ADR, and anthracenedione compounds) to stimulate free radical formation in the above three enzymatic systems. It has been shown that the three oxidoreductases exhibit different structural requirements with respect to their substrate properties for anthraquinones. Therefore, evaluation of the structural factors determining the ability of anthraquinone compounds to generate active oxygen species cannot be limited to a single oxidoreductase system but must include all types of enzymatic systems involved in the catalysis of one-electron transfer reactions.

Key words: Anthraquinone compounds; Free radical formation; NADH dehydrogenase; NADPH cytochrome P450 reductase; Xanthine oxidase

Address correspondence to Jolanta Tarasiuk at his current address: University of Szczecin, Department of Biochemistry, 3a Felczaka St, 71-412 Szczecin, Poland. Tel/Fax: (48) (91) 444-15-50; E-mail: tarasiuk@univ.szczecin.pl

Oncology Research, Volume 13, pp. 253-259
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Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Involvement of ERK1/2 in Invasiveness and Metastatic Development of Rat Prostatic Adenocarcinoma

Tuangporn Suthiphongchai, Piradee Promyart, Sariya Virochrut, Rutaiwan Tohtong, and Prapon Wilairat

Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand

Extracellular signal-regulated kinase (ERK) activation has been implicated in cell motility and invasion. In this study, we demonstrated that the steady-state levels of activated ERK1/2 correlated with the degree of invasiveness and metastatic potential of three Dunning cancer cell lines, originating from the same parental tumor. Inhibition of mitogen-activated protein kinase kinase 1 (MEK1), an upstream regulator of ERK1/2, with PD98059 resulted in a dose-dependent reduction of invasiveness with different IC50 values in the three Dunning cell lines. These results suggest that ERK is, at least in part, responsible for regulating invasiveness and may underlie the differences in the metastatic ability of the cell lines.

Key words: Metastasis; Invasion; Prostate cancer; Extracellular signal-regulated kinase (ERK); Signal transduction

Address correspondence to Tuangporn Suthiphongchai, Department of Biochemistry, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. Tel: 662-245-5195; Fax: 662-248-0375; E-mail: sctsc@mahidol.ac.th

Oncology Research, Volume 13, pp. 261-268
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Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Significant Experimental Decrease of the Hepatocellular Carcinoma Incidence in C3H/Sy Mice After Long-Term Administration of EB1089, a Vitamin D Analogue

Despina Sahpazidou,1 Pelagia Stravoravdi,1 Theano Toliou,1 George Geromichalos,1 George Zafiriou,1 Konstantinos Natsis,2 and Panagiotis Gigis2

1Symeonides Research Center, Theagenio Cancer Hospital, Thessaloniki, Greece
2Department of Anatomy, Medical School, Aristotelian University of Thessaloniki, Greece

EB1089, a vitamin D analogue without the acute side effects of 1a,25(OH)2D3, the physiologically active form of vitamin D, exerts strong antiproliferative activities in malignant cells, including hepatoma cells in vitro, and in experimental hepatomas in animals as well. It also induces cell cycle arrest and apoptosis in premalignant conditions, suggesting its application in chemopreventive trials. We examined the possible chemopreventive effect of EB1089 on hepatocellular carcinoma (HCC) incidence in C3H/Sy virgin female mice, a strain developing an incidence of 58% spontaneous HCCs. A total of 95 mice, 16 weeks old, were used. EB1089 injections of 0.5 mg/kg of body weight were given IP every other day for 2, 4, and 6 months to 51 mice (18, 19, and 14 mice, respectively). The remaining 44 mice were divided into three control groups, accordingly, and injected with the vehicle solution only. The mice were sacrificed when they appeared moribund because of their disease. The rest were sacrificed at the age of at least 80 weeks. A full autopsy was performed and liver tissue was processed for histological examination. The results obtained show that 3.9% of treated mice developed HCC, exclusively in the 2-month group, compared with 36.4% of HCCs in the control group. Our results suggest that the chemopreventive administration of EB1089 causes a very statistically significant (P<0.0001) inhibitory effect on HCC incidence of C3H/Sy mice. This effect could be useful in a potential application on the chemopreventive control of HCCs.

Key words: Preclinical study; Spontaneous tumors; Chemoprevention

Address correspondence to Dr. Despina Sahpazidou, Symeonides Research Center, Theagenio Cancer Hospital, 2, Al. Symeonides str., Thessaloniki 54007, Greece. Tel: ++ 2310 898237; Fax: ++ 2310 845514; E-mail: gerom@chem.auth.gr

Oncology Research, Volume 13, pp. 269-277
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Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Cytotoxicity and Cell Cycle Effects of Novel Indolo[2,3-b]quinoline Derivatives

Rita Humeniuk,1 Lukasz Kaczmarek,2 Wanda Peczynska-Czoch,3 and Ewa Marcinkowska1

1Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolf Weigl St. 12, Wroclaw, Poland
2Pharmaceutical Research Institute, Ludwik Rydygier St. 8, Warszawa, Poland
3Institute of Organic Chemistry, Biochemistry and Biotechnology, Wroclaw Technical University, Wybrzeze Wyspianskiego 27, Wroclaw, Poland

Cellular effects of novel indolo[2,3-b]quinoline derivatives were studied. These compounds are synthetic analogs of plant alkaloid neocryptolepine (5-methyl-5H-indolo[2,3-b]quinoline) present in extracts from Cryptolepis sanguinolenta. They are traditionally used in natural medicine in Central and West Africa. Previous molecular and computational studies indicated that these compounds were DNA intercalators and inhibitors of topoisomerase II. We have extended our studies on their mode of action to the cellular level. Past experiments have shown that these compounds were active in vitro against cell lines derived from solid tumors, so for the present studies we selected leukemic cell lines. Jurkat acute T cell, CCRF-CEM T lymphoblastoid, THP-1 acute monocytic, HL-60 acute promyelocytic leukemias, and HL-60/MX2 subline with reduced expression of topoisomerase II were used. We evaluated the cytotoxicity and cell cycle effects of the indolo[2,3-b]quinoline compounds. We also tested if these compounds were able to induce apoptosis in the cells. Our studies revealed that novel indolo[2,3-b]quinoline derivatives were more cytotoxic to all cell lines than etoposide (used as a reference topoisomerase II inhibitor), and that their cytotoxicity depended on the substituents introduced to the indolo[2,3-b]quinoline core. Surprisingly, our studies have shown that HL-60/MX2 cell line and also THP-1 cell line, resistant to etoposide, were susceptible to methyl-and methoxy-substituted indolo[2,3-b]quinoline derivatives. In parallel to the evaluation of cytotoxicity we studied cell cycle effects of these compounds. Treatment of HL-60 cells with etoposide in subcytotoxic concentrations resulted in a massive accumulation of the cells in the G2/M phase of the cell cycle. When we used subcytotoxic concentrations of our novel indolo[2,3-b]quinoline derivatives the cell cycle progression of HL-60 cells was not affected. Moreover, the cell cycle of HL-60/MX2 cells was not influenced by any of the compounds studied. Indolo[2,3-b]quinoline derivatives induced apoptosis in HL-60 and HL-60/MX2 cells, but only in concentrations close to IC50 determined in cytotoxic assays. Etoposide induced apoptosis in HL-60 parental cell line, but in a very broad range of concentrations. Our results suggest that topoisomerase II may not represent the main cellular target for novel indolo[2,3-b]quinoline derivatives. They show that the cells resistant to topoisomerase II poison, etoposide, were still sensitive to our compounds.

Key words: Indoloquinolines; Cell cycle; Cytotoxicity; Apoptosis; Topoisomerase II; Etoposide

Address correspondence to Ewa Marcinkowska, Laboratory of Reproductive Immunology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigl St. 12, 53-114 Wroclaw, Poland. Tel: 48 (71) 337 11 72; Fax: 48 (71) 337 13 82; E-mail: marcinko@iitd.pan.wroc.pl

Oncology Research, Volume 13, pp. 279-287
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Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Resistance of Decoy PNA-DNA Chimeras to Enzymatic Degradation in Cellular Extracts and Serum

Monica Borgatti,1 Alessandra Romanelli,2 Michele Saviano,2 Carlo Pedone,2 Ilaria Lampronti,1 Laura Breda,1 Claudio Nastruzzi,3 Nicoletta Bianchi,1 Carlo Mischiati,1 and Roberto Gambari1,4

1Department of Biochemistry and Molecular Biology, Ferrara University, Italy
2Institute of Biostructure and Bioimaging, CNR, Napoli, Italy
3Department of Pharmaceutical Chemistry and Technology, University of Perugia, Italy
4Biotechnology Center, Ferrara University, Italy

Double-stranded molecules based on peptide nucleic acids (PNAs)-DNA chimeras carrying binding sites for known transcription factors could be of great interest in decoy pharmacotherapy of neoplastic diseases. For instance, decoy molecules recognizing Sp1 and NF-kB transcription factors were found to inhibit tumor cell growth and invasion activity. In this respect, we have recently found that double-stranded PNA-DNA chimeras carrying NF-kB binding sites inhibit the binding of NF-kB p52 and p50 transcription factors to target DNA molecules. In this article we determined the resistance of double-stranded decoy molecules based on PNA-DNA chimeras to exonucleases (both 3´->5´ and 5´->3´ exonucleases), endonucleases, and 5´-phosphatases. In addition, we performed experiments aimed at determining the resistance of these molecules in cellular extracts and serum. Finally, we used liposomes as protective agents in experimental conditions in which the decoy molecules employed were found to be unstable (high concentrations of enzymes, cellular extracts, or serum). The results obtained demonstrated that decoy molecules based on PNA-DNA chimeras are more resistant than DNA-based decoys to exo-and endonucleases, serum, and cellular extracts. In addition, the resistance of DNA/PNA hybrids in the presence of high concentrations of serum and cellular extracts was increased after complexation to cationic liposomes, due to the fact that double-stranded PNA-DNA-PNA chimeras bind to these delivery systems. The results obtained in the present study support the proposal of molecules based on PNA-DNA chimeras for an efficient decoy treatment of tumor cells both in vitro and in vivo.

Key words: Peptide nucleic acid (PNA); Peptide nucleic acid (PNA)-DNA chimeras; Decoy

Address correspondence to Prof. Roberto Gambari, Dipartimento di Biochimica e Biologia Molecolare, Via L. Borsari n.46, 44100 Ferrara, Italy. Tel: +39-532-291448; Fax: +39-532-202723; E-mail: gam@unife.it

Oncology Research, Volume 13, pp. 289-298
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Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Phosphorylation, But Not Overexpression, of Epidermal Growth Factor Receptor Is Associated With Poor Prognosis of Non-Small Cell Lung Cancer Patients

Takanori Kanematsu,1 Seiji Yano,1 Hisanori Uehara,2 Yoshimi Bando,2 and Saburo Sone1

1Department of Internal Medicine and Molecular Therapeutics and 2Department of Molecular and Environmental Pathology, University of Tokushima School of Medicine, Tokushima, Japan

Epidermal growth factor receptor (EGFR) is commonly overexpressed in non-small cell lung cancer (NSCLC) and its tyrosine kinase phosphorylation is thought to be an ideal target in the treatment of patients with NSCLC. In the present study, we examined surgically obtained specimens from a series of 36 NSCLC patients for expression of EGFR, phosphorylated EGFR (p-EGFR), and HER2 by immunohistochemistry, and also examined the correlation with clinical characteristics. The positive rate of EGFR, p-EGFR, and HER2 was 97.2%, 44.4%, and 88.6%, respectively, and the overexpression rate was 80.6%, 0.0%, and 27.8%, respectively. EGFR overexpression and phosphorylation were seen at almost the same rate in each histological type of squamous and nonsquamous cell carcinoma (squamous vs. nonsquamous; 78.6% vs. 81.8% for EGFR, 35.7% vs. 50.0% for p-EGFR), while HER2 overexpression was seen less frequently in squamous cell carcinoma than in nonsquamous cell carcinoma (0.0% vs. 45.5%, P=0.003). Univariate analysis revealed that EGFR overexpression was related to good performance status (P=0.038) but not related to EGFR phosphorylation. EGFR phosphorylation was correlated to short time to progression (TTP) (P=0.002) and poor prognosis (P=0.002), although EGFR overexpression, HER2 overexpression, or EGFR-HER2 coexpression were not correlated to TTP or survival. Bivariate analysis showed EGFR phosphorylation was related to short TTP and poor prognosis both in early and advanced stages. Multivariate analyses confirmed that clinical stage, performance status, and p-EGFR expression were independently associated with increasing risk of short TTP and poor prognosis. These results suggest that phosphorylation, but not overexpression, of EGFR may be an important predictor for clinical outcome of NSCLCs.

Key words: Epidermal growth factor receptor (EGFR); Phosphorylated EGFR; HER2; Immunohistochemistry; Non-small cell lung cancer; Multivariate analysis

Address correspondence to Saburo Sone, Department of Internal Medicine and Molecular Therapeutics, University of Tokushima School of Medicine, 3-8-15 Kuramoto-cho, Tokushima, Japan. Tel: (81)-88-633-2134; E-mail: ssone@clin.med.tokushima-u.ac.jp