ognizant Communication Corporation



Oncology Research, Volume 15, pp. 333-341
0965-0407/05 $20.00 + .00
E-ISSN 1555-3906
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Overexpression of Extracellular Superoxide Dismutase (EC-SOD) in Mouse Skin Plays a Protective Role in DMBA/TPA-Induced Tumor Formation

Sung-Hyun Kim,1,2 Myoung-Ok Kim,1 Peng Gao,3 Cheng-A Youm,3 Hae-ryoung Park,3 Sang-Ryeul Lee,1 Kil-Soo Kim,4 Jun-Gyo Suh,5 Hoon-Taek Lee,2 Byung-Ju Park,3 Zae-Young Ryoo1* and Tae-Hoon Lee,3*

1School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea
2Department of Animal Science, Graduate School of Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea
3Department of Oral Biochemistry, School of Dentistry, Chonnam National University, 300 Yongbong-Dong, Buk-Ku, Gwangju 500-757, Korea
4Department of Veterinary Medicine, College of Veterinary Medicine, Kyungpook National University, Daegu, 702-701, Republic of Korea
5Experimental Animal Center/Department of Medical Genetics, College of Medicine, Hanllym University, 1-Okchon-dong, Chunchon, Kangwon-do 200-702, Korea

Extracellular superoxide dismutase (EC-SOD, EC is a major antioxidant enzyme that is located in the extracellular matrix and on the cell surface. EC-SOD protects against cell and tissue damage initiated by extracellular-produced reactive oxygen species (ROS). We investigated a major role of EC-SOD in the development of tumor formation. In this study, we reported that skin-specific overexpressed EC-SOD transgenic mice showed half the number of tumors compared with the nontransgenic mice in the dimethylbenzanthracene (DMBA)-initiated and a 12-"O"-tetradecanoylphorbol-13-acetate (TPA)-promoted two-stage skin carcinogenesis model. This model showed a significant increase of the epidermal cell proliferation in the nontransgenic mice, but the proliferative response in the transgenic mice was delayed. The 8-hydroxy-2´-deoxyguanosine (8OH-dG) detection assay showed that the oxidative DNA damage was significantly higher in the nontransgenic mice than in the transgenic mice after TPA treatments. Overall, EC-SOD overexpression inhibited the TPA-induced cell proliferation and DNA damage, and reduced the subsequent formation of tumors. Our data suggest that EC-SOD plays a protective role in DMBA/TPA-induced skin carcinogenesis.

Key words: Extracellular superoxide dismutase (EC-SOD); Transgenic mice; Dimethylbenzanthracene (DMBA); 12-O-Tetradecanoylphorbol-13-acetate (TPA); 5-Bromo-2´-deoxy-uridine (BrdU); 8-Hydroxy-2´-deoxyguanosine (8OH-dG)

Address correspondence to Tae-Hoon Lee, Ph.D, Department of Oral Biochemistry, School of Dentistry, Chonnam National University, 300 Yongbong-Dong, Buk-Ku, Gwangju 500-757, Korea. Tel: 82 62 5304842, Fax: 82 62 5304848; E-mail: thlee83@chonnam.ac.kr

*These authors contributed equally to this work.

Oncology Research, Volume 15, pp. 343-350
0965-0407/05 $20.00 + .00
E-ISSN 1555-3906
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Antitumor Effects of Angiostatin K1-3 and Endostatin Genes Coadministered by the Hydrodynamics-Based Transfection Method

Keun Sik Kim and Yong Serk Park

Department of Biomedical Laboratory Science, College of Health Science, Yonsei University, Wonju 220-710, Republic of Korea

Angiostatin and endostatin are potent endothelial cell growth inhibitors and have been carefully evaluated for antiangiogenic cancer therapy. Previously, we have shown that subcutaneous administration of angiostatin K1-3 and endostatin genes complexed with liposomal vectors is a more practical treatment procedure than administration of angiostatin and endostatin proteins. This study provides additional conclusive evidence supporting the effectiveness of antiangiogenic cancer gene therapy employing angiostatin K1-3 and endostatin genes. Plasmids encoding a mouse angiostatin K1-3 gene (pFLAG-AngioK1/3) and an endostatin gene (pFLAG-Endo) were introduced by the hydrodynamic transduction method into mice carrying Matrigel plugs or B16BL6 mouse melanoma tumors. A single systemic injection of the two genes exhibited potent antiangiogenic and antitumor activity in the mouse model. Hydrodynamic coadministration of the genes inhibited the B16BL6 mouse melanoma growth and pulmonary metastasis more effectively than administration of either gene alone. Compared with the untreated control group, the mice cotreated with pFLAG-AngioK1/3 and pFLAG-Endo exhibited 75% reduction of tumor growth while those treated with pFLAG-AngioK1/3 or pFLAG-Endo showed 46% and 52% reduction, respectively. The cotreatment inhibited B16BL6 pulmonary metastasis formation by 80% while the inhibition induced by individual treatment with pFLAG-AngioK1/3 or pFLAG-Endo was 68% and 71%, respectively. These results provide additional evidence that systemic expression of angiostatin K1-3 and/or endostatin genes is a viable alternative procedure for antiangiogenic cancer therapy.

Key words: Angiostatin; Endostatin; Antiangiogenesis; Hydrodynamics-based transfection

Address correspondence to Yong Serk Park, Department of Biomedical Laboratory Science and Institute of Health Science, Yonsei University, Wonju 220-710, Republic of Korea. Tel: 82-33-760-2448; Fax: 82-33-763-5224; E-mail: yspark@dragon.yonsei.ac.kr

Oncology Research, Volume 15, pp. 351-364
0965-0407/05 $20.00 + .00
E-ISSN 1555-3906
Copyright © 2005 Cognizant Comm. Corp.
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A Comparison of the Ability of DMXAA and Xanthenone Analogues to Activate NF-kB in Murine and Human Cell Lines

See-Tarn Woon,1 Charu B. Reddy,1 Catherine J. Drummond,1 Mary Ann Schooltink,1 Bruce C. Baguley,1 Claudine Kieda,2 and Lai-Ming Ching1

1Auckland Cancer Society Research Center, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
2Centre de Biophysique Moleculaire, CNRS UPR4301, Orleans, France

DMXAA (5,6-dimethylxanthenone-4-acetic acid), the most potent of a series of xanthenone (XAA) analogues developed in this laboratory, is currently undergoing combination clinical trials as an antivascular agent for cancer treatment. XAAs have a complex mode of action, and in vitro assays that are predictive of in vivo antitumor activity have been difficult to develop. In this study, we have utilized a series including XAA, DMXAA, and mono-substituted XAA derivatives to determine firstly whether in vitro NF-kB activation of mouse cell lines predicts for the in vivo antitumor potential of this class of agents, and secondly whether the relative activity of these analogues is similar in murine and human cell lines. Electromobility shift assays were used to measure NF-kB activation in murine HECPP endothelial and 70Z/3 pre-B cells, and in human HPLNEC.B3 endothelial and Raji B-lymphoma cells. A significant correlation was obtained between NF-kB activation in HECPP cells by a series of XAA analogues at 100 mg/ml (r = 0.78, p = 0.008) and at 300 mg/ml (r = 0.75, p = 0.01) and the amount of hemorrhagic necrosis induced in Colon 38 tumors. Different structure-activity relationships were observed in human and murine cell lines. 8-MeXAA, which was inactive in HECPP and 70Z/3 murine cell lines, showed similar NF-kB activation to DMXAA in human HPLNEC.B3 cells and Raji B-lymphoma cells. These results suggest that the receptor protein(s) in human cells that mediate the human response may have a lower stringency to that for murine cells. We also noted differences in the dose-response relationships for NF-kB activation between lymphoid and endothelial lines that were species independent. With increasing concentrations of DMXAA, NF-kB activation in both murine and human lymphoid lines showed a reproducible fluctuation, while in endothelial lines, the intensity of NF-kB activation was relatively constant above a threshold concentration. The results demonstrate interspecies differences in the NF-kB response to XAA analogues, and may also reflect the complex nature of NF-kB regulation.

Key words: DMXAA; NF-kB; Endothelial; Lymphoid; Antitumor

Address correspondence to Lai-Ming Ching, Auckland Cancer Society Research Center, Faculty of Medical and Health Sciences, University of Auckland, Private Bag 92019, Auckland, New Zealand. Tel: (64 9) 3737 599; Fax: (64 9) 3737 502; E-mail: l.ching@auckland.ac.nz

Oncology Research, Volume 15, pp. 365-372
0965-0407/05 $20.00 + .00
E-ISSN 1555-3906
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

CD9 Overexpression Suppressed the Liver Metastasis and Malignant Ascites via Inhibition of Proliferation and Motility of Small-Cell Lung Cancer Cells in NK Cell-Depleted SCID Mice

Rui Zheng,1,4 Seiji Yano,1 Helong Zhang,1 Emiko Nakataki,1 Isao Tachibana,2 Ichiro Kawase,2 Seiji Hayashi,3 and Saburo Sone1

1Department of Internal Medicine and Molecular Therapeutics, University of Tokushima Graduate School, Tokushima, Japan
2Department of Molecular Medicine, Osaka University Graduate School of Medicine, Osaka, Japan
3National Hospital Organization, Kinki-Chuo Chest Medical Center, Osaka, Japan
4Department of Respiratory Internal Medicine, First Affiliated Hospital, Institute of Respiratory Disease, China Medical University, Shenyang, Liaoning, People's Republic of China

CD9, a transmembrane protein known as motility-related protein-1, plays a pivotal role in regulating cell adhesion, motility, and proliferation, and has been regarded as an important metastasis-inhibitory factor of various human cancers. However, little information has been obtained regarding the highly metastatic human small-cell lung cancer (SCLC). In the present study, an SCLC cell line (OS3-R5), lacking CD9 expression, was transfected with human CD9 gene to assess the role of CD9 on the metastatic potential of SCLC. CD9 gene transfection into OS3-R5 cells resulted in cell proliferation and motility in vitro. Parental and mock-transfected OS3-R5 cells developed liver metastasis and malignant ascites when they were intravenously inoculated into NK cell-depleted SCID mice. CD9 gene transfection into OS3-R5 cells caused suppression of the liver metastasis and malignant ascites. Immunohistochemical analysis revealed that the number of proliferating tumor cells was significantly fewer in liver lesions produced by CD9 gene-transfected OS3-R5 cells than those produced by parental or mock control OS3-R5 cells. In addition, no detectable levels of CD9 were expressed in metastatic tumor cells in mice bearing CD9 gene-transfected OS3-R5 cells, as well as those in mice bearing parental or mock control OS3-R5 cells. These results suggest that the restored expression of CD9 in SCLC cells may reduce the metastatic spread of SCLC cells via the inhibition of cell proliferation and motility.

Key words: CD9; Invasion; Proliferation; Liver metastasis; Malignant ascites

Address correspondence to Seiji Yano, Department of Internal Medicine and Molecular Therapeutics, University of Tokushima Graduate School, Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. Tel: 81-88-633-7127; Fax: 81-88-633-2134; E-mail: manae@clin.med.tokushima-u.ac.jp

Oncology Research, Volume 15, pp. 373-383
0965-0407/05 $20.00 + .00
E-ISSN 1555-3906
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Decoy Molecules Based on PNA-DNA Chimeras and Targeting Sp1 Transcription Factors Inhibit the Activity of Urokinase-Type Plasminogen Activator Receptor (uPAR) Promoter

Monica Borgatti,1 Douglas D. Boyd,2 Ilaria Lampronti,1 Nicoletta Bianchi,1 Enrica Fabbri,1 Michele Saviano,3 Alessandra Romanelli,4 Carlo Pedone,3 and Roberto Gambari1,5

1Department of Biochemistry and Molecular Biology, Section of Molecular Biology, Ferrara University, Ferrara, Italy
2Department of Cancer Biology, M.D. Anderson Cancer Center, University of Texas, Houston, TX, USA
3Institute of Biostructures and Bioimages, CNR, Napoli, Italy
4Department of Biological Sciences, University of Naples "Frederico II," Naples, Italy
5Biotechnology Centre, Ferrara University, Ferrara, Italy

The expression levels of urokinase-type plasminogen activator receptor (uPAR) are strongly correlated with metastatic potential in human cancer cell lines of melanoma, breast, lung, and colon. Therefore, targeting of uPAR could have practical implications in the treatment of neoplastic diseases. Because the expression of uPAR is regulated at the level of transcription in part by Sp1, we designed and tested transcription factors decoy molecules targeting Sp1 with the aim of inhibiting uPAR gene expression. The main objective of the present study was to determine whether decoy molecules based on peptide nucleic acids (PNA)-DNA chimeras mimicking Sp1 binding sites might be proposed as useful reagents to alter expression of Sp1-regulated genes involved in tumor invasion and metastasis. The results obtained firmly indicate that Sp1 binding molecules based on PNA-DNA-PNA chimeras are powerful decoys, as they efficiently inhibit the interactions between Sp1 and the uPAR promoter elements. Experiments performed on hepatoma HepG2 cells transfected with a plasmid containing the firefly luciferase gene reporter under the control of the human uPAR promoter demonstrate that PNA-DNA-PNA-based decoy molecules are potent inhibitors of the transcriptional activity of the uPAR promoter. Our results suggest that these molecules warrant attention for the design of novel antimetastatic drugs.

Key words: Gene transcription; Peptide nucleic acids (PNA); PNA-DNA chimeras; Sp1; Urokinase-type plasminogen activator receptor (uPAR); Transcription factor decoy; Gene therapy

Address correspondence to Professor Roberto Gambari, Department of Biochemistry and Molecular Biology, Ferrara University, Via Fossato di Mortara 74, 44100 Ferrara, Italy. E-mail: gam@unife.it

Oncology Research, Volume 15, pp. 385-392
0965-0407/05 $20.00 + .00
E-ISSN 1555-3906
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Irinotecan/5-Fluorouracil Combination Induces Alterations in Mitochondrial Membrane Potential and Caspases on Colon Cancer Cell Lines

Ivana Grivicich,1,2 Andréa Regner,1,3 Adriana B. da Rocha,1,3 Luciano B. Grass,1,3 Pedro A. G. Alves,1 Guilherme B. Kayser,1 Gilberto Schwartsmann,2,4 and João A. Henriques2,5,6

1Laboratório de Marcadores de Estresse Celular, Centro de Pesquisas em Ciências Médicas, Universidade Luterana do Brasil, Canoas, RS, Brazil
2Programa de Pós-Graduação em Genética e Biologia Molecular, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
3Programa de Pós Graduação em Diagnóstico Genético e Molecular, Universidade Luterana do Brasil, Canoas, RS, Brazil
4South-American Office of Anticancer Drug Development, Porto Alegre, RS, Brazil
5Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brazil
6Faculdade de Farmácia, Universidade Luterana do Brasil, Canoas, RS, Brazil

The combination of irinotecan (CPT-11) and 5-fluorouracil (5-FU) is currently used in the treatment of advanced colorectal carcinoma. When compared to both agents alone, CPT-11 followed by 5-FU treatment demonstrated a synergistic effect. This observation can be related to increased in apoptosis induction after caspase activation. Several studies have demonstrated that changes in mitochondrial membrane potential occur earlier in apoptosis. In this study, we verified whether the collapse in mitochondrial membrane and the activation of caspases is responsible for increased apoptosis observed with CPT-11/5-FU treatment. Thus, HT-29 and SNU-C4 human colon carcinoma cell lines were exposed for 24 h to each drug alone, and to various combinations and treatment sequences, and assessed for colony formation, changes in the mitochondrial membrane potential, and the activities of caspase-3, -8, and -9. The CPT-11/5-FU treatment induced apoptosis in both cell lines; however, the most pronounced effect was observed in HT-29 cells. In these cells, both caspase-3 and -9 were involved in the activation of apoptosis after CPT-11/5-FU treatment. Moreover, in these cells, a reduction of 50% in mitochondrial membrane potential was observed with this treatment. On the other hand, in the SNU-C4 cell line in addition to caspase-3 and -9, caspase-8 seems to be important to apoptosis after CPT-11/5-FU treatment. Furthermore, in this cell line we did not observe alterations in mitochondrial membrane potential. In spite of the differences among the cell lines, these results indicated that the increase in apoptosis in HT-29 cells observed with CPT-11 followed by 5-FU treatment could be explained by a disruption in mitochondria membrane potential that induced caspases activation.

Key words: CPT-11/5-FU combination; Apoptosis; Caspases; Mitochondrial membrane potential; Colon cancer cells

Address correspondence to Ivana Grivicich, Ph.D., Laboratório de Marcadores de Estresse Celular, Centro de Pesquisas em Ciências Médicas, Universidade Luterana do Brasil, Av. Farroupilha, 8001, Prédio 22, 5o andar, 92420-280 Canoas, RS, Brazil. Tel: 55 51 477 9219; Fax: 55 51 478 1747; E-mail: grivicich@terra.com.br

Oncology Research, Volume 15, pp. 393-398
0965-0407/05 $20.00 + .00
E-ISSN 1555-3906
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

There Is No Evidence That the SDHB Gene Is Involved in Neuroblastoma Development

Elena Grau,1 Silvestre Oltra,1 Carmen Orellana,1 Miguel Hernández-Martí,2 Victoria Castel,3 and Francisco Martínez1

1Unidad de Genética y Diagnóstico Prenatal, Hospital Universitario La Fe, Valencia, Spain
2Servico de Anatomía Patológica, Hospital Universitario La Fe, Valencia, Spain
3Unidad de Oncología Pediátrica, Hospital Universitario La Fe, Valencia, Spain

Neuroblastoma and pheochromocytoma have the same embryonal origin. They originate from neural crest cells, and they usually affect suprarenal glands. The SDHB gene encodes the B subunit of succinate dehydrogenase, a protein implicated in the electron transport chain and Krebs cycle. Some mutations have been described in this gene in pheochromocytoma, and this gene could be an appropriate candidate for its study in neuroblastoma given its localization in 1p35-36. The aim of this study was to analyze neuroblastoma tumors in order to assess a possible implication of this gene in neuroblastoma development. We studied 28 neuroblastoma tumor samples from different stages. Mutation research in genomic DNA was carried out after individual amplification of each of the eight SDHB exons by SSCP analysis and sequencing of those samples with migration pattern variants. No variant was found except for three polymorphisms in four neuroblastoma samples. The first polymorphism was a synonymous A -> C change in the third position of codon 6 (exon 1). The other two polymorphisms were a TTC insert at the 5´ flanking intron sequence of exon 5 in a stretch of seven TTC repeats. Upon the basis of posterior microsatellite instability and hypermethylation promoter studies, which were not significant, we can conclude that the SDHB gene, a positional candidate gene, is unlikely to be related to either initiation or tumoral progression in neuroblastoma.

Key words: SDHB gene; Neuroblastoma development; Succinate dehydrogenase

Address correspondence to Dr. Elena Grau, Unidad de Genética, Hospital Universitario La Fe, Av/Capanar, 21, 46009 Valencia, Spain. E-mail: elena_grau@hotmail.com

Oncology Research, Volume 15, pp. 399-408
0965-0407/05 $20.00 + .00
E-ISSN 1555-3906
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Treatment With Stem Cell Differentiation Stage Factors of Intermediate-Advanced Hepatocellular Carcinoma: An Open Randomized Clinical Trial

Tito Livraghi,1 Franca Meloni,1 Alberto Frosi,2 Sergio Lazzaroni,3 Mariano Bizzarri,4 Luigi Frati,4 and Pier Mario Biava5

1Department of Radiology, Civil Hospital of Vimercate, Vimercate, Milan, Italy
2Hepatology-Gastroenterology Unit, Internal Medicine Department, Civil Hospital of Sesto S. Giovanni, Milan, Italy
3Department of Internal Medicine, San Biagio Hospital, Clusone, Bergamo, Italy
4Department of Experimental Medicine and Pathology, University of Rome "La Sapienza", Rome, Italy
5Foundation for Research into the Biological Therapies of Cancer, Civil Hospital of Sesto S. Giovanni, Milan, Italy

There is no standard treatment for patients with advanced hepatocellular carcinoma (HCC). We developed a product containing stem cells differentiation stage factors (SCDSF) that inhibits tumor growth in vivo and in vitro. The aim of this open randomized study was to assess its efficacy in patients with HCC not suitable for resection, transplantation, ablation therapy, or arterial chemoembolization. A total of 179 consecutive patients were enrolled. We randomly assigned the patients to receive either SCDSF or only conservative treatment. Primary end points were tumor response and survival. Secondary end points were performance status and patient tolerance. Randomization was stopped at the second interim analysis (6 months) of the first 32 patients recruited when the inspection detected a significant difference in favor of treatment (p = 0.037). The responses to the therapy obtained in 154 additional patients confirmed previous results. Evaluation of survival showed a significant difference between the group of patients who responded to treatment versus the group with progression of disease (p < 0.001). Of the 23 treated patients with a performance status (PS) of 1, 19 changed to 0. The study indicated the efficacy of SCDSF treatment of the patients with intermediate-advanced HCC.

Key words: Biological response modifiers; Embryo; Stem cells; Differentiation factors; Hepatocellular carcinoma

Address correspondence to Pier Mario Biava, Foundation for Research into the Biological Therapies of Cancer, Sesto S. G. Hospital, Viale Matteotti 83, 20099 Sesto S.G., Milano, Italy. Tel: 39 02 26257365; Fax: 39 02 26257524; E-mail: biava@tiscali.it