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Oncology Research, Volume 12, pp. 355-359
0965-0407/01 $20.00 + .00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Antagonistic Effect of the Combination Gemcitabine/Topotecan in Ovarian Cancer Cells

Mariagrazia Distefano, Cristiano Ferlini, Rosa De Vincenzo, Cristiana Gaggini, Salvatore Mancuso, and Giovanni Scambia

Laboratory of Antineoplastic Pharmacology, Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Rome, Italy

The in vitro interaction between the new antimetabolite gemcitabine (GEM) and topotecan (TPT) was analyzed in A2780 ovarian cancer cells. The growth inhibitory effect was assessed after 3 days of drug exposure. GEM and TPT obtained in vitro IC50 values of 2.1 ± 0.9 and 33.7 ± 10.2 nM, respectively. The interaction between GEM and TPT was evaluated by exposing cancer cells at increasing doses of GEM (0.1, 1, and 10 nM) and TPT (1, 10, 100, and 1000 nM). Analysis of data about the interaction between GEM and TPT was performed by applying the isobole method. An antagonistic effect was noticed when GEM was combined with TPT in the tested concentration range. DNA analysis was also performed and showed an augmentation of cells blocked in the G2/M phase during TPT exposure, while an increase of blocked cells in the G0/1 phase was observed after GEM treatment. This latter effect was predominant when the two drugs were used in combination. We also investigated the effect of sequential exposure to drugs, pretreating A2780 cells for 24 h with TPT and then for 48 h with GEM, and, conversely, pretreating A2780 cells with GEM for 24 h and thereafter with TPT for 48 h. Both these combined sequential treatments showed an antagonist effect of the drugs' combination. Long-term growth inhibition effect was established by clonogenic assay performed after 10 days of culture after drug treatment. Also these data confirmed the antagonistic effect between GEM and TPT in A2780 ovarian cancer cells.

Key words: Topotecan; Gemcitabine; Antagonism; Cell cycle

Address correspondence to Prof. Giovanni Scambia, Department of Obstetrics and Gynecology, Catholic University of the Sacred Heart, Lgo A. Gemelli, 8-00168, Rome, Italy. Tel: 06/35508736; Fax: 06-35508736/06-3051160; E-mail: giovanni.scambia@libero.it

Oncology Research, Volume 12, pp. 361-370
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Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Cytotoxicity, DNA Damage, and Cell Cycle Perturbations Induced by Two Representative Gold(III) Complexes in Human Leukemic Cells With Different Cisplatin Sensitivity

Marcella Coronnello,1 Giordana Marcon,2 Stefania Carotti,1 Barbara Caciagli,1 Enrico Mini,1 Teresita Mazzei,1 Pierluigi Orioli,3 and Luigi Messori3

1Department of Pharmacology, University of Florence, Florence, Italy
2CIRCSMB, Unit of Florence, Florence, Italy
3Department of Chemistry, University of Florence, Florence, Italy

The gold(III) complexes [Au(phen)Cl2]Cl and [Au(dien)Cl]Cl2 were recently shown to exert important cytotoxic effects in vitro on human tumor cell lines. To elucidate the biochemical mechanisms leading to cell death, the effects produced by these gold(III) complexes on the leukemic CCRF-CEM cell line--either sensitive (CCRF-CEM) or resistant to cisplatin (CCRF-CEM/CDDP)--were analyzed in detail by various techniques. For comparison purposes the effects produced by equitoxic concentrations of cisplatin were also analyzed. First, the dependence of the IC50 values of either complex on the incubation time was investigated. Cytotoxicity experiments confirmed that both gold(III) compounds retain their efficacy against the cisplatin-resistant line: only minimal cross-resistance with cisplatin was detected. Notably, [Au(phen)Cl2]Cl is more cytotoxic than [Au(dien)Cl]Cl2, with IC50 values of 7.4 and 6.0 M at 24 and 72 h, respectively, on the resistant line. Results of the COMET assay point out that both gold(III) complexes directly damage nuclear DNA. Remarkably, DNA damage inferred by either gold(III) complex in the two cell lines is larger than that produced by equitoxic cisplatin concentrations. Finally, the effects that either gold(III) complex produces on the cell cycle were investigated by flow cytometry. It was found that both complexes cause only moderate and transient cell cycle perturbations. Larger cell cycle perturbations are induced by equitoxic concentrations of cisplatin. The implications of the present results for the mechanism of action of cytotoxic gold(III) complexes are discussed.

Key words: Gold(III) complexes; Cytotoxicity; COMET assay; Flow cytometry

Address correspondence to Dr. Luigi Messori, Department of Chemistry, University of Florence, via Gino Capponi 7, 50121 Firenze, Italy. Tel: +39-55-2757556; Fax: +39-55-2757555; E-mail: messori@cerm.unifi.it

Oncology Research, Volume 12, pp. 371-381
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Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Cell-Cell Adhesion-Independent Killing Due to Lymphokine-Activated Killer Cells Against Glioblastoma Cell Lines

Fumio Komatsu and Tomoko Masuda

Blood Transfusion Service, School of Medicine, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyoku, Tokyo 113-8519, Japan

Lymphokine-activated killer (LAK) cells can kill several tumor cells. Their killing activity is generally due to cell-cell adhesion. Cell-cell adhesion of the LAK cells to the target cells is essential for LAK lysis. In this report, however, we describe that the LAK cells can also kill the target cells by cell-cell adhesion-independent killing. Killing occurred after the target cells were exposed to the LAK cells. When the LAK cells were added to glioblastoma cell lines T98G and U373MG (which proliferate by adhering to the bottom of a culture flask), the LAK cells killed them by cell-cell adhesion killing within 4 h (early killing). On the other hand, when small numbers of the LAK cells were added, some of the target cells escaped from the early killing. At 4 and 6 h after the adding the LAK cells, when the LAK cells were discarded from the flask by washing with PBS, the escaped cells still adhered and were alive. However, they ultimately died over the next 24-96 h (late killing). The late killing was the cell-cell adhesion-independent killing, because it occurred after the LAK cells were removed. In this killing, numerous granules and vacuoles appeared in the cytoplasm of the cells. The vacuoles enlarged and then the cells died. The cell death was different from apoptosis, because the nucleus was intact until the late stage and no DNA fragment laddering in the degenerated cells was recognized. The vacuoles were stained with acid phosphatase and the cell death was inhibited with 3-methyladenine (an inhibitor of lysosome), suggesting that the late killing may be autophagic cell death due to activated lysosome. Induction of late killing in tumor cells using the LAK cells may become one approach for cancer therapy.

Key words: LAK cells; Cell-cell adhesion-independent killing; Apoptosis; Necrotic-like cell death; Autophagic cell death

Address correspondence to Fumio Komatsu, M.D., Blood Transfusion Service, School of Medicine, Tokyo Medical and Dental University, Yushima 1-5-45, Bunkyoku, Tokyo 113-8519, Japan. Tel: 81-3-5803-5646, Fax: 81-3-5803-5647.

Oncology Research, Volume 12, pp. 383-395
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Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Prostaglandin A1 Inhibits Stress-Induced NF-kB Activation and Reverses Resistance to Topoisomerase II Inhibitors

Yoonkyung C. Boller, Lori M. Brandes, Rosalind L. Russell*, Z. Ping Lin**, Steven R. Patierno, and Katherine A. Kennedy

Department of Pharmacology, The George Washington University Medical Center, Washington, DC 20037

Stress conditions associated with solid tumors lead to the formation of heterogeneous tumor cell subpopulations and insensitivity to cancer chemotherapeutics. In this report, we show that EMT6 mouse mammary tumor cells treated with the chemical stress, brefeldin A (BFA), or the physiological stress, hypoxia, develop resistance to the topoisomerase II (topoII) inhibitors teniposide and etoposide. BFA and hypoxia treatment did not alter intracellular drug concentrations, topoII protein levels, or inhibit topoII activity. BFA and hypoxia did cause the activation of the nuclear transcription factor NF-kB. We demonstrate that pretreatment with the synthetic cyclopentenone prostaglandin A1 (PGA1) inhibits stress-induced NF-kB activation and reverses BFA- and hypoxia-induced resistance. The reversal of BFA-induced resistance can occur when PGA1 is administered either before or several hours after the induction of stress. Taken together, these data support the involvement of NF-kB in stress-induced drug resistance, show that pharmacologic inhibitors of NF-kB can disrupt the biological consequences of stress, and imply that inhibitors of NF-kB may be useful agents to enhance the clinical efficacy of topoII-directed chemotherapeutics.

Key words: Nuclear factor-kB; Prostaglandin A1; Drug resistance; Stress; Etoposide

Address correspondence to Dr. Katherine A. Kennedy, Department of Pharmacology, The George Washington University Medical Center, 2300 I Street N.W., Washington, DC 20037. Tel: (202) 994-2957; Fax: (202) 994-2870; E-mail: phmkak@gwumc.edu

*Current address: Pall Corporation, Long Island, NY 11050.
**Current address: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520.

Oncology Research, Volume 12, pp. 397-408
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Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Induction of Apoptosis by Cidofovir in Human Papillomavirus (HPV)-Positive Cells

Graciela Andrei, Robert Snoeck, Dominique Schols, and Erik De Clercq

Rega Institute for Medical Reseach, K.U.Leuven, B-3000 Leuven, Belgium

HPMPC (cidofovir, CDV) is an acyclic nucleoside phosphonate (ANP) with broad-spectrum activity against DNA viruses, including human papillomavirus (HPV). HPMPC has proved to be effective in the treatment of HPV-associated disease in several clinical investigations. In vitro, treatment of HPV-positive cells (compared with normal primary human keratinocytes) with HPMPC has resulted in a concentration- and time-dependent inhibition of cell proliferation. We have now evaluated the mechanism by which this compound induces cell death. Different parameters of apoptosis, that is, (i) induction of CPP32 (caspase-3) protease activity, (ii) translocation of phosphatidylserine (PS) from the inner part of the plasma membrane to the outer layer, (iii) disintegration of the nuclear matrix protein (NMP), (iv) DNA fragmentation, (v) number of cells in apoptotic phase following cell cycle analysis, showed that the mechanism of cell death following treatment with CDV is based on apoptosis. Annexin V staining showed that induction of apoptosis in HPV-positive cells was correlated with a decrease in the percentage of viable cells, while no significant changes in the percentages of living cells were noted in primary human keratinocytes (PHK) cell cultures. Furthermore, a remarkable accumulation of HPMPC-treated cells in the S phase of the cell cycle was observed. Apoptosis induction and S phase arrest were concentration and time dependent. Induction of apoptosis in HPV-positive cells by HPMPC was associated with accumulation of the tumor suppressor protein p53 and the cyclin-dependent kinase inhibitor p21/WAF-1. As HPMPC has proved to induce apoptosis, in a time- and concentration-dependent manner, in a number of HPV-positive cell lines, the regression of papillomatous lesions observed with HPMPC in patients may be due, at least in part, to the induction of apoptosis.

Key words: HPMPC (cidofovir); Human papillomavirus (HPV); Apoptosis

Address correspondence to Graciela Andrei, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. Tel: +32-16-337395; Fax: +32-16-337340; E-mail: graciela.andrei@rega.kuleuven.ac.be

Oncology Research, Volume 12, pp. 409-418
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Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Effects of Photodynamic Therapy With Hypericin in Mice Bearing Highly Invasive Solid Tumors

Michael Blank,1 Gad Lavie,2,3 Mathilda Mandel,2 and Yona Keisari1

1Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Israel
2Institute of Hematology & Blood Transfusion Center, Sheba Medical Center, Israel
3Department of Pathology, NYU Medical Center, New York, NY 10016

The tumoricidal properties of photodynamic therapy (PDT) with hypericin (HY) were evaluated in a highly metastatic adenocarcinoma (DA3Hi) and anaplastic squamous cell carcinoma (SQ2) tumors in vivo. Photosensitization of the tumor site with hypericin (HY-PDT) reduced primary tumor development and significantly prolonged the survival of tumor-bearing (TB) mice. Of these two tumors the squamous cell carcinoma emerged as more sensitive to HY-PDT compared with DA3Hi adenocarcinoma both in vitro and in vivo. HY-PDT caused extensive tumor necrosis that was followed by local, intratumoral, and systemic inflammatory reactions. Analyses of cytokine mRNA profiles reveal increases in mRNA levels of expression confined to inflammation-related cytokines both within the tumor and also systemically (measured in spleens). However, there was no evidence for any HY-PDT-induced antitumoral immune reactions. Our results suggest that PDT with hypericin can be considered as a supplementary treatment in the management of some invasive and metastatic cancers such as squamous carcinoma and similar tumors.

Key words: Photodynamic therapy; Hypericin; RT-PCR; Cytokines; Inflammation; Metastases

Address correspondence to Prof. Yona Keisari, Department of Human Microbiology, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. Tel: 972-3-6409871; Fax: 972-3-6406098; E-mail: ykeisari@ccsg.tau.ac.il

Oncology Research, Volume 12, pp. 419-427
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Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Synergistic Effects of Clinically Achievable Concentrations of 12-O-Tetradecanoylphorbol-13-acetate in Combination With all-trans Retinoic Acid, 1a,25-Dihydroxyvitamin D3, and Sodium Butyrate on Differentiation in HL-60 Cells

Xi Zheng,1 Richard L. Chang,1 Xiao-Xing Cui,1 Kathleen A. Kelly,1 Weichung Joe Shih,2,3 Yong Lin,2,3 Roger Strair,3 Junghan Suh,4 Zheng Tao Han,5 Arnold Rabson,3,4 and Allan H Conney1,3

1Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854
2Division of Biometrics, School of Public Health, University of Medicine and Dentistry of New Jersey, New Brunswick, NJ 08903
3Cancer Institute of New Jersey, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ 08903
4Center for Advanced Biotechnology and Medicine, University of Medicine and Dentistry of New Jersey - Robert Wood Johnson Medical School, Piscataway, NJ 08854
5Henan Tumor Research Institute, Zheng Zhou, Henan 450000, People's Republic of China

Our recent studies demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA) has pharmacological activity for the treatment of acute myelocytic leukemia patients. In the present study, we investigated the potential synergistic effect of all-trans retinoic acid (RA), 1a,25-dihydroxyvitamin D3 (VD3), and sodium butyrate (NaB) on TPA-induced differentiation in HL-60 human promyelocytic leukemia cells. The cells were treated once with these agents for 48 h or treated every 24 h for 96 h. Treatment of HL-60 cells once with TPA, RA, VD3, or NaB for 48 h resulted in concentration-dependent growth inhibition and cell differentiation. At clinically achievable concentrations, TPA (0.16 nM) increased the number of adherent cells and RA (0.1-1 mM) increased the number of nitroblue tetrazolium (NBT)-positive cells. The combinations of TPA (0.16 nM) with RA (0.1-1 mM), VD3 (1 nM), or NaB (100 mM) for 48 h synergistically increased differentiation as measured by the formation of adherent cells (P < 0.01). Moreover, cells treated with various combinations of low concentrations of TPA, RA, VD3, and NaB every 24 h for 96 h resulted in a further decrease in cell growth and an increase in differentiation. At clinically achievable concentrations, the strongest stimulation of differentiation was achieved in cells treated with a "cocktail" that combined TPA, RA, VD3, and NaB. The synergistic effect of combinations of TPA with RA or NaB at clinically effective concentrations on HL-60 cell differentiation suggests that the combination of these agents may improve the therapeutic efficacy of TPA for the treatment of acute promyelocytic leukemia (APL) patients. A differentiation "cocktail" that combines TPA, RA, VD3, and NaB may provide an even more effective strategy for improving the therapeutic efficacy of TPA and RA.

Key words: TPA; PMA; Leukemia; Growth; Differentiation

Address correspondence to Allan H. Conney, Laboratory for Cancer Research, Department of Chemical Biology, College of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854. Tel: (732) 445-4940; Fax: (732) 445-0687; E-mail: aconney@rci.rutgers.edu

Oncology Research, Volume 12, pp. 429-440
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Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Growth Inhibitory Effects and Radiosensitization Induced by Fatty Aromatic Acids on Human Cervical Cancer Cells

Gabriella Ferrandina,1 Perla Filippini,1 Cristiano Ferlini,1 Nicola Maggiano,2 Andrea B. Stoler,1 Erika Fruscella,1 Simona Mozzetti,1 Salvatore Mancuso,1 Ralph S. Freedman,4 Giovanni Scambia,1 and Franco O. Ranelletti3

Departments of 1Gynecology/Obstetrics, 2Pathology, and 3Histology, Catholic University, Rome, Italy
4Department of Gynecologic Oncology, M.D. Anderson Cancer Center, Houston, TX

Evidences have been reported that phenylacetic (PA) and phenylbutyric (PB) fatty aromatic acids can exert tumor growth inhibition in vitro and in vivo. Moreover, clinical trials also showed some activity for these drugs to modulate the expression of genes implicated in tumors growth, metastasis, immunogenicity, and to potentiate the efficacy of cytotoxic agents. The aim of the study was to examine the effects of PA and PB on the growth as well as sensitization to cisplatin and radiation in human cervical cancer cells. The effects of PA and PB on the proliferative activity and apoptosis induction in cervical tumor tissue was investigated. Both PA and PB exhibited a time- and dose-dependent antiproliferative activity in SW756 and ME180 cell lines: after 72-h treatment, the IC50 (concentration able to inhibit 50% of cell growth) of PB was 1.9 ± 0.2 mM and 1.5 ± 0.2 mM in SW756 and ME180 cells, respectively, while the IC50 of PA was 13.0 ± 1.7 mM and 10.0 ± 1.2 mM in SW756 and ME180 cells, respectively. In tumor tissue biopsies obtained from patients affected by squamous cervical cancer, both drugs resulted in a marked reduction of the percentage of bromodeoxyuridine-labeled cells compared with untreated samples (19.0 ± 1.63% in untreated tissues with respect to 1.30 ± 0.54% and 4.20 ± 2.50% of stained cells after treatment with PA (30 mM) (P < 0.0001) and PB (5 mM) (P < 0.0001), respectively). Moreover, analysis of the staining with M30 monoclonal antibody revealed that PA (30 mM) and PB (5 mM) were able to produce a marked increase in the number of stained apoptotic nuclei with respect to untreated samples. Finally, PB and PA were shown to enhance the sensitivity of SW756 to radiation and to exert an additive effect when combined with cisplatin. A significant reduction of the processed form of p21ras and rhoB proteins in the membrane fraction of cells exposed to PA and PB was observed. When farnesol, which is able to circumvent the enzymatic step inhibited by PA and PB, was added to the medium only a partial reversal of the growth inhibition and potentiation of sensitivity to radiation induced by PA and PB were found. In conclusion, the growth inhibitory properties of fatty aromatic acids suggests that these molecules could represent the prototype of a new class of compounds with some therapeutic potential in cervical cancer.

Key words: Fatty aromatic acids; Cervical cancer; Protein isoprenylation

Address correspondence to Giovanni Scambia, Department of Gynecology/Obstetrics, Catholic University of Rome, L.go A. Gemelli,8-00168-Rome Italy. Tel/Fax: 39-06 35508736; E-mail: GIOVANNI@.SCAMBIA LIBERO.IT