ognizant Communication Corporation



Oncology Research, Volume 15, pp. 1-9
0965-0407/05 $20.00 + .00
Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Zoledronic Acid Mediates Ras-Independent Growth Inhibition of Prostate Cancer Cells

Masaki Nogawa, Takeshi Yuasa, Shinya Kimura, Junya Kuroda, Hidekazu Segawa, Kiyoshi Sato, Asumi Yokota, Mitsutera Koizumi, and Taira Maekawa

Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital

Zoledronic acid (ZOL), the most potent known bisphosphonate, is clinically efficacious against advanced prostate cancer, although the molecular mechanism by which bisphosphonates prevent prostate cancer cell growth remains unknown. Because Ras is the most thoroughly characterized member of the small G-proteins involved in the regulation of many cellular functions including several oncogenic pathways, the aim of this study was to clarify whether Ras is the molecular target of ZOL in prostate cancer cells. The prostate cancer cell lines PC-3, DU145, and LNCaP were used. Cell proliferation was determined by a modified MTT assay. Geranylgeranyol (GGOH) and farnesol (FOH) were used as analogues of geranylgeranyl-pyrophosphate and farnesyl-pyrophosphate, respectively. Changes in expression and/or membrane localization of Ras, Rap1, and phosphorylated MAPK were evaluated by Western blotting. ZOL mediated growth inhibition of prostate cancer cells in a dose- and time-dependent manner. The ZOL-induced growth inhibitory effect was circumvented by the addition of GGOH. In contrast, FOH did not reverse the growth inhibitory effect of ZOL. The amount of membrane-anchored Ras was clearly independent of ZOL-mediated growth inhibition. Unexpectedly, ZOL induced N- and H-Ras expression of the cytosolic fraction. Ras does not appear to be the molecular target for ZOL-induced growth inhibition. Prevention of geranylgeranylation rather than farnesylation is an important therapeutic target in prostate cancer.

Key words: Zoledronic acid; Prostate cancer; Ras; Bisphosphonate; Geranylgeranylation

Address correspondence to Takeshi Yuasa, M.D., Department of Transfusion Medicine and Cell Therapy, Kyoto University Hospital, 54 Kawahara-cho Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. Tel: +81-75-751-3630; Fax: +81-75-751-4283; E-mail: yuasa@kuhp.kyoto-u.ac.jp

Oncology Research, Volume 15, pp. 11-20
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Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Antineoplastic Agents 470. Absolute Configuration of the Marine Sponge Bromopyrrole Agelastatin A*

George R. Pettit, Sylvie Ducki, Delbert L. Herald, Dennis L. Doubek, Jean M. Schmidt, and Jean-Charles Chapuis

Cancer Research Institute and Department of Chemistry and Biochemistry, Arizona State University, P.O. Box 872404, Tempe, AZ

Two bromopyrrole marine alkaloids were isolated from the Mexican sponge, Agelas sp.: hymenidin (1) and agelastatin A (2). The structures were elucidated by analysis of their spectroscopic data and found to correspond to those in the literature. The absolute configuration of agelastatin A (2) was elucidated by single-crystal X-ray diffraction methods. Agelastatin A (2) exhibited strong activity against a panel of human cancer cell lines as well as human umbilical vein endothelial cells.

Key Words: Agelastatin A; Anticancer; Hymenidin; Marine sponge

Address correspondence to Dr. G. R. Pettit, Cancer Research Institute, Arizona State University, P.O. Box 872404, Tempe, AZ 85287-2404. Tel: (480) 965-3351; Fax: (480) 965-8558; E-mail: bpettit@asu.edu.

*Dedicated to the memory of Dr. Daniel G. Miller (May 7, 2003), past President of the Strang Cancer Prevention Center and outstanding advocate of improved cancer treatment and prevention.

Oncology Research, Volume 15, pp. 21-37
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Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Mechanisms of Action of Differentiation Inducers: Detection of Inducer Binding Protein(s) in Murine Erythroleukemia Cells

Ioannis S. Pappas,1* John D. Lambris,2 Ioannis S. Vizirianakis,1 Michael S. Winters,2 and Asterios S. Tsiftsoglou1

1Laboratory of Pharmacology, Department of Pharmaceutical Sciences, School of Health Sciences, Aristotle University of Thessaloniki, GR-54121 Thessaloniki, Macedonia, Greece
2Protein Chemistry Laboratory, Department of Pathology & Laboratory Medicine, University of Pennsylvania, 402 Stellar Chance, Philadelphia, PA 19104

We have shown previously that murine erythroleukemia (MEL) and human neuroectodermal RD/TE-671 cells are induced to differentiate by ureido derivatives of pyridine (UDPs) and may contain inducer binding protein(s). In the present study, we prepared radiolabeled [3H]UDP {2-(3-ethylureido)-6-[3H]-acetylaminopyridine} as ligand and investigated whether it interacts selectively with novel binding proteins. MEL and RD/TE-671 cells, incubated with the inducer [3H]UDP and subsequently fractionated, yielded a radiolabeled postmitochondrial soluble fraction containing the [3H]UDP-protein complex. We purified the UDP binding protein by using UDP-sepharose affinity chromatography, gel filtration, and SDS-PAGE electrophoresis and analyzed its structure. The data presented here indicate for the first time that the inducer UDP interacts with a 38,333 ± 30 Da binding protein(s) (p38), of unknown function, in both cell lines. Microsequencing and sequence alignment search revealed that the p38 protein(s) contains at least two homologous domains, one being part of ABC-type transporters and another found in the Wingless-type (Wnt) proteins. Kinetic analysis revealed that the p38 forms a relatively stable protein complex with [3H]UDP that accumulates within the cytosol and nucleus of MEL cells during the precommitment period. This complex, however, decays later on after commitment to erythroid maturation has been initiated. De novo protein and mRNA synthesis is needed for the UDP-p38 complex to form, as shown by the use of metabolic inhibitors. Purified p38 was used to develop an anti-p38 polyclonal serum, and Western blot analysis revealed that the level of p38 was quite similar in both UDP-inducible and -resistant MEL subclones that we developed. Although only a portion of the primary structure of the p38 is known from microsequencing, the mechanism by which the UDP-p38 complex contributes to induction of differentiation in both UDP-responsive mouse MEL and human RD/TE-671 cells is discussed.

Key words: Inducers; Differentiation; Inducer binding protein; Murine erythroleukemia (MEL) cells; Human RD/TE-671 cells

Address correspondence to Prof. Asterios S. Tsiftsoglou, Laboratory of Pharmacology, Department of Pharmaceutical Sciences, School of Health Sciences, Aristotle University of Thessaloniki, GR-54121 Thessaloniki, Macedonia, Greece. Tel: +30-2310-997631; Fax: +30-2310-997618; E-mail: tsif@pharm.auth.gr

*Present address: Laboratory of Pharmacology, Faculty of Veterinary Medicine, School of Health Sciences, University of Thessaly, 224 Trikalon Str., 43100 Karditsa, Thessaly, Greece.

Oncology Research, Volume 15, pp. 39-48
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Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

In Vitro Study of CI-994, a Histone Deacetylase Inhibitor, in Non-Small Cell Lung Cancer Cell Lines

Maura Loprevite,1 Marcello Tiseo,1 Francesco Grossi,1 Tindaro Scolaro,2 Claudia Semino,3 Alessandra Pandolfi,5 Roberto Favoni,4 and Andrea Ardizzoni6

1Oncologia Medica A, 2Oncologia Radioterapica, 3Immunoterapia Cellulare, 4Farmacologia e Neuroscienze, Istituto Nazionale per la Ricerca sul Cancro, Genoa, Italy
5Pfizer Italia, Rome, Italy
6Azienda Ospedaliera Universitaria, Parma, Italy

CI-994 (N-acetyldinaline) is a novel oral compound with a wide spectrum of antitumor activity in preclinical models, in vitro and in vivo. The mechanism of action may involve inhibition of histone deacetylation and cell cycle arrest. We studied the action of CI-994 on two non-small cell lung cancer (NSCLC) cell lines: A-549 (adenocarcinoma) and LX-1 (squamous cell carcinoma). Different drug concentrations were tested, ranging from 0.01 to 160 mM at 24, 48, and 72 h of treatment, with MTT assay. A concentration-dependent cell survival inhibition was observed, with an IC50 at 80 mM. The effect of CI-994, as demonstrated by recovery experiments, was cytostatic and seemed to be superimposable in both cell lines. Cytofluorimetric analysis to assess cell cycle perturbation and apoptosis was performed after 24 h of treatment, indicating a cell block with concomitant increase at G0/G1 phase, a reduction at S phase level at 20, 40, 80, and 160 mM, and apoptosis at the higher concentration (160 mM). When CI-994 was combined with antineoplastic agents commonly used in NSCLC management, a marked synergism of action (R = 1.8, R = 1.5) was observed between CI-994 (40 mM) and gemcitabine (0.01 mM) at 48 and 72 h of treatment. The same result was obtained with docetaxel (0.001 mM) combination (R = 1.4, R = 1.2), but no synergism of action was noted with paclitaxel. CI-994 showed no radiopotentiating effects, when combined with 100, 200, or 400 cGy irradiation. In conclusion, our experiments indicate that CI-994 is a promising novel cytostatic for the treatment of NSCLC. Its use in combination with standard anticancer agents, such as gemcitabine and docetaxel, is warranted.

Key words: Deacetylase inhibitors; CI-994; Preclinical investigation; Combined therapy; Lung cancer; Cell lines

Address correspondence to Dr. Andrea Ardizzoni, Department of Medical Oncology, University Hospital of Parma, Via Gramsci 14, 43100 Parma, Italy. Tel: +39-0521-702316; Fax: +39-0521-995448; E-mail: aardizzoni@ao.pr.it

Oncology Research, Volume 15, pp. 49-57
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Copyright © 2005 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Frequent Overexpression of STK15/Aurora-A/BTAK and Chromosomal Instability in Tumorigenic Cell Cultures Derived From Human Ovarian Cancer

Wei Hu,1 John J. Kavanagh,1 Michael Deaver,2 Dennis A. Johnston,3 Ralph S. Freedman,4 Claire F. Verschraegen,6 and Subrata Sen5

1Department of Gynecologic Medical Oncology, 2Department of Pathology, 3Department of Biomathematics, 4Department of Gynecology Oncology, and 5Department of Molecular Pathology, The University of Texas M.D. Anderson Cancer Center, Houston, TX 77030
6Experimental Therapeutics, University of New Mexico, Albuquerque, NM 87131

The STK15 (also known as Aurora-A/BTAK) gene localized on chromosome 20q13 and encoding a centrosome-associated serine/threonine kinase is amplified and overexpressed in multiple human tumor cell types. Overexpression of this gene is involved in tumorigenic transformation, induction of centrosome duplication-distribution abnormalities, and aneuploidy in mammalian cells. To examine the potential role of STK15 in ovarian tumorigenesis, its mRNA and protein expression status were examined in cells grown in culture from 15 ovarian cancer specimens using semiquantitative RT-PCR and Western blot analysis. Normal ovarian surface tissues and the near diploid nontumorigenic breast epithelial cell line MCF10 were used as controls. The status of STK15 correlated with transformation-associated cellular phenotypes including tumorigenicity in nude mice, p53 expression level, and chromosomal ploidy. For chromosome ploidy analyses, FISH was carried out with direct fluorescence-labeled a-satellite probes for chromosome 3 and 17. STK15 mRNA was found overexpressed in 10 of the 15 ovarian cancer cell cultures. Five of these cell cultures revealed a truncated form of the STK15 protein with a molecular mass of 36 kDa. When tested for tumorigenicity in nude mice, 9 of the 10 cell cultures that overexpressed STK15 mRNA formed tumors in nude mice, while only one of the five cell cultures with no overexpression did. Cells overexpressing STK15 mRNA showed significant correlation with chromosome 3 polysomy. Six of the 13 (46%) cell cultures analyzed for p53 expression revealed overexpression of p53 and five of these six (83%) also overexpressed STK15. Four of the remaining seven cultures (57%) with overexpression of STK15 revealed minimal or no expression of p53. These results demonstrate that overexpression of STK15 significantly correlates with nude mice tumorigenicity and chromosomal aneuploidy in human ovarian cancer cells grown in vitro. Additionally, cells overexpressing STK15 also revealed frequent coordinate loss of wild-type p53 function manifested either as highly expressed intense staining reflective of a mutant form of p53 or almost complete absence of p53 staining. Overexpression of STK15 with coordinate loss of wild-type p53 function thus appears to play an important role in ovarian tumorigenesis and offers a novel molecular target in designing effective therapy of human ovarian cancer.

Key words: Ovarian neoplasm; STK15/Aurora-A/BTAK; Centrosomal kinase; Aneuploidy

Address correspondence to Wei Hu, Department of Gynecologic Medical Oncology, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Box 401, Houston, TX 77030. Tel: (713) 563-1786; Fax: (713) 745-1541; E-mail: weihu@mdanderson.org or ssen@mdanderson.org