ognizant Communication Corporation



Oncology Research, Volume 14, pp. 1-19
0965-0407/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Modeling the Interaction of Paclitaxel With b-Tubulin

Michael T. G. Ivery and Tuyet Le

Faculty of Pharmacy, University of Sydney, N.S.W. 2006, Australia

The natural product cytotoxic agent, paclitaxel, partly induces cell death through its ability to disrupt mitosis by binding to the microtubule protein b-tubulin. Structural characterization of the paclitaxel-b-tubulin complex, a first stage in the design of new antimitotics, has been complicated by contradictory observations obtained from different experimental techniques [electron crystallography, fluorescence resonance energy transfer (FRET), and photo-affinity labeling (PAL)] used to examine the complex. In this study we have used a range of molecular modeling techniques including restrained conformational searching and computer-assisted docking to propose that these contradictions may be resolved by the hypothesis that the nature of the interaction of paclitaxel with b-tubulin depends on the physical form of the tubulin examined. In particular, our analysis identified a binding mode that is consistent with available data for the interaction of paclitaxel with b-tubulin in polymerized tubulin or microtubules. This orientation is characterized by an alternate conformation (inverted orientation of side chains) and inverted orientation of the taxane core of paclitaxel within its tubulin binding site compared with the electron crystallographic structure. The proposed structure, however, is only marginally consistent with electron crystallographic data for the interaction of paclitaxel with b-tubulin in Zn-induced tubulin sheets. Similarly, the electron crystallographic structure shows poor correlation with FRET, solid-state NMR, and some observed SAR relationships for paclitaxel interacting with polymerized tubulin or microtubules. These observations suggest to us that the interaction of paclitaxel with Zn-induced tubulin sheets may not reflect paclitaxel's interaction with tubulin in microtubules and hence may not be an appropriate guide for rational drug design programs.

Key words: Paclitaxel; b-Tubulin; Structure of interaction; Microtubules; Molecular modeling

Address correspondence to Michael T. G. Ivery, Faculty of Pharmacy, University of Sydney, N.S.W. 2006, Australia. Tel: 61-2-9351-6005; Fax: 61-2-9351-439; E-mail: michaeli@pharm.usyd.edu.au

Oncology Research, Volume 14, pp. 21-29
0965-0407/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Reductive Metabolism of the Nitroimidazole-Based Hypoxia-Selective Cytotoxin NLCQ-1 (NSC 709257)

Maria V. Papadopoulou, Ming Ji, Mira K. Rao, and William D. Bloomer

Department of Radiation Medicine at Evanston Northwestern Healthcare, 2650 Ridge Avenue, Evanston, IL 60201

The enzymatic cell-free metabolism of the novel hypoxia-selective cytotoxin 4-[3-(2-nitro-1-imidazolyl)-propylamino]-7-chloroquinoline hydrochloride (NLCQ-1) was investigated under hypoxic or aerobic conditions in the presence of purified reductive enzymes or isolated rat liver microsomes by monitoring the parent compound with HPLC-UV analysis. Enzymatic reduction of NLCQ-1 with isolated rat liver microsomes and NADPH or NADH showed that, only under hypoxic conditions, ca. 45% and 60% of the parent compound was reduced, respectively, within 1 h of incubation (37°C). Under identical conditions but in the presence of 2´-AMP (a P450 reductase inhibitor), 6-propyl-2-thiouracil or p-hydroxymercuribenzoate (cytochrome b5 reductase inhibitors), NLCQ-1 reduction was inhibited. Enzymatic cell-free metabolism of NLCQ-1 with recombinant human DT-diaphorase (DTD) and NADPH or NADH under hypoxic or aerobic conditions showed that <5% of the compound was reduced within 2 h. Reduction kinetics with human P450 reductase-expressing microsomes showed ca. 75% or 50% reduction of NLCQ-1 under hypoxic or aerobic conditions, respectively, after 2-h incubation. These results suggest that DTD is not involved in the initial steps of the bioreductive metabolism of NLCQ-1, although it could be involved with metabolites of NLCQ-1, and that cytochrome P450 and cytochrome b5 reductases play a significant role in the bioreductive metabolism of NLCQ-1.

Key words: NLCQ-1; Bioreductive drug; Hypoxic cytotoxin; Enzymatic reduction

Address correspondence to Maria V. Papadopoulou, Evanston Northwestern Healthcare, Department of Radiation Medicine, 2650 Ridge Avenue, Evanston, IL 60201. Tel: (847) 570-2262; Fax: (847) 570-1878; E-mail: m-papadopoulou@nwu.edu

Oncology Research, Volume 14, pp. 31-37
0965-0407/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Increased Activity and Expression of Phospholipase D2 in Human Colorectal Cancer

Hirokazu Oshimoto, Shinichi Okamura, Makoto Yoshida, and Masatomo Mori

First Department of Internal Medicine, Gunma University School of Medicine, Gunma 371-8511, Japan

Phospholipase D (PLD) has been reported as relevant to some types of human cancer, but its role in human colorectal cancer still remains to be elucidated. Thus, this study was conducted to determine the activity and the expression of PLD2 in human colorectal cancer. A significant elevation of PLD2 activity and higher expression of PLD2 protein were detected in human colorectal cancer in comparison with corresponding normal mucosa. The tendency of higher expression of PLD2 mRNA was also observed. The ratio of PLD2 activity in cancer to that in corresponding normal mucosa was greater in colorectal cancer with nodal involvement and deeper tumor invasion. Our results indicate that PLD2 has a possible implication in carcinogenesis and progression and would be a new therapeutic target and a potential tumor marker for colorectal cancer.

Key words: Phospholipase D (PLD); Phospholipase D2 (PLD2); Colorectal cancer

Address correspondence to Shinichi Okamura, M.D., First Department of Internal Medicine, Gunma University School of Medicine, 3-31 Showa-machi, Maebashi, Gunma 371-8511, Japan. Tel: +81-27-220-8772; Fax: +81-27-220-8770; E-mail: sokamura@showa.gunma-u.ac.jp

Oncology Research, Volume 14, pp. 39-48
0965-0407/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Binding Site(s) on P-Glycoprotein for a Newly Synthesized Photoaffinity Analog of Agosterol A

Masahiko Mitsuo,1,2 Tomohiro Noguchi,1,3 Yuichi Nakajima,1 Shunji Aoki,5 Xiao-Qin Ren,1 Tomoyuki Sumizawa,1 Misako Haraguchi,1 Motomasa Kobayashi,5 Masanori Baba,4 Yukihiro Nagata,2 Shin-ichi Akiyama,1 and Tatsuhiko Furukawa1

1Department of Cancer Chemotherapy, Institute for Cancer Research, 2Department of Obstetrics and Gynecology, 3Department of Surgery, and 4Center for Chronic Viral Diseases, Faculty of Medicine, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520, Japan
5Graduate School of Pharmaceutical Sciences, Osaka University, Yamada-oka 1-6, Suita, Osaka 565-0871, Japan

Agosterol A (AG-A) is a novel agent that reverses P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP1)-meditated multidrug resistance (MDR). We have synthesized [125I]11-azidophenyl agosterol A (azidoAG-A), a photoaffinity analog of AG-A, and characterized its binding to P-gp in membrane vesicles prepared from multidrug-resistant P-gp-overexpressing KB-C2 cells. The photoanalog photolabeled intact P-gp and both the N- and C-terminal fragments of P-gp. [125I]AzidoAG-A is transported by P-gp and the intracellular accumulation of both [125I]azidoAG-A and [3H]AG-A in KB-C2 cells was lower than that in the parental drug-sensitive KB-3-1 cells. [125I]AzidoAG-A bound to the drug binding site(s) on P-gp because photoaffinity labeling of P-gp was inhibited by a variety of known P-gp substrates, including anticancer, reversing, and anti-human immunodeficiency virus (HIV) agents. The binding of [125I]azidoAG-A to P-gp differs from the binding of other photolabeled probes such as iodoaryl-azidoprazosin (IAAP) to P-gp and from the binding of [125I]azidoAG-A to MRP1 based on the differing effects of flupentixol and glutathione (GSH) on their binding. Thus, [125I]azidoAG-A will be a useful tool to elucidate the structure and function of P-gp because it directly binds to the drug binding site(s) on P-gp, is transported by P-gp, and exhibits different P-gp binding characteristics than IAAP.

Key words: P-Glycoprogein (P-gp); Agosterol A (AG-A); Photoaffinity labeling; Drug binding site

Address correspondence to Tatsuhiko Furukawa, Department of Cancer Chemotherapy, Institute for Cancer Research, Faculty of Medicine, Kagoshima University, Sakuragaoka 8-35-1, Kagoshima 890-8520, Japan. Tel: +81-99-275-5490; Fax: +81-99-265-9687; E-mail: furukawa@m3.kufm.kagoshima-u.ac.jp

Oncology Research, Volume 14, pp. 49-60
0965-0407/03 $20.00 + .00
Copyright © 2003 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Development of a Syngeneic In Vivo Tumor Model and its Use in Evaluating a Novel P-Glycoprotein Modulator, PGP-4008

Brian D. Lee,1 Kevin J. French,2 Yan Zhuang,2 and Charles D. Smith2

1Integrative Biosciences Graduate Program, 2Department of Pharmacology, The Penn State College of Medicine, Hershey, PA 17033

Multidrug resistance (MDR) is a phenomenon by which tumor cells develop reduced sensitivity to anticancer drugs, which often leads to the failure of cancer chemotherapy. A prominent mechanism of MDR is the overexpression of the multidrug efflux pump, P-glycoprotein (P-gp), that decreases the intracellular accumulation of many anticancer drugs, leading to increased tumor growth. Intensive efforts are under way to develop clinically useful MDR modulators that inhibit the function of P-gp for use in combination with established anticancer drugs. Our goal was to develop an improved in vivo solid tumor model utilizing immunocompetent animals to examine the efficacy of P-gp-specific MDR modulators. Using in vitro cytotoxicity and drug accumulation assays, two transformed murine cell lines, JC and TIB-75, were found to demonstrate the P-gp-mediated MDR phenotype. In contrast, two similar lines did not express functional P-gp. Western blot analyses confirmed the expression of P-gp and the lack of expression of the closely related drug efflux protein MRP1 in the JC and TIB-75 cell lines. The JC cell line displayed excellent tumorigenicity and consistent growth kinetics when implanted into immune-competent Balb/c mice. Animals treated with a combination of a known MDR modulator, cyclosporin A, and a cytotoxic drug, doxorubicin, exhibited significantly reduced tumor growth compared with untreated controls or animals treated with either cyclosporin A or doxorubicin alone. Similarly, a novel P-gp-specific MDR modulator, PGP-4008, in combination with doxorubicin showed inhibition of tumor growth. However, in contrast with the significant loss of body weight observed in the animals treated with the combination of cyclosporin A and doxorubicin, those treated with PGP-4008 plus doxorubicin did not experience weight loss. Therefore, this syngeneic solid tumor model provides a new in vivo system that can be used to evaluate the efficacy of P-gp inhibitors in an immune-competent host. This should allow improved prediction of the clinical utility of these compounds.

Key words: P-Glycoprotein; Multidrug resistance; Mouse solid tumor model

Address correspondence to Charles D. Smith, Penn State College of Medicine, Department of Pharmacology, 500 University Drive, H078, Hershey, PA 17033. Tel: (717) 531-1672; Fax: (717) 531-5013; E-mail: cdsmith@psu.edu