ognizant Communication Corporation


VOLUME 12, NUMBER 5, 2000

Oncology Research, Volume 12, pp. 209-217, 2000
0965-0407/00 $20.00 + .00
Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Bone Metastasis Model With Multiorgan Dissemination of Human Small-Cell Lung Cancer (SBC-5) Cells in Natural Killer Cell-Depleted SCID Mice

Toyokazu Miki, Seiji Yano, Masaki Hanibuchi, and Saburo Sone

Third Department of Internal Medicine, University of Tokushima School of Medicine, Tokushima, Japan

Lung cancer is commonly associated with multiorgan metastasis, and bone is a frequent metastatic site for lung cancer. Nevertheless, no bone metastasis model of lung cancer with multiorgan dissemination is available, which could provide opportunity to study the molecular pathogenesis. We examined the abilities of eight human lung cancer cell lines injected intravenously into natural killer (NK) cell-depleted SCID mice to generate metastatic nodules in bone and multiple organs, and explored the correlation of the parathyroid hormone-related protein (PTHrP) with the bone metastasis. Although all the small-cell carcinoma cell lines (SBC-5, SBC-3, SBC-3/ADM, H69, H69/VP) formed metastatic nodules in multiple organs (liver, kidney, and lymph nodes), only SBC-5 cells reproducibly developed bone metastases. Squamous cell carcinoma (RERF-LC-AI) cells metastasized mainly into the liver and kidneys, whereas adenocarcinoma (PC-14, A549) mainly produced colonies in the lungs. As assessed by X-ray photography, the osteolytic bone metastases produced by SBC-5 cells were detected as early as on day 28, and all recipient mice developed bone metastasis by day 35. The expression of PTHrP in eight cell lines was directly correlated with the formation of bone metastasis. No correlation was observed between the formation of bone metastasis and the expression of other metastasis-related cytokines (IL-1, IL-6, IL-8, IL-10, IL-11, TNF-a, VEGF, M-CSF). Consistent with the formation of bone metastasis by SBC-5 cells, the levels of PTHrP and calcium in the mouse serum were increased in a time-dependent manner, suggesting that PTHrP produced by human lung cancer may play a crucial role in the formation of bone metastasis and hypercalcemia. These findings indicate that a bone metastasis model of SBC-5 cells may be useful for clarifying the molecular aspects of the metastatic processes in different organ microenvironments and the development of therapeutic modalities for lung cancer patients with bone metastases.

Key words: Parathyroid hormone-related protein (PTHrP); Small-cell lung cancer; NK cells; SCID mice

Address correspondence to Saburo Sone, Third Department of Internal Medicine, The University of Tokushima School of Medicine, Kuramoto-cho 3-18-15, Tokushima, 770-8503, Japan. Tel: (81)-88-633-9274; Fax: (81)-88-633-2134; E-mail: ssone@clin.med.tokushima-u.ac.jp

Oncology Research, Volume 12, pp. 219-229, 2000
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Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.

Indoloquinoxaline Compounds That Selectively Antagonize P-Glycoprotein

Charles D. Smith, Cynthia B. Myers, Jack T. Zilfou, Staci N. Smith, and David S. Lawrence

Department of Pharmacology, Pennsylvania State University, 500 University Drive, Hershey, PA 17033

Tumor cells often develop drug resistance through overexpression of membrane transport proteins that effectively efflux anticancer agents. The pharmacologies of the two best-studied transporters, P-glycoprotein (Pgp) and MRP1, are partially overlapping but distinct. To improve the therapeutic potential of drug resistance reversing agents, we have developed a program to identify compounds with selectivity for Pgp or MRP1. Screening of a commercial library of compounds identified indoloquinoxaline compounds with transporter selectivity, and certain examples were synthesized and further evaluated. 1,4-Dibutoxy-6H-indolo[2,3-b]quinoxaline and 4,7-dibutoxy-2,3-dihydrobenzimidazole-2-spiro-3-indolin-2-one were synthesized by condensation of 3,6-dibutoxy-1,4-diaminobenzene and isatin. Neither compound was cytotoxic to MCF-7 cells, nor did either one affect the sensitivity of MCF-7/VP or HL-60/ADR cells at doses up to at least 20 mM, indicating that they do not antagonize MRP1. In contrast, each compound, at doses as low as 0.25 mM, sensitized NCI/ADR cells to vinblastine, actinomycin D, Taxol, and doxorubicin, indicating that they effectively reverse Pgp-mediated multidrug resistance (MDR). Furthermore, the compounds sensitized two additional cell lines that overexpress Pgp to this panel of anticancer drugs. However, these compounds did not affect the sensitivities of MCF-7 or T24 cells to these cytotoxic drugs, and did not alter the sensitivities of any of the tested cell lines to cisplatin or 5-fluorouracil. Both compounds enhanced the intracellular accumulation of [3H]vinblastine by NCI/ADR cells, but did not inhibit photoaffinity labeling of Pgp by [3H]azidopine at concentrations up to at least 100 mM. Therefore, these novel nontoxic indoloquinoxalines selectively sensitize Pgp-overexpressing cells to drugs that are subject to transport by this protein, without modulating the sensitivities of MRP1-overexpressing or non-Pgp cells to cytotoxic drugs. Because of this transporter selectivity, we predict that these compounds will be effective MDR modulators in vivo.

Key words: Drug resistance; P-glycoprotein; MRP1; Cancer

Address correspondence to Charles D. Smith, Department of Pharmacology, H078, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033-2390. Tel: (717) 531-1672; Fax: (717) 531-5013; E-mail: cdsmith@psu.edu.

Oncology Research, Volume 12, pp. 231-239, 2000
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Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Differences in the Induction of DNA Damage, Cell Cycle Arrest, and Cell Death by 5-Fluorouracil and Antifolates

H. H. J. Backus, H. M. Pinedo, D. Wouters, C. M. Kuiper, G. Jansen, C. J. van Groeningen, and G. J. Peters

Department of Medical Oncology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands

Thymidylate synthase (TS) is an important target for chemotherapy and can be inhibited by 5-fluorouracil (5-FU) and the antifolates, AG337 (Nolatrexed) and multitargeted antifolate (MTA or Pemetrexed). In addition, 5-FU can be incorporated into RNA and DNA, and MTA can inhibit two other enzymes. It is, however, unclear to what extent these differences in drug action will influence activation of downstream mechanisms mediated via TS inhibition. Therefore, two human colon cancer cell lines, WiDr and Lovo, with a different clonogenic origin, were treated with equitoxic concentrations of 5-FU, AG337, and MTA to determine the induction of DNA damage, cell cycle arrest, downstream protein expression, and cell death. At these concentrations, the specific TS inhibitor AG337 induced more DNA damage (up to 20%) than MTA and 5-FU. FACS analysis showed that all drugs induced S phase arrest in Lovo and WiDr that was most pronounced after 5-FU and AG337 exposure (50-70%). Western blotting showed that p53 induction was not detectable in mutant (mt) p53 WiDr and increased much earlier in wild-type (wt) Lovo cells after 5-FU and MTA (24 h) than after AG337 exposure (72 h). In contrast to 5-FU-treated Lovo cells, the bcl-2/bax ratio decreased after antifolate exposure. Nevertheless, both 5-FU and antifolates induced similar amounts of cell death (up to 60%). These results demonstrate that in human colon cancer cells differences in downstream events between AG337 and 5-FU or MTA are related to the additional effects of 5-FU and MTA, which are not associated with TS inhibition.

Key words: 5-Fluorouracil; AG337; Multitargeted antifolate; Apoptosis; Colon; DNA damage

Address correspondence to Dr. G. J. Peters, Department of Medical Oncology, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Tel: (31)-20-4442633; Fax: (31)-20-4443844; E-mail: gj.peters@azvu.nl

Oncology Research, Volume 12, pp. 241-251, 2000
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Copyright © 2001 Cognizant Comm. Corp.
Printed in the USA. All rights reserved.
Angiogenic and Immune Parameters During Recombinant Interferon-a2b Adjuvant Treatment in Patients With Melanoma

Didier Dréau, Mareva Foster, Melanie Hogg, Jeanene Swiggett, Walter D. Holder, and Richard L. White

Carolinas Medical Center, PO Box 32861, 1000 Blythe Blvd., Charlotte, NC 28203

As an adjuvant therapy for patients with high risk of recurrent melanoma, high-dose interferon (IFN)-a2b therapy has been shown to have some efficacy. We examined 22 patients with resected melanoma who were treated with repeated injections of recombinant IFN-a2b during the treatment. Both angiogenic and immune parameters were measured. White blood cells (WBCs) and lymphocyte numbers, lymphocyte subpopulations, serum concentrations of IFN-a and anti-IFN-a antibodies, and the serum vascular endothelial growth factor (VEGF), interleukin (IL)-8, and basis fibroblast growth factor (bFGF) concentrations were determined over time in resected, recurrence-free patients with American Joint Committee on Cancer (AJCC) stage III melanoma with one or less (LN+ <  1, n = 7) or more than one (LN+ > 1, n = 8) lymph nodes involved, and AJCC stage IV resected disease (n = 7). Follow-up and recurrence-free intervals were longer in stage III (LN+ < 1) patients compared with stage IV patients (P < 0.05). The number of WBCs and lymphocytes decreased during the treatment for all patient groups (P < 0.001). In addition, percentages of CD8 and CD20 were higher in stage IV patients than in stage III (LN+ > 1) and stage III (LN+ < 1) patients at the beginning of therapy (P < 0.05). A significant increase in the percentage of CD20+ cells, mostly B lymphocytes, was observed in the stage III (LN+ > 1) and stage III (LN+ < 1) patients over time but not in stage IV patients (P < 0.001). Low IL-8 and bFGF concentrations at the beginning of therapy were associated with significantly longer recurrence-free survival (P < 0.05). These results warrant a larger trial to determine if the differences observed in patients before treatment can provide prognostic markers in patients receiving IFN-a2b therapy.

Key words: Immunotherapy; Melanoma; Interferon-a2b; CD20; Antibody; HLA-DR; Basic fibroblast growth factor; Vascular endothelial growth factor; Interleukin-8

Address correspondence to Didier Dréau, Ph.D., Department of General Surgery Research, Carolinas Medical Center, PO Box 32861, 1000 Blythe Blvd., Charlotte, NC 28232-2861. Tel: (704) 355 8254; Fax: (704) 355 7203; E-mail: ddreau@carolinas.org