ICANCKR KI-SKAKCM 56. 1303-1308. March 15. 19%] Taxol-dependent Transcriptional Activation of IL-8 Expression in a Subset of Human Ovarian Cancer1 Li-Fen Lee, C-C. Schuerer-Maly, Alan K. Lofquist, Caroline van Haaften-Day, Jenny P-Y. Ting, Charise M. White, Brian K. Martin, and J. Stephen Haskill2 Depurimela of Biology ¡L-F. LI, University of North Carolina Lineherger Comprehensive Cancer Center ¡A. K. L, J. P-Y. T., B. K. M.. J. S. H.l und Departments of Microbiology ami Immunology /J. P-Y. T.. C. M. W.. J. S. H.] and Obstetrics and Gynecology ¡J.S. HJ. University (if North Carolina al Chapel Hill, Chapel Hill. North Carolina 27599-7295; Institute of Physiology ¡C-C.S-M.I, University of Zurich. 8057 Zurich. Swilzerlanu; ami Department of Gynecological Oncology ¡C.v. H-DJ. The Royal Hospital for Women, Neu- South Wales 2021. Australia ABSTRACT Taxol is important in the treatment of both primary and drug-resistant ovarian cancer. Although Taxol is known to stabilize microtubules and block cell mitosis, the effectiveness of this drug exceeds that of other antimitotic agents, suggesting it may have an additional mode of action. Stimulated by murine macrophage studies indicating cytokine induction by Taxol, we have investigated prointlammatory cytokine expression in a series of cell lines and recent expiants of human ovarian cancer. Taxol induced secretion of interleukin (ID 8 but not II.-6, II.-la, or II .-I /! in 4 of 10 samples. Induction was dependent on transcriptional activation, and, in contrast to murine macrophage studies, was apparently independent of an active lipopolysaccharide signaling pathway. Confluent cultures se creted as much II.-8 as proliferating cells. Taxol did not induce IL-8 in breast carcinoma, endometrial stromal, or T-lymphocyte or monocyte cultures. \Yc propose that the local expression of this chcmokine in vivo may elicit a host response similar in effectiveness to that of cytokine gene therupy. These data are the first to suggest that a chemotherapeutic agent may have a direct effect on transcription of cytokine and/or growth factor genes in ovarian cancer, and that this effect may not be restricted to proliferating tumor cells. INTRODUCTION Taxol is the prototype of a new class of drugs with promise in the treatment of several epithelial cancers, including breast, ovary, lung, and colon (1. 2). Taxol, a microtubule stabilizing agent (3. 4). has antimitotic and apoptosis-inducing activity (5), but the therapeutic effect appears to be greater than that of microtubule-disrupting drugs such as vinblastine and colchicine, suggesting that Taxol and recently identified variants such as taxotere, have an additional mode of activity in addition to mitotic arrest. The results of early Phase I trials of Taxol have shown a sporadic influence of Taxol. and Phase II studies were restricted to renal, melanoma, and ovarian cancers (1). The ovarian data were particularly interesting, with 30 and 36% response rates seen with otherwise refractory patients (6, 7). The most promising aspect of these early ovarian studies was the efficacy in platinum-resistant patients, suggesting that Taxol is functioning via a pathway, in part, distinct from that of direct DNA-damaging agents. Subsequently. Holmes et al. (7) carried out a Phase II trial in breast cancer with even more encouraging results: a 56% response rate in metastatic breast cancer. Neutropenia appears to have been the major side effect encouraging the combined therapy with granulocyte colo ny-stimulating factor (8). In breast and ovary, the available data Received 6/15/95; accepted 1/12/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked nilvt'riisi'nifni in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ' This work is supported by National Cancer Institute grant 481X5 and an American Cancer Society Faculty Award to J.P-Y.T. C.M.W. is supported by a Minority Enhance ment Supplement to NCI 48185: B.K.M. is supported by an NCI training grant and by an NCI postdoctoral fellowship. - To whom requests for reprints should be addressed, at Linebergcr Comprehensive Cancer Center. University of North Carolina at Chapel Hill. Chapel Hill. NC 27599-7295. Phone: (919) 966-3098; Fax: (919) 966-3015. suggest a lack of cross-reactivity with either doxorubicin or platinum (9). Several reports have shown that in mice Taxol is a potent stimulator which can induce a series of LPS-dependent macrophage-derived cytokines such as IL-la, IL-lß,TNF-a, and IP-10 (10-15). Macro phages from C3H/HeJ mice which carry a mutation in the LPS ' gene fail to activate microtubule-associated protein kinase and are unable to either produce TNF-a or down-regulate TNF-a receptors in response to LPS or Taxol ( 10, 16). These data demonstrate that Taxol and LPS may share a functionally important signal transduction pathway. Cu riously, human monocytes and macrophages appear to be refractory to Taxol ( 15).4 The experiments reported below demonstrate that Taxol induces IL-8 transcription and secretion but not other cytokines such as IL-la. IL-lß.or IL-6 in long-term human ovarian cancer cell lines as well as ovarian cells recently explanted from tumors. In contrast. Taxol did not induce IL-8 synthesis in any breast carcinoma lines, normal T lymphocytes, or monocytes. The induction of IL-8 in both proliferating and confluent cultures suggests that in vivo Taxol re sponsiveness may depend on direct tumor cell destruction as well as modification of the local tumor environment through release of IL-8, a cytokine with proinflammatory and growth-modulating properties. MATERIALS AND METHODS Taxol Treatment of Carcinoma Cell Lines. Initial studies were carried out with four ovarian cancer cell lines (OVCA 420. 429. 432. and 433) which have been established in Dr. Robert Bast's laboratory and the origins of which have been described previously (17, 18). Cells were seeded in 24-well plates (Costar, Inc.) and grown in DMEM media supplemented with endotoxin-free 10% fetal bovine serum until 60-75% of confluence. Immediately before use, the medium was removed, the cells were washed once with sterile endotoxin- free PBS. and then treated with Taxol or the vehicle. DMSO (or methanol in some experiments), as indicated. Stock Taxol was maintained at —¿ 7()°C at a concentration of 50 HIM. The final concentration of DMSO never exceeded 0.1%. At various times after treatment, culture supernatants were removed for ELISA analysis, or cells were treated for RNA isolation as described below. Confluent cultures were obtained as described above, except cells were al lowed to cease proliferation as judged by the lack of mitotic figures and plate density. Cultures were then left for an additional 2 or 5 days prior to Taxol exposure. Although subconfluent cultures exhibited numerous mitotic cells following Taxol exposure, examination of confluent cultures by phase-contrast microscopy indicated the entire plate was a homogeneous monolayer in which mitoses were entirely absent following exposure to Taxol. Six recently ex- planted ovarian cancer cell lines were cultured in 24-well plates (Corning) and grown in RPMI 1640 supplemented with 15% fetal bovine serum. Cells (5 X IO4) were set up per well, and 30 /AMTaxol was added to the cells in log phase. As a control. 0.1% DMSO was used. To check for possible endotoxin contamination, media were assayed several times and found to contain <3 pg/ml endotoxin using the liinulus assay. These cell lines were used within 4 months of establishment from ascites sources, and they are in the range of 10-20 passages. All were derived from patients who had previously tailed 1The abbreviations used are: LPS. lipopolysaccharide; IL. interleukin; TNF. tumor necrosis factor. 4 S. Vogel, personal communication. 1303 on July 22, 2015. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from