Fas-mediated killing of primary prostate cancer cells is increased by mitoxantrone and docetaxel Juliane C. Symes, 1 Michael Kurin, 1 Neil E. Fleshner, 3 and Jeffrey A. Medin 1,2,4 1 Department of Medical Biophysics and 2 Institute of Medical Sciences, University of Toronto; 3 Division of Urology, University Health Network; 4 Division of Stem Cell and Developmental Biology, Ontario Cancer Institute, Toronto, Ontario, Canada Abstract Therapies for prostate cancer based on Fas (CD95) modulation have been under active development at the preclinical stage using immortalized cell lines. To address clinical applicability, the potential of 11 cultures of primary prostate cancer cells to be killed by Fas- mediated apoptosis was investigated. In addition, the effect of the chemotherapeutic agents mitoxantrone and docetaxel on this killing was determined. Apoptosis was induced in patient-derived, primary prostate cancer cells using effector cells engineered by recombinant lentivirus infection to express Fas ligand (FasL) and measured by 51 Cr release assays. All cultured prostate cells were found to undergo Fas-mediated killing; cytotoxicity ranged from 12% to 87% after 6 h. These cells were significantly more sensitive to FasL-mediated killing than PC-3 cells. The basal expression of Fas or the expression of five inhibitors of apoptosis (c-FLIP, survivin, cellular inhibitors of apoptosis protein 1 and 2, and bcl-2) was not found to correlate with susceptibility to Fas- mediated killing. Both mitoxantrone and docetaxel were able to induce Fas receptor expression on primary prostate cancer cells, which translated into a 1.5- to 3-fold enhancement of apoptosis mediated by FasL. Whereas mitoxantrone increased the Fas-induced apop- totic response of all cultured prostate cells tested, docetaxel pretreatment was found to preferentially enhance the killing of bcl-2-expressing cells. These findings show that cultured primary prostate cancer cells are sensitive to Fas-mediated apoptosis. Further- more, the incidence of apoptosis was found to be improved by combining Fas-mediated therapy with standard chemotherapeutic agents. These findings may have significant implications for prostate cancer therapy. [Mol Cancer Ther 2008;7(9):3018–28] Introduction Since the discovery of Fas-mediated apoptosis, there has been sustained interest in exploiting this pathway in therapeutic strategies against prostate cancer (1 – 7). Apop- totic signaling is induced in cells on binding of Fas ligand (FasL) to its receptor, Fas. A caspase cascade is initiated, leading to death of the Fas-expressing cell. Approaches manipulating this pathway are growing more sophisticated and functionally efficacious while at the same time limiting off-target toxicity mediated by this ligand/receptor axis (8 – 11). Although these advances are bringing FasL-based ther- apy closer to clinical reality, the effectiveness of such therapies against prostate cancer has not been thoroughly investigated. Functional studies conducted to date make use of immortalized prostate cancer cell lines as models, which may not accurately reflect the potential clinical response of prostate tumors to FasL-mediated killing. Indeed, the majority of these cell lines, including PC-3, LNCaP, and DU145 cells, were derived from metastases or were artificially created by oncogene overexpression and are likely intrinsically resistant to apoptosis. In addition to immortalization, extensive culture periods may have further altered the phenotype of these commonly used models. Primary prostate cancer cells are thus a more representative model to study the response of prostate cancer to Fas-mediated killing in a preclinical setting. Immunohistochemical studies of primary human pros- tate cancer tissues have shown expression of key apoptotic pathway proteins, including Fas and caspases (12, 13). On the other hand, abundant expression of inhibitors of apoptosis, such as survivin and cellular inhibitors of apoptosis protein 1 and 2 (cIAP1 and cIAP2, respectively), have also been documented (14, 15). Without functional studies, it is not known whether the Fas pathway is operational in primary malignant prostate cells. In fact, based on expression profiling of such apoptotic mediators, it has been predicted that apoptotic signaling may be dramatically interrupted in prostate cancer tissues (12). To date, no functional studies have been conducted looking at Fas-mediated killing of patient-derived primary prostate tumor cells. Various chemotherapeutic drugs, such as camptothecin compounds, have been shown to enhance the sensitivity of prostate tumor cell lines to Fas-mediated apoptosis (16, 17). Certain other anticancer agents, including mitoxantrone, are known to increase cellular Fas expression in LNCaP but not PC-3 or DU145 cells (18). Reports suggest that Received 4/8/08; revised 6/5/08; accepted 6/9/08. Grant support: Prostate Cancer Research Foundation of Canada. Thecostsofpublicationofthisarticleweredefrayedinpartbythe payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Jeffrey A. Medin, 67 College Street, Room 406, Toronto, Ontario, Canada, M5G 2M1. Phone: 416-340-4745; Fax: 416-340-3453. E-mail: jmedin@uhnres.utoronto.ca Copyright C 2008 American Association for Cancer Research. doi:10.1158/1535-7163.MCT-08-0335 3018 Mol Cancer Ther 2008;7(9). 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