[CANCER RESEARCH 64, 7562–7569, October 15, 2004] Fluoxetine Inhibits Multidrug Resistance Extrusion Pumps and Enhances Responses to Chemotherapy in Syngeneic and in Human Xenograft Mouse Tumor Models Dan Peer, Yaron Dekel, Dina Melikhov, and Rimona Margalit Department of Biochemistry, the George S. Wise Life Science Faculty, Tel Aviv University, Tel Aviv, Israel ABSTRACT Multidrug resistance (MDR) operated by extrusion pumps such as P-glycoprotein and multidrug-resistance-associated-proteins, is a major reason for poor responses and failures in cancer chemotherapy. MDR modulators (chemosensitizers) were found among drugs approved for noncancer indications and their derivatives. Yet toxicity, adverse effects, and poor solubility at doses required for MDR reversal prevent their clinical application. Among newly designed chemosensitizers, some still suffer from toxicity and adverse effects, whereas others progressed to clinical trials. Diversities among tumors and among MDR pumps indicate a need for several clinically approved MDR modulators. Here we report for the first time that fluoxetine (Prozac), the well-known antidepressant, is a highly effective chemosensitizer. In vitro, fluoxetine enhanced (10- to 100-fold) cytotoxicity of anticancer drugs (doxorubicin, mitomycin C, vinblastine, and paclitaxel) in drug-resistant but not in drug-sensitive cells (5 and 3 lines, respectively). Fluoxetine increased drug accumulation within MDR-cells and inhibited drug efflux from those cells. In vivo, fluoxetine enhanced doxorubicin accumulation within tumors (12-fold) with unaltered pharmacokinetics. In four resistant mouse tumor models of both syngeneic and human xenograft, combination treatment of fluoxetine and doxorubicin generated substantial (P < 0.001) improvements in tu- mor responses and in survivals (2- to 3-fold). Moreover, fluoxetine re- versed MDR at doses that are well below its human safety limits, free of the severe dose-related toxicity, adverse effects, and poor solubility that are obstacles to other chemosensitizers. This low-dose range, together with the findings reported here, indicate that fluoxetine has a high potential to join the arsenal of MDR reversal agents that may reach the clinic. INTRODUCTION Chemotherapy frequently fails cancer patients due to inherent or acquired multidrug resistance (MDR; refs. 1–3). In the dominant mechanism, intracellular levels of cytotoxic drugs are reduced below lethal thresholds by active extrusion of the cytotoxic drug(s) from the tumor cell, operated by ATP-dependent pumps such as P-glycoprotein and multidrug resistance-associated protein (1–7). Inhibition of the extrusion is a main approach to overturn MDR (1–9). The search for effective inhibitors (also named chemosensitizers, MDR modulators, and MDR reversal agents) is now into the third generation. First-generation candidates were drugs already approved for other noncancerous indications, such as verapamil, cyclosporine A, and progesterone (8 –13). Unfortunately, although successful in vitro, patients could not benefit from these chemosensitizers, because their clinically relevant doses exceed safety limits resulting in unac- ceptable adverse effects, frequent poor solubility, and toxicity (8 –13). Chemical derivatization of first-generation molecules and combinato- rial chemistry lead to second- and third-generation chemosensitizers, such as VX-710, PSC833, XR9051, XR9576, MS-209, GF120918, R101933, LY335979, and OC144 – 093 (ONT-093; refs. 14 –32), some of which are in clinical trials (25, 27, 28, 30 –32). Several of the latter, whereas more potent and less toxic than first-generation com- pounds, may still be prone to adverse effects, poor solubility, and unfavorable changes in pharmacokinetics of the anticancer drugs. Moreover, given tumor diversity, it is rational to assume that more than one chemosensitizer will be needed in the clinic. Here we report for the first time that fluoxetine (Prozac), the well-known antidepressant (33), acts as a highly effective chemosen- sitizer, joining the arsenal of MDR modulators with prospects of reaching the clinic. Seemingly back to first-generation candidates in terms of using drugs approved for noncancerous indications, we suggest it should be viewed as a fourth-generation chemosensitizer. Unlike first-generation molecules, fluoxetine modulates MDR at low doses, free of the severe dose-related drawbacks experienced with che- mosensitizers of previous generations and free of solubility limitations. This report presents results of in vitro and in vivo studies. Activities and mechanism of chemosensitization by fluoxetine were studied in eight cell lines, covering drug-sensitive and drug-resistant cells (in- herent and acquired MDR) Verapamil and Cyclosporin A were in- cluded as in vitro benchmarks, the rationale for this choice stemming from their acknowledged in vitro activities as MDR reversal agents and their availability. The in vivo studies were conducted in three syngeneic and one human xenograft mouse tumor models, exploring pharmacokinetics, biodistribution, and therapeutic responses. MATERIALS AND METHODS Reagents and Cell Cultures. Paclitaxel, rhodamine-123 (Rh-123), vera- pamil, cyclosporine A, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro- mide (MTT), and Trypan-blue were from Sigma Chemical Co. (St. Louis, MO). Doxorubicin and Vinblastine were a kind gift from TEVA Pharmaceutical Ltd. (Natania, Israel). Mitomycin C was a kind gift from Dexon Ltd. (Or Akiva, Israel). Fluoxetine was a kind gift from Unipharm (Ramat Gan, Israel). Cisplatin was from TEVA Pharmaceutical Ltd. Materials for cell cultures (specified under Materials and Methods) were from Biological Industries (Beit Haemek, Israel). [ 3 H]Vinblastine sulfate (specific activity 17 Ci/mmol) was from Amersham (Buckinghamshire, England). [ 3 H]paclitaxel (specific activity 20 Ci/mmol) was from American Radiolabeled Chemicals Inc., (St. Louis, MO). Cell monolayers or suspensions were grown in 100  20-mm dishes (culture plates and dishes were from Corning Glass, Corning, New York). B16F10.9 and HT29 cells were cultured in DMEM at 37°C in 5% CO 2 supplemented with 10% fetal calf serum, penicillin (10,000 units/mL), strep- tomycin (10 mg/mL), and L-glutamine (200 mmol/L). C-26, P388, and D122 cells were similarly cultured, except the medium was RPMI 1640. MCF-7 and MCF-7/ADR cells were maintained as monolayer cultures in MEM containing 10% fetal bovine serum, 1 mmol/L sodium pyruvate, 2 mmol/L L-glutamine, and 1.5 g/L of sodium bicarbonate. P388/ADR and MCF-7/ADR were grown in the presence of 0.25 g Doxorubicin/mL and 0.5 g Doxorubicin/mL, respectively. Cells were free of Mycoplasma contamination. determined by a Mycoplasma ELISA test (Boehringer Mannheim GmbH, Mannheim, Ger- many) performed every 3 months. Cell viability was determined by the following: (1) the MTT method, recording the absorbencies in a plate reader, at two wavelength 550 and 650 nm, (2) the Trypan-blue method, using a hemocytometer, and (3) total cell protein by the Bradford method. Fluorescence-Activated Cell Sorter Analysis. Suspensions of P388/ADR or C-26 (1  10 6 cells/mL) in RPMI 1640 were incubated at 37°C for 30 minutes with 5 mol/L Rh-123 with and without a chemosensitizer, selected from vera- pamil (15 mol/L), cyclosporine A (15 mol/L), and fluoxetine (5 mol/L). Intracellular fluorescence was determined by Fluorescence Activated Cell Sorter Received 12/25/03; revised 5/26/04; accepted 8/11/04. The costs of publication of this article were defrayed in part by the 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: Rimona Margalit, Department of Biochemistry, Tel Aviv University, Tel Aviv 69978, Israel. Phone: 972-3-640-9822; Fax: 972-3-640-6834; E- mail: rimona@post.tau.ac.il. ©2004 American Association for Cancer Research. 7562 Research. on April 28, 2016. © 2004 American Association for Cancer cancerres.aacrjournals.org Downloaded from