Novel off-target effect of tamoxifen — Inhibition of acid ceramidase activity in cancer cells Samy A.F. Morad a,1,2 , Jonathan C. Levin a , Su-Fern Tan b , Todd E. Fox c , David J. Feith b , Myles C. Cabot a, ⁎ ,2 a John Wayne Cancer Institute at Saint John's Health Center, Department of Experimental Therapeutics, Santa Monica, CA 90404, USA b Penn State Hershey Cancer Institute, Penn State University College of Medicine, Hershey, PA, USA c Department of Pharmacology, Penn State College of Medicine, Hershey, PA, USA abstract article info Article history: Received 21 March 2013 Received in revised form 18 July 2013 Accepted 30 July 2013 Available online 9 August 2013 Keywords: Acid ceramidase Tamoxifen Ceramide Antiestrogen Lysosomal protease Acid ceramidase (AC), EC 3.5.1.23, a lysosomal enzyme, catalyzes the hydrolysis of ceramide to constituent sphingoid base, sphingosine, and fatty acid. Because AC regulates the levels of pro-apoptotic ceramide and mito- genic sphingosine-1-phosphate, it is considered an apt target in cancer therapy. The present study reveals, for the first time, that the prominent antiestrogen, tamoxifen, is a pan-effective AC inhibitor in the low, single digit mi- cromolar range, as demonstrated in a wide spectrum of cancer cell types, prostate, pancreatic, colorectal, and breast. Prostate cancer cells were chosen for the detailed investigations. Treatment of intact PC-3 cells with ta- moxifen produced time- and dose-dependent inhibition of AC activity. Tamoxifen did not impact cell viability nor did it inhibit AC activity in cell-free assays. In pursuit of mechanism of action, we demonstrate that tamoxifen induced time-, as early as 5 min, and dose-dependent, as low as 5 μM, increases in lysosomal membrane perme- ability (LMP), and time- and dose-dependent downregulation of AC protein expression. Assessing various prote- ase inhibitors revealed that a cathepsin B inhibitor blocked tamoxifen-elicited downregulation of AC protein; however, this action failed to restore AC activity unless assayed in a cell-free system at pH 4.5. In addition, pre- treatment with tamoxifen inhibited PC-3 cell migration. Toremifene, an antiestrogen structurally similar to tamoxifen, was also a potent inhibitor of AC activity. This study reveals a new, off-target action of tamoxifen that may be of benefit to enhance anticancer therapies that either incorporate ceramide or target ceramide metabolism. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Acid ceramidase, (AC), EC 3.5.1.23, also known as N-acylsphingosine deacylase and encoded in humans by the ASAH1 gene, was first de- scribed by Shimon Gatt [1]. AC now occupies a noteworthy position in cancer biology. By catalyzing ceramide hydrolysis, AC functions as a ‘sen- try’ removing ceramide and thus blunting a host of ceramide-driven cell death cascades [2]. In addition, ceramide hydrolysis liberates sphingo- sine, a substrate for sphingosine kinase (SK), which generates the mito- genic entity, sphingosine 1-phosphate (S1-P) [3]. Thus, this double- edged sword powers-down apoptotic responses and promotes cancer cell growth, migration, and invasion [4]. AC is optimally active at acid pH, between 4.0 and 5.0 [5], appropriate because of its localization in ly- sosomes [6]. Although neutral ceramidase, which has a role in regulating ceramide-induced apoptosis and functions at pH 7.6 [7], and alkaline ceramidases [8] have been the object of intense investigation, AC ap- pears a dominant force in the cancer field, being designated as a novel target in cancer therapy [9]. Highlighting this designation are studies in prostate cancer wherein high AC expression relates to poor outcome [10] and resistance to radiation [11] and plays a role in carcinogenesis [12]. Moreover, a study in pancreatic cancer cells illustrates a role for AC in conversion of cytostatic to cytotoxic end point [13]. Work in mel- anoma shows that AC expression modulates sensitivity to dacarbazine [14], and a genetic study showed that ASAH1 is among the most impor- tant candidate genes in melanoma diagnostics [15]. Thus, it should not be surprising that design and evaluation of inhibitors of AC are impor- tant priorities in cancer medicine. There are a number of small molecule inhibitors of AC that have been employed in a myriad of studies, although it should be noted that some agents, N-oleoylethanolamine (NOE) for example, demon- strate overlapping efficiencies [16]. DM102 [17] and LCL204 [18],a lysosomotropic analog of B13, display high specificity for AC and exert functional impact at the biological level. For example, DM102 amplifies cytotoxic responses to fenretinide and to PSC 833 in prostate [19] and in pancreatic cancer cells [13] respectively, whereas similar effects are Biochimica et Biophysica Acta 1831 (2013) 1657–1664 Abbreviations: AC, acid ceramidase; LMP, lysosomal membrane permeability; SK, sphingosine kinase; S1-P, sphingosine 1-phosphate; NOE, N-oleoylethanolamine; GC, glucosylceramide; PBS, phosphate-buffered saline; AO, acridine orange; PMSF, phenylmethylsulfonyl fluoride; PBST, phosphate-buffered saline with Tween-20 ⁎ Corresponding author at: John Wayne Cancer Institute at Saint John's Health Center, Department of Experimental Therapeutics, 2200 Santa Monica Blvd, Santa Monica, CA 90404, USA. Tel.: +1 310 998 3924; fax: +1 310 582 7325. E-mail address: cabot@jwci.org (M.C. Cabot). 1 Affiliated with South Valley University, Department of Pharmacology, Qena, Egypt. 2 Current address: Department of Biochemistry & Molecular Biology, Brody School of Medicine, East Carolina University, Greenville, NC, USA. 1388-1981/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbalip.2013.07.016 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip