Novel off-target effect of tamoxifen — Inhibition of acid ceramidase activity in cancer cells

• Published in: Biochimica et biophysica acta Vol. 1831; no. 12; pp. 1657 - 1664
• Main Authors: Morad, Samy A.F., Levin, Jonathan C., Tan, Su-Fern, Fox, Todd E., Feith, David J., Cabot, Myles C.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2013
• Subjects: Acid ceramidase; Acid Ceramidase - antagonists & inhibitors; Acid Ceramidase - genetics; Acid Ceramidase - metabolism; Antiestrogen; Antineoplastic Agents - pharmacology; apoptosis; Apoptosis - drug effects; cathepsin B; Cathepsin B - antagonists & inhibitors; Cathepsin B - metabolism; Cell Line, Tumor; cell movement; Cell Survival - drug effects; cell viability; Cell-Free System; Ceramide; ceramides; Ceramides - metabolism; dose response; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; fatty acids; Female; Gene Expression; Humans; Hydrogen-Ion Concentration; hydrolysis; Lysophospholipids - metabolism; Lysosomal protease; Lysosomes - drug effects; Lysosomes - enzymology; Male; mechanism of action; membrane permeability; neoplasm cells; prostatic neoplasms; protein synthesis; proteinase inhibitors; Selective Estrogen Receptor Modulators - pharmacology; sphingosine; Sphingosine - analogs & derivatives; Sphingosine - metabolism; Tamoxifen; Tamoxifen - pharmacology; therapeutics; Toremifene - pharmacology; PBS; AC; NOE; S1-P; Antiestrogen; Acid ceramidase; Tamoxifen; Lysosomal protease; LMP; AO; PMSF; PBST; SK; Ceramide; GC; glucosylceramide; sphingosine kinase; phenylmethylsulfonyl fluoride; phosphate-buffered saline; acridine orange; phosphate-buffered saline with Tween-20; N-oleoylethanolamine; lysosomal membrane permeability; sphingosine 1-phosphate
• ISSN: 1388-1981; 0006-3002; 1879-2618
• DOI: 10.1016/j.bbalip.2013.07.016

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 mitogenic 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 micromolar 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 tamoxifen 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 5min, and dose-dependent, as low as 5μM, increases in lysosomal membrane permeability (LMP), and time- and dose-dependent downregulation of AC protein expression. Assessing various protease 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 pH4.5. In addition, pretreatment 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. •Tamoxifen inhibits acid ceramidase (AC) activity in intact cancer cells.•Tamoxifen causes lysosomal membrane destabilization.•Tamoxifen downregulates AC protein expression.•Lysosomal cathepsin B is responsible for AC proteolysis.•Inhibition of AC indirectly leads to decreased cancer cell migration.