Cancer cells release glutamate via the cystine/glutamate antiporter Mohit K. Sharma 1 , Eric P. Seidlitz 1 , Gurmit Singh * Department of Pathology & Molecular Medicine, McMaster University, Ont., Canada Research Department, Juravinski Cancer Centre, Hamilton, Ont., Canada article info Article history: Received 26 October 2009 Available online 5 November 2009 Keywords: Bone metastasis Breast cancer Cell lines Cystine Glutamate Glutamate transporters Intercellular communication Metabolism Prostate cancer abstract Although the amino acid glutamate is used as an intercellular signaling molecule for normal bone homeo- stasis, little is known regarding its possible role in the metabolic disruption characteristic of bone metas- tasis. We have previously shown in vitro that cancer cell lines relevant to bone metastasis release glutamate into the extracellular environment. This study demonstrates the expression of multiple gluta- mate transporters in cancer cell lines of non-central nervous system origin. Furthermore, we identify the molecular mechanism responsible for glutamate export and show that this system can be inhibited phar- macologically. By highlighting that glutamate secretion is a common biological feature of cancer cells, this study suggests that tumor-derived glutamate could interfere with glutamate-dependent intercellular signaling in normal bone. Pharmacological interference with cancer cell glutamate release may be a via- ble option for limiting host bone response to invading tumor cells in bone metastasis. Ó 2009 Elsevier Inc. All rights reserved. Introduction Breast and prostate cancer cells that metastasize to bone fre- quently provoke significant alterations in bone metabolic pro- cesses that result in pain, hypercalcaemia, and weakening of bone structure [1]. The mechanisms that generate these effects are unknown, although factors released by invading cancer cells are likely involved in signaling these responses [2,3]. The amino acid glutamate has been shown to be critical for normal bone met- abolic processes [4–6], and we have shown that a variety of cancer cells secrete glutamate into their extracellular environment [7]. Tumor cell derived glutamate may have important functional implications for the bone microenvironment. Although the expression of both vesicular and non-vesicular glutamate transporters have been demonstrated in tumors derived from central nervous system (CNS) and peripheral sites, it is not clear whether a common secretion mechanism is used between different tumor types. Although glial-derived cancers release glu- tamate via the glutamate/cystine antiporter mechanism known as system x c À [8–12], it is not known whether non-CNS derived cancers use the same mechanism. Other candidate glutamate transporters include the glutamate–aspartate transporter (GLAST- 1 or the human homolog EAAT1) [13], glutamate transporter 1 (GLT-1 or the human homolog EAAT2) [14], and the vesicular glu- tamate transporters (VGLUT) [15–17]. This study was designed to evaluate the mRNA expression of GLAST, GLT-1, VGLUT1, and system x c À in a series of cancer cell lines. Secondly, protein expression of the transporters primarily in- volved in glutamate secretion was assessed in vitro and in a mouse model of breast cancer bone metastasis. As a functional measure, we quantified the amount of glutamate released from cancer cells in vitro using glutamate-free culture media and with specific phar- macological inhibition of vesicular and non-vesicular glutamate transporters. Materials and methods All protocols for animal studies were reviewed and approved by the Animal Research Ethics Board of McMaster University, Hamil- ton, Ontario, Canada, and adhered to the guidelines of the Canadian Council on Animal Care. Cell lines. Mycoplasma-free cancer cells (obtained from the American Type Culture Collection, Manassas, VA, unless otherwise stated) were maintained in a humidified incubator with 5% CO 2 in room air using DMEM, DMEM/F12 (Ham), MEM, or RPMI-1640 (Invitrogen Canada, Inc., Burlington, Ontario, Canada) supple- mented with 10% fetal bovine serum and antibiotics (100 U/mL 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.10.168 Abbreviations: CNS, central nervous system; EAAT1, excitatory amino acid transporter 1; EAAT2, excitatory amino acid transporter 2; GLAST-1, glutamate/ aspartate transporter 1; GLT-1, glutamate transporter 1; VGLUT1, vesicular glutamate transporter 1; xCT, light-chain subunit of system x c À glutamate/cystine antiporter. * Corresponding author. Address: Research Department, Juravinski Cancer Cen- tre, 699 Concession Street, Hamilton, Ont., Canada L8V 5C2. Fax: +1 905 575 6368. E-mail address: gurmit.singh@jcc.hhsc.ca (G. Singh). 1 These authors contributed equally to this work. Biochemical and Biophysical Research Communications 391 (2010) 91–95 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc