We first hypothesized that the rescue of oxaliplatin-treated cells by DL-TBOA might reflect a dependence of oxaliplatin influx pathway(s) on glutamate transporter activity. The protein level of the major such pathway, CTR1, was modestly decreased by oxaliplatin treatment, yet was not regulated by DL-TBOA. Another possibility was that SLC1A1 might modulate cellular GSH levels, which also regulate oxaliplatin uptake via CTR1 [
35]. SLC1A1 can directly transport
l-cysteine and plays a major role in supporting GSH production [
16,
19,
20], and also SLC1A1-mediated changes in extracellular glutamate signaling could contribute, under conditions where extracellular glutamate availability is a limiting factor. Multiple components of the glutamate signaling machinery are expressed in the colon epithelium, including, in addition to glutamate transporters [
17], NMDA receptors (NMDARs) [
36] and metabotropic glutamate receptors [
37]. Indeed, NMDAR- and mGluR-antagonists inhibit the proliferation of HT29 cells [
37]. On the other hand, excessive NMDAR activation induces increased [Ca
2+]
i and consequent cell death in CRC cells [
36]. Similarly, glutamate release autocrinely stimulates gliomas, yet is toxic to surrounding neurons [
21‐
23]. Our microarray data [
13] support the notion that glutamate- and glutathione homeostasis are broadly altered in the SN38- and oxaliplatin resistant CRC cells, in agreement with previous studies showing increased GSH levels and γ-GCS up-regulation in drug-resistant cancer cells [
38]. Of note, the SN38-resistant LoVo cells, which exhibited a 4-fold increase in SLC1A1 expression, also show up-regulation of both mGluR and iGluR [
13]. Also glutamate decarboxylase, which is rate-limiting for GABA production and was recently assigned important roles in small-cell lung cancer [
39], shows increased expression in several of the resistant cell lines, as does the GSH-dependent ABCC2/MRP2 GS-X drug efflux pump [
13]. Further supporting the notion that a change in basal GSH metabolism contributes to the resistant phenotype, cellular GSH was increased in SN38- and oxaliplatin-resistant HCT116, yet this did not correlate with the effects of DL-TBOA treatment on viability.
p53 induction by chemotherapeutic treatment was reduced by DL-TBOA in both HCT116 and LoVo cells. This differs from the opposite effects of DL-TBOA on viability in HCT116 and LoVo cells, thus, the specific mechanisms involved in the latter must be cell type-dependent and/or upstream of p53. Effects related to the cotransport of Na
+ and H
+ by the glutamate transporters may also be envisaged. Thus, in mouse astrocytes, glutamate uptake reduced cytosolic and mitochondrial pH and inhibited oxidative metabolism in a manner inhibited by DL-TBOA and only in cells expressing the glutamate transporters [
40]. Other mechanisms previously implicated in oxaliplatin resistance include upregulation of Breast Cancer Resistance Protein (BCRP, ATPG2) and increased DNA-damage repair via up-regulation of Excision Repair Cross Complementing Protein 1 (ERCC1) [
41], whereas SN38 resistance was proposed to involve down-regulation of topoisomerase-I [
41]. Future studies should establish the possible link of these mechanisms to altered glutamate transporter activity.
Finally, the subcellular localization of SLC1A1 was altered in chemotherapy-resistant cells as well as by treatment with chemotherapy or DL-TBOA. Chemotherapy treatment (a reduction in viability), was associated with a shift of SLC1A1 towards a perinuclear/nuclear localization, except in HCT116-Oxa cells, in which SLC1A1 was perinuclear/nuclear already prior to treatment. Notably, the effects of DL-TBOA on SLC1A1 localization and cell viability correlated: In SN38-resistant cells, DL-TBOA augmented both SN38-induced loss of viability and chemotherapy-induced nuclear/perinuclear shift of SLC1A1, and in oxaliplatin-resistant cells, DL-TBOA counteracted both oxaliplatin-induced loss of viability and nuclear/perinuclear SLC1A1 localization. The mechanism involved cannot be deduced from the present studies, yet it is notable that regulated nuclear localization of SLC1A3 (GLAST-1) in cancer cells has been reported independently by two groups [
18,
21]. Ye et al. [
21] showed that SLC1A3 localized to the nucleus in glioma cells and glioblastoma patient brain tissue, but to the plasma membrane in normal astrocytes and normal brain tissue. Varini et al. [
18] showed that nuclear localization of SLC1A3 was associated with reduced cell density/loss of cell-cell contacts. Neither study provided direct evidence to the mechanisms involved in this phenomenon, but if SLC1A1 localization is similarly regulated by cell-cell contacts, this might suggest that the translocation is downstream of reduced cell numbers in response to chemotherapy treatment, and also this interpretation is consistent with the precise correlation between the effect of DL-TBOA on viability and on SLC1A1 localization.