title: The glutamate transport inhibitor DL-Threo-β-Benzyloxyaspartic acid (DL-TBOA) differentially affects SN38- and oxaliplatin-induced death of drug-resistant colorectal cancer cells.
abstract: BACKGROUND
  Colorectal cancer (CRC) is a leading cause of cancer death globally and new biomarkers and treatments are severely needed.
  
  
  METHODS
  Here, we employed HCT116 and LoVo human CRC cells made resistant to either SN38 or oxaliplatin, to investigate whether altered expression of the high affinity glutamate transporters Solute Carrier (SLC)-1A1 and -1A3 (EAAT3, EAAT1) is associated with the resistant phenotypes. Analyses included real-time quantitative PCR, immunoblotting and immunofluorescence analyses, radioactive tracer flux measurements, and biochemical analyses of cell viability and glutathione content. Results were evaluated using one- and two-way ANOVA and Students two-tailed t-test, as relevant.
  
  
  RESULTS
  In SN38-resistant HCT116 and LoVo cells, SLC1A1 expression was down-regulated ~60 % and up-regulated ~4-fold, respectively, at both mRNA and protein level, whereas SLC1A3 protein was undetectable. The changes in SLC1A1 expression were accompanied by parallel changes in DL-Threo-β-Benzyloxyaspartic acid (TBOA)-sensitive, UCPH101-insensitive [(3)H]-D-Aspartate uptake, consistent with increased activity of SLC1A1 (or other family members), yet not of SLC1A3. DL-TBOA co-treatment concentration-dependently augmented loss of cell viability induced by SN38, while strongly counteracting that induced by oxaliplatin, in both HCT116 and LoVo cells. This reflected neither altered expression of the oxaliplatin transporter Cu(2+)-transporter-1 (CTR1), nor changes in cellular reduced glutathione (GSH), although HCT116 cell resistance per se correlated with increased cellular GSH. DL-TBOA did not significantly alter cellular levels of p21, cleaved PARP-1, or phospho-Retinoblastoma protein, yet altered SLC1A1 subcellular localization, and reduced chemotherapy-induced p53 induction.
  
  
  CONCLUSIONS
  SLC1A1 expression and glutamate transporter activity are altered in SN38-resistant CRC cells. Importantly, the non-selective glutamate transporter inhibitor DL-TBOA reduces chemotherapy-induced p53 induction and augments CRC cell death induced by SN38, while attenuating that induced by oxaliplatin. These findings may point to novel treatment options in treatment-resistant CRC.
authors.affiliation: Department of Biology, Faculty of Science, University of Copenhagen, 13, Universitetsparken, DK-2100, Copenhagen, Denmark. elenapedraz@gmail.com.
authors.forename: Elena
authors.initials: E
authors.lastname: Pedraz-Cuesta
authors.affiliation: Department of Biology, Faculty of Science, University of Copenhagen, 13, Universitetsparken, DK-2100, Copenhagen, Denmark. sandra.christensen@outlook.com.
authors.forename: Sandra
authors.initials: S
authors.lastname: Christensen
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, 13, Universitetsparken, DK-2100, Copenhagen, Denmark. aaj@sund.ku.dk.
authors.forename: Anders A
authors.initials: AA
authors.lastname: Jensen
authors.affiliation: Faculty of Health and Medical Sciences, Institute of Veterinary Disease Biology, University of Copenhagen, Copenhagen, Denmark. nielsfrankjensen@gmail.com.
authors.forename: Niels Frank
authors.initials: NF
authors.lastname: Jensen
authors.affiliation: Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, 13, Universitetsparken, DK-2100, Copenhagen, Denmark. lebu@sund.ku.dk.
authors.forename: Lennart
authors.initials: L
authors.lastname: Bunch
authors.affiliation: Faculty of Health and Medical Sciences, Institute of Veterinary Disease Biology, University of Copenhagen, Copenhagen, Denmark. maria.unni.romer@gmail.com.|Department of Clinical Physiology, Nuclear Medicine and PET, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark. maria.unni.romer@gmail.com.
authors.forename: Maria Unni
authors.initials: MU
authors.lastname: Romer
authors.affiliation: Faculty of Health and Medical Sciences, Institute of Veterinary Disease Biology, University of Copenhagen, Copenhagen, Denmark. nbr@sund.ku.dk.
authors.forename: Nils
authors.initials: N
authors.lastname: Brünner
authors.affiliation: Faculty of Health and Medical Sciences, Institute of Veterinary Disease Biology, University of Copenhagen, Copenhagen, Denmark. stenvang@sund.ku.dk.
authors.forename: Jan
authors.initials: J
authors.lastname: Stenvang
authors.affiliation: Department of Biology, Faculty of Science, University of Copenhagen, 13, Universitetsparken, DK-2100, Copenhagen, Denmark. sfpedersen@bio.ku.dk.
authors.forename: Stine Falsig
authors.initials: SF
authors.lastname: Pedersen
mesh_terms: D027322:Amino Acid Transport System X-AG / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D001224:Aspartic Acid / Q000494:pharmacology*; D002166:Camptothecin / Q000031:analogs & derivatives* / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism*; D003300:Copper / Q000378:metabolism; D019008:Drug Resistance, Neoplasm; D027341:Excitatory Amino Acid Transporter 1 / Q000235:genetics / Q000378:metabolism; D050580:Excitatory Amino Acid Transporter 3 / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D005978:Glutathione / Q000378:metabolism; D045325:HCT116 Cells; D006801:Humans; D000077146:Irinotecan; D009944:Organoplatinum Compounds / Q000494:pharmacology*; D000077150:Oxaliplatin; D021381:Protein Transport; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: D027322:Amino Acid Transport System X-AG; D000970:Antineoplastic Agents; D027341:Excitatory Amino Acid Transporter 1; D050580:Excitatory Amino Acid Transporter 3; D009944:Organoplatinum Compounds; C493400:SLC1A1 protein, human; C493394:SLC1A3 protein, human; D016159:Tumor Suppressor Protein p53; C120673:benzyloxyaspartate; D000077150:Oxaliplatin; D001224:Aspartic Acid; D000077146:Irinotecan; D003300:Copper; D005978:Glutathione; D002166:Camptothecin