1. Glutamate transporter SLC1A1 is dysregulated in SN38- and Oxaliplatin-resistant colorectal cancer cells Elena Pedraz-Cuesta 1, Sandra Christensen 1, Anders A. Jensen 2, Niels F. Jensen 3, Maria U. Romer 3, Nils Brünner 3, Jan Stenvang 3, and Stine F. Pedersen 1 Expression of SLC1A1 is altered in SN38- and Oxa-resistant CRC lines Consistent with the microarray data, quantitative real time PCR (qPCR) analysis showed that, compared to that in parental cells, the SLC1A1 mRNA level was increased in Oxa resistant HCT116 cells and SN38 resistant LoVo cells, yet tended to be downregulated in SN38 resistant HCT116 cells and not altered in Oxa resistant LoVo cells. The protein level of SLC1A1 followed the same pattern. Glutamate transporter activity is altered in SN38- and Oxa-resistant CRC lines Glutamate transporter activity was measured as uptake of the SLC1A1 and SLC1A3 substrate [ 3 H] Aspartate. Comparing the basal [ 3 H] Aspartate uptake in the resistant and parental cells, it is seen that uptake was decreased by about 60% in SN38 resistant HCT116 cells and, conversely, nearly tripled in SN38 resistant LoVo cells. In Oxa resistant cell lines, uptake was not significantly affected neither in the HCT116 or the LoVo model. Introduction and aim Colorectal cancer (CRC) is the 3rd most common cancer worldwide and has a survival rate of <50% (1). The response rates to standard treatment with Oxaliplatin (Oxa) and the Irinotecan metabolite, SN38, are 31-56% and drug resistance is a major problem (2). To study mechanisms of resistance in CRC, Oxa- and SN38-resistant cell lines were established from HCT116 and LoVo CRC cell lines. Microarray analysis shows an altered expression pattern in many ion transporter and channel proteins. The glutamate transporter SLC1A1 (EAAT3) was markedly up regulated at mRNA and protein levels in both cell lines. As this transporter has not previously been implicated in SN38 or Oxa resistance and is generally restricted to the central nervous system, it is a potential novel biomarker for resistance and an interesting candidate for therapeutic targeting. Therefore, the aim of our study was to determine whether an altered expression pattern and activity of glutamate transporter SLC1A1 contributes to resistance towards Oxa and SN38 in CRC. U N I V E R S I T Y O F C O P E N H A G E N Results C SN38 (µM) * * * * * TBOA (µM) A HCT116-PAR Oxaliplatin (µM) * * * * * 0 0.8 20 B HCT116-PAR Viability (%) HCT116-Oxa Oxaliplatin (µM) * # * D Viability (%) SN38 (µM) * * * * HCT116-SN38 C Viability (%) LoVo-PAR * * # * * * * E SN38 (µM) Viability (%) * * * * # F LoVo-PAR Oxaliplatin (µM) LoVo-SN38 G Viability (%) * ** * SN38 (µM) LoVo-Oxa * # * * Viability (%) H Oxaliplatin (µM) The SLC1A1 and -3 inhibitor TBOA dose-dependently augments cell death induced by SN38 in SN38 resistant cell lines, but protects Oxa-resistant cells from Oxa-induced cell death Viability of the parental HCT116 (Fig. 3A-B) and LoVo (Fig. 3E-F) cells was dose-dependently reduced by exposure to SN38 or Oxa after 48 h at the highest dose tested. When DL -TBOA was added concomitantly with the chemotherapeutic drugs, this had no significant effect in SN38 treated parental cell lines, although a tendency for increased loss of viability was seen (Fig. 3A, E). In contrast, DL -TBOA reversed, in a dose-dependent manner, the effect of Oxa treatment (Fig. 3B, F). The study of markers of death pathways in parental cells showed p53 and p21 were markedly induced by 24 h of treatment with either SN38- or Oxa. When cells were treated with DL -TBOA concomitant with the chemotherapeutic compounds, there was no detectable effect on the induction of p53, p21 and PARP cleavage in HCT116 cells (Fig. 4A), however, p21 after SN38 treatment was increased in LoVo and LoVo-Oxa resistant cells, and p53 and PARP cleavage after Oxa treatment decreased, in LoVo and LoVo SN38 resistant cells (Fig. 4B). Figure 4: Representative blot data of 3 independent experiments of HCT116 (A) or LoVo (B) cell lines with prior 24h chemotreatment (0,8µM SN38 or 20µM Oxa). Protein levels of SLC1A1, p53, p21 and PARP. p150 is shown as a loading control. Figure 3: MTT data HCT116 and LoVo parental treated with 48h of SN38 (A, E) and Oxa (B, F), SN38-resistance HCT116 (C, G) and Oxa-resistance HCT116 (D, H) +/- increasing concentrations of TBOA. *p < 0.05, **p < 0.01, ***p < 0.001 vs control group (without quemo and TBOA treatment) (N = 3). # p<0.05 vs control condition (without TBOA treatment) Tukey’s test following ANOVA. Cellular GSH content is altered in resistant cell lines The cellular content of glutathione (GSH) was measured in parental and resistant cell lines (Fig.5A- C). Preliminary results suggested that compared to the level in parental cells, cellular GSH content is decreased in SN38-resistant and increased in Oxa-resistant HCT116 cells (Fig 5D).. References (1) Pohl, A., Lurje, G., Manegold, P. C., & Lenz, H.-J. (2009). Pharmacogenomics and -genetics in colorectal cancer. Advanced Drug Delivery Reviews, 61(5), 375–380. (2) Mechetner, E., Brünner, N., & Parker, R. J. (2010). In vitro drug responses in primary and metastatic colorectal cancers. Scandinavian Journal of Gastroenterology, 46(1), 70–78. Conclusions  SN38- and Oxa-resistance in CRC cells is associated with SLC1A1 dysregulation.  Inhibition of SLC1A1 sensitizes SN38-resistant CRC cell lines to SN38-induced cell death, but conversely attenuates Oxa-induced cell death in Oxa-resistant cell lines.  Studies to address the possible role of GSH changes in the effect of SLC1A1 on chemotherapy-induced death are ongoing. 1) Department of Biology, Faculty of Science, University of Copenhagen; 2) Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen; 3) Institute of Veterinary Disease Biology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark. Figure1: Relative mRNA levels of SLC1A1 in parental (PAR), SN38- and Oxa-resistant HCT116 (A) and LoVo cells (C), determined by qPCR. B-D. Protein level of SLC1A1 in parental, SN38- and Oxa- resistant HCT116 (B) and LoVo (D) cells relative to that in their parental counterparts. p150 is shown as a loading control. Top: representative Western blots, bottom: densitometric quantification of the Western blot data. qPCR and Western blot data represent 3 independent experiments per condition. *p < 0.05, **p < 0.01, and ***p < 0.001, compared to parental cells by One-way ANOVA and Tukey post-test. PAR SN38 Oxa * ** Relative SLC1A1 expression levels - 60 kDa SLC1A1 p150 - 160 kDa PAR SN38 Oxa B PAR SN38 Oxa * Relative SLC1A1 expression levels -160 kDa - 60 kDa PAR SN38 Oxa SLC1A1 p150 D Relative mRNA expression level HCT116 * A PAR SN38Oxa LoVo *** Relative mRNA expression level C PAR SN38Oxa Figure 2: [ 3 H]-D-Asp uptake level in parental (PAR), SN38 and Oxaliplatin- resistant cell lines after 15 minutes of incubation with [ 3 H]-D-Asp. A. Basal uptake level. B. Treatment with different concentrations of L-Glu, TBOA or UCPH-101. Figure 5: Representative graphic data and summary GSH data (D) of 2 independent experiments of HCT116-PAR (A), HCT116-SN38 (B) or HCT116-OXA (C) cell lines with prior 24h chemotreatment (0,8µM SN38 or 20µM Oxa). AB AB [Ligand] (uM) D E F [ 3 H]-D-Asp uptake (CPM) G HCT116-PARHCT116-SN38HCT116-OXA A B C D