Inhibition of xCT by sulfasalazine results in improved radioresponse in hypoxic human colorectal cancer cells.

Research output: Unpublished contribution to conferencePoster

Abstract

Purpose/objective
Hypoxia is a well-known characteristic of solid tumors that contributes to radioresistance. Furthermore, (hypoxic) cancer cells are characterized by a dysregulated redox homeostasis. It is hypothesized that colorectal cancers (CRC) upregulate the expression of system Xc- to cope with the increased amounts of reactive oxygen species (ROS). This antiporter is responsible for the uptake of cystine, the rate-limiting precursor of glutathione (GSH), one of the main antioxidants. Anti-inflammatory drug sulfasalazine (SSZ) is a known inhibitor of xCT and thus increases oxidative stress. Furthermore, it has been described that xCT-inhibitors induce ferroptosis, which is being investigated in the context of ionizing radiation. Hence, we hypothesized that targeting xCT is an attractive strategy to induce radiomodulatory effects in CRC.
Material/Methods
mRNA levels of SLC7A11 (gene encoding xCT) in CRC patients were examined using the cBioPortal tool utilizing data from the TCGA database. Human CRC cell lines DLD1 and HCT116 were subsequently evaluated. Levels of xCT were determined by western Blot and non-toxic doses of SSZ were determined. The levels of GSH, ROS, Thioredoxin reductase (TrxR), superoxide dismutase (SOD), DNA damage and ferroptosis were examined after SSZ treatment. Induction of ROS and ferroptosis was additionally examined in 3D models. The radio-modulatory effects of SSZ were determined under normoxic and radiobiological relevant hypoxic conditions (0.1%) in 2D and 3D models. Nu/Nu nude mice were injected with DLD1 cells and treated with SSZ and fractionated radiotherapy (3x4Gy). Tumor growth was followed up and 4-HNE expression in tumors analyzed by IHC.
Results
mRNA levels of SLC7A11 were upregulated in microsatellite instable (MSI) tumors compared to microsatellite stable (MSS) tumors. Additionally, a significant correlation could be observed between expression of SLC7A11 and validated hypoxia scores. xCT expression could be detected in DLD1 and HCT116 cells and this was further increased under hypoxic conditions. Treatment with non-toxic doses of SSZ under hypoxic conditions decreased the levels of GSH and TrxR significantly, with a more pronounced effect in DLD1 cells. SOD levels remained unaltered. SSZ radiosensitized human CRC cell line DLD1 and HCT116 under hypoxic conditions with enhancement ratio (ER) of 1.9 and 1.6 respectively, while only a marginal effect was observed under normoxic conditions. ROS levels in both cell lines were significantly upregulated (up to 4 and 7-fold increase), which could be confirmed in 3D models, inducing DNA damage in both cell lines (up to 1.5-fold). High ferroptosis induction resulting from SSZ treatment could only be observed in DLD1 cells in 2D and 3D models. In vivo experiments are still ongoing.
Conclusion
Our results demonstrated that SSZ significantly decreased GSH and TrxR, leading to increased ROS levels within the 2D and 3D models. SSZ radiosensitized DLD1 and HCT116 both in 2D and 3D models. The main mechanisms that could be related to this radiosensitizing effect were the induction of ferroptosis in DLD1 cells and induction of DNA damage through increases in ROS in both DLD1 and HCT116 cells. In vivo confirmation is still necessary before SSZ can be translated into the clinic.
Original languageEnglish
Publication statusUnpublished - 2022
EventBACR Annual meeting 2022 -
Duration: 30 Sep 202230 Sep 2022

Conference

ConferenceBACR Annual meeting 2022
Period30/09/2230/09/22

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