Session Item

Hypoxia is well-known characteristic of solid tumors that contributes to radioresistance. Furthermore, (hypoxic) cancer cells are characterized by a dysregulated redox homeostasis. Cells heavily rely on the antioxidant system to sustain the balance between reactive oxygen species (ROS) production and scavenging. It is hypothesized that colorectal cancers (CRC), among others, upregulate the expression of system Xc- to cope with the increased amounts of ROS. This antiporter is responsible for the uptake of cystine, the rate-limiting precursor of glutathione (GSH) and one of the main antioxidants in the cell. Anti-rheumatoid arthritis drug sulfasalazine (SSZ) is a known inhibitor of xCT (the light subunit of system Xc-) and thus increases oxidative stress. Furthermore, it has been described that xCT inhibitors can induce ferroptosis, which is being investigated in the context of ionizing radiation. Hence, we hypothesized that targeting xCT, by FDA-approved drug SSZ, is an attractive strategy to induce radiomodulatory effects in CRC by disrupting the redox homeostasis and inducing ferroptosis. 

mRNA levels of xCT were examined in CRC using the cBioPortal tool utilizing data from the TCGA database. Human CRC cell lines DLD-1 and HCT116 were further evaluated. Levels of xCT were determined by RT-PCR and western Blot and non-toxic doses of SSZ were determined. The levels of GSH, ROS and ferroptosis were examined after SSZ treatment. xCT knock out (KO) cell lines were generated as a proof of concept.  The radio-modulatory effects of SSZ were determined under normoxic and radiobiological relevant hypoxic conditions (0.1%). 

Treatment with non-toxic doses of SSZ decreased the levels of GSH significantly, both in normoxic and hypoxic conditions, with a more pronounced effect in DLD-1 cells. SSZ slightly radiosensitized human CRC cell line DLD-1 under normoxic conditions with an enhancement ratio (ER) of 1.4.  Under hypoxic conditions, the radiosensitizing effect of SSZ on DLD-1 was further increased (ER: 2.0), while only a marginal effect was observed in HCT116 (ER: 1.4). ROS levels in both cell lines were significantly upregulated (up to 10-fold increase), while induction of ferroptosis was only observed in DLD-1 cells. 

Very little evidence of the radiomodulatory effect of SSZ has been described before and no evidence has been generated under hypoxic conditions. Our preliminary results suggest that SSZ significantly decreased GSH and increased ROS levels within the cells and radiosensitized DLD-1 cells through induction of ferroptosis. Underlying mechanistical pathways by using xCT KO cells and the role of ferroptosis in hypoxic radioresponses are still under investigation. Elucidating the cell line specific sensitivity towards the drug and in vivo confirmation are still necessary before SSZ, alone or in combination therapies, can be used in the clinic as a radiosensitizer.