Samenvatting
Colorectal cancer (CRC) remains a global health burden, ranking as the third most prevalent malignancy worldwide and second leading cause of cancer-related mortality. Radiotherapy (RT) is a cornerstone treatment modality in the battle against cancer, with at least 50% of all cancer patients receiving RT during their treatment course. Combination with additional modalities, such as chemotherapy, is a widely used strategy to enhance therapeutic efficacy.
These combined therapies have the ability to induce cell death through multiple mechanisms, including apoptosis. Nevertheless, the principal hurdle of RT remains the development of resistance to tumor cell death. Targeting novel pathways to induce cell death represents a potential strategy for overcoming this limitation. Ferroptosis, an iron-dependent type of regulated cell death (RCD), has recently emerged as a mode of RCD linked to RT efficacy. However, the exact role of ferroptosis in RT-induced RCD has yet to be elucidated.
In this thesis, we first focussed on elucidating the importance of ferroptosis in RT-induced cell death and assessed whether this mode of cell death is as important as other wellacknowledged types of RCD, such as apoptosis and necroptosis. Results confirmed that ferroptosis is at least as involved in RT-induced cell death as apoptosis and necroptosis. Furthermore, clinical parameters, including hypoxia and fractionated RT increased cellular
resistance to RCD in CRC cell lines; however, the reduction in ferroptosis was less pronounced compared to apoptosis and necroptosis. These observations supported the potential use of ferroptosis inducers (FINs) as radiosensitizers in clinical settings.
Secondly, we investigated the radiosensitizing effects of sulfasalazine (SSZ), a known FIN. SSZ increased the levels of reactive oxygen species, by decreasing intracellular glutathione levels, a major antioxidant. This reduction led to elevated lipid peroxidation in the colorectal cancer cell line DLD-1, which further synergized with the lipid peroxidation induced by RT. SSZ was able to radiosensitize both hypoxic DLD-1 and HCT116 CRC cell lines; however it was more
effective in DLD-1 cells, possibly due to enhanced ferroptosis induction.
Additionally, we aimed to target this pathway further downstream, as GPX4 targeting is expected to be more potent. Direct targeting of GPX4 through RSL3 treatment did not radiosensitize hypoxic CRC cells. Potentially, due to the activation of alternative defence mechanisms as a compensatory response.
Lastly, the influence of SSZ on normal intestinal tissue was briefly examined. SSZ induced similar effects in human intestinal epithelial cells as in cancer cells. Therefore, caution is warranted before clinical translation is considered feasible.
Taken together, we have demonstrated that ferroptosis is a pivotal player in the efficacy of RT. Furthermore, FIN SSZ was able to radiosensitize hypoxic CRC cells. Therefore, the prospect of translating SSZ into clinical practice as radiosensitizer is promising. However, several limitations associated with FINs must be addressed before clinical translation is feasible. These include identifying the most effective FINs, since GPX4 inhibitor, RSL3, exhibited no radiosensitizing effect, and investigating the heterogeneous responses observed. Additionally, SSZ had similar effects on human intestinal epithelial cells, possibly indicative for off-target adverse events. Consequently, further research into biomarkers is required to select patient populations who may benefit from this type of therapy. Moreover, specific tumor targeting could improve the applicability of FINs as clinical radiosensitizers.
These combined therapies have the ability to induce cell death through multiple mechanisms, including apoptosis. Nevertheless, the principal hurdle of RT remains the development of resistance to tumor cell death. Targeting novel pathways to induce cell death represents a potential strategy for overcoming this limitation. Ferroptosis, an iron-dependent type of regulated cell death (RCD), has recently emerged as a mode of RCD linked to RT efficacy. However, the exact role of ferroptosis in RT-induced RCD has yet to be elucidated.
In this thesis, we first focussed on elucidating the importance of ferroptosis in RT-induced cell death and assessed whether this mode of cell death is as important as other wellacknowledged types of RCD, such as apoptosis and necroptosis. Results confirmed that ferroptosis is at least as involved in RT-induced cell death as apoptosis and necroptosis. Furthermore, clinical parameters, including hypoxia and fractionated RT increased cellular
resistance to RCD in CRC cell lines; however, the reduction in ferroptosis was less pronounced compared to apoptosis and necroptosis. These observations supported the potential use of ferroptosis inducers (FINs) as radiosensitizers in clinical settings.
Secondly, we investigated the radiosensitizing effects of sulfasalazine (SSZ), a known FIN. SSZ increased the levels of reactive oxygen species, by decreasing intracellular glutathione levels, a major antioxidant. This reduction led to elevated lipid peroxidation in the colorectal cancer cell line DLD-1, which further synergized with the lipid peroxidation induced by RT. SSZ was able to radiosensitize both hypoxic DLD-1 and HCT116 CRC cell lines; however it was more
effective in DLD-1 cells, possibly due to enhanced ferroptosis induction.
Additionally, we aimed to target this pathway further downstream, as GPX4 targeting is expected to be more potent. Direct targeting of GPX4 through RSL3 treatment did not radiosensitize hypoxic CRC cells. Potentially, due to the activation of alternative defence mechanisms as a compensatory response.
Lastly, the influence of SSZ on normal intestinal tissue was briefly examined. SSZ induced similar effects in human intestinal epithelial cells as in cancer cells. Therefore, caution is warranted before clinical translation is considered feasible.
Taken together, we have demonstrated that ferroptosis is a pivotal player in the efficacy of RT. Furthermore, FIN SSZ was able to radiosensitize hypoxic CRC cells. Therefore, the prospect of translating SSZ into clinical practice as radiosensitizer is promising. However, several limitations associated with FINs must be addressed before clinical translation is feasible. These include identifying the most effective FINs, since GPX4 inhibitor, RSL3, exhibited no radiosensitizing effect, and investigating the heterogeneous responses observed. Additionally, SSZ had similar effects on human intestinal epithelial cells, possibly indicative for off-target adverse events. Consequently, further research into biomarkers is required to select patient populations who may benefit from this type of therapy. Moreover, specific tumor targeting could improve the applicability of FINs as clinical radiosensitizers.
| Originele taal-2 | English |
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| Toekennende instantie |
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| Begeleider(s)/adviseur |
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| Datum van toekenning | 12 mei 2025 |
| Status | Published - 2025 |