Introduction
Colorectal cancer (CRC) therapy faces significant challenges, including drug resistance, tumour heterogeneity, metastatic progression, and treatment-associated toxicity. CRC and other cancers exhibit increased levels of Fe(II) due to dysregulated iron metabolism, which can act as a trigger for selective prodrug activation. In this study, we explore the therapeutic potential of a novel tumour-activated prodrug platform based on 1,2,4,5-tetraoxane scaffold that is selectively activated in cancer cells with high levels of labile Fe(II). As a proof-of-concept, we evaluated the therapeutic potential of 5b, a prodrug coupled with doxorubicin (DOX), which releases this cytotoxic payload upon activation by the labile Fe(II) pool in the cytoplasm of cancer cells.
Aims & Methods
The therapeutic potential and mechanism of action of 5b was evaluated both in vitro and in vivo. IC50 values were determined across multiple cancer cell lines and compared to non-tumorigenic cells. To confirm iron-dependency, HCT116 cell viability was assessed upon co-incubation with 5b and iron chelator deferoxamine (DFO). Co-incubation with apoptosis inhibitor Z-VAD-FMK (Zvad) or ferroptosis inhibitor liproxstatin-1 was used to assess cell death mechanism. The sub-cellular location of 5b activation was evaluated by fluorescence microscopy. In vivo efficacy was evaluated in a xenograft mouse model using immunocompromised mice implanted with HT29 cells and intraperitoneal administrations of 5b, assessing tumour burden and systemic toxicity.
Results
Prodrug 5b effectively reduced cell viability in CRC cells HCT116 and HT29, as well as in glioblastoma (U251), prostate cancer (PC3) and breast cancer (MCF7) cells, with IC50 values ranging from 1.64 to 6.69 µM. Notably, 5b was over 2000 times less toxic than DOX in non-tumorigenic cells AML12 (5b IC50 = 398.2 µM vs. DOX IC50 = 0.18 µM). Taking advantage of DOX intrinsic fluorescence properties (while 5b pro-drug is non-fluorescent), we used fluorescence microscopy analysis to confirm that 5b released its fluorescent DOX payload in the cytoplasm, after activation by labile Fe(II). The significant decrease in cell viability induced by 5b (p < 0.0001) was rescued by the iron chelator DFO and by the ferroptosis inhibitor liproxstatin-1 (p < 0.01) but not by pan-caspase inhibitor Zvad, suggesting that ferroptosis may be the primary mechanism of cell death. In vivo, 5b significantly reduced the relative tumour volume (p < 0.05), demonstrating greater efficacy than DOX, with only minor cardiotoxicity side-effects (multifocal loci of fibrosis affecting of 0.2% of left ventricle volume).
Conclusion
Prodrug 5b efficiently released its DOX payload in response to the cytoplasmatic labile Fe(II), effectively reducing cancer cell viability both in vitro and in vivo, while demonstrating significantly lower toxicity compared to DOX itself. Given its Fe(II)-dependent activation, 5b represents a promising strategy to exploit cancer cell iron addiction for cancer therapy. Future work should further validate ferroptosis as cell death mechanism, optimize the selection of cytotoxic payloads for tumours with dysregulated iron metabolism, and assess its efficacy in relation to KRAS mutational status. Funding: CMPR: PTDC/MED-FAR/3492/2021, LCF/PR/HR21/52410028; RM: 2022.07857.PTDC, PTDC/MED-QUI/30021/2017; VM: 10.54499/2023.11531.PEX; DMS: LCF/TR/CI22/52660003
Disclosure
The authors declare that this work is associated with intellectual property protection under the following patents/patent requests:
- Diogo Magalhães e Silva, Rui Moreira, Francisca Lopes, Cecília Rodrigues, Vanda Marques, “1,2,4,5-tetraoxane compounds, a drug delivery system based in said compounds useful in diagnoses and therapy, and methods thereof”, Portuguese Patent Application nº PT117644 from 16-12-2021;
- Diogo Magalhães e Silva, Rui Moreira, Francisca Lopes, Cecília Rodrigues, Vanda Marques, “Drug delivery systems based on endoperoxides useful in diagnosis and therapy, and methods thereof”, PCT Application nº PCT/IB2022/062107 from 13-12-2022;
- Diogo Magalhães e Silva, Rui Moreira, Francisca Lopes, Cecília Rodrigues, Vanda Marques, “Drug delivery systems based on endoperoxides useful in diagnosis and therapy, and methods thereof”, European Patent Office Application nº EP4448107 from 23-10-2024;
- Diogo Magalhães e Silva, Rui Moreira, Francisca Lopes, Cecília Rodrigues, Vanda Marques, “Drug delivery systems based on endoperoxides useful in diagnosis and therapy, and methods thereof”, United States Patent and Trademark Office patent application nº US20250064840 from 27-02-2025.