DNA-protein cross-links emerge as major contributors to chemotherapeutic cytotoxicity at physiological equitoxic doses
Abstract Chemotherapeutic drugs induce DNA damage, including double-strand breaks (DSBs), interstrand cross-links (ICLs), and DNA-protein cross-links (DPCs), to inhibit cancer cell proliferation. Understanding the relative contributions of these damages is essential for optimizing therapeutic strate...
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Nature Portfolio
2025-07-01
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| Online Access: | https://doi.org/10.1038/s41598-025-06705-3 |
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| author | Mahmoud I. Shoulkamy Tareg Omer Mohammed Hiroshi Ide Toshiaki Nakano |
| author_facet | Mahmoud I. Shoulkamy Tareg Omer Mohammed Hiroshi Ide Toshiaki Nakano |
| author_sort | Mahmoud I. Shoulkamy |
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| description | Abstract Chemotherapeutic drugs induce DNA damage, including double-strand breaks (DSBs), interstrand cross-links (ICLs), and DNA-protein cross-links (DPCs), to inhibit cancer cell proliferation. Understanding the relative contributions of these damages is essential for optimizing therapeutic strategies. To achieve physiologically relevant conditions, we determined the LD20 for four classes of chemotherapeutic agents and treated HeLa cells accordingly. Topoisomerase inhibitors (CPT, ETO) primarily induced DSBs and DPCs, whereas platinum-based agents (CisPt, OXA) predominantly caused DPCs and ICLs. The DNMT inhibitor AzadC was strongly associated with DPC formation. Although both L-PAM and MMC are bifunctional alkylating agents, their cytotoxic mechanisms differed; L-PAM induced DSBs, DPCs, and ICLs, while MMC primarily caused ICLs. DPCs were consistently detected across all drug treatments except MMC, with a half-life of 4.7 to 8.4 h, suggesting their prolonged impact on cytotoxicity. To assess apoptosis induction, we performed Annexin-V assays, which revealed significant apoptotic responses in all treated cells. CPT exhibited the highest proportion of early apoptotic cells (~ 80%) at 24 h, with all drug treatments shifting from early to late apoptosis over time. By 48 h, late apoptotic fractions exceeded 60% in CPT-, ETO-, and AzadC-treated cells. These findings highlight the critical role of DPCs in chemotherapeutic cytotoxicity and suggest that targeting apoptotic pathways could enhance cancer treatment efficacy. |
| format | Article |
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| institution | DOAJ |
| issn | 2045-2322 |
| language | English |
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| spelling | doaj-art-5f90f91189364939bab158301f2eaec52025-08-20T03:03:27ZengNature PortfolioScientific Reports2045-23222025-07-0115111410.1038/s41598-025-06705-3DNA-protein cross-links emerge as major contributors to chemotherapeutic cytotoxicity at physiological equitoxic dosesMahmoud I. Shoulkamy0Tareg Omer Mohammed1Hiroshi Ide2Toshiaki Nakano3WPI Nano Life Science Institute (WPI-Nano LSI), Kanazawa UniversityWPI Nano Life Science Institute (WPI-Nano LSI), Kanazawa UniversityProgram of Mathematical and Life Sciences, Department of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima UniversityProgram of Mathematical and Life Sciences, Department of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima UniversityAbstract Chemotherapeutic drugs induce DNA damage, including double-strand breaks (DSBs), interstrand cross-links (ICLs), and DNA-protein cross-links (DPCs), to inhibit cancer cell proliferation. Understanding the relative contributions of these damages is essential for optimizing therapeutic strategies. To achieve physiologically relevant conditions, we determined the LD20 for four classes of chemotherapeutic agents and treated HeLa cells accordingly. Topoisomerase inhibitors (CPT, ETO) primarily induced DSBs and DPCs, whereas platinum-based agents (CisPt, OXA) predominantly caused DPCs and ICLs. The DNMT inhibitor AzadC was strongly associated with DPC formation. Although both L-PAM and MMC are bifunctional alkylating agents, their cytotoxic mechanisms differed; L-PAM induced DSBs, DPCs, and ICLs, while MMC primarily caused ICLs. DPCs were consistently detected across all drug treatments except MMC, with a half-life of 4.7 to 8.4 h, suggesting their prolonged impact on cytotoxicity. To assess apoptosis induction, we performed Annexin-V assays, which revealed significant apoptotic responses in all treated cells. CPT exhibited the highest proportion of early apoptotic cells (~ 80%) at 24 h, with all drug treatments shifting from early to late apoptosis over time. By 48 h, late apoptotic fractions exceeded 60% in CPT-, ETO-, and AzadC-treated cells. These findings highlight the critical role of DPCs in chemotherapeutic cytotoxicity and suggest that targeting apoptotic pathways could enhance cancer treatment efficacy.https://doi.org/10.1038/s41598-025-06705-3DNA damageChemotherapeutic drugsCytotoxicityEquitoxic doseCancer therapies |
| spellingShingle | Mahmoud I. Shoulkamy Tareg Omer Mohammed Hiroshi Ide Toshiaki Nakano DNA-protein cross-links emerge as major contributors to chemotherapeutic cytotoxicity at physiological equitoxic doses Scientific Reports DNA damage Chemotherapeutic drugs Cytotoxicity Equitoxic dose Cancer therapies |
| title | DNA-protein cross-links emerge as major contributors to chemotherapeutic cytotoxicity at physiological equitoxic doses |
| title_full | DNA-protein cross-links emerge as major contributors to chemotherapeutic cytotoxicity at physiological equitoxic doses |
| title_fullStr | DNA-protein cross-links emerge as major contributors to chemotherapeutic cytotoxicity at physiological equitoxic doses |
| title_full_unstemmed | DNA-protein cross-links emerge as major contributors to chemotherapeutic cytotoxicity at physiological equitoxic doses |
| title_short | DNA-protein cross-links emerge as major contributors to chemotherapeutic cytotoxicity at physiological equitoxic doses |
| title_sort | dna protein cross links emerge as major contributors to chemotherapeutic cytotoxicity at physiological equitoxic doses |
| topic | DNA damage Chemotherapeutic drugs Cytotoxicity Equitoxic dose Cancer therapies |
| url | https://doi.org/10.1038/s41598-025-06705-3 |
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