Abstract
Amaç: Doksorubisin, yaygın olarak kullanılan bir antrasiklin antibiyotiği olup antitümör etkilerini DNA interkalasyonu, topoizomeraz II inhibisyonu ve oksidatif stresin indüklenmesi gibi çeşitli mekanizmalar aracılığıyla gösterir. Bu çalışma, doksorubisinin Kaspaz-1 ve Gasdermin D (GSDMD) aracılığıyla inflamatuar hücre ölümü yolu olan piroptozu aktive etme potansiyelini ve üç boyutlu (3B) hücre kültürü modelinde sferoid morfolojisi üzerindeki etkisini değerlendirmeyi amaçlamaktadır.
Gereç ve Yöntem: Sferoidler 3B hücre kültürü teknikleriyle oluşturulmuş ve farklı dozlardaki doksorubisin (0,1–100 µM) ile 24 ve 48 saat süreyle tedavi edilmiştir. Sferoid sayısı ve çapı ImageJ v1.47 yazılımı ile analiz edilmiştir. Kaspaz-1 ve GSDMD protein düzeyleri, piroptotik yolların aktivasyonunu değerlendirmek için ELISA yöntemiyle ölçülmüştür.
Bulgular: Doksorubisin uygulaması, sferoid sayısı ve çapında doz bağımlı azalmaya yol açmıştır. 100 µM’de, sferoid oluşumu tamamen inhibe edilmiş ve dağılmış hücre kümeleri gözlenmiştir. Kaspaz-1 ve GSDMD protein düzeyleri, yüksek doksorubisin konsantrasyonları ile anlamlı şekilde artmış, maksimum ekspresyon 25 µM’de belirlenmiştir; bu da inflamatuar hücre ölüm yollarının aktive olduğunu göstermektedir. 24 ve 48 saatlik tedavi süreleri arasında anlamlı bir fark saptanmamıştır.
Sonuç: Doksorubisin, 3B sferoid yapısını bozarak ve piroptozla ilişkili proteinlerin ekspresyonunu doz bağımlı şekilde artırmaktadır. Bu bulgular, doksorubisinin tümör yapısını bozan ve inflamatuar hücre ölüm mekanizmalarını aktive eden iki yönlü etkisini vurgulayarak, solid tümörlerdeki terapötik potansiyeline dair önemli ipuçları sunmaktadır.
References
- Ben-Dror J, Shalamov M, Sonnenblick A. The history of early breast cancer treatment. Genes (Basel) 2022;13(6):960.
- Xiong X, Zheng LW, Ding Y, Chen YF, Cai YW, Wang LP, et al. Breast cancer: pathogenesis and treatments. Signal Transduct Target Ther 2025;10(1):49.
- Hou X, Xu J, Wang Y, Zhao J, Guan Y, Yang X, et al. Triggering pyroptosis by doxorubicin-loaded multifunctional nanoparticles in combination with decitabine for breast cancer chemoimmunotherapy. ACS Appl Mater Interfaces 2024;16(43):58392-404.
- Wang Y, Gao W, Shi X, Ding J, Liu W, He H, et al. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature 2017;547(7661):99-103.
- Zhang Z, Zhang Y, Xia S, Kong Q, Li S, Liu X, et al. Gasdermin E suppresses tumor growth by activating anti-tumor immunity. Nature 2020;579(7799):415-20.
- Fang Y, Tian S, Pan Y, Li W, Wang Q, Tang Y, et al. Pyroptosis: a new frontier in cancer. Biomed Pharmacother 2020;121:109595.
- Xia X, Wang X, Cheng Z, Qin W, Lei L, Jiang J, et al. The role of pyroptosis in cancer: pro-cancer or pro-host? Cell Death Dis 2019;10(9):650.
- Yang F, Bettadapura SN, Smeltzer MS, Zhu H, Wang S. Pyroptosis and pyroptosis-inducing cancer drugs. Acta Pharmacol Sin 2022;43(10):2462-73.
- Liu SW, Song WJ, Ma GK, Wang H, Yang L. Pyroptosis and its role in cancer. World J Clin Cases 2023;11(11):2386-95.
- Ma C, Wang Y, Chen W, Hou T, Zhang H, Zhang H, et al. Caspase-1 regulates the apoptosis and pyroptosis induced by phthalocyanine zinc-mediated photodynamic therapy in breast cancer MCF-7 cells. Molecules 2023;28(16):5934.
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- Hamilton G, Rath B. Applicability of tumor spheroids for in vitro chemosensitivity assays. Expert Opin Drug Metab Toxicol 2019;15(1):15-23.
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- Edmondson R, Broglie JJ, Adcock AF, Yang L. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol 2014;12(4):207-18.
- Al-Sisan SM, Zihlif MA, Hammad HM. Differential miRNA expression of hypoxic MCF7 and PANC-1 cells. Front Endocrinol (Lausanne) 2023;14:1110743.
- Aydemir I. The inhibition effect of curcumin on MCF-7 breast cancer cells via GSK-3beta and VEGF signals. Manisa Celal Bayar Univ Health Sci Inst J 2021;8(2):337-42.
- Coelho LL, Vianna MM, da Silva DM, Gonzaga BMS, Ferreira RR, Monteiro AC, et al. Spheroid model of mammary tumor cells: epithelial-mesenchymal transition and doxorubicin response. Biology (Basel) 2024;13(7):463.
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- Yan H, Luo B, Wu X, Guan F, Yu X, Zhao L, Ke X, Wu J, Yuan J. Cisplatin Induces Pyroptosis via Activation of MEG3/NLRP3/caspase-1/GSDMD Pathway in Triple-Negative Breast Cancer. Int J Biol Sci 2021;17(10):2606-21.
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- Xin G, Hou Y, Liu Y, Li H. Tetrandrine and adriamycin reverse multidrug resistance by regulating NLRP3/Caspase-1/GSDMD signaling in human breast cancer cells. Eur J Pharmacol 2025;998:177635.
- Gao K, Liu Y, Sun C, Wang Y, Bao H, Liu G, et al. TNF-ɑ induces mitochondrial dysfunction to drive NLRP3/Caspase-1/GSDMD-mediated pyroptosis in MCF-7 cells. Sci Rep 2024;14(1):25880.
Abstract
Aim: Doxorubicin, a widely used anthracycline antibiotic, exerts its antitumor effects through multiple mechanisms, including DNA intercalation, inhibition of topoisomerase II, and the induction of oxidative stress. This study aimed to evaluate its potential to activate the inflammatory cell death pathway, pyroptosis via Caspase-1 and Gasdermin D (GSDMD), as well as its impact on spheroid morphology in a three-dimensional (3D) cell culture model.
Materials and Methods: Spheroids were formed using 3D cell culture techniques and treated with increasing concentrations of doxorubicin (0.1–100 µM) for 24 and 48 hours. Spheroid number and diameter were analyzed using ImageJ v1.47. Caspase-1 and GSDMD protein levels were quantified by ELISA to assess the activation of pyroptotic pathways.
Results: Doxorubicin treatment led to a dose-dependent reduction in both spheroid number and size. At 100 µM, spheroid formation was completely inhibited and replaced by disorganized cell clusters. Caspase-1 and GSDMD protein levels increased significantly with higher doxorubicin concentrations, with peak expression observed at 25 µM; suggesting activation of inflammatory cell death pathways. No significant differences were found between 24- and 48-hour treatments.
Conclusion: Doxorubicin disrupts 3D spheroid structure and induces the expression of pyroptosis-related proteins in a concentration-dependent manner. These findings highlight its dual role in both impairing tumor architecture and activating inflammatory cell death mechanisms, providing insights into its therapeutic potential in solid tumors.
Ethical Statement
Dear Editor, In our study titled "Effect of doxorubicin on Caspase-1/GSDMD-mediated pyroptosis in MCF-7 breast cancer spheroids", we used a commercially obtained cell line, the name of the company from which the cell line was obtained and the registration number are provided in the ‘’Materials and Methods’’ section of the manuscript. Cells isolated directly from humans were not used; therefore, ethical committee approval is not required.
References
- Ben-Dror J, Shalamov M, Sonnenblick A. The history of early breast cancer treatment. Genes (Basel) 2022;13(6):960.
- Xiong X, Zheng LW, Ding Y, Chen YF, Cai YW, Wang LP, et al. Breast cancer: pathogenesis and treatments. Signal Transduct Target Ther 2025;10(1):49.
- Hou X, Xu J, Wang Y, Zhao J, Guan Y, Yang X, et al. Triggering pyroptosis by doxorubicin-loaded multifunctional nanoparticles in combination with decitabine for breast cancer chemoimmunotherapy. ACS Appl Mater Interfaces 2024;16(43):58392-404.
- Wang Y, Gao W, Shi X, Ding J, Liu W, He H, et al. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin. Nature 2017;547(7661):99-103.
- Zhang Z, Zhang Y, Xia S, Kong Q, Li S, Liu X, et al. Gasdermin E suppresses tumor growth by activating anti-tumor immunity. Nature 2020;579(7799):415-20.
- Fang Y, Tian S, Pan Y, Li W, Wang Q, Tang Y, et al. Pyroptosis: a new frontier in cancer. Biomed Pharmacother 2020;121:109595.
- Xia X, Wang X, Cheng Z, Qin W, Lei L, Jiang J, et al. The role of pyroptosis in cancer: pro-cancer or pro-host? Cell Death Dis 2019;10(9):650.
- Yang F, Bettadapura SN, Smeltzer MS, Zhu H, Wang S. Pyroptosis and pyroptosis-inducing cancer drugs. Acta Pharmacol Sin 2022;43(10):2462-73.
- Liu SW, Song WJ, Ma GK, Wang H, Yang L. Pyroptosis and its role in cancer. World J Clin Cases 2023;11(11):2386-95.
- Ma C, Wang Y, Chen W, Hou T, Zhang H, Zhang H, et al. Caspase-1 regulates the apoptosis and pyroptosis induced by phthalocyanine zinc-mediated photodynamic therapy in breast cancer MCF-7 cells. Molecules 2023;28(16):5934.
- Gielecińska A, Kciuk M, Yahya EB, Ainane T, Mujwar S, Kontek R. Apoptosis, necroptosis, and pyroptosis as alternative cell death pathways induced by chemotherapeutic agents? Biochim Biophys Acta Rev Cancer 2023;1878(6):189024.
- Lee SY, Koo IS, Hwang HJ, Lee DW. In vitro three-dimensional (3D) cell culture tools for spheroid and organoid models. SLAS Discov 2023;28(4):119-37.
- Hamilton G, Rath B. Applicability of tumor spheroids for in vitro chemosensitivity assays. Expert Opin Drug Metab Toxicol 2019;15(1):15-23.
- Su C, Chuah YJ, Ong HB, Tay HM, Dalan R, Hou HW. A facile and scalable hydrogel patterning method for microfluidic 3D cell culture and spheroid-in-gel culture array. Biosensors (Basel) 2021;11(12):509.
- Jinhai Y, Yunxiu C, Qi J, Jiancheng G, Zhida P, Sha W, et al. Three-dimensional cell spheroid culture and cell viability study of uveal melanoma cell line C918 with luteolin treatment. Int Ophthalmol 2024;44(1):385.
- Edmondson R, Broglie JJ, Adcock AF, Yang L. Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors. Assay Drug Dev Technol 2014;12(4):207-18.
- Al-Sisan SM, Zihlif MA, Hammad HM. Differential miRNA expression of hypoxic MCF7 and PANC-1 cells. Front Endocrinol (Lausanne) 2023;14:1110743.
- Aydemir I. The inhibition effect of curcumin on MCF-7 breast cancer cells via GSK-3beta and VEGF signals. Manisa Celal Bayar Univ Health Sci Inst J 2021;8(2):337-42.
- Coelho LL, Vianna MM, da Silva DM, Gonzaga BMS, Ferreira RR, Monteiro AC, et al. Spheroid model of mammary tumor cells: epithelial-mesenchymal transition and doxorubicin response. Biology (Basel) 2024;13(7):463.
- Liu M, Yin H, Qian X, Dong J, Qian Z, Miao J. Xanthohumol, a prenylated chalcone from hops, inhibits the viability and stemness of doxorubicin-resistant MCF-7/ADR cells. Molecules 2016;22(1):36.
- Crispim D, Ramos C, Esteves F, Kranendonk M. The adaptation of MCF-7 breast cancer spheroids to the chemotherapeutic doxorubicin: the dynamic role of phase I drug metabolizing enzymes. Metabolites 2025;15(2):136.
- Yang Y, Liu PY, Bao W, Chen SJ, Wu FS, Zhu PY. Hydrogen inhibits endometrial cancer growth via a ROS/NLRP3/caspase-1/GSDMD-mediated pyroptotic pathway. BMC Cancer 2020;20(1):28.
- Yan H, Luo B, Wu X, Guan F, Yu X, Zhao L, Ke X, Wu J, Yuan J. Cisplatin Induces Pyroptosis via Activation of MEG3/NLRP3/caspase-1/GSDMD Pathway in Triple-Negative Breast Cancer. Int J Biol Sci 2021;17(10):2606-21.
- Chen C, Ye Q, Wang L, Zhou J, Xiang A, Lin X, Guo J, Hu S, Rui T, Liu J. Targeting pyroptosis in breast cancer: biological functions and therapeutic potentials on It. Cell Death Discov 2023;9(1):75.
- Xin G, Hou Y, Liu Y, Li H. Tetrandrine and adriamycin reverse multidrug resistance by regulating NLRP3/Caspase-1/GSDMD signaling in human breast cancer cells. Eur J Pharmacol 2025;998:177635.
- Gao K, Liu Y, Sun C, Wang Y, Bao H, Liu G, et al. TNF-ɑ induces mitochondrial dysfunction to drive NLRP3/Caspase-1/GSDMD-mediated pyroptosis in MCF-7 cells. Sci Rep 2024;14(1):25880.
Details
| Primary Language | English |
|---|---|
| Subjects | Cancer Cell Biology, Histology and Embryology |
| Journal Section | Research Article |
| Authors | |
| Submission Date | April 26, 2025 |
| Acceptance Date | June 19, 2025 |
| Publication Date | December 8, 2025 |
| DOI | https://doi.org/10.19161/etd.1684512 |
| IZ | https://izlik.org/JA66ES67PE |
| Published in Issue | Year 2025 Volume: 64 Issue: 4 |
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