Ferula orientalis Ekstraktı, MDA-MB-231 Üçlü Negatif Meme Kanseri Hücrelerinde Redoks Modülasyonu Yoluyla Mitokondriyal Apoptozu İndükler

Abstract

Amaç: Üçlü negatif meme kanseri (TNBC), östrojen reseptörü, progesteron reseptörü ve HER2 ekspresyonunun olmaması ile karakterize edilen, agresif seyirli bir meme kanseri alt tipidir. Hedefe yönelik tedavi seçeneklerinin sınırlı olması ve tümör hücrelerinin redoks adaptasyonları nedeniyle TNBC hücreleri artmış oksidatif kırılganlık göstermektedir; bu durum redoks modülasyonunu umut verici bir terapötik strateji haline getirmektedir. Fitokimyasal açıdan zengin bir tıbbi bitki olan Ferula orientalis’in potansiyel antikanser özelliklere sahip biyoaktif bileşikler içerdiği bildirilmiştir. Bununla birlikte, TNBC hücrelerinde redoks homeostazı ve mitokondriyal apoptoz üzerindeki etkileri büyük ölçüde aydınlatılmamıştır. Bu çalışma, Ferula orientalis ekstraktının MDA-MB-231 TNBC hücre hattında oksidatif stres parametreleri, antioksidan enzim aktiviteleri ve apoptoz ile ilişkili gen ekspresyonu üzerindeki etkilerini araştırmayı amaçlamıştır.

Gereç ve Yöntemler: MDA-MB-231 hücreleri 24 saat süreyle 10, 50 ve 100 µg/mL konsantrasyonlarında F. orientalis ekstraktı ile muamele edilmiştir. Hücre içi indirgenmiş glutatyon (GSH) ve malondialdehit (MDA) düzeyleri redoks durumunu değerlendirmek amacıyla spektrofotometrik yöntemlerle ölçülmüştür. Süperoksit dismutaz (SOD), katalaz (CAT) ve glutatyon peroksidaz (GPX) aktiviteleri enzimatik analizlerle belirlenmiştir. Apoptozla ilişkili gen ekspresyon düzeyleri (BAX ve BCL2), 2^−ΔΔCt yöntemi kullanılarak RT-qPCR ile analiz edilmiştir. İstatistiksel değerlendirme tek yönlü ANOVA ve Tukey post-hoc testi ile yapılmıştır.

Bulgular: F. orientalis uygulaması GSH düzeylerinde doza bağımlı azalma ve MDA düzeylerinde anlamlı artışa yol açarak oksidatif stresin arttığını göstermiştir. Antioksidan enzim aktiviteleri (SOD, CAT ve GPX) tüm tedavi gruplarında anlamlı şekilde baskılanmıştır. Ayrıca BAX ekspresyonu artarken BCL2 ekspresyonu azalmış ve BAX/BCL2 oranı belirgin şekilde yükselmiştir. Bu bulgular, ekstraktın redoks dengesizliği üzerinden mitokondriyal apoptotik sinyalizasyonu aktive ettiğini göstermektedir.

Sonuç: Ferula orientalis ekstraktı TNBC hücrelerinde redoks homeostazını bozmakta ve oksidatif stres aracılı mitokondriyal apoptozu indüklemektedir. Oksidatif yükün artırılması ve anti-apoptotik savunma mekanizmalarının baskılanmasını içeren bu çift yönlü etki, F. orientalis’in TNBC’de redoks-hedefli tamamlayıcı terapötik bir aday olarak değerlendirilmesini desteklemektedir. Bulguların in vivo modellerde doğrulanması ve aktif bileşenlerin tanımlanması gerekmektedir.

Keywords

• Üçlü negatif meme kanseri
• Ferula orientalis
• Oksidatif stres
• Redoks homeostazı
• Mitokondriyal apoptoz

Ferula orientalis Extract Induces Mitochondrial Apoptosis Through Redox Modulation in MDA-MB-231 Triple-Negative Breast Cancer Cells

Abstract

Objective: Triple-negative breast cancer (TNBC) is an aggressive molecular subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor, and HER2 expression. Due to limited targeted therapeutic options and intrinsic redox adaptations, TNBC cells exhibit increased oxidative vulnerability, making redox modulation a promising therapeutic strategy. Ferula orientalis, a phytochemically rich medicinal plant, has been reported to possess bioactive compounds with potential anticancer properties. However, its effects on redox homeostasis and mitochondrial apoptosis in TNBC cells remain largely unexplored. This study aimed to investigate the impact of Ferula orientalis extract on oxidative stress parameters, antioxidant enzyme activities, and apoptosis-related gene expression in the MDA-MB-231 TNBC cell line.

Materials and Methods: MDA-MB-231 cells were treated with F. orientalis extract at concentrations of 10, 50, and 100 µg/mL for 24 hours. Intracellular reduced glutathione (GSH) and malondialdehyde (MDA) levels were measured spectrophotometrically to assess redox status. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities were determined using enzymatic assays. Apoptosis-related gene expression levels (Bax and Bcl-2) were quantified by RT-qPCR using the 2^−ΔΔCt method. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test.

Results: Treatment with F. orientalis resulted in a dose-dependent depletion of GSH and a significant elevation of MDA levels, indicating enhanced oxidative stress. Antioxidant enzyme activities (SOD, CAT, and GPX) were significantly suppressed across treatment groups. Furthermore, Bax expression was upregulated, whereas Bcl-2 expression was downregulated, leading to a marked increase in the Bax / Bcl-2 ratio. These findings collectively demonstrate activation of mitochondrial apoptotic signaling in response to extract-induced redox imbalance.

Conclusion: Ferula orientalis extract disrupts redox homeostasis and promotes oxidative stress–mediated mitochondrial apoptosis in TNBC cells. The dual mechanism involving enhancement of oxidative burden and suppression of anti-apoptotic defenses highlights the potential of F. orientalis as a redox-targeted complementary therapeutic candidate in TNBC. Further studies are required to identify active constituents and validate these findings in in vivo models.

Keywords

• Triple-negative breast cancer
• Ferula orientalis
• Oxidative stress
• Redox homeostasis
• Mitochondrial apoptosis

References

1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians 2021;71:209–49. https://doi.org/10.3322/caac.21660.
2. Bianchini G, Balko JM, Mayer IA, Sanders ME, Gianni L. Triple-negative breast cancer: challenges and opportunities of a heterogeneous disease. Nature Reviews Clinical Oncology 2016;13:674–90. https://doi.org/10.1038/nrclinonc.2016.66.
3. Minn AJ, Gupta GP, Siegel PM, Bos PD, Shu W, Giri DD, et al. Genes that mediate breast cancer metastasis to lung. Nature 2005;436:518–24. https://doi.org/10.1038/nature03799.
4. Gosslau A, Chen KY. Nutraceuticals, apoptosis, and disease prevention. Nutrition 2004;20:95–102. https://doi.org/10.1016/j.nut.2003.09.017.
5. Park EJ, Pezzuto JM. Botanicals in cancer chemoprevention. Cancer and Metastasis Reviews 2002;21:231–55. https://doi.org/10.1023/A:1021254725842.
6. Sabzehzari M, Naghavi MR, Bozari M, Orafai HM, Johnston TP, Sahebkar A. Pharmacological and therapeutic aspects of plants from the genus Ferula: a comprehensive review. Mini-Reviews in Medicinal Chemistry 2020;20:993–1007. https://doi.org/10.2174/1389557520666200129113131.
7. Ateba SB, Mvondo MA, Njamen D. Natural terpenoids against female breast cancer: a 5-year recent research. Current Medicinal Chemistry 2018;25:4915–51. https://doi.org/10.2174/0929867325666171204111651.
8. Turan M, Sökmen A, Karadayı K, Polat ZA, Şen M. Antineoplastic effects of some plant extracts specific to the Sivas region. Cumhuriyet Medical Journal 2010;32:9–18.

9. Hayes JD, Dinkova-Kostova AT, Tew KD. Oxidative stress in cancer. Cancer Cell 2020;38:167–97. https://doi.org/10.1016/j.ccell.2020.06.001.
10. Barrera G, Pizzimenti S, Daga M, Dianzani C, Arcaro A, Cetrangolo GP, et al. Lipid peroxidation-derived aldehydes, 4-hydroxynonenal and malondialdehyde in aging-related disorders. Antioxidants 2018;7:102. https://doi.org/10.3390/antiox7080102.
11. Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nature Reviews Drug Discovery 2009;8:579–91. https://doi.org/10.1038/nrd2803.
12. Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nature Reviews Cancer 2002;2:647–56. https://doi.org/10.1038/nrc883.
13. Dai J, Mumper RJ. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules 2010;15:7313–52. https://doi.org/10.3390/molecules15107313.
14. Cailleau R, Young R, Olive M, Reeves WJ Jr. Breast tumor cell lines from pleural effusions. Journal of the National Cancer Institute 1974;53:661–74. https://doi.org/10.1093/jnci/53.3.661.
15. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54. https://doi.org/10.1016/0003-2697(76)90527-3.
16. Beauchamp C, Fridovich I. Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971;44:276–87. https://doi.org/10.1016/0003-2697(71)90370-8.
17. Aebi H. Catalase in vitro. Methods in Enzymology 1984;105:121–6. https://doi.org/10.1016/S0076-6879(84)05016-3.
18. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. Journal of Laboratory and Clinical Medicine 1967;70:158–69.
19. Ellman GL. Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics 1959;82:70–7. https://doi.org/10.1016/0003-9861(59)90090-6.
20. Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 1979;95:351–8. https://doi.org/10.1016/0003-2697(79)90738-3.
21. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001;25:402–8. https://doi.org/10.1006/meth.2001.1262.
22. Garrido-Castro AC, Lin NU, Polyak K. Insights into molecular classifications of triple-negative breast cancer: improving patient selection for treatment. Cancer Discovery 2019;9:176–98. https://doi.org/10.1158/2159-8290.CD-18-1126.
23. Liou GY, Storz P. Reactive oxygen species in cancer. Free Radical Research 2010;44:479–96. https://doi.org/10.3109/10715761003667554.
24. Moloney JN, Cotter TG. ROS signalling in the biology of cancer. Seminars in Cell & Developmental Biology 2018;80:50–64. https://doi.org/10.1016/j.semcdb.2017.05.023.
25. Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nature Reviews Drug Discovery 2009;8:579–91. https://doi.org/10.1038/nrd2803.
26. Brigelius-Flohé R, Maiorino M. Glutathione peroxidases. Biochimica et Biophysica Acta – General Subjects 2013;1830:3289–303. https://doi.org/10.1016/j.bbagen.2012.11.020.
27. Bansal A, Simon MC. Glutathione metabolism in cancer progression and treatment resistance. Journal of Cell Biology 2018;217:2291–8. https://doi.org/10.1083/jcb.201804161.
28. Ayala A, Muñoz MF, Argüelles S. Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. Oxidative Medicine and Cellular Longevity 2014;2014:360438. https://doi.org/10.1155/2014/360438.
29. Farhan M, Rizvi A. Understanding the prooxidant action of plant polyphenols in the cellular microenvironment of malignant cells: role of copper and therapeutic implications. Frontiers in Pharmacology 2022;13:929853. https://doi.org/10.3389/fphar.2022.929853.
30. Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews Molecular Cell Biology 2008;9:47–59. https://doi.org/10.1038/nrm2308.
31. Redza-Dutordoir M, Averill-Bates DA. Activation of apoptosis signalling pathways by reactive oxygen species. Biochimica et Biophysica Acta – Molecular Cell Research 2016;1863:2977–92. https://doi.org/10.1016/j.bbamcr.2016.09.012.
32. Forouzanfar F, Sahranavard T, Tsatsakis A, Iranshahi M, Rezaee R. Rutin: a pain-relieving flavonoid. Inflammopharmacology 2025;33:1289–301. https://doi.org/10.1007/s10787-025-01671-8.
33. Sahebkar A, Iranshahi M. Biological activities of essential oils from the genus Ferula (Apiaceae). Asian Biomedicine 2010;4:835–47. https://doi.org/10.2478/abm-2010-0110.