Betülinik asit EAG1 Baskılanması Yoluyla SH-SY5Y Nöroblastom Hücrelerinde Apoptoz ve G2/M Hücre Döngüsü Durmasını İndükler

Year 2025, Volume: 9 Issue: 2, 274 - 284, 31.08.2025

Tuğba Yalçınkaya , Şeyda Kaya , Hatice Kubra Yildiz , Ahmet Çarhan

https://doi.org/10.29058/mjwbs.1743485

Abstract

Amaç: Nöroblastoma, çocuklarda en sık görülen ekstrakraniyal solid tümör olup özellikle ileri evrelerde yüksek mortaliteye sahiptir.
Tekrarlama ve ilaç direnci gibi mevcut tedavi yöntemlerinin sınırlılıkları nedeniyle, tümöre özgü etkisi yüksek ve normal hücrelere minimum
toksisite gösteren yeni ajanlara ihtiyaç vardır. Doğal bir pentasiklik triterpen olan betulinik asit (BA), seçici antitümör özellikleri nedeniyle ilgi
çekmektedir.
Gereç ve Yöntemler: Bu çalışmada, BA’nın SH-SY5Y nöroblastoma ve epitelyal hücre hattı, Vero, üzerindeki sitotoksik ve moleküler etkileri
incelendi. Hücre canlılığı ise MTT testi ile değerlendirildi. Morfolojik değişiklikler mikroskop ile gözlemlendi. Apoptoz (Bax, Bcl-2, Caspase-3,p53, p21) ve hücre döngüsü (CDK1, Cyclin B1, Ki-67, EAG1) ile ilişkili genlerin ekspresyon seviyeleri qPCR ile analiz edildi.
Bulgular: BA, her iki hücre hattında da zaman ve doz bağımlı olarak hücre canlılığını anlamlı şekilde azalttı; SH-SY5Y hücreleri daha
duyarlı bulundu (48 saat için IC₅₀=75 μM). SH-SY5Y hücrelerinde hücre büzülmesi ve hücre yoğunluğunda azalma gibi morfolojik
değişiklikler belirgindi. Bu hücre hattında BA tedavisi pro-apoptotik genlerin (Bax, Caspase-3, p53, p21) ifadesini artırırken, anti-apoptotik ve
proliferasyonla ilişkili genlerin (Bcl-2, CDK1, Cyclin B1, Ki-67, EAG1) ifadesini azalttı (p<0,05). Vero hücrelerinde ise değişiklikler daha ılımlı
olup sınırlı sitotoksisite gözlendi.
Sonuç: Betulinik asit, nöroblastoma hücrelerinde seçici olarak apoptozu indükleyip hücre döngüsünü inhibe ederken, normal hücreleri
korumaktadır. EAG1 genindeki azalma, BA’nın sitotoksik etkisinde voltaj bağımlı potasyum kanallarının rol oynayabileceğini göstermektedir.
Bu bulgular, BA’nın hedefe yönelik nöroblastoma tedavisinde seçici bir ajan olarak terapötik potansiyelini ortaya koymaktadır

Keywords

Betulinik asit , nöroblastoma , SH-SY5Y , apoptoz , hücre döngüsü , EAG1

Betulinic Acid Induces Apoptosis and G2/M Cell Cycle Arrest in SH-SY5Y Neuroblastoma Cells via EAG1 Suppression

Abstract

Aim: Neuroblastoma is the most common extracranial solid tumour in children and is associated with high mortality, particularly in advanced
stages. Given the limitations of current treatment approaches, such as recurrence and drug resistance, novel agents with tumour-specific
effects and minimal toxicity to normal cells are needed. Betulinic Acid (BA), a naturally occurring pentacyclic triterpene, has gained attention
due to its selective antitumor properties.
Material and Methods: This study investigated the cytotoxic and molecular effects of BA on SH-SY5Y neuroblastoma and epithelial
cell lines, Vero. Cell viability was assessed using the MTT assay. Microscopic observations were conducted to evaluate morphological
changes. Quantitative real-time PCR (qPCR) was performed to analyse the gene expression changes associated with apoptosis (Bax, Bcl-2,
Caspase-3, p53, p21) and the cell cycle (CDK1, Cyclin B1, Ki-67, EAG1).
Results: BA significantly reduced cell viability in both cell lines in a time- and dose-dependent manner, with SH-SY5Y cells showing greater
sensitivity (IC₅₀=75 μM at 48 hours). Morphological changes such as cell shrinkage and reduced cell density were more pronounced in
SH-SY5Y cells. In this cell line, BA treatment significantly upregulated pro-apoptotic genes (Bax, Caspase-3, p53, p21) and downregulated
anti-apoptotic and proliferation-related genes (Bcl-2, CDK1, Cyclin B1, Ki-67, EAG1) (p<0.05). In contrast, Vero cells exhibited moderate
changes, indicating limited cytotoxicity.
Conclusion: Betulinic acid selectively induces apoptosis and inhibits cell cycle progression in neuroblastoma cells while sparing normal
cells. The downregulation of EAG1 suggests that voltage-gated potassium channels may be involved in the mechanism of BA-mediated
cytotoxicity. These findings highlight the therapeutic potential of BA as a selective agent for targeted neuroblastoma treatment

Keywords

Betulinic acid , neuroblastoma , SH-SY5Y , apoptosis , cell cycle , EAG1

References

• 1. Turkish Statistical Institute. (2022). Health statistics yearbook 2022 [Data set]. https://data.tuik.gov.tr/Bulten/Index?p=Saglik- Istatistikleri-Yilligi-2022-46749
• 2. Ministry of Health of Türkiye. (2022). Childhood cancers in Türkiye: Incidence and evaluation report [PDF]. Republic of Türkiye Ministry of Health. https://hsgm.saglik.gov.tr/depo/birimler/ kanser-db/Dokumanlar/Raporlar/17.Agustos_2021_Kanser_ Kontrol_Programi_versiyon-1.pdf
• 3. World Health Organization. (2022). Cancer. https://www.who. int/news-room/fact-sheets/detail/cancer
• 4. Banerjee, S., Banerjee, S., Bishayee, A., Da Silva, M. N., Sukocheva, O. A., Tse, E., ... & Bishayee, A. (2024). Cellular and molecular mechanisms underlying the potential of betulinic acid in cancer prevention and treatment. Phytomedicine, 132, 155858. https://doi.org/10.1016/j.phymed.2024.155858
• 5. Aswathy, M., Vijayan, A., Daimary, U. D., Girisa, S., Radhakrishnan, K. V., & Kunnumakkara, A. B. (2022). Betulinic acid: A natural promising anticancer drug, current situation, and future perspectives. Journal of Biochemical and Molecular Toxicology, 36(12), e23206. https://doi.org/10.1002/jbt.23206
• 6. Gao, S., Wang, W., Ye, W., & Wang, K. (2022). The mechanism study of Eag1 potassium channel in gastric cancer. Translational Cancer Research, 11(10), 3827–3835. https://doi. org/10.21037/tcr-22-2276
• 7. Luis, E., Anaya-Hernández, A., León-Sánchez, P., & Durán- Pastén, M. L. (2022). The Kv10.1 channel: A promising target in cancer. International Journal of Molecular Sciences, 23(15), 8458. https://doi.org/10.3390/ijms23158458
• 8. Toplak, Ž., Hendrickx, L. A., Abdelaziz, R., Shi, X., Peigneur, S., Tomašič, T., ... & Pardo, L. A. (2022). Overcoming challenges of HERG potassium channel liability through rational design: Eag1 inhibitors for cancer treatment. Medicinal Research Reviews, 42(1), 183–226. https://doi.org/10.1002/med.21808
• 9. An, T., Zha, W. & Zi, J. (2020). Biotechnological production of betulinic acid and derivatives and their applications. Appl Microbiol Biotechnol 104, 3339–3348. https://doi.org/10.1007/ s00253-020-10495-1
• 10. Hordyjewska, A., Ostapiuk, A., Horecka, A., & Kurzepa, J. (2019). Betulin and betulinic acid: Triterpenoids derivatives with a powerful biological potential. Phytochemistry Reviews, 18, 929–951. https://doi.org/10.1007/s11101-019-09623-1
• 11. Bettariga, F., Taaffe, D. R., Galvão, D. A., Bishop, C., Kim, J. S., & Newton, R. U. (2024). Suppressive effects of exercise- conditioned serum on cancer cells: A narrative review of the influence of exercise mode, volume, and intensity. In Journal of Sport and Health Science (Vol. 13, Issue 4). https://doi. org/10.1016/j.jshs.2023.12.001
• 12. Zeng, A. Q., Yu, Y., Yao, Y. Q., Yang, F. F., Liao, M., Song, L. J., Li, Y. L., Yu, Y., Li, Y. J., Deng, Y. Le, Yang, S. P., Zeng, C. J., Liu, P., Xie, Y. M., Yang, J. L., Zhang, Y. W., Ye, T. H., & Wei, Y. Q. (2018). Betulinic acid impairs metastasis and reduces immunosuppressive cells in breast cancer models. Oncotarget, 9(3). https://doi.org/10.18632/oncotarget.23376
• 13. Lombrea, A., Scurtu, A. D., Avram, S., Pavel, I. Z., Turks, M., Lugiņina, J., ... & Danciu, C. (2021). Anticancer potential of betulonic acid derivatives. International Journal of Molecular Sciences, 22(7), 3676. https://doi.org/10.3390/ijms22073676
• 14. Hordyjewska, A., Ostapiuk, A., & Horecka, A. (2018). Betulin and betulinic acid in cancer research. Journal of Pre-Clinical and Clinical Research, 12(2), 72–75. https://bibliotekanauki.pl/ articles/3735.pdf
• 15. Ali Seyed, M., Dhyani, A., & Csuk, R. (2022). Chemopreventive and chemotherapeutic potential of betulin and betulinic acid: mechanistic insights from in vitro, in vivo and clinical studies. Food Science & Nutrition, 10(11), Article 2903. https://doi. org/10.3390/biomedicines10112903
• 16. Zúñiga, L., Cayo, A., González, W., Vilos, C., & Zúñiga, R. (2022). Potassium channels as a target for cancer therapy: Current perspectives. OncoTargets and Therapy, 15, 783–800. https://doi.org/10.2147/OTT.S346369