Effect of Hypericum Perforatum Extract on cell viability and p53 localization in HepG2 cells

Öz

Aim: This study aims to investigate the potential anticancer effects of Hypericum perforatum on hepatocellular carcinoma cells, with a focus on the role of the p53 protein. Materials and Methods: HepG2 cells cultured in DMEM were subjected to daily live-cell imaging under a phase-contrast microscope to monitor cell viability and morphology. Treatment groups received 0.8% Hypericum perforatum extract for 24 hours, supplemented with complete medium. Proliferation was assessed using the CCK-8 (WST-8) assay, and the resulting samples were analyzed spectrophotometrically using a microplate reader (BioTek Synergy H1) at the specified wavelength, with optical density changes reflecting cell viability. The expression of p53 proteins in HepG2 cells was evaluated through immunohistochemical staining. Cells were fixed with appropriate fixatives during both weak adhesion and adhesion phases and then treated with specific primary and secondary antibodies. Data analysis was performed using GraphPad Prism 10.0. Results: Treatment with 0.8% Hypericum perforatum extract significantly reduced cell viability in HepG2 cells, indicating a cytotoxic effect. Additionally, p53 protein was observed to translocate from the cytoplasm to the nucleus in these cells. Conclusion: Hypericum perforatum extract may activate p53 pathways, leading to DNA damage and cellular stress responses, suggesting its potential as an anticancer agent worthy of further investigation.

Anahtar Kelimeler

• HepG2
• Hypericum Perforatum
• Hepatocellular Carcinoma
• p53 Protein

Destekleyen Kurum

TÜBİTAK

Proje Numarası

1919B012222883

Hypericum Perforatum Ekstraktının HepG2 hücrelerinde hücre canlılığı ve p53 lokalizasyonu üzerine etkisi

Öz

Amaç: Bu çalışmanın amacı, p53 proteininin rolüne odaklanarak, Hypericum perforatum'un hepatosellüler karsinoma hücreleri üzerindeki potansiyel antikanser etkilerini araştırmaktır. Malzemeler ve Yöntemler: DMEM'de kültürlenen HepG2 hücreleri, hücre canlılığını ve morfolojisini izlemek için faz kontrast mikroskobu altında günlük canlı hücre görüntülemesine tabi tutuldu. Tedavi gruplarına 24 saat boyunca tam ortamla desteklenmiş %0,8 Hypericum perforatum özütü verildi. Proliferasyon, CCK-8 (WST-8) testi kullanılarak değerlendirildi ve elde edilen örnekler, optik yoğunluk değişimlerinin hücre canlılığını yansıttığı belirtilen dalga boyunda bir mikro plaka okuyucu (BioTek Synergy H1) kullanılarak spektrofotometrik olarak analiz edildi. HepG2 hücrelerindeki p53 proteinlerinin ekspresyonu, immünohistokimyasal boyama yoluyla değerlendirildi. Hücreler, hem zayıf yapışma hem de yapışma fazlarında uygun fiksatiflerle sabitlendi ve ardından spesifik birincil ve ikincil antikorlarla tedavi edildi. Veri analizi GraphPad Prism 10.0 kullanılarak gerçekleştirildi. Sonuçlar: %0,8 Hypericum perforatum özütü ile tedavi, HepG2 hücrelerinde hücre canlılığını önemli ölçüde azalttı ve bu da sitotoksik bir etki olduğunu gösterdi. Ek olarak, p53 proteininin bu hücrelerde sitoplazmadan çekirdeğe taşındığı gözlemlendi. Sonuç: Hypericum perforatum özütü, p53 yollarını aktive ederek DNA hasarına ve hücresel stres tepkilerine yol açabilir ve bu da daha fazla araştırmaya değer bir antikanser ajanı olma potansiyelini göstermektedir.

Anahtar Kelimeler

• HepG2
• Hypericum Perforatum
• p53 Proteini

Destekleyen Kurum

TÜBİTAK

Proje Numarası

1919B012222883

Kaynakça

1. Wang X, Lee J, Xie C. Autophagy Regulation on Cancer Stem Cell Maintenance, Metastasis, and Therapy Resistance. Vol. 14, Cancers. MDPI AG; 2022. p. 381.
2. ‌Recio-Boiles A, Waheed A, Babiker HM. Cancer, Liver. PubMed. Treasure Island (FL): StatPearls Publishing; 2024.
3. Llovet JM, Kelley RK, Villanueva A, Singal AG, Pikarsky E, Roayaie S, et al. Hepatocellular carcinoma. Vol. 7, Nature Reviews Disease Primers. Springer Science and Business Media LLC; 2021.
4. Wang H, Li W. Recent update on comprehensive therapy for advanced hepatocellular carcinoma. Vol. 13, World Journal of Gastrointestinal Oncology. Baishideng Publishing Group Inc.; 2021. p. 845–55.
5. Kaur P, Robin, Mehta RG, Arora S, Singh B. Progression of conventional hepatic cell culture models to bioengineered HepG2 cells for evaluation of herbal bioactivities. Vol. 40, Biotechnology Letters. Springer Science and Business Media LLC; 2018. p. 881–93.
6. ‌Donato MT, Tolosa L, Gómez-Lechón MJ. Culture and Functional Characterization of Human Hepatoma HepG2 Cells. Methods in Molecular Biology. Springer New York; 2014. p. 77–93.
7. Kazama R, Fujita S, Sakai S. Cell Dome as an Evaluation Platform for Organized HepG2 Cells. Vol. 12, Cells. MDPI AG; 2022. p. 69.
8. Rozman M, Štukovnik Z, Sušnik A, Pakseresht A, Hočevar M, Drobne D, et al. A HepG2 Cell-Based Biosensor That Uses Stainless Steel Electrodes for Hepatotoxin Detection. Vol. 12, Biosensors. MDPI AG; 2022. p. 160.

9. Kitano H, Kawabe Y, Kamihira M. HepG2-Based Designer Cells with Heat-Inducible Enhanced Liver Functions. Vol. 11, Cells. MDPI AG; 2022. p. 1194.
10. Westerink WMA, Schoonen WGEJ. Phase II enzyme levels in HepG2 cells and cryopreserved primary human hepatocytes and their induction in HepG2 cells. Vol. 21, Toxicology in Vitro. Elsevier BV; 2007. p. 1592–602.
11. Arzumanian VA, Kiseleva OI, Poverennaya EV. The Curious Case of the HepG2 Cell Line: 40 Years of Expertise. Vol. 22, International Journal of Molecular Sciences. MDPI AG; 2021. p. 13135.
12. Kayalı D, Akbulut Z, Maraş H, Kahveci R, Gülhan R. Tip I Kollajenin Hepasellular Karsinoma Hücre Hattı (HepG2) Proliferasyonuna Etkisi. Vol. 12, Maltepe Tıp Dergisi. Maltepe Tıp Dergisi; 2020. p. 79–82.
13. Wang H, Guo M, Wei H, Chen Y. Targeting p53 pathways: mechanisms, structures, and advances in therapy. Vol. 8, Signal Transduction and Targeted Therapy. Springer Science and Business Media LLC; 2023.
14. Choudhary HB, Mandlik SK, Mandlik DS. Role of p53 suppression in the pathogenesis of hepatocellular carcinoma. Vol. 14, World Journal of Gastrointestinal Pathophysiology. Baishideng Publishing Group Inc.; 2023. p. 46–70.
15. Nobakht SZ, Akaberi M, Mohammadpour A, Tafazoli Moghadam A, Emami A. Hypericum perforatum: Traditional uses, clinical trials, and drug interactions. Iranian Journal of Basic Medical Sciences. 2022 Sep;25(9).
16. Wölfle U, Seelinger G, Schempp C. Topical Application of St. Johnʼs Wort (Hypericum perforatum). Vol. 80, Planta Medica. Georg Thieme Verlag KG; 2013. p. 109–20.
17. Menegazzi M, Masiello P, Novelli M. Anti-Tumor Activity of Hypericum perforatum L. and Hyperforin through Modulation of Inflammatory Signaling, ROS Generation and Proton Dynamics. Vol. 10, Antioxidants. MDPI AG; 2020. p. 18.
18. Olya M, Zaferani Arani H, Shekarriz A, Zabolian A, Zare Marzouni H, Aryan H, et al. Hypericin Exerts Detrimental Effect on Huh-7 As a Delegacy of Hepatocellular Carcinoma: A P53 Dependent Pathway. Vol. 9, Galen Medical Journal. Salvia Medical Sciences Ltd; 2020. p. e1896.
19. Celik E. Apoptotic and Anti-inflammatory Effects of Hypericum Perforatum Extract in Human Basal Cell Carcinoma TE 354.T Cell Line. Vol. 48, Dicle Tıp Dergisi. Dicle Medical Journal/Dicle Tip Dergisi; 2021. p. 92–8.
20. Montagnani Marelli M, Macchi C, Ruscica M, Sartori P, Moretti RM. Anticancer Activity of Delta-Tocotrienol in Human Hepatocarcinoma: Involvement of Autophagy Induction. Vol. 16, Cancers. MDPI AG; 2024. p. 2654.
21. Juaid N, Amin A, Abdalla A, Reese K, Alamri Z, Moulay M, et al. Anti-Hepatocellular Carcinoma Biomolecules: Molecular Targets Insights. Vol. 22, International Journal of Molecular Sciences. MDPI AG; 2021. p. 10774.
22. Russo E, Scicchitano F, Whalley BJ, Mazzitello C, Ciriaco M, Esposito S, et al. Hypericum perforatum : Pharmacokinetic, Mechanism of Action, Tolerability, and Clinical Drug-Drug Interactions. Vol. 28, Phytotherapy Research. Wiley; 2013. p. 643–55.
23. Choudhary N, Collignon TE, Tewari D, Bishayee A. Hypericin and its anticancer effects: From mechanism of action to potential therapeutic application. Vol. 105, Phytomedicine. Elsevier BV; 2022. p. 154356.
24. Bolat Küçükzeybek B, Taşkaynatan H, Yiğit S, Yıldız Y, Akder Sarı A, Oflazoğlu U, et al. Prognostic Significance of Immunohistochemical P53 Expression in Patients with Breast Cancer. Vol. 51, Acta Oncologica Turcica. Galenos Yayinevi; 2018. p. 125–31.