Ajuga reptans L. Etanol Özütünün HCT116 Kolon Kanseri Hücrelerinde NF-κB ve eNOS Aktivitesinin Inhibisyonu Yoluyla Apoptotik ve Antiproliferatif Etkileri

Öz

Amaç: Bu çalışmanın amacı, Ajuga reptans L. (Lamiaceae) etanol ekstraktının, HCT116 insan kolorektal karsinom hücrelerinde hücre proliferasyonu, apoptotik yanıt, Kaspaz-3, NF-κB ve eNOS proteinlerinin ifade düzeyleri üzerine etkilerini araştırmaktır. Materyal ve Metot: A. reptans bitkisi Soxhlet cihazı kullanılarak etanol ile ekstrakte edilmiştir. Elde edilen ekstrakt, HCT116 ve HUVEC (sağlıklı kontrol) hücrelerine artan konsantrasyonlarda (0–800 µg/mL) uygulanmıştır. Hücre canlılığı, WST-1 testi ile 24 ve 48 saatlik sürelerde değerlendirilmiştir. Morfolojik değişiklikler inverted mikroskopi ile incelenmiştir. Kaspaz-3, NF-κB ve eNOS protein düzeylerinin kantifikasyonu için ELISA analizi uygulanmıştır. Bulgular: A. reptans ekstraktı, HCT116 hücrelerinde konsantrasyona ve süreye bağımlı sitotoksik etki göstermiştir; IC₅₀ değerleri sırasıyla 24 saatte 206.9 µg/mL, 48 saatte 147.5 µg/mL olarak hesaplanmıştır. HUVEC hücrelerinde minimum düzeyde sitotoksisite gözlenmiştir. Mikroskobik incelemeler, HCT116 hücrelerinde apoptozu düşündüren morfolojik değişiklikleri doğrulamıştır. ELISA analizleri, Caspase-3 düzeylerinde artış ve NF-κB ile eNOS düzeylerinde azalma göstermiştir. Sonuç: Ajuga reptans’ın etanolik ekstraktı, kolorektal kanser hücrelerinde seçici proliferasyon baskılanması, apoptoz indüksiyonu ve NF-κB ile eNOS sinyallerinin modülasyonu yoluyla antikanser etki göstermiştir. Bu bulgular, ekstraktın kolorektal kanser tedavisinde bitki kaynaklı tamamlayıcı bir ajan olarak potansiyelini ortaya koymaktadır.

Anahtar Kelimeler

• A. reptans
• endotel nitrik oksit sentaz (eNOS)
• kaspaz-3
• kolon kanseri
• nuclear factor kappa B (NF-κB)

Apoptotic and Antiproliferative Effects of Ajuga reptans L. Ethanol Extract via Inhibition of NF-κB and eNOS Activity in HCT116 Colon Cancer Cells

Abstract

Objective: This study aimed to investigate the ethanol extract of Ajuga reptans L. (Lamiaceae) on cell proliferation, apoptotic response, expression levels of Caspase-3, NF-κB and eNOS proteins on HCT116 human colorectal carcinoma cells. Materials and Methods: A. reptans was extracted using a Soxhlet apparatus with ethanol, and the obtained extract was applied to HCT116 and HUVEC cells (as healthy controls) in increasing concentrations (0–800 µg/mL). Cell viability was analysed by the WST-1 assay at 24 h and 48 h. Morphological changes were observed via inverted microscopy. ELISA was performed to quantify Caspase-3, NF-κB, and eNOS protein levels. Results: A. reptans ethanol extract showed concentration and duration-dependent cytotoxic effects on HCT116 cells, with IC₅₀ values of 206.9 µg/mL (24 h) and 147.5 µg/mL (48 h). Minimal cytotoxicity was observed in HUVEC cells. Microscopy confirmed morphological signs of apoptosis in HCT116 cells. ELISA results demonstrated increased Caspase-3 and decreased NF-κB and eNOS levels, indicating induction of apoptosis and suppression of pro-survival pathways. Conclusions: The ethanol extract of Ajuga reptans selectively inhibited the proliferation of colorectal cancer cells by apoptotic induction and modulation of NF-κB and eNOS signaling, suggesting its potential as a plant-based adjunctive agent in colorectal cancer therapy.

Keywords

• Ajuga reptans
• caspase-3
• colon cancer
• eNOS
• NF-κB

References

1. Cragg GM, Newman DJ. Natural products: a continuing source of novel drug leads. Biochim Biophys Acta. 2013;1830(6):3670-95. doi:10.1016/j.bbagen.2013.02.008
2. Afrin S, Giampieri F, Gasparrini M, et al. Dietary phytochemicals in colorectal cancer prevention and treatment: A focus on the molecular mechanisms involved. Biotechnol Adv. 2020:38:107322. doi:10.1016/j.biotechadv.2018.11.011
3. Rana S, Narinderpal K, Poonam DA. Review on pharmacognostic profile of Ajuga reptans. J. Pharmacogn. 2020;7:9-18.
4. Nagarkoti K, Kanyal J, Prakash O, Kumar R, Rawat DS, Pant,AK. Ajuga L.: A systematic review on chemical composition, phytopharmacological and biological potential. Curr Bioact Compd. 2021;17(9),11-37. doi:10.2174/1573407216999210101230234
5. Maliuvanchuk S, Grytsyk A, Popadynets O, Kotyk T, Raal A, Koshovyi O. Ajuga reptans L. Herb Extracts: Phytochemical composition and pharmacological activity screening. Plants. 2025;14(2),219. doi:10.3390/plants14020219
6. Dziki A, Malinowska MA, Szopa A, Sikora E. Comparative study of the phytochemical profile and biological activity of Ajuga reptans L. leaf and root extracts. Appl Sci. 2024; 14(12),5105. doi:10.3390/app14125105
7. Karin M. Nuclear factor-κB in cancer development and progression. Nature. 2006;441(7092):431-6. doi:10.1038/nature04870
8. Gao Y, Zhou S, Xu Y, Sheng S, Qian SY, Huo X. Nitric oxide synthase inhibitors 1400W and L-NIO inhibit angiogenesis pathway of colorectal cancer. Nitric Oxide. 2019;1:83:33-39. doi:10.1016/j.niox.2018.12.008

9. Newman DJ, Cragg GM. Natural products as sources of new drugs over the nearly four decades from 1981 to 2019. J Nat Prod. 2020;83(3):770-803. doi: 10.1021/acs.jnatprod.9b01285
10. Mukkavilli V, Ramakrishnan G, Gujjula KR, Balachandran S, Chamarthy S, Mekala JR. Molecular understanding and pharmacological potency of plant-derived compounds in colorectal cancer (CRC): A Critical Analysis and Future Perspectives. Cell Biochem Biophys. 2024;82(3):1777-1795. doi:10.1007/s12013-024-01370-1
11. Caskurlu A, Karavus SN, Biltekin SN, Helvaci EZ, Karadag AE. In vitro cytotoxicity evaluation and phytochemical analysis of Ajuga reptans L. extracts. J Res Pharm. 2023;27(5):2115-2123. doi:10.29228/jrp.490
12. Hussar, P. Apoptosis regulators bcl-2 and caspase-3. Encyclopedia. 2022;2(4),1624-1636. doi:10.3390/encyclopedia2040111
13. Martin M, Sun M, Motolan A, Lu T. The pivotal player: components of NF-κB pathway as promising biomarkers in colorectal cancer. Int J Mol Sci. 2021;22(14):7429. doi:10.3390/ijms22147429
14. Xie Y, Liu F, Wu Y, et al. Inflammation in cancer: therapeutic opportunities from new insights. Mol Cancer. 2025;24(1):51. doi:10.1186/s12943-025-02243-8
15. Wang H, Wang L, Xie Z, et al. Nitric oxide (NO) and NO synthases (NOS)-based targeted therapy for colon cancer. Cancers (Basel). 2020;12(7):1881. doi:10.3390/cancers12071881
16. Luan F, Han K, Li M, et al. Ethnomedicinal uses, phytochemistry, pharmacology, and toxicology of species from the genus Ajuga L.: a systematic review. Am J Chin Med. 2019;47(5):959-1003. doi:10.1142/S0192415X19500502
17. Jin Z, Wang B, Ren L, et al. 20-Hydroxyecdysone inhibits inflammation via SIRT6-mediated NF-κB signaling in endothelial cells. Biochim Biophys Acta Mol Cell Res. 2023;1870(5):119460. doi:10.1016/j.bbamcr.2023.119460
18. Romaniuk-Drapała A, Lisiak N, Totoń E, et al. Proapoptotic and proautophagic activity of 20-hydroxyecdysone in breast cancer cells in vitro. Chem Biol Interact. 2021:342:109479. doi:10.1016/j.cbi.2021.109479
19. Arif Y, Singh P, Bajguz A, Hayat S. Phytoecdysteroids: Distribution, structural diversity, biosynthesis, activity, and crosstalk with phytohormones.  Int J Mol Sci. 2022;23(15):8664. doi:10.3390/ijms23158664