Cytotoxicity of Betaine in 4T1 and L929 Cells: In Silico ADMET Profiling

Abstract

Betaine, a naturally occurring zwitterion, was evaluated for its anticancer potential against murine 4T1 breast cancer cells and non-cancerous L929 fibroblast cells. Cells were treated with various concentrations for 48 hours, and viability was assessed using the MTT assay. Betaine showed weak, dose-dependent cytotoxicity with an IC₅₀ value of 859.10 µg/mL. In silico ADME analysis showed high solubility, low lipophilicity (LogP: –2.18), and poor blood-brain barrier permeability. ProTox-II predicted low hepatotoxicity, nephrotoxicity, and immunotoxicity, but possible neurotoxic and cardiotoxic effects. Betaine was non-carcinogenic and showed no significant interaction with major metabolic enzymes. These findings suggest that while betaine has limited direct cytotoxicity, its favorable safety and pharmacokinetic profile support its potential as an adjuvant in cancer therapy.

Keywords

• Antiproliferative activity
• betaine
• breast cancer
• toxicity

Ethical Statement

Since it involved only in vitro cell culture experiments and did not include human participants or animal subjects, formal ethical approval was not required.

References

1. [1]. Wang, L, Chen, L, et al. 2013. Betaine supplement alleviates hepatic triglyceride accumulation of apolipoprotein E deficient mice via reducing methylation of peroxisomal proliferator-activated receptor alpha promoter. Lipids in Health and Disease; 12(1): 34.
2. [2]. Lu, MS, Fang, YJ, et al. 2015. Choline and betaine intake and colorectal cancer risk in Chinese population: A case-control study. PLOS ONE; 10(3): e0118661.
3. [3]. Sun, S, Li, X, et al. 2016. Choline and betaine consumption lowers cancer risk: A meta-analysis of epidemiologic studies. Scientific Reports; 6: 35547.
4. [4]. Ying, J, Rahbar, MH, et al. 2013. Associations between dietary intake of choline and betaine and lung cancer risk. PLOS ONE; 8(2): e54561.
5. [5]. Youn, J, Cho, E, Lee, JE. 2019. Association of choline and betaine levels with cancer incidence and survival: A meta-analysis. Clinical Nutrition; 38(1): 100–109.
6. [6]. Hassan, MS, Khalid, T, et al. 2025. Therapeutic potential of betaine and its derivatives in cancer treatment: A comprehensive review. RSC Advances; 15(26): 20605–20622.
7. [7]. Owumi, SE, Oluwawibe, BJ, et al. 2024. An in vivo and in silico probing of the protective potential of betaine against sodium fluoride induced neurotoxicity. BMC Pharmacology and Toxicology; 25(1): 87.
8. [8]. Kola, I, Landis, J. 2004. Can the pharmaceutical industry reduce attrition rates? Nature Reviews Drug Discovery; 3(8): 711–715.

9. [9]. Banerjee, P, Eckert, AO, Schrey, AK, Preissner, R. 2018. ProTox-II: A webserver for the prediction of toxicity of chemicals. Nucleic Acids Research; 46(W1): W257–W263.
10. [10]. Tao, K, Fang, M, Alroy, J, Sahagian, GG. 2008. Imagable 4T1 model for the study of late stage breast cancer. BMC Cancer; 8(1): 228.
11. [11]. Bahar, D, Muhammed, MT, Akkoc, S. 2025. In vitro cytotoxic activity, cells staining, DFT computation, docking, and MD simulation of silver N-heterocyclic carbene complexes. Journal of the Indian Chemical Society; 102(3): 101602.
12. [12]. Sahin, D, Kepekci, RA, et al. 2025. Biological evaluations and computational studies of newly synthesized thymol-based Schiff bases as anticancer, antimicrobial and antioxidant agents. Journal of Biomolecular Structure and Dynamics; 43(7): 3375–3389.
13. [13]. Tapera, M, Muhammed, MT, et al. 2025. Cytotoxic activity, caspase-3 mediated apoptosis, and PARP-1 inhibition targeted molecular modeling studies of novel imidazole-fused hydrazones. Journal of Molecular Liquids; 419: 127637
14. [14]. Daina, A, Michielin, O, Zoete, V. 2017. SwissADME: A free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific Reports; 7(1): 42717.
15. [15]. Bilici, E, Türkoğlu, BG, Akkoç, S. 2025. Using the MTT Cell Viability Test and Immunocytochemistry Techniques, Benzimidazole’s Cytotoxicity and Apoptosis Activation in Mouse 4T1 Cell Culture. Dicle Üniversitesi Veteriner Fakültesi Dergisi; 18(1): 33–37.
16. [16]. Fahmy, SA, Ponte, F, et al. 2021. Betaine host–guest complexation with a calixarene receptor: Enhanced in vitro anticancer effect. RSC Advances; 11(40): 24673–24680.
17. [17]. Kar, F, Hacioglu, C, et al. 2019. Betaine suppresses cell proliferation by increasing oxidative stress-mediated apoptosis and inflammation in DU-145 human prostate cancer cell line. Cell Stress and Chaperones; 24(5): 871–881.
18. [18]. Kulthanaamondhita, P, Kornsuthisopon, C, et al. 2024. Betaine induces apoptosis and inhibits invasion in OSCC cell lines. International Journal of Molecular Sciences; 25(19): 10295.
19. [19]. Im, AR, Kim, YH, et al. 2013. Betaine protects against rotenone-induced neurotoxicity in PC12 cells. Cellular and Molecular Neurobiology; 33(5): 625–635.
20. [20]. Hashim, AR, Bashir, DW, et al. 2024. Neuroprotective assessment of betaine against copper oxide nanoparticle-induced neurotoxicity in the brains of albino rats: A histopathological, neurochemical, and molecular investigation. ACS Chemical Neuroscience; 15(8): 1684–1702.