Effects of Allantoin Combined with Bevacizumab on Estrogen Receptor-Positive and Triple-Negative Breast Cancer Cells

Objective: This study aimed to evaluate the modulatory effect of Allantoin on the response to Bevacizumab in estrogen receptor-positive (MCF-7) and triple-negative (MDA-MB-231) breast cancer cell lines, with a focus on proliferation, angiogenesis-related markers, inflammatory responses, and wound closure under in vitro conditions. Methods: MCF-7 and MDA-MB-231 cell lines were treated with Allantoin, Bevacizumab, and their combination for 24 and 48 hours. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to determine effective dose ranges. Wound closure was evaluated using a wound-healing (scratch) assay. Immunocytochemical analyses were performed to assess relative changes in the immunoreactivity of Ki-67, vascular endothelial growth factor (VEGF), nuclear factor kappa B (NF-κB), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) using a semi-quantitative H-score method. In addition, potential interactions between Allantoin and the Bevacizumab Fab fragment were analyzed using in silico molecular docking. Results: Treatment with Allantoin and Bevacizumab resulted in a time- and dose-dependent decrease in cell viability in both cell lines. The combination treatment was associated with decreased Ki-67 immunoreactivity, indicating reduced proliferative activity. The levels of VEGF protein dropped in MCF-7 cells, whereas in MDA-MB-231 cells VEGF immunoreactivity increased over time in all groups except the combination group, which showed lower VEGF immunoreactivity than the time-matched control at 48 hours. Allantoin increased wound closure in both cell lines, whereas the Bevacizumab and combination groups showed widening of the wound area with cell detachment, reflecting loss of monolayer integrity and reduced viability rather than inhibition of migration. Inflammatory markers exhibited a variable pattern across treatment groups, with changes observed in NF-κB, TNF-α, and IL-6 immunoreactivity. Docking analysis predicted only a weak interaction between Allantoin and the Bevacizumab Fab fragment (−5.4 kcal/mol), suggesting that direct drug-drug binding is unlikely to explain the observed effects. Conclusion: Co-treating breast cancer cells with Allantoin and Bevacizumab induced distinct behavioral shifts in vitro; nevertheless, these preliminary observations necessitate subsequent validation through in vivo and translational models. The magnitude of these effects differed across cell lines, while the fluctuating inflammatory patterns highlight a multifaceted regulatory response. Taken together, these exploratory findings show that the combination of Allantoin and Bevacizumab produced cell line-dependent changes compared with either agent alone; whether Allantoin modulates the response to Bevacizumab requires mechanistic and in vivo confirmation.