
Investigating the Effect of Ethanolic Extract of Pomegranate Seed on Breast Cancer Cell line(MCF-7)in a Fibrin_based Hydrogel Scaffold
Ali Honarvar 1 ©, Mehrzad Jafari Barmak 1, Zahra Gholami 2, Zahra Bostani 2 ℗
Abstract
Abstract: Introduction and Objective: Breast cancer is the most common cancer worldwide and the second leading cause of cancer-related death after lung cancer. Recently, three-dimensional scaffold-based models have been developed for anticancer drug research. The aim of this study was to investigate the antiproliferative effects of ethanolic pomegranate seed extract within a fibrin hydrogel scaffold on the MCF7 breast cancer cell line. Materials and Methods: Ethanolic pomegranate seed extract was prepared using the maceration method. A fibrin hydrogel scaffold was fabricated by combining fibrinogen (obtained from cryoprecipitate bags) and thrombin (from FFP bags) according to a defined protocol. The scaffold’s biodegradability properties, including water absorption and tensile strength, were assessed using laboratory tests. MCF7 breast cancer cells were purchased and cultured in DMEM medium supplemented with 10% fetal bovine serum and antibiotics. Cells were incubated at 37°C with 5% CO2 until reaching over 80% confluency. Then, 10^4 cells were seeded in 24-well plates in triplicate. Treatment groups included 100 µL of pomegranate seed extract (100 µg/mL) and 100 µL of 5-fluorouracil (5FU) (250 µg/mL). After 24 hours incubation under the same conditions, cell viability was assessed by the MTT assay. Apoptosis stages were evaluated by staining with PI/Annexin V followed by flow cytometry analysis. Data normality was checked using the Kolmogorov-Smirnov test, and statistical analysis was performed by one-way ANOVA and Tukey’s post hoc test, with P0.05 considered statistically significant. Results: • Water absorption was significantly lower in the fibrin hydrogel scaffold alone compared to scaffolds containing pomegranate seed extract or 5FU (P=0.0001). • Tensile testing confirmed that the fibrin hydrogel scaffold had sufficient mechanical strength to support cell maintenance. • Cell viability significantly decreased in groups treated with 5FU and 5FU+fibrin compared to fibrin alone (P=0.0001). Viability in the 5FU+fibrin group was also significantly lower than in the pomegranate seed extract+fibrin group (P=0.001). Although viability in the pomegranate seed extract+fibrin group was lower than fibrin alone, this reduction was not statistically significant. • Flow cytometry results showed that early and late apoptosis percentages were higher in the 5FU and 5FU+fibrin groups compared to the control group (MCF7+fibrin). The pomegranate seed extract group also showed increased apoptosis compared to control. Conclusion: The fibrin hydrogel scaffold, due to its three-dimensional structure and hydrophilic nature as a natural polymer, likely provides a suitable environment for cell growth. The pomegranate seed extract, owing to its antioxidant properties, may serve as a potential therapeutic candidate for breast cancer treatment by inducing apoptosis and inhibiting cell proliferation.
Keywords: Breast cancer, MCF7, fibrin hydrogel, three-dimensional culture, pomegranate extract