Integrated Experimental and Computational Evaluation of Silymarin Nanoparticles: Apoptosis Induction, EMT Reversal, and Cell Cycle Arrest in A431 Epidermoid Carcinoma Cells
Enas Abdallah Elsayed, Mohammed Abdalla Hussein, Aysam Fayed, Mohamed F. ElshalIntroduction:
Cancer remains a major global health challenge. Silymarin-based nanoparticles offer a promising, safer platform for next-generation anticancer therapies. The present study was designed to investigate the cytotoxic and apoptotic potential of silymarin nanoparticles (Sil-NPs) in A431 epidermoid carcinoma cells and to elucidate the underlying mechanisms using an integrated experimental and computational approach.
Methods:
Sil-NPs were prepared and characterized by TEM, DLS, Zeta potential, UV-Vis, and FT-IR spectroscopy. IC50 values were determined by cytotoxicity against A431 cells, and apoptosis was assessed by Annexin V staining. The levels of apoptotic proteins (Bax, Bcl-2, and caspase- 3), cell cycle regulators (Cyclin D1 and Cyclin E2), metabolic enzymes (HKI, HPI, and GLUT1), oxidative stress marker (MDA), and antioxidant enzymes (GR, SOD, and GST) were determined. All three genes were analyzed by qPCR for gene expression: E-cadherin (A), Ncadherin (B), and P35 (C). The binding modes of silymarin to E-cadherin, N-cadherin, and P35 were predicted by molecular docking.
Results:
Characterization confirmed the preparation of spherical Sil-NPs with a mean size of 145.5 ± 14.9 nm, a low polydispersity index (0.16), and a negative zeta potential (-10.6 ± 0.11 mV), indicating good colloidal stability. Drug entrapment and chemical stability were confirmed by UV-Vis and FT-IR spectra. Sil-NPs can reduce cell viability (IC50 = 84.52 μg/mL), induce apoptosis (as evidenced by increased Annexin V, Bax, and caspase-3, and decreased Bcl-2), cause G0/G1 cell cycle arrest by inhibiting cyclin D1 and E2, and induce cytotoxicity. Declines in HKI, HPI, and GLUT1 suggest a metabolic disorder, while increased MDA levels and fluctuations in enzyme activity indicate oxidative stress. The ability of Sil-NPs to reverse Epithelial- Mesenchymal Transition (EMT) was also confirmed by significant upregulation of E-cadherin and P35, and downregulation of N-cadherin. Molecular docking analyses showed that silymarin interacts strongly with E-cadherin (ΔG = -6.85 kcal/mol) and N-cadherin (ΔG = -7.12 kcal/mol), and moderately with P35 (ΔG = -5.94 kcal/mol), which are consistent with the changes observed in vivo following exercise modification.
Discussion:
The current results show that Sil-NPs mediate cytotoxic effects in A431 cells by promoting oxidative stress, inhibiting glycolysis and the TCA cycle, and inducing mitochondrial apoptosis. In addition, Sil-NPs most likely inhibit Epithelial-Mesenchymal Transition (EMT) by modulating intercellular cadherin expression, thereby reducing metastatic potential. The coordinated effects on cell death, metabolism, and adhesion illustrate both the strength and complexity of the chemopreventive potential of silymarin nanoparticles.
Conclusion:
Our results show that Sil-NPs have a significant anticancer effect against A431 cells via oxidative stress, induction of apoptosis, and modulation of cell cycle–associated proteins. The results thus provide evidence for the hypothesis that silymarin exerts its effects through direct binding to functional proteins, as suggested by both experimental data and molecular docking analyses. Taken together, these findings identify SiL-NPs as a potential multi-targeted nanotherapeutic agent that should be evaluated further in more advanced preclinical in vivo models.