Synergistic effects of andrographolide and berbamine in glioblastoma: insights from proteomic and metabolomic pathway analysis.

Glioblastoma (GB) is one of the most aggressive and highly invasive brain cancer associated with poor prognosis and frequent recurrence. Although standard therapeutic strategies, including temozolomide (TMZ)-based chemotherapy and radiotherapy, remain the mainstay of treatment, their associated toxicity and long-term adverse effects have prompted increasing interest in plant-derived therapeutic alternatives. This study explores the effects of two phytocompounds: andrographolide, a diterpenoid, and berbamine, an alkaloid. Their cytotoxic effects were tested on GB cell lines LN229 and U-87 MG using MTT and wound healing assays, with HEK 293 cells used to assess safety. Additionally, the impact of these compounds on apoptosis (via Annexin V), cell cycle progression, oxidative stress, and mitochondrial dysfunction was evaluated through flow cytometry in GB cells. The combination of andrographolide and berbamine markedly enhanced cell death, impaired wound healing capacity, and increased oxidative stress following treatment. The mechanism behind this growth inhibition was further supported by in silico analysis: Swiss Target Prediction identified specific molecular targets for both compounds, and WebGestalt data revealed their involvement in pathways related to metabolism, cell communication, and biological regulation, providing insights into their roles in apoptosis and cell cycle regulation. After getting a broad idea on impact of the phytocompound combination on pathways, to study the impact in detail, DSC, mass spectrometry and NMR were used to analyze the changes in protein structure and metabolite changes in metabolism. The combined treatment was found to impose significant metabolic stress, highlighting its potential as a therapeutic approach in targeting glioblastoma by simultaneously disrupting multiple cellular pathways.