Antiproliferative activities of panduratin A and its derivatives against U-251 MG glioblastoma cells and U-251 MG cancer stem-like cells: structure-activity relationships and identification of PHB2 as a candidate binding protein.

Panduratin A (1), a cyclohexenyl chalcone isolated from Boesenbergia rotunda, has been reported to exhibit various biological activities, including antitumor effects. However, its antiproliferative activity against glioblastoma cells and glioblastoma-derived cancer stem-like cells (CSCs), and the molecular basis of this activity, remain incompletely elucidated. In this study, we evaluated the antiproliferative activity of 1 against U-251 MG glioblastoma cells (non-CSCs) and U-251 MG CSCs and found that 1 exhibited potent activity against both cell populations. To investigate the structural features associated with this activity, cyclohexenyl chalcone derivatives of 1 (2-11) were synthesized through a chalcone intermediate generated from an acetophenone precursor, followed by Diels-Alder reactions with dienes. Structure-activity relationship analysis, particularly in U-251 MG non-CSCs, suggested that the phenolic oxygen functionalities of 1 contribute to its potent activity, whereas some acetylated derivatives retained moderate activity. Based on this observation, affinity-based target identification using U-251 MG non-CSC extracts identified prohibitin 2 (PHB2), an inner mitochondrial membrane protein, as a candidate binding protein for 1. In these cells, 1 also markedly altered the mitochondrial morphology and reduced the mitochondrial membrane potential. Together, these findings suggest that the antiproliferative activity of 1 in U-251 MG glioblastoma cells may be associated with PHB2 and mitochondrial alterations.