Synergistic Anticancer Effect of a Combination of Berbamine and Arcyriaflavin A against Glioblastoma Stem-like Cells

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumor. Relapse is frequent and rapid due to glioblastoma stem-like cells (GSCs) that induce tumor initiation, drug resistance, high cancer invasion, immune evasion, and recurrence. Therefore, suppression of GSCs is a powerful therapeutic approach for GBM treatment. Natural compounds berbamine and arcyriaflavin A (ArcA) are known to possess anticancer activity by targeting calcium/calmodulin-dependent protein kinase II gamma (CaMKII gamma) and cyclin-dependent kinase 4 (CDK4), respectively. In this study, we evaluated the effects of concurrent treatment with both compounds on GSCs. Combined treatment with berbamine and ArcA synergistically inhibited cell viability and tumorsphere formation in U87MG- and C6-drived GSCs. Furthermore, simultaneous administration of both compounds potently inhibited tumor growth in a U87MG GSC-grafted chick embryo chorioallantoic membrane (CAM) model. Notably, the synergistic anticancer effect of berbamine and ArcA on GSC growth is associated with the promotion of reactive oxygen species (ROS)- and calcium-dependent apoptosis via strong activation of the p53-mediated caspase cascade. Moreover, co-treatment with both compounds significantly reduced the expression levels of key GSC markers, including CD133, integrin alpha 6, aldehyde dehydrogenase 1A1 (ALDH1A1), Nanog, Sox2, and Oct4. The combined effect of berbamine and ArcA on GSC growth also resulted in downregulation of cell cycle regulatory proteins, such as cyclins and CDKs, by potent inactivation of the CaMKII gamma-mediated STAT3/AKT/ERK1/2 signaling pathway. In addition, a genetic knockdown study using small interfering RNAs (siRNAs) targeting either CaMKII gamma or CDK4 demonstrated that the synergistic anticancer effect of the two compounds on GSCs resulted from dual inhibition of CaMKII gamma and CDK4. Collectively, our findings suggest that a novel combination therapy involving berbamine and ArcA could effectively eradicate GSCs.

Automated summary

This study investigated the therapeutic effects of two natural compounds, berbamine and arcyriaflavin A, on glioblastoma stem-like cells (GSCs). The combined treatment synergistically inhibited GSC viability and tumorsphere formation and induced ROS- and calcium-dependent apoptosis. Furthermore, it downregulated the expression of key GSC markers and cell cycle regulatory proteins through inactivation of the CaMKII gamma-mediated signaling pathway.