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

Abstract

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 (CaMKIIγ) 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 α6, 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 CaMKIIγ-mediated STAT3/AKT/ERK1/2 signaling pathway. In addition, a genetic knockdown study using small interfering RNAs (siRNAs) targeting either CaMKIIγ or CDK4 demonstrated that the synergistic anticancer effect of the two compounds on GSCs resulted from dual inhibition of CaMKIIγ and CDK4. Collectively, our findings suggest that a novel combination therapy involving berbamine and ArcA could effectively eradicate GSCs.

Keywords: arcyriaflavin A; berbamine; calcium/calmodulin-dependent protein kinase II gamma; cyclin-dependent kinase 4; glioblastoma stem-like cells.

Figure 1

Combined treatment of berbamine and ArcA synergistically suppresses GSCs viability. (A) The chemical structures of berbamine and ArcA. (B,C) U87MG- and C6-derived GSCs were treated with the indicated concentrations of berbamine and ArcA for 7 days. Cell viability was measured using the CellTiter-Glo^® luminescent assay system. The number of formed tumorspheres was counted under an optical microscope. ** p < 0.01, *** p < 0.001 vs. the compound alone.

Figure 2

Combined treatment of berbamine and ArcA strongly promotes GSC apoptosis. (A–D) U87MG- and C6-derived GSCs were treated with the indicated concentrations of berbamine and ArcA for 24 h. (A) Effect of combined treatment of berbamine and ArcA on the nuclear morphology. Changes in nuclear morphology were monitored by DAPI staining under a fluorescence microscope. (B) Effect of combined treatment of berbamine and ArcA on the intracellular ROS generation. ROS levels were detected with H₂DCFDA using a fluorescence microscope and were further quantified by densitometry. The level of DCF fluorescence for untreated control was normalized to 1-fold. (C) Effect of combined treatment of berbamine and ArcA on the intracellular calcium level. The levels of calcium were detected with Fluo-4 AM using a fluorescence microscope and were further quantified by densitometry. The level of Fluo-4 AM fluorescence for untreated control was normalized to 1-fold. (D) Effect of combined treatment of berbamine and ArcA on the expression of apoptosis regulators. Protein levels were detected by Western blot analysis using specific antibodies and were further quantified by densitometry. β-Actin levels were used as an internal control. The ratio of each target protein to β-actin for untreated control was normalized to 1-fold. * p < 0.05, *** p < 0.001 vs. the compound alone.

Figure 2

Combined treatment of berbamine and ArcA strongly promotes GSC apoptosis. (A–D) U87MG- and C6-derived GSCs were treated with the indicated concentrations of berbamine and ArcA for 24 h. (A) Effect of combined treatment of berbamine and ArcA on the nuclear morphology. Changes in nuclear morphology were monitored by DAPI staining under a fluorescence microscope. (B) Effect of combined treatment of berbamine and ArcA on the intracellular ROS generation. ROS levels were detected with H₂DCFDA using a fluorescence microscope and were further quantified by densitometry. The level of DCF fluorescence for untreated control was normalized to 1-fold. (C) Effect of combined treatment of berbamine and ArcA on the intracellular calcium level. The levels of calcium were detected with Fluo-4 AM using a fluorescence microscope and were further quantified by densitometry. The level of Fluo-4 AM fluorescence for untreated control was normalized to 1-fold. (D) Effect of combined treatment of berbamine and ArcA on the expression of apoptosis regulators. Protein levels were detected by Western blot analysis using specific antibodies and were further quantified by densitometry. β-Actin levels were used as an internal control. The ratio of each target protein to β-actin for untreated control was normalized to 1-fold. * p < 0.05, *** p < 0.001 vs. the compound alone.

Figure 3

Combined treatment of berbamine and ArcA potently downregulates CaMKIIγ-mediated growth signaling pathway. (A–D) U87MG- and C6-derived GSCs were treated with the indicated concentrations of berbamine and ArcA for 24 h. (A,B) Effect of combined treatment of berbamine and ArcA on the cell cycle in both GSCs. Cell cycle distribution was detected using a Muse Cell Analyzer with Muse^® Cell Cycle kit. (C) Effect of combined treatment of berbamine and ArcA on the CaMKIIγ-mediated STAT3/AKT/ERK1/2 signaling pathway in U87MG-derived GSCs. (D) Effect of combined treatment of berbamine and ArcA on the expression of cell cycle regulatory proteins in U87MG-derived GSCs. (C,D) Protein levels were detected by Western blot analysis using specific antibodies and were further quantified by densitometry. β-Actin levels were used as an internal control. * p < 0.05 vs. the compound alone or the control.

Figure 4

Combined treatment of berbamine and ArcA synergistically suppresses expression of GSC markers. U87MG-derived GSCs were treated with the indicated concentrations of berbamine and ArcA for 24 h. Protein levels were detected by Western blot analysis using specific antibodies and were further quantified by densitometry. β-Actin levels were used as an internal control. * p < 0.05 vs. the compound alone or the control.

Figure 5

Synergistic anticancer effect of berbamine and ArcA on GSCs is related to dual inhibition of CaMKIIγ and CDK4. U87MG cells were transfected with either CaMKIIγ siRNA or CDK4 siRNA. Knockdown of (A) CaMKIIγ and (B) CDK4 genes was confirmed by Western blot analysis. Protein levels were detected by Western blot analysis using specific antibodies and were further quantified by densitometry. β-Actin levels were used as an internal control. * p < 0.05 vs. the control siRNA. Following genetic knockdown, U87MG-derived GSCs were treated with the indicated concentrations of (C) ArcA and (D) berbamine for 7 days. (E) Effect of simultaneous knockdown of CaMKIIγ and CDK4 genes on the cell viability and tumorsphere formation of U87MG-derived GSCs. (C–E) Cell viability was measured using the CellTiter-Glo^® luminescent assay system. The number of formed tumorspheres was counted under an optical microscope. ** p < 0.01, *** p < 0.001 vs. the compound alone or the single gene knockdown.

Figure 6

Combined treatment of berbamine and ArcA potently suppresses tumor growth derived by GSCs in vivo. Fertilized chick eggs were incubated in a humidified incubator at 37 °C. At embryonic day seven, U87MG-derived GSCs were mixed with ECM gel in the absence or presence of the indicated compounds (5 μg/egg) and were grafted onto the CAM surface. Seven days later, the CAMs were observed, the formed tumors were retrieved, and the tumor weight was calculated. ** p < 0.01 vs. the compound alone.