Amentoflavone Induces Apoptosis and Inhibits NF-ĸB-modulated Anti-apoptotic Signaling in Glioblastoma Cells

Abstract

The goal of the present study was to investigate anticancer effect of amentoflavone on glioblastoma cells in vitro. Our results demonstrated that amentoflavone not only significantly reduced cell viability, nuclear factor-ĸappa B (NF-ĸB) activation, and protein expression of cellular Fas-associated protein with death domain-like interleukin 1 beta-converting enzyme inhibitory protein (C-FLIP) and myeloid cell leukemia 1 (MCL1), but significantly triggered cell accumulation at the sub-G₁ phase, loss of mitochondrial membrane potential, and expression of active caspase-3 and -8. In order to verify the effect of NF-ĸB inhibitor on expression of anti-apoptotic proteins, we performed western blotting. We found that the of NF-ĸB inhibitor or amentoflavone markedly diminished protein levels of MCL1 and C-FLIP. Taken all together, our findings show that amentoflavone induces intrinsic and extrinsic apoptosis and inhibits NF-ĸB-modulated anti-apoptotic signaling in U-87 MG cells in vitro.

Keywords: Glioblastoma; NF-ĸB; amentoflavone; apoptosis.

Figure 1. U-87 MG cells (2×104) were seeded in 96-well plates and treated with 0, 50, 100, 150, 200 μM amentoflavone in 0.1% dimethylsulfoxide for 48 h. The viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide assay. **Significantly different at p<0.01 vs.0 μM.

Figure 2. U-87 MG cells were treated with 0, 50, 100 μM amentoflavone for 48 h and prepared for cell cycle and caspase-3 flow cytometry (A, B),or analyzed for mitochondrial membrane potential and caspase-8 (C, D). **Significantly different at p<0.01 vs. 0 μM.

Figure 3. U-87 MG cells were treated with 0-1 μM of the nuclear factor-ĸappa B (NF-ĸB) inhibitor QNZ for 48 h and NF-ĸB activation was evaluated by bioluminescence imaging. B: U-87 MG cells were treated with 0 and 0.3 μM QNZ for 48 h and harvested for western blotting assay using Image J system to quantify the protein level of myeloid cell leukemia 1 (MCL1) and cellular Fas-associated protein with death domain-like interleukin 1 beta-converting enzyme inhibitory protein (C-FLIP). **Significantly different at p<0.01 vs. 0 μM.

Figure 4. U-87 MG cells (2×104) were seeded in 96-well plates and treated with 0-200 μM amentoflavone in 0.1% dimethylsulfoxide (DMSO) for 48 h. Nuclear factor-ĸappa B (NF-ĸB) activation was evaluated by reporter gene assay. Relative NF-ĸB activity of each group was normalized with viability and calculated through dividing by the value for the control group treated with 0.1% DMSO. B: Cells were treated with 0, 50, 100 μM amentoflavone for 48h and harvested for western blotting assay. The quantification of protein expression of myeloid cell leukemia 1 (MCL1) and cellular Fas-associated protein with death domain-like interleukin 1 beta-converting enzyme inhibitory protein (C-FLIP) was measured by Image J. **Significantly different at p<0.01 vs. 0 μM.