PMC

Chrysin decreases migration (A) and invasion (B) of glioblastoma cells (U251 and U87) in transwell migration and Matrigel invasion assays. (C) Quantification of the number of migrated and invasive cells based on four independent experiments. *p<0.05, **p<0.01, and ^#p<0.001 versus the vehicle group.

Chrysin inhibits tumor growth in in vivo tumorigenicity experiments. Chrysin treatment inhibited tumor volume (A and B) and weight (D) of U87 tumor xenografts, but did not affect body weight (C). Western blot analysis (E and F) of p-ERK1/2, ERK1/2, and Nrf2 in tumors of mice treated with the refined olive oil (vehicle) or chrysin (40 and 80 mg/kg). *p<0.05, **p<0.01, and ^#p<0.001.
Abbreviations: ERK, extracellular signal-regulated kinase; Nrf2, nuclear factor erythroid 2 (NF-E2)-related factor 2; DMSO, dimethyl sulfoxide.

Chrysin suppresses the migration capacity of glioblastoma cells in vitro wound healing assay. U251 (A) and U87 (B) cells were treated with vehicle or chrysin (10, 20, and 30 µM) for 24 hours, and at time intervals of 0, 12, and 24 hours, cells migrated into the wound areas were observed and imaged. (C) Quantification of the wound healing ability. The wound healing ability of vehicle-treated group was adjusted to the value of 1. The values are expressed as the mean ± SD from four independent experiments. *p<0.05, **p<0.01, and ^#p<0.001 versus the control.

Chrysin inhibits proliferation of glioblastoma cells. (A–C) The cell viability was analyzed by a CCK-8 assay. 0.1% DMSO was used as a vehicle control. Chrysin suppressed the growth of human glioblastoma cell lines (T98, U251, and U87) in both dose- and time-dependent manners without affecting NHAs. However, it did not demonstrate any significant effect on the cell viability at the concentrations of up to 30 µM. (D) Cells were incubated for 10 days with increasing concentrations of chrysin (0, 30, 60, and 120 µM) and chrysin reduced colony formation in all tested human glioblastoma cell lines.
Abbreviations: CCK-8, Cell Counting Kit-8; DMSO, dimethyl sulfoxide; NHA, normal human astrocyte.

Chrysin downregulates Nrf2 pathway via inhibition of ERK signaling. (A and B) Reduced expression of p-ERK1/2 was observed after exposure to chrysin (30 and 60 µM) for 24 hours. (C and E) U87 cells were treated with chrysin (30 µM) and U0126 (50 µM) individually or in combination. The protein expression levels of Nrf2 were decreased after exposure to U0126; however, it had no significant change in combination with chrysin. (D) Immunofluorescence staining proved that chrysin and U0126 inhibited Nrf2 translocation into the nucleus, and there was no statistical difference between the U0126 and the combined treatment group (p>0.05, data not shown).
Notes: *p<0.05, **p<0.01.
Abbreviations: JNK, Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; Nrf2, nuclear factor erythroid 2 (NF-E2)-related factor 2; DMSO, dimethyl sulfoxide.

Chrysin deactivates Nrf2 signaling pathway in a Keap1-independent manner. (A and B) The relative protein levels of chrysin-treated cells were expressed compared with the vehicle-treated group. (C) Cells were processed with shRNA (Sc) or Nrf2 shRNA (Nrf2i). Reduced expression of Nrf2 was observed after exposure to Nrf2 shRNA. (E and F) Chrysin was unable to change protein levels of Nrf2 and Nrf2-target genes in U87 cells with Nrf2 knockdown. The cells were pretreated with Nrf2 shRNA (Nrf2i), followed by chrysin treatment for 24 hours. (D) Nrf2 knockdown decreased the sensitivity of cells to chrysin. Relative cell numbers were monitored by a CCK-8 assay. The seeded cells were adjusted to the value of 1. Data are expressed as mean ± SD (n=4). *p<0.05, **p<0.01, and ^#p<0.001 versus the vehicle-treated group.
Abbreviations: Nrf2, nuclear factor erythroid 2 (NF-E2)-related factor 2; CCK-8, Cell Counting Kit-8; HO-1, hemeoxygenase-1; NQO-1, NAD(P)H quinine oxidoreductase 1; Keap1, Kelch-like erythroid cell-derived protein with cap’n’collar homology-associated protein 1.