Fig 1: Chrysin interacts with DGKa to inhibit the activation of FAK/AKT signaling. The structure of chrysin (A) and 3-dimensional structure of DGKa catalytic domain (B). 2- (C) or 3-Dimensional (D) structure of docking of chrysin to the Asp435 site in the catalytic domain of DGKa. (E) The indicated ESCC cells were treated with chrysin (10, 25, and 50 µmol/L) for 2 h. The interaction between DGKa and FAK, or the activation of FAK in DGKa/FAK complex was assayed using IP (IP: FAK) and IB (IB: FAK, pFAK, or DGKa) analysis (E). (F) The indicated ESCC cells were treated with chrysin (10, 25, and 50 µmol/L) for 2 h. The interaction between DGKa and FAK was evaluated using confocal assay. Cells were stained with DAPI to visualize the nucleus. Scale bar, 20 µm as indicated. (G) and (H) The indicated ESCC cells were treated with 50 µmol/L chrysin. The interaction between DGKa and FAK was evaluated using IP (IP: FAK) and IB (IB: FAK, or DGKa) assay after 2-h treatment (G), and the phosphorylation of FAK Tyr397 was evaluated using quantitative ELISA assay after 4-h treatment (H). ***P < 0.001. Error bars, mean ± SD of five independent experiments.
Fig 2: Proposed model of chrysin-mediated anti-ESCC effect. Chrysin inhibited several malignant phenotypes, including proliferation, invasion, stemness, and glycolysis, in ESCC cells. Mechanistically, chrysin disrupted the formation of DGKa/FAK signalosome via interacting with the Asp435 site in the catalytic domain of DGKa and subsequently inhibited the phosphorylation of FAK Tyr397 site, suppressed the activation of FAK/AKT pathway and its controlled downstream tumor-promoting effectors in ESCC cells.
Fig 3: Chrysin inhibits the expression of downstream malignant effectors via DGKa/FAK complex. (A)–(L) The indicated control or DGKa shRNA or 1 µmol/L PF562271-incubated ESCC cells were treated with 50 µmol/L chrysin. The intratumoral expression of C-MYC (A), cyclin D1 (B), survivin (C), SOX2 (D), NANOG (E), OCT4 (F), BMI1 (G), PKM2 (H), HKII (I), LDHA (J), GLUT1 (K), and the secretion of MMP9 in supernatant (L) were evaluated using quantitative ELISA assay. n.s, no significant difference. Error bars, mean ± SD of five independent experiments.
Fig 4: DGKa is critical for chrysin-mediated tumor inhibitory effect and FAK/AKT signaling inhibition. Stable silencing DGKa in 2 specific shRNA-transduced ESCC cell lines analyzed by immunoblotting. GAPDH was used as a loading control. (B)–(D) The indicated control or DGKa shRNA ESCC cells were treated with 50 µmol/L chrysin. Then, the growth ability of ESCC cells was observed by MTS assay (B). The activation of FAK (C) and AKT (D) was evaluated using quantitative ELISA assay. n.s, no significant difference. Error bars, mean ± SD of five independent experiments.
Fig 5: Chrysin inhibits ESCC malignancy via suppressing the activation of FAK/AKT signaling. The indicated ESCC cells were treated with 50 µmol/L chrysin, and then the activation of AKT or RAF/ERK signaling were analyzed using antibody array. No difference of pRAF1 Ser301 expression was indicated between control group and 50 µmol/L chrysin group. (B)–(E) The indicated ESCC cells were treated with chrysin (10, 25, and 50 µmol/L) for 4 h, the activation of pAKT Ser473 (B), pPRAS40 Thr246 (C), pRPS6 Ser235/236 (D) and pRAF1 Ser301 (E) was evaluated by quantitative ELISA assay. (F) Total protein lysates from the indicated cells treated with 50 µmol/L chrysin were analyzed using antibody array against 71 tyrosine kinases. (G) The indicated ESCC cells were treated with chrysin (10, 25, and 50 µmol/L) for 4 h, the activation of pFAK Tyr397 was evaluated by quantitative ELISA assay. n.s, no significant difference, **P < 0.01, ***P < 0.001. Error bars, mean ± SD of five independent experiments.
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