Research Paper Volume 12, Issue 7 pp 6240—6259

Figure 6. Synergistic effects of genistein in combination with GLPG0634 or MK-2206 on the proliferation of Eca-109 cells. (A) A CCK-8 assay was conducted to assess the effects of the JAK1 pathway inhibitor GLPG0634 or the Akt pathway inhibitor MK-2206 on the proliferation of Eca-109 cells. (B) The effects of genistein (4 μM) in combination with the JAK1 pathway inhibitor GLPG0634 (16 nM) or the Akt pathway inhibitor MK-2206 (32 nM) on the proliferation of Eca-109 cells was measured through a CCK-8 assay. The effects of genistein (4 μM) in combination with GLPG0634 (16 nM) or MK-2206 (32 nM) on (C) apoptosis, (D) ROS levels and (E) the cell cycle in Eca-109 cells were measured through flow cytometry. (F) qPCR analysis was performed to quantify the expression of CyclinD1, CDK4, P53, STAT3 and MDM2 in Eca-109 cells co-treated with genistein (4 μM) and GLPG0634 (16 nM) or MK-2206 (32 nM) for 72 h. (G) The effects of genistein (4 μM) or MK-2206 (32 nM) or co-treatment on the protein levels of STAT3, p-STAT3, MDM2 and p-MDM2 in Eca-109 cells. (H) The effects of genistein (4 μM) or GLPG0634 (16 nM) or co-treatment on the protein levels of STAT3, p-STAT3, MDM2 and p-MDM2 in Eca-109 cells. (I, J) Genistein (5 mg/kg) treatment alone or in combination with MK-2206 (1 mg/kg) suppresses tumor growth and tumor volume in xenograft nude mice (n = 6 in each group). (J) Representative images of tumor volume. (K, L) Genistein (5 mg/kg) treatment alone or in combination with GLPG0634 (1 mg/kg) inhibits tumor growth and tumor volume in xenograft nude mice (n = 6 per group). (J, L) Representative images of tumor volume. All in vitro experiments were independently repeated three times. Data are analyzed using one-way ANOVA with Dunnett’s test and presented as the mean ± SD. *P<0.05; **P<0.01. Gen or G, genistein. OV, sodium orthovanadate. p-, phosphorylated.