Icaritin Synergizes with Imatinib against Philadelphia Chromosome-Positive B-Cell Acute Lymphoblastic Leukemia by Inhibiting PI3K/AKT Signaling

Figure 1. Icaritin inhibits Ph+ B-ALL cell proliferation in a concentration-dependent manner and synergizes with imatinib. (A) Cells were treated with different concentrations of imatinib (IM, 3, 6, 12, and 24 μM), and cell viability was measured by CCK-8 assay over 0–100 h. (B) Cells were treated with different concentrations of icaritin (ICA, 10, 20, 40, and 80 μM), and cell viability was measured by CCK-8 assay over 0–100 h. (C) Cells were treated with 20 μM icaritin, 6 μM imatinib, or their combination, and cell viability was measured by CCK-8 assay. Data are presented as mean ± SD. **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. the NC group.

Figure 2. Icaritin combined with imatinib inhibits migration and induces mitochondria-mediated apoptosis in Ph+ B-ALL cells. (A) Transwell migration assay and quantitative analysis. (B) JC-1 staining showing mitochondrial membrane potential changes. Scale bar = 20 μm. (C) Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. (D) Western blot analysis of Bax and Bcl-2 protein levels. β-actin was used as the loading control. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the NC group.

Figure 3. Icaritin (ICA) combined with imatinib (IM) synergistically suppresses the PI3K/AKT/FOXO1 signaling pathway, and the PI3K agonist 740Y‑P reverses this inhibitory effect in SUP-B15 cells. (A) SUP-B15 cells were treated with 6 μM imatinib (IM), 20 μM icaritin (ICA), or their combination for 48 h. Western blot assay was performed to detect the protein expression levels of total and phosphorylated PI3K, AKT and FOXO1, with β-actin as the internal reference. The relative phosphorylation levels of p-PI3K, p-AKT and p-FOXO1 were quantified and shown in the bar graphs. (B) Cells were treated with 3 μM IM, 20 μM ICA, or their combination in the presence or absence of 20 μM PI3K agonist 740Y-P for 48 h. Western blot analysis was used to determine the phosphorylation levels of PI3K and AKT, and the relative protein levels were quantitatively analyzed. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. the NC group; ns, no significant difference.

Figure 4. Activation of the PI3K/AKT pathway by740Y-P reverses the synergistic effects of icaritin and imatinib. Ph+ B-ALL cells were treated with imatinib (IM, 6 μM), icaritin (ICA, 20 μM), or their combination in the presence or absence of the AKT agonist 740Y-P (20 μM). (A) Cell viability was assessed by CCK-8 assay. (B) Cell migration was evaluated by Transwell assay. (C) Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. (D) Mitochondrial membrane potential was analyzed by JC-1 staining. Scale bar = 20 μm. (E) Western blot analysis of Bax and Bcl-2 protein levels. β-actin was used as the loading control. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.