Article Figures & Data
Figures
- Figure 1.
Scutellarin strongly suppresses the AKT signaling pathway. A, The effect of scutellarin on the in vitro kinase activity of various kinases. The effect of scutellarin on the activity of 26 kinases was determined using active protein kinases and the specific substrate for each kinase. Data are shown as means values ± SD. B and C, Effect of scutellarin on EGF- or hydrogen peroxide (H2O2)-induced kinase signaling in JB6 cells. Serum-starved (0.1% FBS; 48 hours) cells were treated with different doses of scutellarin for 1 hour followed by treatment with EGF or H2O2 for 30 minutes. D, Effect of scutellarin on kinase signaling in KYSE30 ESCC cells. Cells were treated with scutellarin and then various signaling proteins were examined by Western blotting. For B–D, band density was measured using the Image J (NIH) software program. All results of Western blotting are shown as mean values ± SD for 3 independent experiments.
- Figure 2.
Scutellarin is a novel AKT1 and AKT2 inhibitor. A, Modeling of scutellarin with the AKT1 or AKT2 protein. Modeling of the binding of scutellarin with AKT1 (left) or AKT2 (right) and an enlarged view of the binding. The AKT1 and AKT2 proteins are shown as ribbon representation and scutellarin is shown as stick. Hydrogen bonds are shown as blue lines. Scutellarin directly binds to AKT1 and AKT2 in a KYSE30 ESCC cell lysate (B) or recombinant proteins (C). The cell lysate or recombinant protein was incubated with scutellarin-conjugated Sepharose 4B beads or with Sepharose 4B beads alone. Proteins were pulled down and then analyzed by Western blotting. For B and C, similar results were observed from 3 independent experiments. D, Effect of scutellarin on AKT1 (top) and AKT2 (bottom) kinase activities. AKT1 or AKT2 kinase activity was assessed by an in vitro kinase assay using active AKT1 or active AKT2 and inactive GSK3β proteins. The effect of scutellarin was determined by Western blotting using a phosphorylation GSK3β antibody. Similar results were observed from 3 independent experiments, and band density was measured using the Image J (NIH) software program. The asterisk (*) indicates a significant (P < 0.05) difference.
- Figure 3.
AKT is a therapeutic target in ESCC cells. Expression of the phosphorylated AKT (Ser473) protein in adjacent tumor tissues and ESCC tissues (A) or Shee normal esophageal and ESCC cells (B). Adjacent tumor or tumor tissues were stained with antibodies to detect phosphorylated AKT by using IHC. Cells were seeded and incubated for 48 hours and expression of the total or phosphorylated AKT protein was analyzed by Western blotting. C, Effect of knockdown of AKT1 or AKT2 on total or phosphorylated AKT protein. KYSE450 or KYSE510 ESCC cells stably expressing knockdown AKT1 and AKT2 or control were established. The expression of total AKT1, total AKT2, or phosphorylated AKT was determined by Western blotting. For B and C, similar results were observed from 3 independent experiments and band density was measured using the Image J (NIH) software program. D, Effect of AKT1 and AKT2 knockdown on growth of ESCC cells. Cells were seeded and incubated for 72 hours or 2 weeks and cell growth was determined by MTT assay (D) or soft agar assay (E). For E, colonies were counted using a microscope and the Image-Pro PLUS (v.6) computer software program. For D and E, data are shown as means ± SD of triplicate values from 3 independent experiments. The asterisk (*) indicates a significant difference (P < 0.05).
- Figure 4.
Scutellarin exerts anticancer activities. A, Effect of scutellarin on the viability of Shee normal esophageal cells. Cells were seeded for 24 hours and treated with scutellarin for 48 hours. B, Effect of scutellarin on ESCC cell growth. For A and B, cells were treated with scutellarin at various concentrations and then incubated for 48 hours and growth was determined by the MTT assay. C, Effect of scutellarin on cell cycle. Cells were treated with scutellarin for 48 hours in medium supplemented with 2% (KYSE30) or 10% (KYSE450) FBS. Cells were stained with propidium iodide (PI) and cell cycle was analyzed by FACS. For A–C, data are shown as means ± SD of triplicate values from 3 independent experiments and the asterisk (*) indicates a significant (P < 0.05) difference. D, Effect of scutellarin on the expression of cell cycle marker proteins was determined by Western blotting. Band density was measured using the Image J (NIH) software program. For D, similar results were observed from 3 independent experiments. E, Effect of scutellarin on anchorage-independent growth of ESCC cells. Cells were treated with scutellarin and incubated for 2 weeks and then colonies were counted using a microscope and the Image-Pro PLUS (v.6) computer software program. For E, data are shown as means ± SD of values from 3 independent experiments each with triplicate samples and the asterisk (*) indicates a significant (P < 0.05) difference.
- Figure 5.
Inhibition of cell growth by scutellarin is dependent on the expression of AKT1 and AKT2. A, The effect of scutellarin on esophageal cancer cell growth was assessed in cells stably expressing shAKT1 and shAKT2 or cells stably expressing shControl. Cells were seeded for 24 hours and treated or not treated with scutellarin at various concentrations and then incubated for 48 hours and growth was determined by the MTT assay. B, The effect of scutellarin on anchorage-independent esophageal cancer cell growth was assessed in cells stably expressing shAKT1 and shAKT2 or cells stably expressing shControl. Cells were treated with scutellarin and incubated for 2 weeks, and then, colonies were counted using a microscope and the Image-Pro PLUS (v.6) computer software program. All data are represented as means ± SD of triplicate values from 3 independent experiments. The asterisk (*) indicates a significant (P < 0.05) difference.
- Figure 6.
Scutellarin inhibits ESCC PDX tumor growth in vivo. A, Effect of scutellarin on ESCC tumor growth. Tumor-bearing mice were orally administered (by gavage) scutellarin or vehicle once a day Monday through Friday for 21 days. Tumor volumes were measured on the days indicated. B, Effect of scutellarin on mouse body weight. Body weights from treated or untreated groups of mice were obtained once a week. C, Effect of scutellarin on Ki-67 expression. Treated or untreated groups of tumor tissues were stained with antibodies to detect Ki-67. The number of Ki-67–stained cells was counted from IHC results (n = 8). D, Effect of scutellarin on the AKT signaling pathway. Tumor tissues from each group were immunoblotted with antibodies to detect total AKT, total GSK3β, phosphorylated AKT, phosphorylated GSK3β, and β-actin. Band density was measured using the Image J (NIH) software program. All data are shown as means ± SE of values obtained from the experiment groups. The asterisk (*) indicates a significant difference between tumors from untreated or treated mice as determined by t test (P < 0.05).
Additional Files
Supplementary Data
- Figure S1 - Chemical structure of scutellarin
- Figure S2 - The effect of scutellarin on mouse body weight
- Figure S3 - Expression of phosphorylated or total AKT in ESCC tissues
- Figure S4 - Assessing the toxicity of scutellarin in vivo
- Supplemental figure legends - Supplemental figure legends
- Table S1 - Clinical information of PDX tissue
Volume 12, Issue 12
