Fig. 7: Sorafenib combined with shikonin can more strongly inhibit angiogenesis and macrophage polarization by targeting GP73 and PKM2.

A Shikonin attenuates the intracellular interaction of GP73 and PKM2 in HCC cells. HCC cells co-transfected with HA-GP73 and FLAG-PKM2 and treated with or without shikonin (10 μmol/L, 24 h) were collected to conduct the Co-IP assay. B Protein levels of GP73 and PKM2 in cell lysates and supernatants of HCC cells after incubation with or without shikonin were measured by western blotting. β-actin was used as an intracellular loading control and GST as an extracellular loading control. C Schematic illustration for the drug combination therapy. D, E The combination therapy of sorafenib and shikonin shows a more significant inhibition on HUVEC migration and invasion. D Left, representative images of the Transwell assay showed the migration and invasion potential of HUVEC after incubation with the indicated supernatant from HepG2 cells. Scale bar, 200 μm. Right, the statistical graph of cell migration and invasion number. n = 3 per each group. E Left, representative images of the wound healing assay revealed the migration potential of HUVEC after incubation with the indicated supernatant from HepG2 cells. Scale bar, 500 μm. Right, the quantitative statistic of cell repaired ratio was measured by Image J. n = 3 per each group. F The protein levels of VEGF in HUVEC after incubation with the indicated supernatant from HepG2 cells were analyzed using western blotting. G Flow cytometry showed that the combination therapy of sorafenib and shikonin had a more significant inhibition on the MFI of the M2 macrophage gene CD206. n = 3 per each group. H Schematic description of the establishment of the animal model. I–L The combined treatment of sorafenib with shikonin suppresses tumor growth in vivo. I Sorafenib combined with shikonin had more significant inhibition on tumorigenesis of HepG2 cells in nude mice (five mice per group). J The tumors were weighed after injection for 31 days (five mice per group). K The tumor growth curve was shown. L Left, representative IHC images of the Ki67 expression in subcutaneous tumors in mice. Scale bar, 50 μm. Right, the statistical graph of the positive area of Ki67 per field. n = 3 per each group. M Left, representative IHC images of the CD31, CD206, GP73, and PKM2 expressions in subcutaneous tumors in mice. Scale bar, 50 μm. Right, the statistical graph of the positive area of CD31, CD206, GP73, and PKM2 per field. n = 3 per each group. N Content of ALT, AST, CREA, and UA from the blood of mice in the indicated groups (five mice per group). Error bars represent mean ± SD. *p <0.05, ** p <0.01, *** p <0.001; n.s., no significance.