Xanthohumol suppresses glioblastoma via modulation of Hexokinase 2 -mediated glycolysis

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  Figure 1. Inhibitory effects of xanthohumol on the growth of GBM cells. A, Chemical structure of Xanthohumol. B, Xanthohumol inhibits anchorage-dependent growth in a panel of human GBM cells, including U87 (left), T98G (middle), and LN229 (right). Cell proliferation assay was performed as described in the “Material and Methods”. Data shown are the proliferation ability of human GBM cells treated with different concentrations of xanthohumol compared with the DMSO-treated group, asterisk, significant suppression (*p<0.05, **p<0.01, ***p<0.001) of proliferation by Xanthohumol. C. The soft agar colony formation assay was performed as described in the “Material and Methods”. Data shown are the colony formation ability of U87 (up), T98G (middle), and LN229 (bottom) cells treated with different concentrations of Xanthohumol or irinotecan. The average colony number was calculated from three separate experiments. Iri, Irinotecan. Asterisk, significant suppression (*p<0.05, **p<0.01, ***p<0.001) of colony formation by xanthohumol compared with DMSO-treated group.

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  Figure 2. Xanthohumol suppresses glycolysis in GBM cells. GBM cells, including U87 (A), T98G (B) and LN229 (C) were treated with xanthohumol as indicated for 24 h. Western blotting was performed to determine the protein level of HK2, β-actin was used as a loading control (A, B and C, left). The levels of glucose consumption (A, B and C, middle) and lactate production (A, B, and C, right) were examined in these cells. The asterisk, significant suppression (*p<0.05, **p<0.01, ***p<0.001) of glycolysis by xanthohumol compared with DMSO-treated group. D, xanthohumol inhibits HK2 localization on mitochondria and up-regulates apoptosis. Human GBM cells, including U87 (left), T98G (middle) and LN229 (right) cells were treated with xanthohumol for 24 h, the mitochondrial fractions and whole-cell extracts were isolated, western blot was conducted to detect target proteins as indicated.

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  Figure 3. Xanthohumol promotes the proteasome-mediated degradation of c-Myc in GBM cells. A, xanthohumol decreases the protein levels of c-Myc in GBM cells. U87 (left), T98G (middle), and LN229 (right) cells were treated with xanthohumol for 24 h. Western blot was conducted to detect target proteins as indicated. B, the effect of xanthohumol on c-Myc mRNA expression in GBM cells, U87 (left), T98G (middle) and LN229 (right) cells were treated with xanthohumol for 24 h, qPCR was conducted to determine the mRNA level of c-Myc. C, U87 (left), T98G (middle) and LN229 (right) cells were treated with xanthohumol for 24 h as indicated, 20 μM of MG132 was added to the cell culture medium and incubated for another 6 h. The whole-cell lysate was extracted, and western blot was conducted to detect the target proteins. D, The U87 cells were treated with 20 μM CHX at multiple time points, whole-cell lysate was extracted, and western blot was conducted to detect the target proteins. E, HA-c-Myc, and Flag-FBW7 plasmids were co-transfected into 293T cells for 48 h. The whole-cell extract was incubated with the HA antibody (left) or Flag antibody (right) for co-immunoprecipitation. Western blot was…

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  Figure 4. Down-regulation of Akt-GSK3β-FBW7 signaling axis results in the destabilization of c-Myc and inhibition of glycolysis. A, Stable knockdown of c-Myc in U87 (left), T98G (middle), and LN229 (right) cells were performed. Western blotting was conducted to detect the target proteins. B, U87 cells were treated with xanthohumol for 24 h. Western blotting was performed. C, U87 (left) and T98G (right) cells were treated with wortmannin for 24 h, western blotting was performed. D, E, and F, the Myr-Akt1 or vehicle plasmid was transfected into U87 cells, followed by treated with xanthohumol for another 24 h as indicated. Western blot analysis was conducted to detect the protein expression levels (D), and glucose consumption (E) and lactate production (F) were examined in these cells. Asterisk, significant suppression (*p<0.05, **p<0.01) of glycolysis between xanthohumol and DMSO treated group or Myr-Akt1 transfected group. G, the Myr-Akt1 or vehicle plasmid was transfected into U87 cells. These cells were treated with xanthohumol for another 24 h as indicated, followed by incubated with MG132 for 6 h, immunoprecipitation and western blot were used to detect the ubiquitination of c-Myc. H…

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  Figure 5. Xanthohumol inhibits tumor growth in xenograft mouse model. A and B, LN229 (A) and U87 (B) cells were subcutaneously injected into the flank of mice, after tumor volumes reached at 100 mm3, the mice were treated with vehicle or xanthohumol, tumor volumes were measured every two or three days. C and D, The average tumor weight from LN229 (C) and U87 (D) xenograft models treated with vehicle or xanthohumol were measured. E and F, During the treatment period, the bodyweight of LN229 (E) and U87 (F) xenograft mice was measured twice a week to determine the effect of xanthohumol. For A, B, C, and D, data are shown as mean values ± S.D. obtained from 5 mice in each group. G, Immunohistochemical staining examination of Ki67 and HK2 expression in tumor sections from vehicle- or xanthohumol-treated group mice.