Fig 2: Effect of GANT61 and IR in vivo(A) Relative tumor growth of 22Rv1 (left) and PC3 (right) xenograft mice treated with concomitant GANT61 and IR (6 Gy) (6 mice per treatment group, = 12 tumors). (B) Immunohistochemical analysis of Ki67 (upper) and cleaved caspase 3 (lower) immunostaining in the 22Rv1 (left) and PC3 (right) tumors. (C) qPCR analysis of Hh target genes (GLI1, GLI2 and PTCH1) in the 22Rv1 (left) and PC3 (right) tumors. Human and murine primers were used to assess gene expression in the tumor cells and surrounding stromal cells, respectively.

Fig 3: Link between metformin and Hedgehog signaling. (A) GLI1, GLI2 and PTCH1 gene expression after 72-h metformin treatment. Means ± SEM of two independent experiments. * p < 0.05 vs. control; (B) PTCH1, GLI1 and GLI2 protein expression after 72-h metformin treatment; (C) (p)AMPK protein and GLI1 expression in 22Rv1 cells transfected with AMPK siRNA and treated with metformin (5 mM) 72-h prior to protein lysis. GLI1, glioma-associated oncogene homolog 1; GLI2, glioma-associated oncogene homolog 2; PTCH1, patched 1.

Fig 4: Effect of the combination of metformin and GANT61 on radiosensitivity in a 22Rv1 xenograft model. (A) Relative tumor growth of 22Rv1 xenograft mice treated with metformin (250 mg/kg), GANT61 (50 mg/kg) or combination. At treatment day 5, tumors were irradiated with a single dose of 6Gy (six mice/group); (B) Immunohistochemical analyses of necrosis, Ki67, cleaved caspase 3, mean vessel density (MVD), hypoxia and (C) Hedgehog (Hh) target proteins GLI1, GLI2 and PTCH1 (six mice/group, n = 12 tumors). * p < 0.05 vs. control; (D) Protein expression of pAMPK and AMPK in 22Rv1 xenograft tumors (n = 3 tumors/treatment group).