Fig 1.
Metformin decreased clonogenic survival in p53-deficient compared with p53 wild-type cells.
HCT116 p53+/+ and p53-/- cells were treated with 1–10 mM metformin for 48 h, cultured and used in (A) clonogenic and (B) WST-1 (cell viability) assays; *p < 0.001 for HCT116 p53+/+ cells, †p < 0.001 for HCT116 p53-/- cells. §p < 0.001, between HCT116 p53+/+ and p53-/- cells, compared to control. WST, water-soluble tetrazolium.
Fig 2.
Metformin significantly inhibited tumor growth and enhanced radiosensitivity in p53-deficient cells and xenografts.
Radiosensitivity of (A) HCT116 p53+/+ and (B) p53-/- cells with and without exposure to metformin (2.5 mM) after varying doses of 60Co γ-ray radiation was measured using a clonogenic assay. HCT116 p53+/+ and p53-/- xenografted mice were randomly divided into four groups including control (Con), metformin (Met), ionizing radiation (IR), and combination of metformin and IR (Met+IR). Graphs indicate the tumor volume in (C) HCT116 p53+/+ and (D) p53-/- xenograft model; *p < 0.05.
Fig 3.
Metformin prevented cell cycle progression by significantly prolonging IR-induced G2/M arrest in HCT116 p53-/- cells.
HCT116 p53+/+ and p53-/- cells were pretreated with 2.5 mM metformin for 24 h, and then irradiated with 6 Gy. Cell cycle measured by flow cytometry (A) 24 and (B) 48 h after IR. (C) Expression of G2/M checkpoint regulators including cyclin B1, phosphorylated cdc2 (Tyr15), phosphorylated histone H3 (Ser10), and phosphorylated Chk2 (Thr68) were measured using immunoblotting in HCT116 p53+/+ and p53-/- cells 48 h after irradiation. Densitometric quantification was normalized to β-actin. Values are mean ± SEM. of three experiments, *p < 0.001. IR, ionizing radiation
Fig 4.
Metformin delayed repair of IR-induced DNA damage in p53-deficient cells compared with p53 wild-type cells.
HCT116 p53+/+ and p53-/- cells were pretreated with 2.5 mM metformin for 24 h, and then irradiated with 6 Gy. (A) Immunofluorescence staining at 6 and 24 h after IR, showed γ-H2AX, a marker for DNA damage; Rad51, a marker of DNA repair; and nuclear DNA stained with DAPI using image analysis in three (green/red/blue) fluorescence channels. (B) For quantitative analysis, γ-H2AX or Rad51 foci-positive cells were counted in at least 100 cells from randomly captured images. Values are mean ± S.E.M, *p < 0.001. IR, ionizing radiation; γ-H2AX, γ-H2A histone family, member X.
Fig 5.
Metformin enhanced radiosensitivity by reducing p53-related HR repair proteins in vitro, especially in p53-deficient cells.
HCT116 p53+/+ and p53-/- cells were pretreated with 2.5 mM metformin for 24 h, and then irradiated with 6 Gy. Cells from both groups were immunoblotted with p53-related HR repair proteins such as MRE11-Rad50-p95/NBS1 complex, BRCA1, BRCA2, Rad51, Rad52, and ERCC1 24 h after ionizing radiation (IR). Densitometric quantification was normalized to β-actin. HR, homologous recombination; MRE11, meiotic recombination 11; NBSI, nijmegen breakage syndrome protein 1; BRCA, breast cancer early onset; ERCC 1, excision repair cross-complementation group 1.
Fig 6.
Metformin enhanced radiosensitivity by reducing Rad51 and ERCC1 proteins in vivo, especially in p53-deficient xenografts.
In vivo, confirmation of (A) Rad51 and (B) ERCC1 expression in HCT116 p53+/+ and p53-/- tumor tissues analyzed by immunohistochemistry and graphs indicate histological score for each group. (C) Tumor tissues were lysed and immunoblotted with Rad51 and ERCC1. Densitometric quantification was normalized to β-actin. Data are mean ± S.E.M, *p < 0.05.