Metformin kills and radiosensitizes cancer cells and preferentially kills cancer stem cells

Abstract

The anti-cancer effects of metformin, the most widely used drug for type 2 diabetes, alone or in combination with ionizing radiation were studied with MCF-7 human breast cancer cells and FSaII mouse fibrosarcoma cells. Clinically achievable concentrations of metformin caused significant clonogenic death in cancer cells. Importantly, metformin was preferentially cytotoxic to cancer stem cells relative to non-cancer stem cells. Metformin increased the radiosensitivity of cancer cells in vitro, and significantly enhanced the radiation-induced growth delay of FSaII tumors (s.c.) in the legs of C3H mice. Both metformin and ionizing radiation activated AMPK leading to inactivation of mTOR and suppression of its downstream effectors such as S6K1 and 4EBP1, a crucial signaling pathway for proliferation and survival of cancer cells, in vitro as well as in the in vivo tumors.

Conclusion: Metformin kills and radiosensitizes cancer cells and eradicates radioresistant cancer stem cells by activating AMPK and suppressing mTOR.

Figures

Figure 1. Clonogenic death of FSaII and MCF-7 cells treated with metformin alone or with radiation.

(A, B) Cells were incubated for 1–48 h with 0.5–10 mM metformin and the clonogenic survival was determined. (C) Cells were cultured in media containing 5–100 µM of metformin for 10 days for FSaII cells and 20 days for MCF-7 days. (D, E) Cells were incubated with 1 or 5 mM metformin for 24 h, irradiated with various doses of X-rays, further incubated with metformin for 24 h and then the clonogenic survival was determined. The radiation survival curves were normalized for the cell death caused by metformin alone. All data points are means of 5–7 independent experiments ± 1 S.E.

Figure 2. Western blotting for the effect of metformin, 4 Gy irradiation or combined on the signals associated with AMPK/mTOR pathway in MCF-7 and FSaII cells.

Whole cell lysates were prepared and Western blot analysis was conducted using anti-p-AMPK, -AMPK, -p-ACC, -ACC, -p-mTOR,-mTOR, -p-S6K1, -S6K1, -p-4EBP1 and -4EBP1 antibodies. Experiments were repeated 4-5 times and the representative results are shown. The numbers under each blot are intensity of the blot relative to that of untreated control.

Figure 3. Effects of AMPK siRNA on the cytotoxic and radiosensitizing effects of metformin.

(A) Transfection of MCF-7 and FSaII ells with siRNA against AMPK markedly reduced the AMPK levels in the cells. (B) Transfection of cells with AMPK siRNA markedly reduced the clonogenic cell death caused by 48 h incubation with various concentrations of metformin. (C) Transfection of cells with AMPK siRNA reduced the efficacy of metformin to sensitize the cells to radiation. Cells were incubated with 1 or 5 mM metformin for 24 h, irradiated with various doses of X-rays, further incubated with metformin for 24 h and then cultured for colony formation. The radiation survival curves were normalized for the cell death caused by metformin alone.

Figure 4. Effect of metformin on the CSCs or CS-like cells.

Cells were incubated with different concentrations of metformin for 48 h, dispersed to single cells, washed, and the fraction of CSCs or CS-like cells were analyzed. (A) FACS for CD44high/CD24low cells of MCF-7 cells treated with 0.5–5.0 mM metformin for 48 h. (B) % of CD44high/CD24low MCF-7 cells in the surviving cells after treating with different concentrations of metformin for 48 h. Means of 7 experiments ± 1 S.E are shown. The declines in % of CD44high/CD24low cells by 0.5–10 mM metformin were statistically significant (P < 0.05). (C) FACS for ALDH1-positive FSaII cells treated with metformin for 48 h. (D) % of ALDH1-positive cells of FSaII cells treated with different concentrations of metformin for 48 h. Means of 5–7 experiments ± 1 S.E are shown. The declines in % of ALDH1-positive cells by 0.1–1.0 mM metformin were statistically significant (P < 0.05). (E) Changes in % of SP cells in FSaII cells treated with different concentrations of metformin for 48 h.

Figure 5. Effect of metformin on the growth of spheres of MCF-7 and FSaII cells.

Cells were cultured for 8 days in ultralow attachment wells with sphere media containing different concentrations of metformin. (A) MCF-7 spheres grown with or without 0.1 mM metformin. (B) The numbers of sphere with > 50 µm in diameter obtained from 103 cells. Means of 5 experiments ± 1 S.E are shown. *P < 0.05 between control and metformin treated groups.

Figure 6. Effects of metformin on the radiosensitivity of CSCs or CS-like cells.

(A) MCF-7 cells were irradiated, incubated with or without 1 mM metformin for 48 h and analyzed for CD44high/CD24low cells. (B) % of CD44high/CD24low MCF-7 cells after irradiation and 48 h incubation with 1 mM metformin. Means of 5 experiments ± 1 S.E. *P < 0.05 for IR-induced increases. **P < 0.05 between IR and IR + Metformin. (C) % of ALDH1-positive FSaII cells after irradiation and 48 h incubation with 1 mM metformin. Means of 5 experiment ± 1 S.E. *P < 0.05 for IR-induced increases. **P < 0.05 between IR and IR + 1 mM Metformin groups.

Figure 7. Growth and immunohistochemistry of FSaII tumors.

(A) Tumors grown (s.c.) to 150 mm3 in the hind legs of C3H mice were treated as follows. (a) Control. (b) Metformin: mice were injected i.p. twice a day with metformin at 25 mg/kg/dose from Day 0. (c) Tumors were irradiated with 20 Gy of X-rays in a single exposure. (d) 20 Gy + Metformin: Tumors were irradiated with 20 Gy of X-rays in a single exposure 1 h after the first metformin treatment. Data points are means of 7–10 tumors ± 1 S.E. (B–E) Immunohistological study for the expression of p-AMPK and p-mTOR in FSaII tumors. Tumors were treated with metformin and 20 Gy alone or combined as the tumors were treated for the tumor growth study (A), and tumors were excised on day 6. The cross-sections were stained and quantified for p-AMPK (B, C) and p-mTOR (D, E). All results are expressed as mean pixel counts per image ± standard error from 20 images derived from 3 tumors per group (magnification 200x). Representative images illustrating the average amount of staining are depicted in panels B and D for each treatment modality as indicated. *P < 0.001 treatment group vs. vehicle; #P < 0.05 metformin and radiation group vs. metformin alone.