Figure 1.
Western blotting of AMPK/mTOR pathway in MCF-7 and MDA-MB-231 cells treated with hyperthermia and metformin.
(A, C) Cells were heated at 42°C for 1 h and then incubated at 37°C for 47 h (total 48 h treatment). (B, D) The effects of metformin alone were studied by incubating cells with 5 mM metformin for 48 h at 37°C. The combined effects of metformin and heating were studied by heating the cells at 42°C for 1 h with 5 mM metformin and then incubating at 37°C for 47 h. (E) The combined effects of metformin and heating were studied by heating the cells at 39.5–41°C for 1 h with 5 mM metformin and then incubating at 37°C for 47 h. (F) The combined effects of metformin and heating were studied by heating the cells at 39.5°C for 6 h with 30 µM metformin and then incubating at 37°C for 47 h. Experiments were repeated 4–5 times and the representative results are shown. The Fig 1C S6K panel was previously published in https://doi.org/10.1038/srep00362 and is excluded from this article's CC BY 4.0 license.
Figure 2.
Clonogenic surviving fraction of MCF-7 and MDA-MB-231 cells after treated with hyperthermia and metformin.
(A, C) Cells were incubated with 0–10 mM of metformin for 48 h at 37°C, or cells were heated at 42°C for 1 h with 0–10 mM metformin or without metformin and then incubated for 47 h at 37°C. After the 48 h metformin treatments, cells were washed and cultured for 18 days in regular media and the colonies formed were counted. Means of 6 experiments ±1 S.E. are shown. The decrease in cell survival by heating alone was statistically significant, and the survival of cells treated with the combination of heating and metformin was statistically smaller than that by metformin alone at all the metformin concentrations tested. (B, D) To elucidate the role of AMPK in the cell death caused by metformin alone or in combination with heating, cells were transfected with AMPK siRNA or control siRNA. The insets are Western blots for AMPK after transfection with control siRNA or AMPK siRNA. The transfected cells were treated with metfomin alone, heat alone or in combination with heating as described above and their clonogenic survival was determined. Cell death caused by the combined treatment was normalized for the death caused by heating alone. Means of 5 experiments ±1 S.E. are shown. The decreases in the effect of metformin by siRNA transfection were statistically significant both at 37°C and 42°C. (E) MCF-7 cells in medium containing 30 µM metformin were heated at 39.5°C for 6 h and then cultured at 37°C for 18 days. The effects of heating alone, metformin alone and combined on % survival of cells are shown. Means of 7 experiments ±1 S.E. are shown. The survival of cells treated with metformin in combination with heating was statistically smaller than that caused by metformin alone and heating alone.
Figure 3.
Proliferation of MCF-7 human breast cancer cells treated with hyperthermia and metformin.
Numbers of viable cells (trypan blue excluding cells) were counted with a hemocytometer after incubations for 0–72 h. (A) 37°C; incubated in regular medium. 37°C + Met; incubated in medium containing 5 mM metformin. 42°C; heated at 42°C for 1 h in regular medium and incubated. 42°C + Met; heated at 42°C for 1 h and incubated in medium containing 5 mM metformin. The variances in cell numbers among the 4 groups at 72 h were statistically significant (ANOVA). The cell number of 42°C + Met group was statistically smaller than that of 37°C + Met group by student t-test. (B, C) MCF-7 cells were transfected with control siRNA or AMPK siRNA and the effects of metformin and heating or combined on cell proliferation were studied. Effect of siRNA to suppress the effect of metformin alone or in combination with heating was statistically significant.
Figure 4.
Cell proliferation was studied with immunostaining for PCNA and DAPI in MCF-7 cells.
(A) Cells transfected with AMPK siRNA or control siRNA were incubated for 48 h at 37°C with or without 5 mM metformin. The effect of heating at 42°C for the first 1 h of 48 h incubation with or without 5 mM metformin was also studied. Following treatments, cells were immunostained for PCNA and DAPI, and the immunofluorescence intensity of PCNA and DAPI was determined with immunofluorescence microscopy. (B) Ratio of PCNA/DAPI fluorescence intensity observed in A. Means of 5 experiments ±1 S.E. is shown. The decrease in PCNA/DAPI fluorescence intensity by metformin was statistically significant at both 37°C and 42°C, and siRNA transfection significantly reduced the effect of metformin to decrease the PCNA/DAPI fluorescence intensity.
Figure 5.
Western blotting of cyclins expression in MCF-7 and MDA-MB-231 cells treated with hyperthermia and metformin.
(A) 42°C; Cells were heated at 42°C for 1 h and then incubated at 37°C for 24–72 h. Met; Cells were incubated with or without 5 mM metformin for 24–72 h at 37°C. 42°C + Met; Cells were heated at 42°C for 1 h with or without 5 mM metformin and then incubated at 37°C for 24–72 h. (B) Cells were transfected with AMPK siRNA or control siRNA, and incubated with or without 5 mM metformin for 72 h at 37°C. Effects of 1 h heating at 42°C at the inception for 72 h treatment with or without 5 mM metformin were also studied. Representative results out of several repeated studies are shown. (C) Changes in cell cycle distribution after 42°C heating for 1 h followed by 47 h incubation at 37°C, 48 h treatment with 5 mM metformin at 37°C, or combination of heating and metformin. The increase in G2/M cells, decreased in S cells and increase in apoptotic cells by metformin treatment was statistically significant. The increase in apoptotic cell population in the Heat + Met group as compared with that in Met group was statistically significant.
Figure 6.
Percent of CSCs in MCF-7 human breast cancer cells treated with hyperthermia and metformin.
(A) Cells were incubated with 0–5 mM metformin for 48 h at 37°C, dispersed to single cells and analyzed for CD44high/CD24low cells (CSCs) with FACS. The effect of heating alone was studied by heating the cells at 42°C for 1 h followed by 47 h incubation at 37°C. The combined effect of heating and metformin was studied by heating the cells for 1 h at 42°C with metformin and then incubating at 37°C for 47 h. (B) Experiments described in A were repeated four times and the average of % of CSCs was obtained. Averages of 5 experiments ±1 S.E. are shown. The decreases in CSCs by 1 mM and 5 mM alone were statistically significant. The decrease in CSCs by heating at 42°C for 1 h was also statistically significant. The combinations of heating and metformin were statistically more effective than metformin alone. (C) MIA PaCa-2 cells were incubated with 0–10 mM metformin for 48 h at 37°C, dispersed to single cells and analyzed for CD44high/CD24high cells (CSCs) with FACS. The effect of heating alone was studied by heating the cells at 42.5°C for 1 h followed by 47 h incubation at 37°C. The combined effect of heating and metformin was studied by heating the cells for 1 h at 42.5°C with metformin and then incubating at 37°C for 47 h. (D, E) MCF-7 cells were plated in ultralow attachment plate (1,000 cells/plate) in sphere media. Metformin was added to the media (0.5–5 mM), then heating at 42°C for 1 h. Thereafter, the cells were incubated for 8 days under the standard culture conditions. The numbers of spheres with diameter >50 µm were counted under a microscope. The combinations of heating and metformin were statistically more effective than metformin alone. The Fig 6A 37°C panel was previously published in https://doi.org/10.1038/srep00362 and is excluded from this article's CC BY 4.0 license.