Combined 2-deoxy glucose and metformin improves therapeutic efficacy of sodium-iodide symporter-mediated targeted radioiodine therapy in breast cancer cells

Figures & data

Figure 1 Effect of 2-DG, metformin, and their combination with Cobalt60 radiation on MCF-7 cells.

Notes: (A and B) MCF-7 cells were treated with different dose of 2-DG or metformin with or without radiation exposure for 24 hours, and clonogenic assay was performed. (C) Combination of 20 mM 2-DG and 2 mM metformin with 2 Gy radiation dose showing further decrease in survival fraction. *P<0.05; **P<0.01. (D) Immunofluorescence analysis of γH2A.X foci showing increased expression in combination therapy as compared with control. Scale bars represent 10 μm. (E) Metformin, but not 2-DG, treatment shows significant decrease in ATP content of the cells. Error bars indicate SEM. **P<0.01.
Abbreviations: 2-DG, 2-deoxy-d-glucose; ATP, adenosine triphosphate; Gy, grey; Met F, metformin; SEM, standard error of mean.

Figure 2 Cell cycle status at different time points of treatment of MCF-7 cell.

Notes: Data showing elongation of S phase in combination treatment at 24 and 48 hours. However, when treated with radiation alone, S phase elongation is relieved after 12 hours.
Abbreviations: 2-DG, 2-deoxy-d-glucose; Gy, grey; Met F, metformin.

Figure 3 Characterization of MCF-7 cells stably expressing NIS.

Notes: (A) Diagrammatic representation of AttB-CAG-NIS DNA vector used for establishing NIS expressing cell lines. (B) Western blot analysis data showing major band of NIS at 97 kDa for membrane NIS expressing clone indicating dominance of completely glycosylated form of NIS. However, in the clone where NIS is localized in cytoplasm, the band intensity of glycosylated form of NIS (97 kDa band) is less. (C) NIS expression in MCF-7 control cells showing faint staining, and strong cytoplasmic or membrane expression in NIS overexpressing cells. Scale bar represents 20 μm. (D) Chart showing significantly high ¹²⁵I uptake in MCF-7 NIS cells than in cytoplasmic NIS expressing or parental cells. Blocking by 30 μM KClO₄ shows complete inhibition of iodine uptake. **P<0.01. (E) Efflux assay showing maximum efflux of iodine within 15 minutes. Error bars indicate SEM.
Abbreviations: AttB, attachment site of bacteria; CAG, synthetic promoter made by fusion of various elements; Cyto, cytoplasmic; DAPI, 4′,6-diamidino-2-phenylindole; NIS, sodium-iodide symporter; SEM, standard error of mean.

Figure 4 Cytotoxic effect of radioiodine therapy in MCF-7 cells stably expressing NIS.

Notes: (A) Clonogenic assay showing significant decrease in colony formation post ¹³¹I treatment. Error bars indicate SEM. **P<0.01. (B) Apoptosis assay showing significant loss in live cell population. (C) Immunofluorescence staining for γH2A.X foci showing increased number of foci formation in ¹³¹I treated cells than in untreated controls.
Abbreviations: NIS, sodium-iodide symporter; SEM, standard error of mean.

Figure 5 Effect of 2-DG, metformin, and their combination with ¹³¹I treatment on different prospects of MCF-7 NIS cells.

Notes: (A) MCF-7 NIS cell population were treated with 20 mM 2-DG and 4 mM metformin with or without ¹³¹I (50 μCi) for 24 hours, and clonogenic assay was performed. Graph showing significant decrease in survival fraction in case of combination therapy as compared with control or only ¹³¹I treated cells. **P<0.01; ***P<0.001. (B) Immunofluorescence analysis of γH2A.X foci showing increased expression in combination therapy than various treatment controls as marked. Scale bars represent 10 μm. (C) Apoptotic assay showing significant decrease in live cell population in combination therapy than other treatment controls. (D) Graph showing radiosensitization effect of the combination therapy on Cobalt60 or ¹³¹I. Radiosensitization effect by these drugs is more pronounced on radioiodine therapy than Cobalt60 radiation exposure. *P<0.05.
Abbreviations: 2-DG, 2-deoxy-d-glucose; Met F, metformin; NIS, sodium-iodide symporter; ns, not significant.

Figure 6 Effect of 2-DG, metformin, and their combination with ¹³¹I treatment on survival of plain MCF-7 cells.

(A) MCF-7 cells were treated with 20 mM 2-DG and 4 mM metformin with or without ¹³¹I (50 µCi) for 24 hours, and clonogenic assay was performed. Treatment with ¹³¹I alone shows only 30% decrease in cell survival; however, its combination with 2-DG and metformin results in significant loss in live cells (30%, PI =0.0110, ¹³¹I vs¹³¹I+2-DG + Met F). *P<0.05. (B) Immunofluorescence analysis of γH2A.X foci showing increased expression in combination therapy than various treatment controls as marked. Scale bars represent 10 µm. It is to be noted that the intensity and frequency of foci is lesser in MCF-7 plain cells when compared with MCF-7 NIS clonal population.
Abbreviations: 2-DG, 2-deoxy-d-glucose; Met F, metformin; NIS, sodium-iodide symporter; PI, propidium iodide.

Figure S1 Cytotoxic effect of 2-DG and metformin in (A) MCF-7 and (B) MDA MB 231 breast cancer cell lines.

Abbreviations: 2-DG, 2-deoxy-d-glucose; Met F, metformin.

Figure S2 Effect of 2-DG, metformin, and their combination with Cobalt60 radiation on MDA MB 231 cells.

Notes: (A and B) MDA MB231 cells were treated with different dose of 2-DG or metformin, with or without radiation exposure for 24 hours, and clonogenic assay was performed. (C) Combination of 5 mM 2-DG and 4 mM metformin with 2 Gy radiation dose showing further decrease in survival fraction. **P<0.01.
Abbreviations: 2-DG, 2-deoxy-d-glucose; Ctrl, control; Gy, grey; Met F, metformin; ns, not significant.

Figure S3 Effect of Cobalt60 radiation and it’s combination with 2-DG and Met F on foci formation in MCF-7 cells.

Notes: (A) Foci assay images from cells treated with Cobalt60 radiation and combination with radio sensitizers showing foci staining with dylight 633 secondary antibody in red channel, nucleus stained with DAPI in blue channel, and merged image showing colocalization of foci with nucleus. Scale bars represent 10 μm. (B) Quantification of foci from the IF images. In each sample, 50 cells were quantified for intensity of staining from various fields of the cover slip.
Abbreviations: 2-DG, 2-Deoxy-d-glucose; DAPI, 4′,6-diamidino-2-phenylindole; Gy, grey; IF, immunofluorescence; Met F, metformin; MFI, mean fluorescence intensity.

Figure S4 (A) Foci assay images from Figure 4 (MCF-7 NIS cells treated with ¹³¹I and its combination with radio sensitizers) showing foci staining with dylight 633 secondary antibody in red channel, nucleus stained with DAPI in blue channel, and merged image showing colocalization of foci with nucleus. Scale bars represent 10 μm. (B) Quantification of foci from the IF images. In each sample, 50 cells were quantified for intensity of staining from various fields of the cover slip.

Abbreviations: 2-DG, 2-Deoxy-d-glucose; DAPI, 4′,6-diamidino-2-phenylindole; IF, immunofluorescence; MET, metformin; NIS, sodium-iodide symporter; MFI, mean fluorescence intensity.

Figure S5 (A) Foci assay images from Figure 5 (MCF-7 plain cells treated with ¹³¹I and its combination with radio sensitizers) showing foci staining with dylight 633 secondary antibody in red channel, nucleus stained with DAPI in blue channel, and merged image showing colocalization of foci with nucleus. Scale bars represent 10 μm. (B) Quantification of foci from the IF images. In each sample, 50 cells were quantified for intensity of staining from various fields of the cover slip.

Abbreviations: 2-DG, 2-deoxy-D-glucose; DAPI, 4′,6-diamidino-2-phenylindole; IF, immunofluorescence; MET, metformin; MFI, mean fluorescence intensity.