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

Abstract

Radiosensitization using either metformin or 2-deoxy-d-glucose (2-DG) in various cancer cells has been reported. The present study reveals novel information on combining these drugs to enhance radiosensitization effect in breast cancer (BC) cells. Responses to low-dose Cobalt60 radiation, as well as a newly emerged radioiodine therapy target for BC, that is, sodium-iodide symporter (NIS or SLC5A5) protein, are tested. As therapeutic potential of NIS in BC is often limited due to low uptake and fast efflux rate of iodine, the scope of these two radiosensitizers to further improve NIS-mediated ¹³¹I therapeutic efficacy is explored. Two BC cell lines, MCF-7, and MDA MB231 are tested to optimize minimal drug doses required for radiosensitization. A combination of 2 mM metformin and 20 mM 2-DG with 2 grey (Gy) Cobalt60 radiation shows significant radiosensitization effect (P=0.0002). In cells treated with the combination therapy, increased γH2A.X foci formation was noted. Further, MCF-7 BC cells overexpressing NIS (MCF-7 NIS) was established, and using the optimized drug concentrations, significant radiosensitization (P=0.0019) by 50 μ Ci ¹³¹I usage was found to be the case as well. Apoptosis data corroborates with the result of clonogenic assay showing significant increase in apoptotic population upon dual drug-mediated radiosensitization. In case of metformin treatment, lowered adenosine triphosphate (ATP) content of the cell has been observed. The encouraging radiosensitization effect observed using combined 2-DG and metformin may aid in reducing Cobalt60 radiation exposure or for targeted radioiodine therapy in BC cells with NIS expression. This study indicates high potential of this drug combination in sensitizing BC cells for NIS-mediated-targeted radioiodine therapy, which otherwise may have lacked efficacy.

Keywords:

• sodium-iodide symporter
• 2-DG
• ¹³¹I
• radiosensitizers

Supplementary materials

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.

Acknowledgments

Radioactive iodine-related experiments were carried out in the Radiation Medicine Centre, TATA Memorial Hospital campus, Mumbai, India. Research funding from the Indian Council for Medical Research, New Delhi, to AD (reference number 5/13/25/10/NCD-III); and personal support from the Advanced Centre for Treatment, Research and Education in Cancer, Tata Memorial Centre, to SC for graduate study, is also acknowledged.

Disclosure

The authors report no conflicts of interest in this work.