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Metformin inhibits human breast cancer cell growth by promoting apoptosis via a ROS-independent pathway involving mitochondrial dysfunction: pivotal role of superoxide dismutase (SOD)

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Abstract

Purpose

Despite a growing body of evidence indicating a potential efficacy of the anti-diabetic metformin as anti-cancer agent, the exact mechanism underlying this efficacy has remained largely unknown. Here, we aimed at assessing putative mechanisms associated with the ability of metformin to reduce the proliferation and migration of breast cancer cells.

Methods

A battery of in vitro assays including MTT, colony formation, NBT and scratch wound healing assays were performed to assess the viability, proliferation, anti-oxidative potential and migration of breast cancer-derived MCF-7, MDA-MB-231 and T47D cells, respectively. Reactive oxygen species (ROS) assays along with fluorescence microscopy were used to assess apoptotic parameters. Quantification of SOD, Bcl-2, Bax, MMPs, miR-21 and miR-155 expression was performed using qRT-PCR.

Results

We found that metformin inhibited the growth, proliferation and clonogenic potential of the breast cancer-derived cells tested. ROS levels were found to be significantly reduced by metformin and, concomitantly, superoxide dismutase (SOD) isoforms were found to be upregulated. Mitochondrial dysfunction was observed in metformin treated cells, indicating apoptosis. In metastatic MDA-MB-231 cells, migration was found to be suppressed by metformin through deregulation of the matrix metalloproteinases MMP-2 and MMP-9. The oncogenic microRNAs miR-21 and miR-155 were found to be downregulated by metformin, which may be correlated with the suppression of cell proliferation and/or migration.

Conclusions

Our data indicate that metformin may play a pivotal role in modulating the anti-oxidant system, including the SOD machinery, in breast cancer-derived cells. Our observations were validated by in silico analyses, indicating a close interaction between SOD and metformin. We also found that metformin may inhibit breast cancer-derived cell proliferation through apoptosis induction via the mitochondrial pathway. Finally, we found that metformin may modulate the pro-apoptotic Bax, anti-apoptotic Bcl-2, MMP-2, MMP-9, miR-21 and miR-155 expression levels. These findings may be instrumental for the clinical management and/or (targeted) treatment of breast cancer.

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Fig. 1: Effect of metformin on viability and proliferation/colony formation of breast cancer cells
Fig. 2: Effect of metformin on ROS generation in breast cancer cells
Fig. 3: Effect of metformin on SOD isoform expression and SOD activity in breast cancer cells
Fig. 4: Metformin treatment enhances apoptosis in breast cancer cells
Fig. 5: Effect of metformin on mitochondrial membrane potential (ΔΨm) in breast cancer cells
Fig. 6: Effect of metformin on migration and MMP-2/MMP-9 expression in breast cancer cells
Fig. 7: Effect of metformin on miR-21 and miR-155 expression in breast cancer cells
Fig. 8: Interaction of metformin with SOD 1 and SOD 2

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Acknowledgements

The authors express their sincere thanks to Ranbaxy Research Laboratories Ltd. (Mumbai, Maharashtra, India) for providing metformin as a gift sample. This research was funded by the Central University of Punjab, Bathinda as part of Ph.D Thesis. The authors remain indebted to journal editorial board for improving the quality of  the manuscript. The authors would like to kindly acknowledge their efforts. We gratefully acknowledge Dr. Alpana Saini, Head, Department of classical and modern languages, Central University of Punjab, Bathinda, India, and Dr. Raju Gautam for their help and support in editing and polishing the language of the manuscript.  

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  1. Centre for Biosciences, School of Basic and Applied Science, Central University of Punjab, Bathinda, Punjab, 151001, India

    Prateek Sharma & Sanjeev Kumar

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Supplementary Fig. 1

Effect of metformin on BC cells viability. MCF-7, MDA-MB-231 and T47D cells were exposed to the culture medium (control) and metformin for 48 hours. Cell viability was estimated after staining with 0.4% (w/v) trypan blue dye. All data are represented as means ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 versus control. (PNG 804 kb)

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Supplementary Fig. 2

Effect of SOD inhibitor Silver diethyl thiocarbamate on the ROS levels, SOD expression levels, and growth of BC cells. (A) Representative pictures in panel display alterations in DCFH-DA fluorescence intensity after SOD inhibitor treatment. (B) DCFH-DA fluorescence intensities were quantified spectrophotometrically using microplate reader. (C) Silver diethyl thiocarbamate reduces SOD mRNA expression levels in cells. (D) Silver diethyl thiocarbamate reduces proliferation of BC cells. All data are represented as means ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 versus control. (PNG 18884 kb)

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Supplementary Fig. 3

Quantification of apoptotic nuclei in MCF-7, MDA-MB-231 and T47D BC cells. The apoptotic nuclei were quantified by fluorescence microscopic analysis. All data are represented as means ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 versus control. (PNG 355 kb)

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Sharma, P., Kumar, S. Metformin inhibits human breast cancer cell growth by promoting apoptosis via a ROS-independent pathway involving mitochondrial dysfunction: pivotal role of superoxide dismutase (SOD). Cell Oncol. 41, 637–650 (2018). https://doi.org/10.1007/s13402-018-0398-0

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