Cell Biochemistry and Biophysics

, Volume 75, Issue 3–4, pp 311–317 | Cite as

Modified Metformin as a More Potent Anticancer Drug: Mitochondrial Inhibition, Redox Signaling, Antiproliferative Effects and Future EPR Studies

  • Balaraman Kalyanaraman
  • Gang Cheng
  • Micael Hardy
  • Olivier Ouari
  • Adam Sikora
  • Jacek Zielonka
  • Michael B. Dwinell
Original Paper


Metformin, one of the most widely prescribed antidiabetic drugs in the world, is being repurposed as a potential drug in cancer treatment. Epidemiological studies suggest that metformin exerts anticancer effects in diabetic patients with pancreatic cancer. However, at typical antidiabetic doses the bioavailability of metformin is presumably too low to exert antitumor effects. Thus, more potent analogs of metformin are needed in order to increase its anticancer efficacy. To this end, a new class of mitochondria-targeted metformin analogs (or mito-metformins) containing a positively-charged lipophilic triphenylphosphonium group was synthesized and tested for their antitumor efficacy in pancreatic cancer cells. Results indicate that the lead compound, mito-metformin10, was nearly 1000-fold more potent than metformin in inhibiting mitochondrial complex I activity, inducing reactive oxygen species (superoxide and hydrogen peroxide) that stimulate redox signaling mechanisms, including the activation of adenosinemonophosphate kinase and inhibition of proliferation of pancreatic cancer cells. The potential use of the low-temperature electron paramagnetic resonance technique in assessing the role of mitochondrial complexes including complex I in tumor regression in response to metformin and mito-metformins in the in vivo setting is discussed.


Modified metformin Reactive oxygen species Redox signaling Mitochondrial metabolism Pancreatic cancer 



This work was supported by a grant from NIH (U01 CA178960) to M.D. and B.K. A.S. was supported by a grant from Polish National Science Centre No. 2015/18/E/ST4/00235.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests.


  1. 1.
    Bailey, C., & Day, C. (2004). Metformin: Its botanical background. Practical Diabetes International, 21, 115–117.CrossRefGoogle Scholar
  2. 2.
    Thomas, I., & Gregg, B. (2017). Metformin; A review of its history and future: From lilac to longevity. Pediatric Diabetes, 18, 10–16.CrossRefPubMedGoogle Scholar
  3. 3.
    Evans, J. M., Donnelly, L. A., Emslie-Smith, A. M., Alessi, D. R., & Morris, A. D. (2005). Metformin and reduced risk of cancer in diabetic patients. BMJ (Clinical Research Ed.), 330, 1304–1305.CrossRefGoogle Scholar
  4. 4.
    Heckman-Stoddard, B. M., Gandini, S., Puntoni, M., Dunn, B. K., Decensi, A., & Szabo, E. (2016). Repurposing old drugs to chemoprevention: The case of metformin. Seminars in Oncology, 43, 123–133.CrossRefPubMedGoogle Scholar
  5. 5.
    Kordes, S., Pollak, M. N., Zwinderman, A. H., Mathot, R. A., Weterman, M. J., Beeker, A., Punt, C. J., Richel, D. J., & Wilmink, J. W. (2015). Metformin in patients with advanced pancreatic cancer: A double-blind, randomised, placebo-controlled phase 2 trial. The Lancet Oncology, 16, 839–847.CrossRefPubMedGoogle Scholar
  6. 6.
    Chandel, N. S., Avizonis, D., Reczek, C. R., Weinberg, S. E., Menz, S., Neuhaus, R., Christian, S., Haegebarth, A., Algire, C., & Pollak, M. (2016). Are metformin doses used in murine cancer models clinically relevant? Cell Metabolism, 23, 569–570.CrossRefPubMedGoogle Scholar
  7. 7.
    Cheng, G., Zielonka, J., Ouari, O., Lopez, M., McAllister, D., Boyle, K., Barrios, C. S., Weber, J. J., Johnson, B. D., Hardy, M., Dwinell, M. B., & Kalyanaraman, B. (2016). Mitochondria-targeted analogues of metformin exhibit enhanced antiproliferative and radiosensitizing effects in pancreatic cancer cells. Cancer Research, 76, 3904–3915.CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Graham, G. G., Punt, J., Arora, M., Day, R. O., Doogue, M. P., Duong, J. K., Furlong, T. J., Greenfield, J. R., Greenup, L. C., Kirkpatrick, C. M., Ray, J. E., Timmins, P., & Williams, K. M. (2011). Clinical pharmacokinetics of metformin. Clinical Pharmacokinetics, 50, 81–98.CrossRefPubMedGoogle Scholar
  9. 9.
    Foretz, M., Guigas, B., Bertrand, L., Pollak, M., & Viollet, B. (2014). Metformin: From mechanisms of action to therapies. Cell Metabolism, 20, 953–966.CrossRefPubMedGoogle Scholar
  10. 10.
    Bridges, H., Jones, A., Pollak, M., & Hirst, J. (2014). Effects of metformin and other biguanides on oxidative phosphorylation in mitochondria. The Biochemical Journal, 462, 475–487.CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Liu, X., Romero, I. L., Litchfield, L. M., Lengyel, E., & Locasale, J. W. (2016). Metformin targets central carbon metabolism and reveals mitochondrial requirements in human cancers. Cell Metabolism, 24, 728–739.CrossRefPubMedGoogle Scholar
  12. 12.
    Wheaton, W. W., Weinberg, S. E., Hamanaka, R. B., Soberanes, S., Sullivan, L. B., Anso, E., Glasauer, A., Dufour, E., Mutlu, G. M., Budigner, G. S., & Chandel, N. S. (2014). Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis. eLife, 3, e02242.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Birsoy, K., Possemato, R., Lorbeer, F. K., Bayraktar, E. C., Thiru, P., Yucel, B., Wang, T., Chen, W. W., Clish, C. B., & Sabatini, D. M. (2014). Metabolic determinants of cancer cell sensitivity to glucose limitation and biguanides. Nature, 508, 108–112.CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Pollak, M. (2014). Overcoming drug development bottlenecks with repurposing: Repurposing biguanides to target energy metabolism for cancer treatment. Nature Medicine, 20, 591–593.CrossRefPubMedGoogle Scholar
  15. 15.
    Eikawa, S., Nishida, M., Mizukami, S., Yamazaki, C., Nakayama, E., & Udono, H. (2015). Immune-mediated antitumor effect by type 2 diabetes drug, metformin. Proceedings of the National Academy of Sciences, 112, 1809–1814.CrossRefGoogle Scholar
  16. 16.
    Webb, T. J., Carey, G. B., East, J. E., Sun, W., Bollino, D. R., Kimball, A. S., Brutkiewicz, R. R., & Flajnik, M. (2016). Alterations in cellular metabolism modulate CD1d-mediated NKT-cell responses. Pathogens and Disease, 74, ftw055–ftw055.CrossRefPubMedGoogle Scholar
  17. 17.
    Delmastro-Greenwood, M. M., & Piganelli, J. D. (2013). Changing the energy of an immune response. American Journal of Clinical and Experimental Immunology, 2, 30–54.PubMedPubMedCentralGoogle Scholar
  18. 18.
    Cheng, G., Zielonka, J., McAllister, D., Hardy, M., Ouari, O., Joseph, J., Dwinell, M. B., & Kalyanaraman, B. (2015). Antiproliferative effects of mitochondria-targeted cationic antioxidants and analogs: Role of mitochondrial bioenergetics and energy-sensing mechanism. Cancer Letters, 365, 96–106.CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Cheng, G., Zielonka, J., Dranka, B. P., McAllister, D., Mackinnon, Jr., A. C., Joseph, J., & Kalyanaraman, B. (2012). Mitochondria-targeted drugs synergize with 2-deoxyglucose to trigger breast cancer cell death. Cancer Research, 72, 2634–2644.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Cheng, G., Zielonka, J., McAllister, D., Tsai, S., Dwinell, M. B., & Kalyanaraman, B. (2014). Profiling and targeting of cellular bioenergetics: Inhibition of pancreatic cancer cell proliferation. British Journal of Cancer, 111, 85–93.CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Cheng, G., Zielonka, J., McAllister, D. M., Mackinnon, Jr., A. C., Joseph, J., Dwinell, M. B., & Kalyanaraman, B. (2013). Mitochondria-targeted vitamin E analogs inhibit breast cancer cell energy metabolism and promote cell death. BMC Cancer, 13, 285.CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Dickey, J. S., Gonzalez, Y., Aryal, B., Mog, S., Nakamura, A. J., Redon, C. E., Baxa, U., Rosen, E., Cheng, G., Zielonka, J., Parekh, P., Mason, K. P., Joseph, J., Kalyanaraman, B., Bonner, W., Herman, E., Shacter, E., & Rao, V. A. (2013). Mito-tempol and dexrazoxane exhibit cardioprotective and chemotherapeutic effects through specific protein oxidation and autophagy in a syngeneic breast tumor preclinical model. PLoS ONE, 8, e70575.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Rao, V. A., Klein, S. R., Bonar, S. J., Zielonka, J., Mizuno, N., Dickey, J. S., Keller, P. W., Joseph, J., Kalyanaraman, B., & Shacter, E. (2010). The antioxidant transcription factor Nrf2 negatively regulates autophagy and growth arrest induced by the anticancer redox agent mitoquinone. The Journal of Biological Chemistry, 285, 34447–34459.CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Zhao, H., Joseph, J., Fales, H. M., Sokoloski, E. A., Levine, R. L., Vasquez-Vivar, J., & Kalyanaraman, B. (2005). Detection and characterization of the product of hydroethidine and intracellular superoxide by HPLC and limitations of fluorescence. Proceedings of the National Academy of Sciences of the United States of America, 102, 5727–5732.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Zielonka, J., & Kalyanaraman, B. (2010). Hydroethidine- and MitoSOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: Another inconvenient truth. Free Radical Biology & Medicine, 48, 983–1001.CrossRefGoogle Scholar
  26. 26.
    Zielonka, J., Vasquez-Vivar, J., & Kalyanaraman, B. (2008). Detection of 2-hydroxyethidium in cellular systems: A unique marker product of superoxide and hydroethidine. Nature Protocols, 3, 8–21.CrossRefPubMedGoogle Scholar
  27. 27.
    Zielonka, J., Hardy, M., & Kalyanaraman, B. (2009). HPLC study of oxidation products of hydroethidine in chemical and biological systems: Ramifications in superoxide measurements. Free Radical Biology and Medicine, 46, 329–338.CrossRefPubMedGoogle Scholar
  28. 28.
    Zielonka, J., Srinivasan, S., Hardy, M., Ouari, O., Lopez, M., Vasquez-Vivar, J., Avadhani, N. G., & Kalyanaraman, B. (2008). Cytochrome c-mediated oxidation of hydroethidine and mito-hydroethidine in mitochondria: Identification of homo- and heterodimers. Free Radical Biology and Medicine, 44, 835–846.CrossRefPubMedGoogle Scholar
  29. 29.
    Sikora, A., Zielonka, J., Adamus, J., Debski, D., Dybala-Defratyka, A., Michalowski, B., Joseph, J., Hartley, R. C., Murphy, M. P., & Kalyanaraman, B. (2013). Reaction between peroxynitrite and triphenylphosphonium-substituted arylboronic acid isomers: Identification of diagnostic marker products and biological implications. Chemical Research in Toxicology, 26, 856–867.CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Zielonka, J., Sikora, A., Adamus, J., & Kalyanaraman, B. (2015). Detection and differentiation between peroxynitrite and hydroperoxides using mitochondria-targeted arylboronic acid. Methods in Molecular Biology, 1264, 171–181.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Zielonka, J., Zielonka, M., VerPlank, L., Cheng, G., Hardy, M., Ouari, O., Ayhan, M. M., Podsiadly, R., Sikora, A., Lambeth, J. D., & Kalyanaraman, B. (2016). Mitigation of NADPH oxidase 2 activity as a strategy to inhibit peroxynitrite formation. The Journal of Biological chemistry, 291, 7029–7044.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Hardie, D. G., & Ashford, M. L. (2014). AMPK: Regulating energy balance at the cellular and whole body levels. Physiology (Bethesda, Md.), 29, 99–107.Google Scholar
  33. 33.
    Mackenzie, R. M., Salt, I. P., Miller, W. H., Logan, A., Ibrahim, H. A., Degasperi, A., Dymott, J. A., Hamilton, C. A., Murphy, M. P., Delles, C., & Dominiczak, A. F. (2013). Mitochondrial reactive oxygen species enhance AMP-activated protein kinase activation in the endothelium of patients with coronary artery disease and diabetes. Clinical Science 124, 403–411.Google Scholar
  34. 34.
    Quintero, M., Colombo, S. L., Godfrey, A., & Moncada, S. (2006). Mitochondria as signaling organelles in the vascular endothelium. Proceedings of the National Academy of Sciences of the United States of America, 103, 5379–5384.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Boukalova, S., Stursa, J., Werner, L., Ezrova, Z., Cerny, J., Bezawork-Geleta, A., Pecinova, A., Dong, L., Drahota, Z., & Neuzil, J. (2016). Mitochondrial targeting of metformin enhances its activity against pancreatic cancer. Molecular Cancer Therapeutics, 15, 2875–2886.CrossRefPubMedGoogle Scholar
  36. 36.
    Orme-Johnson, N. R., Hansen, R. E., & Beinert, H. (1974). Electron paramagnetic resonance-detectable electron acceptors in beef heart mitochondria. Reduced diphosphopyridine nucleotide ubiquinone reductase segment of the electron transfer system. The Journal of Biological Chemistry, 249, 1922–1927.PubMedGoogle Scholar
  37. 37.
    Chandran, K., Aggarwal, D., Migrino, R. Q., Joseph, J., McAllister, D., Konorev, E. A., Antholine, W. E., Zielonka, J., Srinivasan, S., Avadhani, N. G., & Kalyanaraman, B. (2009). Doxorubicin inactivates myocardial cytochrome c oxidase in rats: Cardioprotection by Mito-Q. Biophysical Journal, 96, 1388–1398.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Bennett, B., Helbling, D., Meng, H., Jarzembowski, J., Geurts, A. M., Friederich, M. W., Van Hove, J. L. K., Lawlor, M. W., & Dimmock, D. P. (2016). Potentially diagnostic electron paramagnetic resonance spectra elucidate the underlying mechanism of mitochondrial dysfunction in the deoxyguanosine kinase deficient rat model of a genetic mitochondrial DNA depletion syndrome. Free Radical Biology and Medicine, 92, 141–151.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Ghosh, A., Chandran, K., Kalivendi, S. V., Joseph, J., Antholine, W. E., Hillard, C. J., Kanthasamy, A., Kanthasamy, A., & Kalyanaraman, B. (2010). Neuroprotection by a mitochondria-targeted drug in a Parkinson’s disease model. Free Radical Biology & Medicine, 49, 1674–1684.CrossRefGoogle Scholar
  40. 40.
    Sobotta, M. C., Liou, W., Stöcker, S., Talwar, D., Oehler, M., Ruppert, T., Scharf, A. N. D., & Dick, T. P. (2015). Peroxiredoxin-2 and STAT3 form a redox relay for H2O2 signaling. Nature Chemical Biology, 11, 64–70.CrossRefPubMedGoogle Scholar
  41. 41.
    Babot, M., Birch, A., Labarbuta, P., & Galkin, A. (2014). Characterisation of the active/de-active transition of mitochondrial complex I. Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1837, 1083–1092.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Balaraman Kalyanaraman
    • 1
  • Gang Cheng
    • 1
  • Micael Hardy
    • 2
  • Olivier Ouari
    • 2
  • Adam Sikora
    • 3
  • Jacek Zielonka
    • 1
  • Michael B. Dwinell
    • 4
  1. 1.Department of Biophysics and Free Radical Research CenterMedical College of WisconsinMilwaukeeUSA
  2. 2.CNRS, Institut de Chimie Radicalaire (ICR)Aix-Marseille UnivMarseilleFrance
  3. 3.Institute of Applied Radiation Chemistry Faculty of ChemistryLodz University of Technology, Zeromskiego 116LodzPoland
  4. 4.Department of Microbiology and Molecular GeneticsMedical College of WisconsinMilwaukeeUSA

Personalised recommendations