Advertisement

Breast Cancer Research and Treatment

, Volume 147, Issue 3, pp 539–550 | Cite as

Dichloroacetate enhances apoptotic cell death via oxidative damage and attenuates lactate production in metformin-treated breast cancer cells

  • Allison B. HaugrudEmail author
  • Yongxian Zhuang
  • Joseph D. Coppock
  • W. Keith Miskimins
Preclinical study

Abstract

The unique metabolism of breast cancer cells provides interest in exploiting this phenomenon therapeutically. Metformin, a promising breast cancer therapeutic, targets complex I of the electron transport chain leading to an accumulation of reactive oxygen species (ROS) that eventually lead to cell death. Inhibition of complex I leads to lactate production, a metabolic byproduct already highly produced by reprogrammed cancer cells and associated with a poor prognosis. While metformin remains a promising cancer therapeutic, we sought a complementary agent to increase apoptotic promoting effects of metformin while attenuating lactate production possibly leading to greatly improved efficacy. Dichloroacetate (DCA) is a well-established drug used in the treatment of lactic acidosis which functions through inhibition of pyruvate dehydrogenase kinase (PDK) promoting mitochondrial metabolism. Our purpose was to examine the synergy and mechanisms by which these two drugs kill breast cancer cells. Cell lines were subjected to the indicated treatments and analyzed for cell death and various aspects of metabolism. Cell death and ROS production were analyzed using flow cytometry, Western blot analysis, and cell counting methods. Images of cells were taken with phase contrast microscopy or confocal microscopy. Metabolism of cells was analyzed using the Seahorse XF24 analyzer, lactate assays, and pH analysis. We show that when DCA and metformin are used in combination, synergistic induction of apoptosis of breast cancer cells occurs. Metformin-induced oxidative damage is enhanced by DCA through PDK1 inhibition which also diminishes metformin promoted lactate production. We demonstrate that DCA and metformin combine to synergistically induce caspase-dependent apoptosis involving oxidative damage with simultaneous attenuation of metformin promoted lactate production. Innovative combinations such as metformin and DCA show promise in expanding breast cancer therapies.

Keywords

Metformin Dichloroacetate Breast cancer Lactate Apoptosis 

Notes

Acknowledgments

This article was supported by grants from Susan G. Komen for the Cure (KG100497) and the National Cancer Institute of the National Institutes of Health (1R01CA180033). Flow Cytometry and Imaging Cores were supported by COBRE Grants P20GM103548 and P20GM103620 from the National Institute of General Medical Sciences at the National Institutes of Health.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical standards

The authors declare that the experiments comply with the current laws of the country in which they were performed.

References

  1. 1.
    Warburg O (1956) On the origin of cancer cells. Science 123(80):309–314. doi: 10.1016/S0306-9877(96)90136-X
  2. 2.
    DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB (2008) The biology of cancer: metabolic reprogramming fuels cell growth and proliferation. Cell Metab 7:11–20. doi: 10.1016/j.cmet.2007.10.002 PubMedCrossRefGoogle Scholar
  3. 3.
    Zamzami N, Kroemer G (2001) The mitochondrion in apoptosis : how Pandora’ s box opens. Nat Rev Mol Cell Biol 2:67–71. doi: 10.1038/35048073
  4. 4.
    Gottfried E, Kunz-Schughart LA, Ebner S et al (2006) Tumor-derived lactic acid modulates dendritic cell activation and antigen expression. Blood 107:2013–2021. doi: 10.1182/blood-2005-05-1795 PubMedCrossRefGoogle Scholar
  5. 5.
    Fischer K, Hoffmann P, Voelkl S et al (2007) Inhibitory effect of tumor cell-derived lactic acid on human T cells. Blood 109:3812–3819. doi: 10.1182/blood-2006-07-035972 PubMedCrossRefGoogle Scholar
  6. 6.
    Jiralerspong S, Palla SL, Giordano SH et al (2009) Metformin and pathologic complete responses to neoadjuvant chemotherapy in diabetic patients with breast cancer. J Clin Oncol 27:3297–3302. doi: 10.1200/JCO.2009.19.6410 PubMedCrossRefPubMedCentralGoogle Scholar
  7. 7.
    Evans JMM, Donnelly LA, Emslie-Smith AM et al (2005) Metformin and reduced risk of cancer in diabetic patients. BMJ 330:1303–1304. doi: 10.1136/bmj.38393.572188.EB PubMedCrossRefPubMedCentralGoogle Scholar
  8. 8.
    Libby G, Donnelly LA, Donnan PT et al (2009) New users of Metformin are at low risk of incident cancer. Diabetes Care 32:1620–1625. doi: 10.2337/dc08-2175 PubMedCrossRefPubMedCentralGoogle Scholar
  9. 9.
    Zakikhani M, Dowling R, Fantus IG et al (2006) Metformin is an AMP kinase-dependent growth inhibitor for breast cancer cells. Cancer Res 66:10269–10273. doi: 10.1158/0008-5472.CAN-06-1500 PubMedCrossRefGoogle Scholar
  10. 10.
    Isakovic A, Harhaji L, Stevanovic D et al (2007) Dual antiglioma action of metformin: cell cycle arrest and mitochondria-dependent apoptosis. Cell Mol Life Sci 64:1290–1302. doi: 10.1007/s00018-007-7080-4 PubMedCrossRefGoogle Scholar
  11. 11.
    Hadad SM, Appleyard V, Thompson AM (2008) Therapeutic metformin/AMPK activation promotes the angiogenic phenotype in the ERα negative MDA-MB-435 breast cancer model. Breast Cancer Res Treat 114:391. doi: 10.1007/s10549-008-0016-3 PubMedCrossRefGoogle Scholar
  12. 12.
    Buzzai M, Jones RG, Amaravadi RK et al (2007) Systemic treatment with the antidiabetic drug metformin selectively impairs p53-deficient tumor cell growth. Cancer Res 67:6745–6752. doi: 10.1158/0008-5472.CAN-06-4447 PubMedCrossRefGoogle Scholar
  13. 13.
    Sarbassov DD, Ali SM, Sabatini DM (2005) Growing roles for the mTOR pathway. Curr Opin Cell Biol 17:596–603. doi: 10.1016/j.ceb.2005.09.009 PubMedCrossRefGoogle Scholar
  14. 14.
    Zhuang Y, Miskimins WK (2008) Cell cycle arrest in Metformin treated breast cancer cells involves activation of AMPK, downregulation of cyclin D1, and requires p27Kip1 or p21Cip1. J Mol Signal 3:1–11. doi: 10.1186/1750-2187-3-18 CrossRefGoogle Scholar
  15. 15.
    Hinke SA, Martens GA, Cai Y et al (2007) Methyl succinate antagonises biguanide-induced AMPK-activation and death of pancreatic beta-cells through restoration of mitochondrial electron transfer. Br J Pharmacol 150:1031–1043. doi: 10.1038/sj.bjp.0707189 PubMedCrossRefPubMedCentralGoogle Scholar
  16. 16.
    Zou M-H, Kirkpatrick SS, Davis BJ et al (2004) Activation of the AMP-activated protein kinase by the anti-diabetic drug metformin in vivo. Role of mitochondrial reactive nitrogen species. J Biol Chem 279:43940–43951. doi: 10.1074/jbc.M404421200 PubMedCrossRefGoogle Scholar
  17. 17.
    Owen MR, Doran E, Halestrap AP (2000) Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem J 614:607–614. doi: 10.1042/0264-6021:3480607
  18. 18.
    Carvalho C, Correia S, Santos MS et al (2008) Metformin promotes isolated rat liver mitochondria impairment. Mol Cell Biochem 308:75–83. doi: 10.1007/s11010-007-9614-3 PubMedCrossRefGoogle Scholar
  19. 19.
    Kefas BA, Cai Y, Kerckhofs K et al (2004) Metformin-induced stimulation of AMP-activated protein kinase in beta-cells impairs their glucose responsiveness and can lead to apoptosis. Biochem Pharmacol 68:409–416. doi: 10.1016/j.bcp.2004.04.003 PubMedCrossRefGoogle Scholar
  20. 20.
    Zhuang Y, Miskimins WK (2012) Metformin induces both caspase-dependent and poly(ADP-ribose) polymerase-dependent cell death in breast cancer cells. Mol Cancer Res 9:603–615. doi: 10.1158/1541-7786.MCR-10-0343.Metformin CrossRefGoogle Scholar
  21. 21.
    Bailey CJ, Wilcock C, Day C (1992) Effect of metformin on glucose metabolism in the splanchnic bed. Br J Pharmacol 105:1009–1013PubMedCrossRefPubMedCentralGoogle Scholar
  22. 22.
    Stacpoole PW, Lorenz AC, Thomas RG, Harman EM (1988) Dichloroacetate in the treatment of lactic acidosis. Ann Intern Med 108:58–63. doi: 10.1056/NEJM198308183090702 PubMedCrossRefGoogle Scholar
  23. 23.
    Whitehouse BSUE, Cooper RH, Randle PJ (1974) Mechanism of activation of pyruvate dehydrogenase by dichloroacetate and other halogenated carboxylic acids. Biochem J 141:761–774. doi: 10.1056/NEJM198308183090702
  24. 24.
    Hussien R, Brooks GA (2011) Mitochondrial and plasma membrane lactate transporter and lactate dehydrogenase isoform expression in breast cancer cell lines. Physiol Genomics 43:255–264. doi: 10.1152/physiolgenomics.00177.2010 PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Kim J, Tchernyshyov I, Semenza GL, Dang CV (2006) HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab 3:177–185. doi: 10.1016/j.cmet.2006.02.002 PubMedCrossRefGoogle Scholar
  26. 26.
    Wigfield SM, Winter SC, Giatromanolaki A et al (2008) PDK-1 regulates lactate production in hypoxia and is associated with poor prognosis in head and neck squamous cancer. Br J Cancer 98:1975–1984. doi: 10.1038/sj.bjc.6604356 PubMedCrossRefPubMedCentralGoogle Scholar
  27. 27.
    Stacpoole PW, Kurtz TL, Han Z, Langaee T (2008) Role of dichloroacetate in the treatment of genetic mitochondrial diseases. Adv Drug Deliv Rev 60:1478–1487. doi: 10.1016/j.addr.2008.02.014 PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Michelakis ED, Sutendra G, Dromparis P et al (2010) Metabolic modulation of glioblastoma with dichloroacetate. Sci Transl Med 2:31ra34. doi: 10.1126/scitranslmed.3000677 PubMedCrossRefGoogle Scholar
  29. 29.
    Mori M, Yamagata T, Goto T et al (2004) Dichloroacetate treatment for mitochondrial cytopathy: long-term effects in MELAS. Brain Develop 26:453–458. doi: 10.1016/j.braindev.2003.12.009 CrossRefGoogle Scholar
  30. 30.
    Chou T (2010) drug combination studies and their synergy quantification using the Chou-Talalay Method drug combination studies and their synergy quantification using the Chou-Talalay Method. Cancer Res 70:440–446. doi: 10.1158/0008-5472.CAN-09-1947 PubMedCrossRefGoogle Scholar
  31. 31.
    Mah L, Karagiannis TC (2010) γH2AX: a sensitive molecular marker of DNA damage and repair. Leukemia 24:679–686. doi: 10.1038/leu.2010.6
  32. 32.
    Alimova IN, Liu B, Fan Z et al (2009) Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro. Cell Cycle 8:909–915. doi:7933 [pii]Google Scholar
  33. 33.
    Soini Y, Vakkala M, Kahlos K et al (2001) MnSOD expression is less frequent in tumour cells of invasive breast carcinomas than in in situ carcinomas or non-neoplastic breast epithelial cells. J Pathol 195:156–162. doi: 10.1002/path.946 PubMedCrossRefGoogle Scholar
  34. 34.
    Walenta S, Wetterling M, Lehrke M et al (2000) High lactate levels predict likelihood of metastases, tumor recurrence, and restricted patient survival in human cervical cancers high lactate levels predict likelihood of metastases, tumor recurrence, and restricted patient survival in human cervical. Cancer Res 60:916–921PubMedGoogle Scholar
  35. 35.
    Leek R, Harris AL (2009) Lactate dehydrogenase 5 expression in squamous cell head and neck cancer relates to prognosis following radical or postoperative radiotherapy. Oncology 77:285–292. doi: 10.1159/000259260 PubMedCrossRefGoogle Scholar
  36. 36.
    Isidoro A, Casado E, Redondo A et al (2005) Breast carcinomas fulfill the Warburg hypothesis and provide metabolic markers of cancer prognosis. Carcinogenesis 26:2095–2104. doi: 10.1093/carcin/bgi188 PubMedCrossRefGoogle Scholar
  37. 37.
    Isidoro A, Martínez M, Fernández PL et al (2004) Alteration of the bioenergetic phenotype of mitochondria is a hallmark of breast, gastric, lung and oesophageal cancer. Biochem J 378:17–20. doi: 10.1042/BJ20031541 PubMedCrossRefPubMedCentralGoogle Scholar
  38. 38.
    Walenta S, Schroeder T (2004) Lactate in solid malignant tumors: potential basis of a metabolic classification in clinical oncology. Curr Med Chem 11:2195–2204PubMedCrossRefGoogle Scholar
  39. 39.
    Choi YW, Lim IK (2014) Sensitization of metformin-cytotoxicity by dichloroacetate via reprogramming glucose metabolism in cancer cells. Cancer Lett 346:300–308. doi: 10.1016/j.canlet.2014.01.015 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Allison B. Haugrud
    • 1
    Email author
  • Yongxian Zhuang
    • 1
  • Joseph D. Coppock
    • 1
  • W. Keith Miskimins
    • 1
  1. 1.Cancer Biology Research CenterSanford ResearchSioux FallsUSA

Personalised recommendations