Hormones and Cancer

, 2:272 | Cite as

A Switch Between Cytoprotective and Cytotoxic Autophagy in the Radiosensitization of Breast Tumor Cells by Chloroquine and Vitamin D

  • Eden N. Wilson
  • Molly L. Bristol
  • Xu Di
  • William A. Maltese
  • Kristen Koterba
  • Matthew J. Beckman
  • David A. Gewirtz
Article

Abstract

Calcitriol or 1,25-dihydroxyvitamin D3, the hormonally active form of vitamin D, as well as vitamin D analogs, has been shown to increase sensitivity to ionizing radiation in breast tumor cells. The current studies indicate that the combination of 1,25-dihydroxyvitamin D3 with radiation appears to kill p53 wild-type, estrogen receptor-positive ZR-75-1 breast tumor cells through autophagy. Minimal apoptosis was observed based on cell morphology by DAPI and TUNEL staining, annexin/PI analysis, caspase-3, and PARP cleavage as well as cell cycle analysis. Induction of autophagy was indicated by increased acridine orange staining, RFP-LC3 redistribution, and detection of autophagic vesicles by electron microscopy, while autophagic flux was monitored based on p62 degradation. The autophagy inhibitors, chloroquine and bafilomycin A1, as well as genetic suppression of the autophagic signaling proteins Atg5 or Atg 7 attenuated the impact of the combination treatment of 1,25 D3 with radiation. In contrast to autophagy mediating the effects of the combination treatment, the autophagy induced by radiation alone was apparently cytoprotective in that either pharmacological or genetic inhibition increased sensitivity to radiation. These studies support the potential utility of vitamin D for improving the impact of radiation for breast cancer therapy, support the feasibility of combining chloroquine with radiation for the treatment of breast cancer, and demonstrate the existence of an “autophagic switch” from cytoprotective autophagy with radiation alone to cytotoxic autophagy with the 1,25 D3–radiation combination.

Keywords

Breast cancer Vitamin D Radiation Autophagy Chloroquine 

Abbreviations

1,25 D3

1,25 dihydroxyvitamin D3

DAPI

4′,6-diamidino-2-phenylindole

AO

Acridine orange

AVOs

Acidic vacuolar organelles

CQ

Chloroquine

IR

Ionizing radiation

TUNEL

Terminal deoxynucleotidyl transferase dUTP nick end labeling

TEM

Transmission electron microscopy

BAF

Bafilomycin A1

SS

Serum starvation

PI

Propidium iodide

FACS

Fluorescence-activated cell sorting

Supplementary material

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12672_2011_81_MOESM4_ESM.ppt (52 kb)
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References

  1. 1.
    Chen DJ, Nirodi CS (2007) The epidermal growth factor receptor: a role in repair of radiation-induced DNA damage. Clin Cancer Res 13:6555–6560PubMedCrossRefGoogle Scholar
  2. 2.
    Spitz DR, Azzam EI, Li JJ et al (2004) Metabolic oxidation/reduction reactions and cellular responses to ionizing radiation: a unifying concept in stress response biology. Cancer Metastasis Rev 23:311–322PubMedCrossRefGoogle Scholar
  3. 3.
    Dent P, Yacoub A, Contessa J et al (2003) Stress and radiation-induced activation of multiple intracellular signaling pathways. Radiat Res 159:283–300PubMedCrossRefGoogle Scholar
  4. 4.
    Sundaram S, Gewirtz DA (1999) The vitamin D3 analog EB 1089 enhances the response of human breast tumor cells to radiation. Radiat Res 152:479–486PubMedCrossRefGoogle Scholar
  5. 5.
    Demasters G, Di X, Newsham I et al (2006) Potentiation of radiation sensitivity in breast tumor cells by the vitamin D3 analogue, EB 1089, through promotion of autophagy and interference with proliferative recovery. Mol Cancer Ther 5:2786–2797PubMedCrossRefGoogle Scholar
  6. 6.
    Light BW, Yu WD, McElwain MC et al (1997) Potentiation of cisplatin antitumor activity using a vitamin D analogue in a murine squamous cell carcinoma model system. Cancer Res 57:3759–3764PubMedGoogle Scholar
  7. 7.
    Ravid A, Rocker D, Machlenkin A et al (1999) 1,25-Dihydroxyvitamin D3 enhances the susceptibility of breast cancer cells to doxorubicin-induced oxidative damage. Cancer Res 59:862–867PubMedGoogle Scholar
  8. 8.
    Hershberger PA, Yu WD, Modzelewski RA et al (2001) Calcitriol (1,25-dihydroxycholecalciferol) enhances paclitaxel antitumor activity in vitro and in vivo and accelerates paclitaxel-induced apoptosis. Clin Cancer Res 7:1043–1051PubMedGoogle Scholar
  9. 9.
    Chaudhry M, Sundaram S, Gennings C et al (2001) The vitamin D3 analog, ILX-23-7553, enhances the response to adriamycin and irradiation in MCF-7 breast tumor cells. Cancer Chemother Pharmacol 47:429–436PubMedCrossRefGoogle Scholar
  10. 10.
    Chen N, Karantza-Wadsworth V (2009) Role and regulation of autophagy in cancer. Biochim Biophys Acta 1793:1516–1523PubMedCrossRefGoogle Scholar
  11. 11.
    Shintani T, Klionsky DJ (2004) Autophagy in health and disease: a double-edged sword. Science 306:990–995PubMedCrossRefGoogle Scholar
  12. 12.
    Ito H, Daido S, Kanzawa T et al (2005) Radiation-induced autophagy is associated with LC3 and its inhibition sensitizes malignant glioma cells. Int J Oncol 26:1401–1410PubMedGoogle Scholar
  13. 13.
    Fung C, Lock R, Gao S et al (2008) Induction of autophagy during extracellular matrix detachment promotes cell survival. Mol Biol Cell 19:797–806PubMedCrossRefGoogle Scholar
  14. 14.
    Wu YT, Tan HL, Huang Q et al (2008) Autophagy plays a protective role during zVAD-induced necrotic cell death. Autophagy 4:457–466PubMedGoogle Scholar
  15. 15.
    Maiuri MC, Zalckvar E, Kimchi A et al (2007) Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol 8:741–752PubMedCrossRefGoogle Scholar
  16. 16.
    Gorka M, Daniewski WM, Gajkowska B et al (2005) Autophagy is the dominant type of programmed cell death in breast cancer MCF-7 cells exposed to AGS 115 and EFDAC, new sesquiterpene analogs of paclitaxel. Anticancer Drugs 16:777–788PubMedCrossRefGoogle Scholar
  17. 17.
    Oh SH, Kim YS, Lim SC et al (2008) Dihydrocapsaicin (DHC), a saturated structural analog of capsaicin, induces autophagy in human cancer cells in a catalase-regulated manner. Autophagy 4:1009–1019PubMedGoogle Scholar
  18. 18.
    Paglin S, Hollister T, Delohery T et al (2001) A novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles. Cancer Res 61:439–444PubMedGoogle Scholar
  19. 19.
    Gozuacik D, Kimchi A (2007) Autophagy and cell death. Curr Top Dev Biol 78:217–245PubMedCrossRefGoogle Scholar
  20. 20.
    Gewirtz DA, Hilliker ML, Wilson EN (2009) Promotion of autophagy as a mechanism for radiation sensitization of breast tumor cells. Radiother Oncol 92:323–328PubMedCrossRefGoogle Scholar
  21. 21.
    Livesey KM, Tang D, Zeh HJ et al (2009) Autophagy inhibition in combination cancer treatment. Curr Opin Invest Drugs 10:1269–1279Google Scholar
  22. 22.
    Lomonaco SL, Finniss S, Xiang C et al (2009) The induction of autophagy by gamma-radiation contributes to the radioresistance of glioma stem cells. Int J Cancer 125:717–722PubMedCrossRefGoogle Scholar
  23. 23.
    Apel A, Herr I, Schwarz H et al (2008) Blocked autophagy sensitizes resistant carcinoma cells to radiation therapy. Cancer Res 68:1485–1494PubMedCrossRefGoogle Scholar
  24. 24.
    Kim KW, Mutter RW, Cao C et al (2006) Autophagy for cancer therapy through inhibition of pro-apoptotic proteins and mammalian target of rapamycin signaling. J Biol Chem 281:36883–36890PubMedCrossRefGoogle Scholar
  25. 25.
    Cao C, Subhawong T, Albert JM et al (2006) Inhibition of mammalian target of rapamycin or apoptotic pathway induces autophagy and radiosensitizes PTEN null prostate cancer cells. Cancer Res 66:10040–10047PubMedCrossRefGoogle Scholar
  26. 26.
    Peng PL, Kuo WH, Tseng HC et al (2008) Synergistic tumor-killing effect of radiation and berberine combined treatment in lung cancer: the contribution of autophagic cell death. Int J Radiat Oncol Biol Phys 70:529–542PubMedCrossRefGoogle Scholar
  27. 27.
    Shen HM, Codogno P (2011) Autophagic cell death: Loch Ness monster or endangered species? Autophagy 7:457–465Google Scholar
  28. 28.
    Ingraham BA, Bragdon B, Nohe A (2008) Molecular basis of the potential of vitamin D to prevent cancer. Curr Med Res Opin 24:139–149PubMedGoogle Scholar
  29. 29.
    Wu G, Fan RS, Li W et al (1997) Modulation of cell cycle control by vitamin D3 and its analogue, EB1089, in human breast cancer cells. Oncogene 15:1555–1563PubMedCrossRefGoogle Scholar
  30. 30.
    Kwong J, Kulbe H, Wong D et al (2009) An antagonist of the chemokine receptor CXCR4 induces mitotic catastrophe in ovarian cancer cells. Mol Cancer Ther 8:1893–1905PubMedCrossRefGoogle Scholar
  31. 31.
    Portugal J, Mansilla S, Bataller M (2010) Mechanisms of drug-induced mitotic catastrophe in cancer cells. Curr Pharm Des 16:69–78PubMedCrossRefGoogle Scholar
  32. 32.
    Pyo JO, Jang MH, Kwon YK et al (2005) Essential roles of Atg5 and FADD in autophagic cell death: dissection of autophagic cell death into vacuole formation and cell death. J Biol Chem 280:20722–20729PubMedCrossRefGoogle Scholar
  33. 33.
    Eskelinen EL, Saftig P (2009) Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochim Biophys Acta 1793:664–673PubMedCrossRefGoogle Scholar
  34. 34.
    Zakeri Z, Melendez A, Lockshin RA (2008) Detection of autophagy in cell death. Methods Enzymol 442:289–306PubMedCrossRefGoogle Scholar
  35. 35.
    Kirisako T, Ichimura Y, Okada H et al (2000) The reversible modification regulates the membrane-binding state of Apg8/Aut7 essential for autophagy and the cytoplasm to vacuole targeting pathway. J Cell Biol 151:263–276PubMedCrossRefGoogle Scholar
  36. 36.
    Pankiv S, Clausen TH, Lamark T et al (2007) p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Chem 282:24131–24145PubMedCrossRefGoogle Scholar
  37. 37.
    Larsen KB, Lamark T, Overvatn A et al (2010) A reporter cell system to monitor autophagy based on p62/SQSTM1. Autophagy 6:784–793PubMedCrossRefGoogle Scholar
  38. 38.
    Shvets E, Fass E, Scherz-Shouval R et al (2008) The N-terminus and Phe52 residue of LC3 recruit p62/SQSTM1 into autophagosomes. J Cell Sci 121:2685–2695PubMedCrossRefGoogle Scholar
  39. 39.
    Debacq-Chainiaux F, Erusalimsky JD, Campisi J et al (2009) Protocols to detect senescence-associated beta-galactosidase (SA-betagal) activity, a biomarker of senescent cells in culture and in vivo. Nat Protoc 4:1798–1806PubMedCrossRefGoogle Scholar
  40. 40.
    Shacka JJ, Klocke BJ, Roth KA (2006) Autophagy, bafilomycin and cell death: the “a-B-cs” of plecomacrolide-induced neuroprotection. Autophagy 2:228–230PubMedGoogle Scholar
  41. 41.
    Carew JS, Medina EC, Esquivel JA 2nd et al (2010) Autophagy inhibition enhances vorinostat-induced apoptosis via ubiquitinated protein accumulation. J Cell Mol Med 14:2448–2459PubMedCrossRefGoogle Scholar
  42. 42.
    Klionsky DJ (2005) The molecular machinery of autophagy: unanswered questions. J Cell Sci 118:7–18PubMedCrossRefGoogle Scholar
  43. 43.
    Jones KR, Elmore LW, Jackson-Cook C et al (2005) p53-Dependent accelerated senescence induced by ionizing radiation in breast tumour cells. Int J Radiat Biol 81:445–458PubMedCrossRefGoogle Scholar
  44. 44.
    Mirzayans R, Scott A, Cameron M et al (2005) Induction of accelerated senescence by gamma radiation in human solid tumor-derived cell lines expressing wild-type TP53. Radiat Res 163:53–62PubMedCrossRefGoogle Scholar
  45. 45.
    Botti J, Djavaheri-Mergny M, Pilatte Y et al (2006) Autophagy signaling and the cogwheels of cancer. Autophagy 2:67–73PubMedGoogle Scholar
  46. 46.
    Hoyer-Hansen M, Jaattela M (2007) Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium. Cell Death Differ 14:1576–1582PubMedCrossRefGoogle Scholar
  47. 47.
    Qin L, Wang Z, Tao L et al (2010) ER stress negatively regulates AKT/TSC/mTOR pathway to enhance autophagy. Autophagy 6:239–247PubMedCrossRefGoogle Scholar
  48. 48.
    Kim KW, Moretti L, Mitchell LR et al (2010) Endoplasmic reticulum stress mediates radiation-induced autophagy by perk-eIF2alpha in caspase-3/7-deficient cells. Oncogene 29:3241–3251PubMedCrossRefGoogle Scholar
  49. 49.
    Zhang Y, Zhang J, Studzinski GP (2006) AKT pathway is activated by 1, 25-dihydroxyvitamin D3 and participates in its anti-apoptotic effect and cell cycle control in differentiating HL60 cells. Cell Cycle 5:447–451PubMedCrossRefGoogle Scholar
  50. 50.
    Lisse TS, Hewison M (2011) Vitamin D: a new player in the world of mTOR signaling. Cell Cycle 10:1888–1889PubMedCrossRefGoogle Scholar
  51. 51.
    Crighton D, Wilkinson S, O’Prey J et al (2006) DRAM, a p53-induced modulator of autophagy, is critical for apoptosis. Cell 126:121–134PubMedCrossRefGoogle Scholar
  52. 52.
    Gavrieli Y, Sherman Y, Ben-Sasson SA (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. J Cell Biol 119:493–501PubMedCrossRefGoogle Scholar
  53. 53.
    Darzynkiewicz, Z, Li X, Gong J (1994) PI flow for apoptosis. Methods in Cell Biology 41:26–29Google Scholar
  54. 54.
    Dimri GP, Lee X, Basile G et al (1995) A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci USA 92:9363–9367PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Eden N. Wilson
    • 1
  • Molly L. Bristol
    • 1
  • Xu Di
    • 1
  • William A. Maltese
    • 2
  • Kristen Koterba
    • 2
  • Matthew J. Beckman
    • 1
  • David A. Gewirtz
    • 1
  1. 1.Department of Pharmacology and Toxicology, Massey Cancer CenterVirginia Commonwealth UniversityRichmondUSA
  2. 2.Department of Biochemistry and Cancer BiologyUniversity of Toledo College of MedicineToledoUSA

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