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Chloroquine-Mediated Lysosomal Dysfunction Enhances the Anticancer Effect of Nutrient Deprivation

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ABSTRACT

Purpose

To investigate the ability of chloroquine, a lysosomotropic autophagy inhibitor, to enhance the anticancer effect of nutrient deprivation.

Methods

Serum-deprived U251 glioma, B16 melanoma and L929 fibrosarcoma cells were treated with chloroquine in vitro. Cell viability was measured by crystal violet and MTT assay. Oxidative stress, apoptosis/necrosis and intracellular acidification were analyzed by flow cytometry. Cell morphology was examined by light and electron microscopy. Activation of AMP-activated protein kinase (AMPK) and autophagy were monitored by immunoblotting. RNA interference was used for AMPK and LC3b knockdown. The anticancer efficiency of intraperitoneal chloroquine in calorie-restricted mice was assessed using a B16 mouse melanoma model.

Results

Chloroquine rapidly killed serum-starved cancer cells in vitro. This effect was not mimicked by autophagy inhibitors or LC3b shRNA, indicating autophagy-independent mechanism. Chloroquine-induced lysosomal accumulation and oxidative stress, leading to mitochondrial depolarization, caspase activation and mixed apoptotic/necrotic cell death, were prevented by lysosomal acidification inhibitor bafilomycin. AMPK downregulation participated in chloroquine action, as AMPK activation reduced, and AMPK shRNA mimicked chloroquine toxicity. Chloroquine inhibited melanoma growth in calorie-restricted mice, causing lysosomal accumulation, mitochondrial disintegration and selective necrosis of tumor cells.

Conclusion

Combined treatment with chloroquine and calorie restriction might be useful in cancer therapy.

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Abbreviations

AICAR:

5-aminoimidazole-4-carboxamide riboside

AMPK:

AMP-activated protein kinase

DHR:

dihydrorhodamine

FCS:

fetal calf serum

FITC:

fluorescein isothyocyanate

LC3:

microtubule-associated protein 1 light-chain 3

MEM:

Eagle’s Minimum Essential Medium

MTT:

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

PBS:

phosphate buffered saline

PI:

propidium iodide

ROS:

reactive oxygen species

shRNA:

short hairpin RNA

REFERENCES

  1. Kennedy BK, Steffen KK, Kaeberlein M. Ruminations on dietary restriction and aging. Cell Mol Life Sci. 2007;64:1323–8.

    Article  PubMed  CAS  Google Scholar 

  2. Omodei D, Fontana L. Calorie restriction and prevention of age-associated chronic disease. FEBS Lett. 2011;585:1537–42.

    Article  PubMed  CAS  Google Scholar 

  3. Grifantini K. Understanding pathways of calorie restriction: a way to prevent cancer? J Natl Cancer Inst. 2008;100:619–21.

    Article  PubMed  Google Scholar 

  4. Longo VD, Fontana L. Calorie restriction and cancer prevention: metabolic and molecular mechanisms. Trends Pharmacol Sci. 2010;31:89–98.

    Article  PubMed  CAS  Google Scholar 

  5. Yorimitsu T, Klionsky DJ. Autophagy: molecular machinery for self-eating. Cell Death Differ. 2005;12 Suppl 2:1542–52.

    Article  PubMed  CAS  Google Scholar 

  6. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature. 2008;451:1069–75.

    Article  PubMed  CAS  Google Scholar 

  7. Gump JM, Thorburn A. Autophagy and apoptosis: what is the connection? Trends Cell Biol. 2011;21:387–92.

    Article  PubMed  CAS  Google Scholar 

  8. Thorburn A. Apoptosis and autophagy: regulatory connections between two supposedly different processes. Apoptosis. 2008;13:1–9.

    Article  PubMed  CAS  Google Scholar 

  9. Warburg O. On the origin of cancer cells. Science. 1956;123:309–14.

    Article  PubMed  CAS  Google Scholar 

  10. Levy JM, Thorburn A. Targeting autophagy during cancer therapy to improve clinical outcomes. Pharmacol Ther. 2011;131:130–41.

    Article  PubMed  CAS  Google Scholar 

  11. Maycotte P, Thorburn A. Autophagy and cancer therapy. Cancer Biol Ther. 2011;11:127–37.

    Article  PubMed  CAS  Google Scholar 

  12. Fan C, Wang W, Zhao B, Zhang S, Miao J. Chloroquine inhibits cell growth and induces cell death in A549 lung cancer cells. Bioorg Med Chem. 2006;14:3218–22.

    Article  PubMed  CAS  Google Scholar 

  13. Jiang PD, Zhao YL, Deng XQ, Mao YQ, Shi W, Tang QQ, et al. Antitumor and antimetastatic activities of chloroquine diphosphate in a murine model of breast cancer. Biomed Pharmacother. 2010;64:609–14.

    Article  PubMed  CAS  Google Scholar 

  14. Munshi A. Chloroquine in glioblastoma–new horizons for an old drug. Cancer. 2009;115:2380–3.

    Article  PubMed  CAS  Google Scholar 

  15. Rahim R, Strobl JS. Hydroxychloroquine, chloroquine, and all-trans retinoic acid regulate growth, survival, and histone acetylation in breast cancer cells. Anticancer Drugs. 2009;20:736–45.

    Article  PubMed  CAS  Google Scholar 

  16. Solomon VR, Hu C, Lee H. Design and synthesis of chloroquine analogs with anti-breast cancer property. Eur J Med Chem. 2010;45:3916–23.

    Article  PubMed  CAS  Google Scholar 

  17. Zheng Y, Zhao YL, Deng X, Yang S, Mao Y, Li Z, et al. Chloroquine inhibits colon cancer cell growth in vitro and tumor growth in vivo via induction of apoptosis. Cancer Invest. 2009;27:286–92.

    Article  PubMed  CAS  Google Scholar 

  18. Hu C, Solomon VR, Ulibarri G, Lee H. The efficacy and selectivity of tumor cell killing by Akt inhibitors are substantially increased by chloroquine. Bioorg Med Chem. 2008;16:7888–93.

    Article  PubMed  CAS  Google Scholar 

  19. Solomon VR, Lee H. Chloroquine and its analogs: a new promise of an old drug for effective and safe cancer therapies. Eur J Pharmacol. 2009;625:220–33.

    Article  PubMed  CAS  Google Scholar 

  20. Poole B, Ohkuma S. Effect of weak bases on the intralysosomal pH in mouse peritoneal macrophages. J Cell Biol. 1981;90:665–9.

    Article  PubMed  CAS  Google Scholar 

  21. Glaumann H, Ahlberg J. Comparison of different autophagic vacuoles with regard to ultrastructure, enzymatic composition, and degradation capacity–formation of crinosomes. Exp Mol Pathol. 1987;47:346–62.

    Article  PubMed  CAS  Google Scholar 

  22. Lum JJ, Bauer DE, Kong M, Harris MH, Li C, Lindsten T, et al. Growth factor regulation of autophagy and cell survival in the absence of apoptosis. Cell. 2005;120:237–48.

    Article  PubMed  CAS  Google Scholar 

  23. Carew JS, Medina EC, Esquivel 2nd JA, Mahalingam D, Swords R, Kelly K, et al. Autophagy inhibition enhances vorinostat-induced apoptosis via ubiquitinated protein accumulation. J Cell Mol Med. 2010;14:2448–59.

    Article  PubMed  CAS  Google Scholar 

  24. Kaludjerovic GN, Miljkovic D, Momcilovic M, Djinovic VM, Mostarica Stojkovic M, Sabo TJ, et al. Novel platinum(IV) complexes induce rapid tumor cell death in vitro. Int J Cancer. 2005;116:479–86.

    Article  PubMed  CAS  Google Scholar 

  25. Harhaji L, Isakovic A, Vucicevic L, Janjetovic K, Misirkic M, Markovic Z, et al. Modulation of tumor necrosis factor-mediated cell death by fullerenes. Pharm Res. 2008;25:1365–76.

    Article  PubMed  CAS  Google Scholar 

  26. Bastien-Dionne PO, Valenti L, Kon N, Gu W, Buteau J. Glucagon-like peptide 1 inhibits the sirtuin deacetylase SirT1 to stimulate pancreatic beta-cell mass expansion. Diabetes. 2011;60:3217–22.

    Google Scholar 

  27. Kaushik S, Rodriguez-Navarro JA, Arias E, Kiffin R, Sahu S, Schwartz GJ, et al. Autophagy in hypothalamic AgRP neurons regulates food intake and energy balance. Cell Metab. 2011;14:173–83.

    Article  PubMed  CAS  Google Scholar 

  28. Bjorkoy G, Lamark T, Brech A, Outzen H, Perander M, Overvatn A, et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J Cell Biol. 2005;171:603–14.

    Article  PubMed  Google Scholar 

  29. Blommaart EF, Krause U, Schellens JP, Vreeling-Sindelarova H, Meijer AJ. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem. 1997;243:240–6.

    Article  PubMed  CAS  Google Scholar 

  30. Klionsky DJ, Elazar Z, Seglen PO, Rubinsztein DC. Does bafilomycin A1 block the fusion of autophagosomes with lysosomes? Autophagy. 2008;4:849–950.

    PubMed  CAS  Google Scholar 

  31. Rogers SW, Rechsteiner M. Degradation of structurally characterized proteins injected into HeLa cells. Effects of intracellular location and the involvement of lysosomes. J Biol Chem. 1988;263:19843–9.

    PubMed  CAS  Google Scholar 

  32. van Es HH, Renkema H, Aerts H, Schurr E. Enhanced lysosomal acidification leads to increased chloroquine accumulation in CHO cells expressing the pfmdr1 gene. Mol Biochem Parasitol. 1994;68:209–19.

    Article  PubMed  Google Scholar 

  33. Hardie DG, Hawley SA, Scott JW. AMP-activated protein kinase - development of the energy sensor concept. J Physiol. 2006;574:7–15.

    Article  PubMed  CAS  Google Scholar 

  34. Boya P, Gonzalez-Polo RA, Casares N, Perfettini JL, Dessen P, Larochette N, et al. Inhibition of macroautophagy triggers apoptosis. Mol Cell Biol. 2005;25:1025–40.

    Article  PubMed  CAS  Google Scholar 

  35. Maycotte P, Aryal S, Cummings CT, Thorburn J, Morgan MJ, Thorburn A. Chloroquine sensitizes breast cancer cells to chemotherapy independent of autophagy. Autophagy. 2012;8:200–12.

    Article  PubMed  CAS  Google Scholar 

  36. Yoon YH, Cho KS, Hwang JJ, Lee SJ, Choi JA, Koh JY. Induction of lysosomal dilatation, arrested autophagy, and cell death by chloroquine in cultured ARPE-19 cells. Invest Ophthalmol Vis Sci. 2010;51:6030–7.

    Article  PubMed  Google Scholar 

  37. Michihara A, Toda K, Kubo T, Fujiwara Y, Akasaki K, Tsuji H. Disruptive effect of chloroquine on lysosomes in cultured rat hepatocytes. Biol Pharm Bull. 2005;28:947–51.

    Article  PubMed  CAS  Google Scholar 

  38. Jiang PD, Zhao YL, Shi W, Deng XQ, Xie G, Mao YQ, et al. Cell growth inhibition, G2/M cell cycle arrest, and apoptosis induced by chloroquine in human breast cancer cell line Bcap-37. Cell Physiol Biochem. 2008;22:431–40.

    Article  PubMed  CAS  Google Scholar 

  39. Loeffler M, Kroemer G. The mitochondrion in cell death control: certainties and incognita. Exp Cell Res. 2000;256:19–26.

    Article  PubMed  CAS  Google Scholar 

  40. Zorov DB, Juhaszova M, Sollott SJ. Mitochondrial ROS-induced ROS release: an update and review. Biochim Biophys Acta. 2006;1757:509–17.

    Article  PubMed  CAS  Google Scholar 

  41. Kim JS, He L, Lemasters JJ. Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. Biochem Biophys Res Commun. 2003;304:463–70.

    Article  PubMed  CAS  Google Scholar 

  42. Ono K, Wang X, Han J. Resistance to tumor necrosis factor-induced cell death mediated by PMCA4 deficiency. Mol Cell Biol. 2001;21:8276–88.

    Article  PubMed  CAS  Google Scholar 

  43. Ershler WB, Berman E, Moore AL. Slower B16 melanoma growth but greater pulmonary colonization in calorie-restricted mice. J Natl Cancer Inst. 1986;76:81–5.

    PubMed  CAS  Google Scholar 

  44. Inoue S, Hasegawa K, Ito S, Wakamatsu K, Fujita K. Antimelanoma activity of chloroquine, an antimalarial agent with high affinity for melanin. Pigment Cell Res. 1993;6:354–8.

    Article  PubMed  CAS  Google Scholar 

  45. Vogler C, Rosenberg HS, Williams JC, Butler I. Electron microscopy in the diagnosis of lysosomal storage diseases. Am J Med Genet Suppl. 1987;3:243–55.

    Article  PubMed  CAS  Google Scholar 

  46. Lin CL, Huang HC, Lin JK. Theaflavins attenuate hepatic lipid accumulation through activating AMPK in human HepG2 cells. J Lipid Res. 2007;48:2334–43.

    Article  PubMed  CAS  Google Scholar 

  47. Jarzyna R, Lenarcik E, Bryla J. Chloroquine is a potent inhibitor of glutamate dehydrogenase in liver and kidney-cortex of rabbit. Pharmacol Res. 1997;35:79–84.

    Article  PubMed  CAS  Google Scholar 

  48. Mastorodemos V, Zaganas I, Spanaki C, Bessa M, Plaitakis A. Molecular basis of human glutamate dehydrogenase regulation under changing energy demands. J Neurosci Res. 2005;79:65–73.

    Article  PubMed  CAS  Google Scholar 

  49. Sheen JH, Zoncu R, Kim D, Sabatini DM. Defective regulation of autophagy upon leucine deprivation reveals a targetable liability of human melanoma cells in vitro and in vivo. Cancer Cell. 2011;19:613–28.

    Article  PubMed  CAS  Google Scholar 

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ACKNOWLEDGMENTS & DISCLOSURES

The study was supported by the Ministry of Science and Technological Development of the Republic of Serbia (grants 173053 to LHT and 41025 to VT). Ljubica Harhaji-Trajkovic is a recipient of the UNESCO L’OREAL national scholarship program “For Women in Science” (contract number 403F). The authors declare that there are no conflicts of interest.

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Authors and Affiliations

  1. Institute for Biological Research, University of Belgrade, Despot Stefan Blvd. 142, 11060, Belgrade, Serbia

    Ljubica Harhaji-Trajkovic, Gordana Tovilovic, Nevena Zogovic & Kristina Janjetovic

  2. Institute of Microbiology and Immunology, School of Medicine, University of Belgrade, Dr. Subotica 1, 11000, Belgrade, Serbia

    Katarina Arsikin, Aleksandar Pantovic, Biljana Ristic, Kristina Janjetovic & Vladimir Trajkovic

  3. Institute of Histology and Embryology, School of Medicine, University of Belgrade, Belgrade, Serbia

    Tamara Kravic-Stevovic & Vladimir Bumbasirevic

  4. Institute of Biomedical Research, Galenika a.d., Belgrade, Serbia

    Sasa Petricevic

Authors
  1. Ljubica Harhaji-Trajkovic
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Corresponding authors

Correspondence to Ljubica Harhaji-Trajkovic or Vladimir Trajkovic.

Additional information

Ljubica Harhaji-Trajkovic and Katarina Arsikin contributed equally to the work.

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Harhaji-Trajkovic, L., Arsikin, K., Kravic-Stevovic, T. et al. Chloroquine-Mediated Lysosomal Dysfunction Enhances the Anticancer Effect of Nutrient Deprivation. Pharm Res 29, 2249–2263 (2012). https://doi.org/10.1007/s11095-012-0753-1

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  • DOI: https://doi.org/10.1007/s11095-012-0753-1

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