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Phloretin increases the anti-tumor efficacy of intratumorally delivered heat-shock protein 70 kDa (HSP70) in a murine model of melanoma

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Abstract

Recombinant HSP70 chaperone exerts a profound anticancer effect when administered intratumorally. This action is based on the ability of HSP70 to penetrate tumor cells and extract its endogenous homolog. To enhance the efficacy of HSP70 cycling, we employed phloretin, a flavonoid that enhances the pore-forming activity of the chaperone on artificial membranes. Phloretin increased the efficacy of HSP70 penetration in B16 mouse melanoma cells and K-562 human erythroblasts; this was accompanied with increased transport of the endogenous HSP70 to the plasma membrane. Importantly, treatment with HSP70 combined with phloretin led to the elevation of cell sensitivity to cytotoxic lymphocytes by 16–18 % compared to treatment with the chaperone alone. The incubation of K-562 cells with biotinylated HSP70 and phloretin increased the amount of the chaperone released from cells, suggesting that chaperone cycling could trigger a specific anti-tumor response. We studied the effect of the combination of HSP70 and phloretin using B16 melanoma and a novel method of HSP70-gel application. We found that the addition of phloretin to the gel reduced tumor weight almost fivefold compared with untreated mice, while the life span of the animals extended from 25 to 39 days. The increased survival was corroborated by the activation of innate and adaptive immunity; interestingly, HSP70 was more active in induction of CD8+ cell-mediated toxicity and γIFN production while phloretin contributed largely to the CD56+ cell response. In conclusion, the combination of HSP70 with phloretin could be a novel treatment for efficient immunotherapy of intractable cancers such as skin melanoma.

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Abbreviations

CD:

Cluster of differentiation

CTL:

Cytotoxic T lymphocyte

DAPI:

4′,6-Diamidino-2-phenylindole

DOPE:

1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine

DOPS:

1,2-Dioleoyl-sn-glycero-3-phospho-l-serine

ELISA:

Enzyme-linked immunosorbent assay

HSP:

Heat-shock protein

γIFN:

Gamma-interferon

GLUT1 and GLUT2:

Glucose transporters of 1 and 2 type

NK cell:

Natural killer cell

PBS:

Phosphate-buffered saline

PrG-Sepharose:

ProteinG-Sepharose

SDS:

Sodium dodecylsulfate

SE:

Standard error

References

  1. Ito A, Matsuoka F, Honda H, Kobayashi T (2004) Antitumor effects of combined therapy of recombinant heat shock protein 70 and hyperthermia using magnetic nanoparticles in an experimental subcutaneous murine melanoma. Cancer Immunol Immunother 53:26–32

    Article  PubMed  CAS  Google Scholar 

  2. Abkin SV, Pankratova KM, Komarova EY, Guzhova IV, Margulis BA (2013) Hsp70 chaperone-based gel composition as a novel immunotherapeutic anti-tumor tool. Cell Stress Chaperones 18:391–396

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  3. Shevtsov MA, Pozdnyakov AV, Mikhrina AL, Yakovleva LY, Nikolaev BP, DobrodumovAV Komarova EY, Meshalkina DA, Ischenko AM, Pitkin E, Guzhova IV, Margulis BA (2014) Effective immunotherapy of rat glioblastoma with prolonged intratumoral delivery of exogenous heat shock protein Hsp70. Int J Cancer 135:2118–2128

    Article  PubMed  CAS  Google Scholar 

  4. Shevtsov MA, Komarova EY, Meshalkina DA, Bychkova NV, Aksenov ND, Abkin SV, Margulis BA, Guzhova IV (2014) Exogenously delivered heat shock protein 70 displaces its endogenous analogue and sensitizes cancer cells to lymphocytes-mediated cytotoxicity. Oncotarget 5:3101–3114

    Article  PubMed  PubMed Central  Google Scholar 

  5. Gastpar R, Gross C, Rossbacher L, Ellwart J, Riegger J, Multhoff G (2004) The cell surface-localized heat shock protein 70 epitope TKD induces migration and cytolytic activity selectively in human NK cells. J Immunol 172:972–980

    Article  PubMed  CAS  Google Scholar 

  6. Murshid A, Gong J, Stevenson MA, Calderwood SK (2011) Heat shock proteins and cancer vaccines: developments in the past decade and chaperoning in the decade to come. Expert Rev Vaccines 10:1553–1568

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  7. Geng H, Zhang GM, Xiao H, Yuan Y, Li D, Zhang H, Qiu H, He YF, Feng ZH (2006) HSP70 vaccine in combination with gene therapy with plasmid DNA encoding sPD-1 overcomes immune resistance and suppresses the progression of pulmonary metastatic melanoma. Int J Cancer 118:2657–2664

    Article  PubMed  CAS  Google Scholar 

  8. Arispe N, Doh M, Simakova O, Kurganov B, De Maio A (2004) Hsc70 and Hsp70 interact with phosphatidylserine on the surface of PC12 cells resulting in a decrease of viability. FASEB J 18:1636–1645

    Article  PubMed  CAS  Google Scholar 

  9. Arispe N, De Maio A (2000) ATP and ADP modulate a cation channel formed by Hsc70 in acidic phospholipid membranes. J Biol Chem 275:30839–30843

    Article  PubMed  CAS  Google Scholar 

  10. Komarova EY, Meshalkina DA, Aksenov ND, Pchelin IM, Martynova E, Margulis BA, Guzhova IV (2015) The discovery of Hsp70 domain with cell-penetrating activity. Cell Stress Chaperones 20:343–354

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  11. Andersen OS, Finkelstein A, Katz I, Cass A (1976) Effect of phloretin on the permeability of thin lipid membranes. J Gen Physiol 67:749–771

    Article  PubMed  CAS  Google Scholar 

  12. Franklin JC, Cafiso DS (1993) Internal electrostatic potentials in bilayers: measuring and controlling dipole potentials in lipid vesicles. Biophys J 65:289–299

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  13. Cseh R, Hetzer M, Wolf K, Kraus J, Bringmann G, Benz R (2000) Interaction of phloretin with membranes: on the mode of action of phloretin at the water-lipid interface. EurBiophys J 29:172–183

    CAS  Google Scholar 

  14. Rokitskaya TI, Antonenko YN, Kotova EA (1997) Effect of the dipole potential of a bilayer lipid membrane on gramicidin channel dissociation kinetics. Biophys J 73:850–854

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  15. Luchian T, Mereuta L (2006) Phlorizin- and 6-ketocholestanol-mediated antagonistic modulation of alamethicin activity in phospholipid planar membranes. Langmuir 22:8452–8457

    Article  PubMed  CAS  Google Scholar 

  16. Ostroumova OS, Malev VV, Bessonov AN, Takemoto JY, Schagina LV (2008) Altering the activity of syringomycin E via the membrane dipole potential. Langmuir 24:2987–2991

    Article  PubMed  CAS  Google Scholar 

  17. Ostroumova OS, Malev VV, Ilin MG, Schagina LV (2010) Surfactin activity depends on the membrane dipole potential. Langmuir 26:15092–15097

    Article  PubMed  CAS  Google Scholar 

  18. Ostroumova OS, Efimova SS, Mikhailova EV, Schagina LV (2014) The interaction of dipole modifiers with amphotericin-ergosterol complexes. Effects of phospholipid and sphingolipid membrane composition. Eur Biophys J 43:207–215

    Article  PubMed  CAS  Google Scholar 

  19. Guzhova IV, Lazarev VF, Kaznacheeva AV, Ippolitova MV, Muronetz VI, Kinev AV, Margulis BA (2011) Novel mechanism of Hsp70 chaperone-mediated prevention of polyglutamine aggregates in a cellular model of huntington disease. Hum Mol Genet 20:3953–3963

    Article  PubMed  CAS  Google Scholar 

  20. Multhoff G, Botzler C, Wiesnet M, Müller E, Meier T, Wilmanns W, Issels RD (1995) A stress-inducible 72-kDa heat-shock protein (HSP72) is expressed on the surface of human tumor cells, but not on normal cells. Int J Cancer 61:272–279

    Article  PubMed  CAS  Google Scholar 

  21. Kobori M, Shinmoto H, Tsushida T, Shinohara K (1997) Phloretin-induced apoptosis in B16 melanoma 4A5 cells by inhibition of glucose transmembrane transport. Cancer Lett 119:207–212

    Article  PubMed  CAS  Google Scholar 

  22. Srivastava PK, Udono H, Blachere NE, Li Z (1994) Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics 39:93–98

    Article  PubMed  CAS  Google Scholar 

  23. Multhoff G, Pfister K, Gehrmann M, Hantschel M, Gross C, Hafner M, Hiddemann W (2001) A 14-mer Hsp70 peptide stimulates natural killer (NK) cell activity. Cell Stress Chaperones 6:337–344

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  24. Calderwood SK, Theriault JR, Gong J (2005) How is the immune response affected by hyperthermia and heat shock proteins? Int J Hyperthermia 21:713–716

    Article  PubMed  CAS  Google Scholar 

  25. Arispe N, Doh M, De Maio A (2002) Lipid interaction differentiates the constitutive and stress-induced heat shock proteins Hsc70 and Hsp70. Cell Stress Chaperones 7:330–338

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  26. Armijo G, Okerblom J, Cauvi DM, Lopez V, Schlamadinger DE, Kim J, Arispe N, De Maio A (2014) Interaction of heat shock protein 70 with membranes depends on the lipid environment. Cell Stress Chaperones 19:877–886

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  27. Noessner E, Gastpar R, Milani V, Brandl A, Hutzler PJ, Kuppner MC, Roos M, Kremmer E, Asea A, Calderwood SK, Issels RD (2002) Tumor-derived heat shock protein 70 peptide complexes are cross-presented by human dendritic cells. J Immunol 169:5424–5432

    Article  PubMed  CAS  Google Scholar 

  28. Zhu SP, Liu G, Wu XT, Chen FX, Liu JQ, Zhou ZH, Zhang JF, Fei SJ (2013) The effect of phloretin on human γδ T cells killing colon cancer SW-1116 cells. Int Immunopharmacol 15:6–14

    Article  PubMed  CAS  Google Scholar 

  29. Kim MS, Kwon JY, Kang NJ, Lee KW, Lee HJ (2009) Phloretin induces apoptosis in H-Ras MCF10A human breast tumor cells through the activation of p53 via JNK and p38 mitogen-activated protein kinase signaling. Ann NY Acad Sci 1171:479–483

    Article  PubMed  CAS  Google Scholar 

  30. Cao X, Fang L, Gibbs S, Huang Y, Dai Z, Wen P, Zheng X, Sadee W, Sun D (2007) Glucose uptake inhibitor sensitizes cancer cells to daunorubicin and overcomes drug resistance in hypoxia. Cancer Chemother Pharmacol 59:495–505

    Article  PubMed  CAS  Google Scholar 

  31. Yang KC, Tsai CY, Wang YJ, Wei PL, Lee CH, Chen JH, Wu CH, Ho YS (2009) Apple polyphenol phloretin potentiates the anticancer actions of paclitaxel through induction of apoptosis in human hep G2 cells. Mol Carcinog 48:420–431

    Article  PubMed  CAS  Google Scholar 

  32. Wu CH, Ho YS, Tsai CY, Wang YJ, Tseng H, Wei PL, Lee CH, Liu RS, Lin SY (2009) In vitro and in vivo study of phloretin-induced apoptosis in human liver cancer cells involving inhibition of type II glucose transporter. Int J Cancer 124:2210–2219

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

Authors thank Prof. Gabriele Multhoff for cmHsp70.1-FITC antibody and Katerina Pankratova for technical assistance. This study was funded by a grant from the Russian Science Foundation (No. 14-50-00068) for Elena Komarova, Maxim Shevtsov, Darya Meshalkina, Boris Margulis and Irina Guzhova and by a grant from the Russian Academy of Sciences “Molecular and Cell Biology” Program No 1.7P for Olga Ostroumova and Sergey Abkin.

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Authors and Affiliations

  1. Institute of Cytology of Russian Academy of Sciences, Tikhoretsky Prospect, 4, St. Petersburg, Russia, 194064

    Sergey V. Abkin, Olga S. Ostroumova, Elena Y. Komarova, Darya A. Meshalkina, Maxim A. Shevtsov, Boris A. Margulis & Irina V. Guzhova

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  1. Sergey V. Abkin
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  2. Olga S. Ostroumova
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  3. Elena Y. Komarova
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  4. Darya A. Meshalkina
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  5. Maxim A. Shevtsov
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  6. Boris A. Margulis
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  7. Irina V. Guzhova
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Correspondence to Irina V. Guzhova.

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Abkin, S.V., Ostroumova, O.S., Komarova, E.Y. et al. Phloretin increases the anti-tumor efficacy of intratumorally delivered heat-shock protein 70 kDa (HSP70) in a murine model of melanoma. Cancer Immunol Immunother 65, 83–92 (2016). https://doi.org/10.1007/s00262-015-1778-1

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  • DOI: https://doi.org/10.1007/s00262-015-1778-1

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