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Apoptosis

, Volume 24, Issue 1–2, pp 119–134 | Cite as

Oxidative stress generated by irradiation of a zinc(II) phthalocyanine induces a dual apoptotic and necrotic response in melanoma cells

  • Federico Valli
  • María C. García Vior
  • Leonor P. Roguin
  • Julieta MarinoEmail author
Article

Abstract

Melanoma is an aggressive form of skin carcinoma, highly resistant to traditional therapies. Photodynamic therapy (PDT) is a non-invasive therapeutic procedure that can exert a selective cytotoxic activity toward malignant cells. In this work we evaluated the effect of a cationic zinc(II) phthalocyanine (Pc13) as photosensitizer on a panel of melanoma cells. Incubation with Pc13 and irradiation induced a concentration and light dose-dependent phototoxicity. In order to study the mechanism underlying Pc13-related cell death and to compare the effect of different doses of PDT, the most sensitive melanoma B16F0 cells were employed. By confocal imaging we showed that Pc13 targeted lysosomes and mitochondria. After irradiation, a marked increase in intracellular reactive oxygen species was observed and a complete protection from Pc13 phototoxicity was reached in the presence of the antioxidant trolox. Acridine orange/ethidium bromide staining showed morphological changes indicative of both apoptosis and necrosis. Biochemical hallmarks of apoptosis, including a significant decrease in the expression levels of Bcl-2, Bcl-xL and Bid and mitochondrial membrane permeabilization, were observed at short times post irradiation. The consequent release of cytochrome c to cytosol and caspase-3 activation led to PARP-1 cleavage and DNA fragmentation. Simultaneously, a dose dependent increase of lactate dehydrogenase in the extracellular compartment of treated cells revealed plasma membrane damage characteristic of necrosis. Taken together, these results indicate that a dual apoptotic and necrotic response is triggered by Pc13 PDT-induced oxidative stress, suggesting that combined mechanisms of cell death could result in a potent alternative for melanoma treatment.

Keywords

Reactive oxygen species Cationic phthalocyanine Mitochondrial membrane permeabilization Apoptosis Necrosis Photodynamic therapy 

Abbreviations

AO

Acridine orange

DCFH-DA

2′,7′-Dichlorodihydrofluorescein diacetate

DiOC6

3,3′-Dihexyloxacarbocyanine iodide

EB

Ethidium bromide

Trolox

6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

LDH

Lactate dehydrogenase

LMP

Lysosomal membrane permeabilization

Δψm

Mitochondrial transmembrane potential

Pc

Phthalocyanine

PDT

Photodynamic therapy

PS

Photosensitizer

PARP-1

Poly(ADP-ribose) polymerase

PI

Propidium iodide

ROS

Reactive oxygen species

1O2

Singlet oxygen

Notes

Acknowledgements

This work was supported by grants of Agencia Nacional de Promoción Científica y Tecnológica PICT 2013-0144, Consejo Nacional de Investigaciones Científicas y Técnicas PIP 0154 and Secretaria de Ciencia y Técnica de la Universidad de Buenos Aires (UBACyT 20020130100024), Argentina. Authors are grateful to Dr. Marcela Villaverde for the generous gift of A375 cells.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10495_2018_1512_MOESM1_ESM.pdf (40 kb)
Online Resource 1 Phototoxic effect of Pc13 in melanoma cell lines. (a) In order to explore Pc13 cytotoxicity in the dark, B16F0 cells were incubated with increasing concentrations (up to 32 μM) of Pc13. (b) Melanoma cells or (c) normal keratinocytes HaCat were incubated with different concentrations of Pc13 and maintained in the dark or irradiated with 340 mJ cm-2. Phototoxicity was evaluated 24 h after irradiation. Results are expressed relative to control obtained in the absence of Pc13 and represent the mean ± S.E.M. of three different experiments (PDF 40 KB)
10495_2018_1512_MOESM2_ESM.pdf (146 kb)
Online Resource 2 Lysosomal membrane permeabilization induced by photodynamic treatment. (a) B16F0 cells grown on coverslips were incubated in the absence or presence of 0.2 µM Pc13, irradiated (340 mJ cm-2) and incubated at 37°C for 1h. Then, lysosomes were stained with LysoTracker Green and examined by fluorescence microscopy. Scale bar 50 µm. (b) B16F0 cells exposed to 0.2 µM Pc13 were irradiated and then incubated for 1 h or 3 h. Cathepsin D was detected in cytosolic fractions by Western blot assay and actin was used as loading control. Results from one representative experiment are shown and densitometric analyses correspond to mean ± S.E.M. of three different experiments. ANOVA-Dunnet **p < 0.01, significantly different from control (irradiated cells without Pc13) (PDF 146 KB)

References

  1. 1.
    Fecher LA, Cummings SD, Keefe MJ, Alani RM (2007) Toward a molecular classification of melanoma. J Clin Oncol 25(12):1606–1620Google Scholar
  2. 2.
    Ferlay J, Soerjomataram I, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray F (2015) Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 136(5):E359–E386Google Scholar
  3. 3.
    Ossio R, Roldán-Marín R, Martínez-Said H, Adams DJ, Robles-Espinoza CD (2017) Melanoma: a global perspective. Nat Rev Cancer 17(7):393–394Google Scholar
  4. 4.
    Davies H, Bignell GR, Cox C, Stephens P, Edkins S, Clegg S, Teague J, Woffendin H, Garnett MJ, Bottomley W, Davis N, Dicks E, Ewing R, Floyd Y, Gray K, Hall S, Hawes R, Hughes J, Kosmidou V, Menzies A, Mould C, Parker A, Stevens C, Watt S, Hooper S, Wilson R, Jayatilake H, Gusterson BA, Cooper C, Shipley J, Hargrave D, Pritchard-Jones K, Maitland N, Chenevix-Trench G, Riggins GJ, Bigner DD, Palmieri G, Cossu A, Flanagan A, Nicholson A, Ho JW, Leung SY, Yuen ST, Weber BL, Seigler HF, Darrow TL, Paterson H, Marais R, Marshall CJ, Wooster R, Stratton MR, Futreal PA (2002) Mutations of the BRAF gene in human cancer. Nature 417(6892):949–954Google Scholar
  5. 5.
    Rajkumar S, Watson IR (2016) Molecular characterisation of cutaneous melanoma: creating a framework for targeted and immune therapies. Br J Cancer 115(2):145–155Google Scholar
  6. 6.
    Bollag G, Hirth P, Tsai J, Zhang J, Ibrahim PN, Cho H, Spevak W, Zhang C, Zhang Y, Habets G, Burton EA, Wong B, Tsang G, West BL, Powell B, Shellooe R, Marimuthu A, Nguyen H, Zhang KY, Artis DR, Schlessinger J, Su F, Higgins B, Iyer R, D’Andrea K, Koehler A, Stumm M, Lin PS, Lee RJ, Grippo J, Puzanov I, Kim KB, Ribas A, McArthur GA, Sosman JA, Chapman PB, Flaherty KT, Xu X, Nathanson KL, Nolop K (2010) Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 467:596–599Google Scholar
  7. 7.
    Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, Powderly JD, Carvajal RD, Sosman JA, Atkins MB, Leming PD, Spigel DR, Antonia SJ, Horn L, Drake CG, Pardoll DM, Chen L, Sharfman WH, Anders RA, Taube JM, McMiller TL, Xu H, Korman AJ, Jure-Kunkel M, Agrawal S, McDonald D, Kollia GD, Gupta A, Wigginton JM, Sznol M (2012) Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 366(26):2443–2454Google Scholar
  8. 8.
    Wong DJ, Ribas A (2016) Targeted therapy for melanoma. Cancer Treat Res 167:251–262Google Scholar
  9. 9.
    Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H, Cho KH, Aiba S, Bröcker EB, LeBoit PE, Pinkel D, Bastian BC (2005) Distinct sets of genetic alterations in melanoma. N Engl J Med 353(20):2135–2147Google Scholar
  10. 10.
    Chapman PB, Hauschild A, Robert C, Haanen JB, Ascierto P, Larkin J, Dummer R, Garbe C, Testori A, Maio M, Hogg D, Lorigan P, Lebbe C, Jouary T, Schadendorf D, Ribas A, O’Day SJ, Sosman JA, Kirkwood JM, Eggermont AM, Dreno B, Nolop K, Li J, Nelson B, Hou J, Lee RJ, Flaherty KT, McArthur GA (2011) BRIM-3 Study Group. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N Engl J Med 364(26):2507–2516Google Scholar
  11. 11.
    Hugo W, Zaretsky JM, Sun L, Song C, Moreno BH, Hu-Lieskovan S, Berent-Maoz B, Pang J, Chmielowski B, Cherry G, Seja E, Lomeli S, Kong X, Kelley MC, Sosman JA, Johnson DB, Ribas A, Lo RS (2016) Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma. Cell 165(1):35–44Google Scholar
  12. 12.
    Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, Moan J, Peng Q (1998) Photodynamic therapy. J Natl Cancer Inst 90:889–905Google Scholar
  13. 13.
    Agostinis P, Berg K, Cengel KA, Foster TH, Girotti AW, Gollnick SO, Hahn SM, Hamblin MR, Juzeniene A, Kessel D, Korbelik M, Moan J, Mroz P, Nowis D, Piette J, Wilson BC, Golab J (2011) Photodynamic therapy of cancer: an update. CA Cancer J Clin 61(4):250–281Google Scholar
  14. 14.
    Castano AP, Demidova TN, Hamblin MR (2004) Mechanisms in photodynamic therapy. Part one: photosensitizers, photochemistry and cellular localization. Photodiag Photodyn Ther 1:279–293Google Scholar
  15. 15.
    van Straten D, Mashayekhi V, de Bruijn HS, Oliveira S, Robinson DJ (2017) Oncologic photodynamic therapy: basic principles, current clinical status and future directions. Cancers 9(2):1–54Google Scholar
  16. 16.
    Hasan T, Ortel B, Moor A, Pogue B (2003) Holland-Frei cancer medicine, eds Kufe, D. et al. Ch. 40 BC Decker, Inc., Hamilton, OntarioGoogle Scholar
  17. 17.
    Dolmans DE, Fukumura D, Jain RK (2003) Photodynamic therapy for cancer. Nat Rev Cancer 3(5):380–387Google Scholar
  18. 18.
    Castano AP, Mroz P, Hamblin MR (2006) Photodynamic therapy and anti-tumour immunity. Nat Rev Cancer 6:535–545Google Scholar
  19. 19.
    Dimofte A, Zhu TC, Hahn SM, Lustig RA (2002) In vivo light dosimetry for motexafin lutetium-mediated PDT of breast cancer. Lasers Surg Med 31:305–312Google Scholar
  20. 20.
    Rosenthal MA, Kavar B, Hill JS, Morgan DJ, Nation RL, Stylli SS, Basser RL, Uren S, Geldard H, Green MD, Kahl SB, Kaye AH (2001) Phase I and pharmacokinetic study of photodynamic therapy for high-grade gliomas using a novel boronated porphyrin. J Clin Oncol 19(2):519–524Google Scholar
  21. 21.
    Bown SG, Rogowska AZ, Whitelaw DE, Lees WR, Lovat LB, Ripley P, Jones L, Wyld P, Gillams A, Hatfield AWR (2002) Photodynamic therapy for cancer of the pancreas. Gut 50:549–557Google Scholar
  22. 22.
    Favilla I, Favilla ML, Gosbell AD, Barry WR, Ellims P, Hill JS, Byrne JR (1995) Photodynamic therapy: a 5-year study of its effectiveness in the treatment of posterior uveal melanoma, and evaluation of haematoporphyrin uptake and photocytotoxicity of melanoma cells in tissue culture. Melanoma Res 5(5):355–364Google Scholar
  23. 23.
    Sibata CH, Colussi VC, Oleinick NL, Kinsella TJ (2001) Photodynamic therapy in oncology. Expert Opin Pharmacother (6):917–927Google Scholar
  24. 24.
    Braathen LR, Szeimies RM, Basset-Seguin N et al (2007) Guidelines on the use of photodynamic therapy for nonmelanoma skin cancer: an international consensus. International Society for Photodynamic Therapy in Dermatology, 2005. J Am Acad Dermatol 56:125–143Google Scholar
  25. 25.
    Brown SB, Brown EA, Walker I (2004) The present and future role of photodynamic therapy in cancer treatment. Lancet Oncol 5(8):497–508Google Scholar
  26. 26.
    Davids LM, Kleemann B, Kacerovská D, Pizinger K, Kidson SH (2008) Hypericin phototoxicity induces different modes of cell death in melanoma and human skin cells. J Photochem Photobiol B 91(2–3):67–76Google Scholar
  27. 27.
    Wainwright M (2008) Photodynamic therapy: the development of new photosensitisers. Anticancer Agents Med Chem 8(3):280–291Google Scholar
  28. 28.
    O’Connor AE, Gallagher WM, Byrne AT (2009) Porphyrin and nonporphyrin photosensitizers in oncology: preclinical and clinical advances in photodynamic therapy. Photochem Photobiol 85:1053–1074Google Scholar
  29. 29.
    Mfouo-Tynga I, Abrahamse H (2015) Cell death pathways and phthalocyanine as an efficient agent for photodynamic cancer therapy. Int J Mol Sci 16(5):10228–10241Google Scholar
  30. 30.
    Tudor D, Nenu I, Filip GA, Olteanu D, Cenariu M, Tabaran F, Ion RM, Gligor L, Baldea I (2017) Combined regimen of photodynamic therapy mediated by Gallium phthalocyanine chloride and Metformin enhances anti-melanoma efficacy. PLoS ONE 12(3):e0173241 (eCollection 2017)Google Scholar
  31. 31.
    Marino J, García Vior MC, Dicelio LE, Roguin LP, Awruch J (2010) Photodynamic effects of isosteric water-soluble phthalocyanines on human nasopharynx KB carcinomacells. Eur J Med Chem 45:4129–4139Google Scholar
  32. 32.
    Dysart JS, Patterson MS (2005) Characterization of photofrin photobleaching for singlet oxygen dose estimation during photodynamic therapy of mll cells in vitro. Phys Med Biol 50:2597–2616Google Scholar
  33. 33.
    Abrahamse H, Hamblin MR (2016) New photosensitizers for photodynamic therapy. Biochem J 473(4):347–364Google Scholar
  34. 34.
    Marino J, García Vior MC, Furmento VA, Blank VC, Awruch J, Roguin LP (2013) Lysosomal and mitochondrial permeabilization mediates zinc(II) cationic phthalocyanine phototoxicity. Int J Biochem Cell Biol 45(11):2553–2562Google Scholar
  35. 35.
    Chan FK, Moriwaki K, De Rosa MJ (2013) Detection of necrosis by release of lactate dehydrogenase activity. Methods Mol Biol 979:65–70Google Scholar
  36. 36.
    Adler J, Parmryd I (2010) Quantifying colocalization by correlation: the Pearson correlation coefficient is superior to the Mander’s overlap coefficient. Cytometry A 77(8):733–742Google Scholar
  37. 37.
    Kasibhatla S, Amarante-Mendes GP, Finucane D, Brunner T, Bossy-Wetzel E, Green DR (2006) Acridine orange/ethidium bromide (AO/EB) staining to detect apoptosis. CSH Protoc.  https://doi.org/10.1101/pdb.prot4493 Google Scholar
  38. 38.
    Liu K, Liu PC, Liu R, Wu X (2015) Dual AO/EB staining to detect apoptosis in osteosarcoma cells compared with flow cytometry. Med Sci Monit Basic Res 21:15–20Google Scholar
  39. 39.
    Kågedal K, Zhao M, Svensson I, Brunk UT (2001) Sphingosine-induced apoptosis is dependent on lysosomal proteases. Biochem J 359(Pt 2):335–343Google Scholar
  40. 40.
    Martinou JC, Youle RJ (2011) Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. Dev Cell 21(1):92–101Google Scholar
  41. 41.
    Kim H, Du F, Fang M, Wang X (2005) Formation of apoptosome is initiated by cytochrome c-induced dATP hydrolysis and subsequent nucleotide exchange on Apaf-1. Proc Natl Acad Sci USA 102(49):17545–17550Google Scholar
  42. 42.
    Huang YY, Vecchio D, Avci P, Yin R, Garcia-Diaz M, Hamblin MR (2013) Melanoma resistance to photodynamic therapy: new insights. Biol Chem 394(2):239–250Google Scholar
  43. 43.
    Sharma KV, Bowers N, Davids LM (2011) Photodynamic therapy-induced killing is enhanced in depigmented metastatic melanoma cells. Cell Biol Int 35:939–944Google Scholar
  44. 44.
    Suzukawa AA, Vieira A, Winnischofer SM, Scalfo AC, Di Mascio P, Da Costa Ferreira AM, Ravanat JL, De Luna Martins D, Rocha ME, Martinez GR (2012) Novel properties of melanins include promotion of DNA strand breaks, impairment of repair, and reduced ability to damage DNA after quenching of singlet oxygen. Free Radic Biol Med 52:1945–1953Google Scholar
  45. 45.
    Baldea I, Ion RM, Olteanu DE, Nenu I, Tudor D, Filip AG (2015) Photodynamic therapy of melanoma using new, synthetic porphyrins and phthalocyanines as photosensitisers—a comparative study. Clujul Med 88(2):175–180Google Scholar
  46. 46.
    Bucheit AD, Davies MA (2014) Emerging insights into resistance to BRAF inhibitors in melanoma. Biochem Pharmacol 87(3):381–389Google Scholar
  47. 47.
    Lei W, Xie J, Hou Y, Jiang G, Zhang H, Wang P, Wang X, Zhang B (2010) Mitochondria-targeting properties and photodynamic activities of porphyrin derivatives bearing cationic pendant. J Photochem Photobiol B 98(2):167–171Google Scholar
  48. 48.
    Kramer-Marek G, Serpa C, Szurko A, Widel M, Sochanik A, Snietura M, Kus P, Nunes RM, Arnaut LG, Ratuszna A (2006) Spectroscopic properties and photodynamic effects of new lipophilic porphyrin derivatives: efficacy, localisation and cell death pathways. J Photochem Photobiol B 84(1):1–14Google Scholar
  49. 49.
    Buytaert E, Dewaele M, Agostinis P (2007) Molecular effectors of multiple cell death pathways initiated by photodynamic therapy. Biochim Biophys Acta 1776(1):86–107Google Scholar
  50. 50.
    Ezzeddine R, Al-Banaw A, Tovmasyan A, Craik JD, Batinic-Haberle I, Benov LT (2013) Effect of molecular characteristics on cellular uptake, subcellular localization, and phototoxicity of Zn(II) N-alkylpyridylporphyrins. J Biol Chem 288(51):36579–36588Google Scholar
  51. 51.
    Kessel D, Oleinick NL (2018) Cell death pathways associated with photodynamic therapy: an update. Photochem Photobiol 94(2):213–218Google Scholar
  52. 52.
    Ricchelli F, Franchi L, Miotto G, Borsetto L, Gobbo S, Nikolov P, Bommer JC, Reddi E (2005) Meso-substituted tetra-cationic porphyrins photosensitize the death of human fibrosarcoma cells via lysosomal targeting. Int J Biochem Cell Biol 37(2):306–319Google Scholar
  53. 53.
    Engelmann FM, Rocha SV, Toma HE, Araki K, Baptista MS (2007) Determination of n-octanol/water partition and membrane binding of cationic porphyrins. Int J Pharm 329:12–18Google Scholar
  54. 54.
    Perry SW, Norman JP, Barbieri J, Brown EB, Gelbard HA (2011) Mitochondrial membrane potential probes and the proton gradient: a practical usage guide. Biotechniques 50(2):98–115Google Scholar
  55. 55.
    Kessel D, Luo YJ (1998) Mitochondrial photodamage and PDT-induced apoptosis. Photochem Photobiol B 42(2):89–95Google Scholar
  56. 56.
    Oleinick NL, Morris RL, Belichenko I (2002) The role of apoptosis in response to photodynamic therapy: what, where, why, and how. Photochem Photobiol Sci 1(1):1–21Google Scholar
  57. 57.
    Kessel D, Castelli M (2001) Evidence that bcl-2 is the target of three photosensitizers that induce a rapid apoptotic response. Photochem Photobiol 74(2):318–322Google Scholar
  58. 58.
    Xue LY, Chiu SM, Oleinick NL (2001) Photochemical destruction of the Bcl-2 oncoprotein during photodynamic therapy with the phthalocyanine photosensitizer Pc 4. Oncogene 20(26):3420–3427Google Scholar
  59. 59.
    Fabris C, Valduga G, Miotto G, Borsetto L, Jori G, Garbisa S, Reddi E (2001) Photosensitization with zinc (II) phthalocyanine as a switch in the decision between apoptosis and necrosis. Cancer Res 61(20):7495–7500Google Scholar
  60. 60.
    Luo Y, Kessel D (1997) Initiation of apoptosis versus necrosis by photodynamic therapy with chloroaluminum phthalocyanine. Photochem Photobiol 66(4):479–483Google Scholar
  61. 61.
    Nagata S, Obana A, Gohto Y, Nakajima S (2003) Necrotic and apoptotic cell death of human malignant melanoma cells following photodynamic therapy using an amphiphilic photosensitizer, ATX-S10(Na). Lasers Surg Med 33(1):64–70Google Scholar
  62. 62.
    Oleinick NL, Nieminen AL, Chiu SM (2008) Cell killing by photodynamic therapy. In: Hamblin MR, Mroz P (eds) Advances in photodynamic therapy: basic, translational and clinical. Artech House, Boston, pp 115–133Google Scholar
  63. 63.
    Sharma KV, Davids LM (2012) Hypericin-PDT-induced rapid necrotic death in human squamous cell carcinoma cultures after multiple treatment. Cell Biol Int 36(12):1261–1266Google Scholar
  64. 64.
    Igney FH, Krammer PH (2002) Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer (4):277–288Google Scholar
  65. 65.
    Garg AD, Krysko DV, Vandenabeele P, Agostinis P (2011) DAMPs and PDT-mediated photo-oxidative stress: exploring the unknown. Photochem Photobiol Sci 10(5):670–680Google Scholar
  66. 66.
    Korbelik M (1996) Induction of tumor immunity by photodynamic therapy. J Clin Laser Med Surg 14(5):329–334Google Scholar

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Authors and Affiliations

  1. 1.Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológicas (IQUIFIB)Universidad de Buenos Aires, CONICET-UBABuenos AiresArgentina
  2. 2.Departamento de Química Orgánica, Facultad de Farmacia y BioquímicaUniversidad de Buenos Aires, CONICETBuenos AiresArgentina

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