, 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


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.


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



Acridine orange


2′,7′-Dichlorodihydrofluorescein diacetate


3,3′-Dihexyloxacarbocyanine iodide


Ethidium bromide


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


Lactate dehydrogenase


Lysosomal membrane permeabilization


Mitochondrial transmembrane potential




Photodynamic therapy




Poly(ADP-ribose) polymerase


Propidium iodide


Reactive oxygen species


Singlet oxygen



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)


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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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