Eficienta antitumorala a terapieifotodinamice mediate de o mesoporfirinade sinteza pe modele de melanom in vitro

Ioana Baldea, Diana Olteanu, FlaviuTabaran, Rodica Mariana Ion, Pompei Bolfa, Mihai Cenariu , Nicoleta Decea, RemusOrăsan, Adriana Filip

Purpose

� To study in vitro the mechanism of action and efficacy of a synthetic porphyrin: meso-5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin (THOPP) as photosensitizer for PDT, against cocultures of 3 melanoma cell lines: WM35 (human, RGP), M1-15 (human, metastatic), B16-F10 (mouse, metastatic) and endotelial cells: HUVEC cell line.

Introduction

Experimental model

Utilility/efficiency of THOPP mediated PDT against WM35 melanoma cells in culture

Method and Material

� Melanoma cells: WM35 - (Wistar Institute, Philadelphia, PA, USA), M1-15-donated by dr Eva Fisher- Fodor, Oncology Institute, Cluj, B16-F10, HUVEC (Promocell, Hamburg, Germany)

� Irradiation was done with red light (wave length 630 nm, lamp power 2.5mW/cm2) provided by a Philips LED red light system, with dose of 6 J/cm2.

� 1. Cytotoxicity assay – MTS (Promega)� 2. Cell death investigation � - Flow cytometric analysis Annexin V-fluorescein isothiocyanate (FITC)+propidium iodide

(PI) (BD Pharmingen Biosciences). � - ELISA � - caspase-3 (CPP32) and tumor necrosis factor alpha (TNFα), Superoxide

dismutase (SOD), sICAM1 Immunoassay kits from R&D Systems� - Western blotting � - NFkB, pNFkB (Santa Cruz Biotechnology), γH2AX (Stressgen Bioreagents) and

GADPH (Trevigen Biotechnology). � -Mitochondrial lesions� MitoID Kit (Invitrogen)

� Statistical method� two-way ANOVA and paired Student TTEST, followed by Bonferroni post test using GraphPad;

results were considered significant for p≤0.05.

Results

Cell viability

WM35 M1-15 B16-F10

FACS results

Apoptosis/ necrosis in

WM35 and M1-15

necrosis B16-F10

Apoptosis mechanism

� Mithocondrial

� Necroptosis

� Mediat de γH2Ax

Oxidative stress

NF-kB level increased

pNFkB not significantly modified

Angiogenesis

sICAM1 increased in WM35 and M1-15;

decreased VEGF-inhibition of angiogenesis

Conclusion

� PDT mediated by THOPP was efficient in inducing a dose related melanoma cell killing, with no dark toxicity at effective doses

� The best results in what concerns the efficiency of tumor cell killing were obtained in low pigmented WM35, followed by M1-15 human melanomas, while B16-F10 murinpigmented melanoma seems to be more resistant to THOPP mediated PDT.

References

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