Cytotoxic effect and apoptosis pathways activated by methylene blue-mediated photodynamic therapy in fibroblasts

• Published in: Photodiagnosis and photodynamic therapy 2020-03, Vol.29, p.101654, Article 101654
• Main Authors: Campos Chaves Lamarque, Giuliana, Cusicanqui Méndez, Daniela Alejandra, Arruda Matos, Adriana, José Dionísio, Thiago, Andrade Moreira Machado, Maria Aparecida, Magalhães, Ana Carolina, Cardoso Oliveira, Rodrigo, Cruvinel, Thiago
• Format: Article
• Language: English
• Subjects: Animals; Antimicrobial Photodynamic therapy; Apoptosis; Apoptosis - drug effects; Apoptosis - genetics; Bcl-2; Cell Survival; Cell viability; Fibroblasts - drug effects; Genes, bcl-2; In Vitro Techniques; Methylene Blue; Methylene Blue - pharmacology; Methylene Blue - toxicity; Mice; Photochemotherapy; Photochemotherapy - methods; Photosensitizing Agents - pharmacology; Photosensitizing Agents - toxicity
• ISSN: 1572-1000; 1873-1597
• DOI: 10.1016/j.pdpdt.2020.101654

•MB-mediated aPDT induced a significant dose-dependent cytotoxic effect on mouse fibroblasts.•The apoptosis mechanisms were related to mitochondrial photodamage.•The cytotoxicity occurred by the activation of Bcl-2 apoptotic genes. Antimicrobial photodynamic therapy (aPDT) has been used as an adjuvant treatment of oral infections as a minimal intervention clinical approach. Its antimicrobial efficacy was demonstrated in several studies; however, there is a lack of evidence on its cytotoxic effect on mouse fibroblasts (NIH/3T3). The aim of this study was to evaluate the cytotoxicity and apoptotic pathways of methylene blue-mediated aPDT on mouse fibroblasts. Cells were treated with 0.1 or 1.0 mg.L−1 methylene blue (MB), and 0.075 or 7.5 J.cm-² LED at 630 nm. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet (CV) assays, while cDNA expression for Bax, Bad, Bcl-2, VDAC-1, cytochrome C and Fas-L was assessed by qRT-PCR (1, 3, 6 and 24 h). The differences between groups were detected by Kruskal-Wallis and post-hoc Dunn's tests for MTT and CV assays, and by ANOVA and post-hoc Tukey test for qPCR (P < 0.05). The combination of 1.0 mg.L−1 MB and 7.5 J.cm-² LED significantly reduced the cellular viability, whereas MB and LED alone were innocuous to fibroblasts. MB-mediated aPDT increased the expression of cytochrome C and Fas-L after 3 h, and Bax/Bcl-2, Bad/Bcl-2, and VDAC-1 after 6 h from treatment. Based on these results, MB-mediated aPDT induced cytotoxicity on mouse fibroblasts, with consequent activation of Bcl-2 apoptosis signaling pathways. Further studies are needed to determine the adequate parameters of aPDT to inactivate microorganisms without damaging fibroblasts.