The online version of this article (doi:10.1186/s12885-017-3179-7) contains supplementary material, which is available to authorized users.
Breast cancer is the main cause of mortality among women. The disease presents high recurrence mainly due to incomplete efficacy of primary treatment in killing all cancer cells. Photodynamic therapy (PDT), an approach that causes tissue destruction by visible light in the presence of a photosensitizer (Ps) and oxygen, appears as a promising alternative therapy that could be used adjunct to chemotherapy and surgery for curing cancer. However, the efficacy of PDT to treat breast tumours as well as the molecular mechanisms that lead to cell death remain unclear.
In this study, we assessed the cell-killing potential of PDT using methylene blue (MB-PDT) in three breast epithelial cell lines that represent non-malignant conditions and different molecular subtypes of breast tumours. Cells were incubated in the absence or presence of MB and irradiated or not at 640 nm with 4.5 J/cm2. We used a combination of imaging and biochemistry approaches to assess the involvement of classical autophagic and apoptotic pathways in mediating the cell-deletion induced by MB-PDT. The role of these pathways was investigated using specific inhibitors, activators and gene silencing.
We observed that MB-PDT differentially induces massive cell death of tumour cells. Non-malignant cells were significantly more resistant to the therapy compared to malignant cells. Morphological and biochemical analysis of dying cells pointed to alternative mechanisms rather than classical apoptosis. MB-PDT-induced autophagy modulated cell viability depending on the cell model used. However, impairment of one of these pathways did not prevent the fatal destination of MB-PDT treated cells. Additionally, when using a physiological 3D culture model that recapitulates relevant features of normal and tumorous breast tissue morphology, we found that MB-PDT differential action in killing tumour cells was even higher than what was detected in 2D cultures.
Finally, our observations underscore the potential of MB-PDT as a highly efficient strategy which could use as a powerful adjunct therapy to surgery of breast tumours, and possibly other types of tumours, to safely increase the eradication rate of microscopic residual disease and thus minimizing the chance of both local and metastatic recurrence.
Additional file 1: Figure S1. MB-PDT induced massive cell death in MDA-MB-231 without dark cytotoxicity. (a) Representative graph showing the viability of MCF-10A, MCF-7 and MDA-MB-231 cells after MB-PDT with 20 μM of MB followed by 4.5 J/cm2 irradiation and the experimental controls: irradiation alone (λ); dark toxicity of MB, that means incubation without irradiation (MB); or not treated cells (control). These results were obtained after 24 h (n = 3 independent experiments); *: p < 0.05 versus control. (b) Viability of MDA-MB-231 cells after MB-PDT with MB (20 μM) followed by 4.5 J/cm2 or 1.5 J/cm2 irradiation after 24 h. Results are shown as mean ± s.e.m. (n = 3 independent experiments); *: p < 0.05 versus 4.5 J/cm2. Figure S2. MB is preferentially localized in the lysosomes. (a) Low magnification images of the data shown in Fig. 5 of the cells simultaneously incubated with LysoTracker green (LysoTG) and MB. Merged images of the following two images (yellow; left column). MB Fluorescence (red; middle column) and fluorescence arising from LysoTracker (green; right column). ( b) Confocal microscopy images of the cells simultaneously incubated with MitoTracker green (MitoTG) and MB. Merged images of the following two images (yellow; left column). MB Fluorescence (red; middle column) and fluorescence arising from MitoTracker (green; right column). Hoechst 33342, indicating nuclei. [MB] = 20 μM; [LysoTG] = 300 nM; [MitoTG] = 300 nM; nucleus (HO 3334 = 300 nM). Size bar: 10μm. Figure S3. No evidences of apoptotic nuclei after MB-PDT. ( a) Representative nuclei of human mammary cells treated for 1 h, 3 h and 24 h with MB-PDT with 0.2 μM of MB. Nucleus stained with Hoechst 33342 (blue) and propidium iodide (red). Size bar: 20μm ( b) Viability time curves after MB-PDT of cell cultures with 2 (a) or 20 μM of MB (b) followed by of 4.5 J/cm2 irradiation obtained after 1 h, 3 h and 24 h post-irradiation (n = 3 independent experiments) * p < 0.05 versus MCF-10A. Results are shown as mean ± s.e.m. (DOCX 2074 kb)
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- Methylene blue photodynamic therapy induces selective and massive cell death in human breast cancer cells
Ancély F. dos Santos
Letícia F. Terra
Rosangela A. M. Wailemann
Talita C. Oliveira
Vinícius de Morais Gomes
Marcela Franco Mineiro
Flávia Carla Meotti
Maurício S. Baptista
- BioMed Central
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