Apoptosis induced by methylene‐blue‐mediated photodynamic therapy in melanomas and the involvement of mitochondrial dysfunction revealed by proteomics

Abstract

Methylene blue (MB) is a widely studied agent currently under investigation for its properties relating to photodynamic therapy (PDT). Recent studies have demonstrated that MB exhibits profound phototoxicity affecting a variety of tumor cell lines. However, the mechanistic explanation for methylene-blue-mediated photodynamic therapy (MB-PDT) in the context of melanoma therapy is still obscure. In the present study, B16F1 melanoma cells were treated by MB-PDT under different conditions, and thereafter subjected to cell viability detection assays. MB-PDT could induce intense apoptotic cell death through a series of steps beginning with the photochemical generation of reactive oxygen species that activate the caspase-9/ caspase-3 apoptosis pathway. Blocking activation of caspase-3 and induction of oxidative stress by caspase inhibitor and by glutathione, respectively, markedly reduced apoptotic cell death in vitro. P hotodynamic therapy (PDT) involves the systemic or local administration of a tumor-localizing photosensitizer followed by irradiation of the targeted lesion with light of the appropriate wavelength. This multistep procedure initiates a complex photochemical reaction to generate reactive oxygen species (ROS). The cytotoxic products generated by PDT trigger a cascade of molecular events that lead to apoptosis and/or necrosis of cells responsible for tumor destruction.(1,2) Therefore PDT is considered to be a promising technology approved as an alternative treatment for a diverse array of cancers, such as cervical cancer, oesophageal cancer, head and neck cancer and skin malignancies. (3,4) Melanoma is a cancer developing from malignant transformation of melanocytes, the pigment-producing cells that are found predominantly in the skin.(5) It is worth noting that melanocytes originating from highly motile cells have enhanced survival properties and are relatively resistant to drug-induced apoptosis and chemotherapy.(6,7) Previous studies have investigated the application of PDT with several photosensitizers, such as lutetium texaphyrin, (8) chlorin e 6 , (9) and 5-aminolaevulinic acid,to induce cell death of melanocytes in vivo and in vitro. Although initial treatment responses after PDT are routinely positive; the difficulty in achieving good light excitation of these sensitizers and ethical considerations concerning the aggressiveness of the disease hinder the development of the application of PDT for treating melanomas. Methylene blue (MB), from the phenothiazinium family, is a photosensitizer which has excellent photochemical properties. MB is well known to have a high quantum yield of intersystem crossing and singlet oxygen ( 1 O 2 ) generation, and can produce radical species in the presence of reducing agents.(11,12) Importantly, MB possesses exceptional affinity to melanocyteproduced melanin, which contributes to selective absorption of this compound by cutaneous melanomas.(13) Compared with other photosensitizers, MB showed enhanced penetration efficiency into melanoma cells. It has been reported ...

Photodynamic therapy: illuminating the road from cell death towards anti-tumour immunity

“…Here, DCs being the principle antigen-presenting cells could take up the tumourassociated antigens (TAAs), become mature and present the same to the cells adaptive immune system in order to instigate an anti-tumour immune response. Also, the production of various cytokines and chemokines in the tumour microenvironment plays an important role in modulating a plethora of innate and adaptive immune processes Apoptosis (2010) 15:1050-1071 1051 which have been made available in the last years have confirmed indeed that apoptosis is a major pathway of PDT-mediated cell death in vivo, thus justifying major efforts bestowed in understanding the biochemistry of the apoptotic photokilling process [7][8][9][10]. The mechanisms and signalling pathways activated by PDT are remarkably complex and have been the subjects of recent extensive reviews [1,11,12].…”

Can Photodynamic Therapy Be an Alternative Method in Melanoma Treatment?

“…Marchetti et al demonstrated that Ph is a ligand for the mitochondrial peripheral benzodiazepine receptor, which is responsible for triggering pore transition , Marchetti et al, 1996. The mitochondrial apoptotic pathway was also shown by Chen et al in therapy with methylene blue (MB) (Chen et al, 2008). It is a photosensitizer which has excellent photochemical properties.…”

“…MB showed enhanced penetration efficiency into melanoma cells and also in mitochondrial membranes, which induced apoptotic cell death by causing mitochondrial dysfunction (Ball et al, 1998). Chen et al showed that MB-PDT could induce apoptotic cell death through the photochemical generation of reactive oxygen species that activate the caspase-9 and caspase-3 apoptosis pathway (Chen et al, 2008). A transplantable mouse melanoma model showed that the tumor size in treated mice decreased, which was associated with enhanced apoptotic cell death.…”

Mycoplasma Removal from Cell Culture Using Antimicrobial Photodynamic Therapy

“…It was speculated that apoptosis was triggered by aPDT, because it has been reported that MB-mediated PDT can induce apoptosis to some cells. 10,11 Uninfected HEK293 cells were prepared in DME base medium and then aPDT was performed in the presence of MB (100 ng/mL) with red LED irradiation (0-480 sec). It was found that the ratio of apoptotic cells increased in a time-dependent manner, and the difference of ratio of apoptotic cells between unirradiated cells and 60 sec irradiated cells was *4%, whereas that of unirradiated cells and 120 sec irradiated cells was *13% (Fig.…”