Synergistic effect of NO and reactive oxygen species as a new approach in PDT: Photobiological and biochemical studies involving phthalocyanine-ruthenium complexes as new photosensitizer

Light irradiation has been used in clinical therapy, including Photodynamic Therapy (PDT), for several decades. PDT favors cell death by combining the effect of a photosensitizer, light, and oxygen to generate reactive oxygen species (ROS). However, when one of these components is a limiting factor, instead of dying, the treated cells can activate biochemical defense pathways that promote cell growth, causing the tumor to recur. This work presents the development of new photosensitizers able to increase oxidative and nitrosative stress in addition to PDT as a strategy to potentiate tumor cell death. We evaluate the anticancer effects of a synergistic approach using a nitrosyl ruthenium phthalocyanine, trans-[Ru(Pc)(NO)(NO₂)], that combine the production of reactive oxygen species (ROS) with the release of nitric oxide (NO) and compare it with its precursor [Ru(Pc)]. Photochemical and photophysical aspects were examined in terms of ROS and NO production by light irradiation and reductimetric processes. The cytotoxicity of the photosensitizers was assessed in both the absence and presence of irradiation against human melanoma cells, A375, and the non-cancer cells, 3 T3. The cytotoxic evaluation in A375 showed 1.0 μM of [Ru(Pc] killed 63 % while trans-[Ru(NO)(Pc)(NO₂)] eradicated 90 %, leading to a synergistic effect. The studies were extended regarding the relevance of the photosensitizer cellular sub-localization assessed by confocal microscopy, indicating their presence in the endoplasmatic reticulum (ER). The cell death biochemistry mechanism was investigated by Western blotting, which revealed expression of caspase-3-cleaved proteins, BAX, and cytochrome c, suggesting that apoptosis was stimulated by the intrinsic pathway initiated by oxidative stress in ER and mitochondria. The NO synergistic effect on PDT was studied by evaluating the cell recurrence by the Scratch Wound assay and the regulation of survival mechanisms related to the expression of the anti-apoptotic transcription factor NF-kB. NO appeared to inhibit NF-kB, suggesting that the synergistic effect was due to pro-apoptotic protein stimulation and anti-apoptotic protein inhibition.