Photobiomodulation and nitric oxide as strategies to potentiate the antitumor action of photodynamic therapy mediated by ruthenium-phthalocyanines: Chemical, biochemical and photobiological aspects

Author(s)

Laísa Bonafim Negri

• Document type: Doctoral Thesis
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-09-20

Examining board members

Roberto Santana da Silva; Mauricio da Silva Baptista

• Advisor: Roberto Santana da Silva

Abstract

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 aims to present strategies that can potentiate the tumor cell death elicited by PDT, avoiding cellular fragment survival and possible disease recurrence. One of the possibilities is to develop photosensitizers that can produce not only ROS, but also nitric oxide (NO), a radical molecule that underlies oxidative and nitrosative cellular stress and which is an important biological messenger that plays a dual role in regulating biochemical pathways controlling cell recovery. This work has improved the synthesis the ruthenium-phthalocyanine photosensitizers trans- [RuCl(Pc)(DMSO)] and trans-[Ru(NO)(Pc)(NO2)], which were characterized by different analytical techniques such as UV-Vis, Infrared, NMR, EPR, and mass spectrometry. Together, these techniques helped to evaluate the efficiency of the employed synthetic routes. Photochemical and photophysical aspects were examined in terms of ERO and NO production by light irradiation at 660 nm and reductimetric processes. The cytotoxicity of the photosensitizers was assessed in both the absence and presence of irradiation at 660 nm against the tumor lines B16F10, A375, and MCF7 and the non-tumor cell lines 3T3, L929, and MCF10. The synergistic effect between NO and ERO elicited by trans- [Ru(NO)(Pc)(NO2)] photostimulation was evaluated and compared to the cytotoxic effect of trans-[RuCl(Pc)(DMSO)]--when photostimulated, the latter complex produces only ROS. The cell viability studies showed that 1.0 µM trans- [RuCl(Pc)(DMSO)] and trans- [Ru(NO)(Pc)(NO2)] complexes under 3.0 J.cm-2 PDT promoted 63% and 90% A375 cell death and 64% and 72% MCF7 cell death, respectively. The studies were extended regarding the relevance of the photosensitizer cellular sub-localization as assessed by confocal microscopy with different biological probes. The results implied cell death. The cell death mechanism was investigated by Western blot, which revealed expression of caspase-3-cleaved proteins, BAX, and cytochrome c when the tumor cells were submitted to PDT in the presence of the synthesized photosensitizers. This suggested that apoptosis was stimulated by the intrinsic pathway. The NO synergistic effect NO on PDT was studied by evaluating the role that NO plays in cell recurrence by the Scratch Wound assays and the regulation of survival mechanisms related to the expression of the anti-apoptotic transcription factor NFkB. NO appeared to inhibit NF-kB, suggesting that the synergistic effect was due to proapoptotic protein stimulation and anti-apoptotic protein inhibition. Another strategy that seemed to potentiate PDT was double wavelength photostimulation by using a new therapy called Photobiomodulation (PBM) at 850 nm, followed by PDT at 660 nm. The way PBM can modulate the signaling pathway via ROS, ATP, and NO production and how the photosensitizer cellular internalization combined with PDT favors the synergistic effect on cell death are also discussed. (AU)

• FAPESP's process: 15/03746-9 - Chemo-phototherapeutic process of phthalocyanine-ruthenium system and cisplatin: chemical and photobiological evaluation in aqueous solution and in drug delivery system
• Grantee: Laísa Bonafim Negri
• Support Opportunities: Scholarships in Brazil - Doctorate