bacteriochlorophylls and Melanoma

Reactive oxygen species (ROS) comprise a group of noxious byproducts of oxidative processes which participate in the induction of many common diseases. However, understanding their role in the regulation of normal physiological redox signaling is currently evolving. Detailed study of the dynamic functions of ROS within the biological milieu is difficult because of their high chemical reactivity, short lifetime, minute concentrations and cytotoxicity at high concentrations. In this study, we show that increasing intracellular ROS levels, set off by controlled in situ photogeneration of a nontoxic bacteriochlorophyll-based sensitizer initiate responses in cultured melanoma cells. Using hydroethidine as detector, we determined light-dependent generation of superoxide and hydroxyl radicals in cell-free and cell culture models. Monitoring the ROS-induced responses revealed individual and differential behavior of protein kinases [p38, mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and Akt] as well as effects on the subcellular distribution of phosphorylated p38. Furthermore, alterations in morphology and motility and effects on cell viability as a function of time and photosensitizer doses were observed. Following mild ROS challenge, enzymatic and cellular changes were observed in the majority of the cells, without inducing extensive cell death. However, upon vigorous ROS challenge, a similar profile of the overall responses was observed, terminating in cell death. This study shows that precisely controlled photogeneration of ROS can provide simple, fine-tuned, noninvasive manipulation of ROS-sensitive cellular responses ranging from individual enzymes to gross behavior of target cells. The observations made with this tool enable a dynamic and causal correlation, presenting a new alternative for studying the role of ROS in cellular redox signaling.

Topics: Animals; Bacteriochlorophylls; Cell Line, Tumor; Cell Survival; Cell-Free System; Cells, Cultured; Dose-Response Relationship, Drug; Extracellular Signal-Regulated MAP Kinases; Flow Cytometry; Hydrogen Peroxide; Image Processing, Computer-Assisted; Immunoprecipitation; Iron; JNK Mitogen-Activated Protein Kinases; Light; MAP Kinase Signaling System; Melanoma; Mice; Microscopy, Fluorescence; Microscopy, Video; Neutral Red; Oxidation-Reduction; Oxygen; p38 Mitogen-Activated Protein Kinases; Phenanthridines; Phosphorylation; Photochemotherapy; Propidium; Protein Binding; Reactive Oxygen Species; Serine; Signal Transduction; Spectrometry, Fluorescence; Spectrophotometry; Time Factors

Antivascular photodynamic therapy (PDT) of tumors with palladium-bacteriopheophorbide (TOOKAD) relies on in situ photosensitization of the circulating drug by local generation of cytotoxic reactive oxygen species, which leads to rapid vascular occlusion, stasis, necrosis and tumor eradication. Intravascular production of reactive oxygen species is associated with photoconsumption of O(2) and consequent evolution of paramagnetic deoxyhemoglobin. In this study we evaluate the use of blood oxygenation level-dependent (BOLD) contrast magnetic resonance imaging (MRI) for real-time monitoring of PDT efficacy. Using a solid tumor model, we show that TOOKAD-PDT generates appreciable attenuation (25-40%) of the magnetic resonance signal, solely at the illuminated tumor site. This phenomenon is independent of, though augmented by, ensuing changes in blood flow. These results were validated by immunohistochemistry and intravital microscopy. The concept of photosensitized BOLD-contrast MRI may have intraoperative applications in interactive guidance and monitoring of antivascular cancer therapy, PDT treatment of macular degeneration, interventional cardiology and possibly other biomedical disciplines.

Topics: Animals; Bacteriochlorophylls; Contrast Media; Disease Models, Animal; Hemoglobins; Humans; Image Processing, Computer-Assisted; Magnetic Resonance Imaging; Male; Melanoma; Mice; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species; Skin Neoplasms