Secondary Singlet Oxygen Research Articles

Cellular Mechanisms of Singlet Oxygen in Photodynamic Therapy

In this review, we delve into the realm of photodynamic therapy (PDT), an established method for combating cancer. The foundation of PDT lies in the activation of a photosensitizing agent using specific wavelengths of light, resulting in the generation of reactive oxygen species (ROS), notably singlet oxygen (1O2). We explore PDT’s intricacies, emphasizing its precise targeting of cancer cells while sparing healthy tissue. We examine the pivotal role of singlet oxygen in initiating apoptosis and other cell death pathways, highlighting its potential for minimally invasive cancer treatment. Additionally, we delve into the complex interplay of cellular components, including catalase and NOX1, in defending cancer cells against PDT-induced oxidative and nitrative stress. We unveil an intriguing auto-amplifying mechanism involving secondary singlet oxygen production and catalase inactivation, offering promising avenues for enhancing PDT’s effectiveness. In conclusion, our review unravels PDT’s inner workings and underscores the importance of selective illumination and photosensitizer properties for achieving precision in cancer therapy. The exploration of cellular responses and interactions reveals opportunities for refining and optimizing PDT, which holds significant potential in the ongoing fight against cancer.

Dynamics of Singlet Oxygen-Triggered, RONS-Based Apoptosis Induction after Treatment of Tumor Cells with Cold Atmospheric Plasma or Plasma-Activated Medium

Treatment of tumor cells with cold atmospheric plasma (CAP) or with plasma-activated medium (PAM) leads to a biochemical imprint on these cells. This imprint is mediated by primary singlet oxygen, which is mainly generated through the interaction between CAP-derived H2O2 and NO2−. This imprint is induced with a low efficiency as local inactivation of a few membrane-associated catalase molecules. As sustained generation of secondary singlet oxygen by the tumor cells is activated at the site of the imprint, a rapid bystander effect-like spreading of secondary singlet oxygen generation and catalase inactivation within the cell population is thus induced. This highly dynamic process is essentially driven by NOX1 and NOS of the tumor cells, and finally leads to intercellular RONS-driven apoptosis induction. This dynamic process can be studied by kinetic analysis, combined with the use of specific inhibitors at defined time intervals. Alternatively, it can be demonstrated and quantified by transfer experiments, where pretreated cells are mixed with untreated cells and bystander signaling is determined. These studies allow to conclude that the specific response of tumor cells to generate secondary singlet oxygen is the essential motor for their self-destruction, after a singlet oxygen-mediated triggering process by CAP or PAM.

Intercellular singlet oxygen-mediated bystander signaling triggered by long-lived species of cold atmospheric plasma and plasma-activated medium

Treatment of tumor cells with H2O2 and nitrite, two long-lived species derived from cold atmospheric plasma, induces a complex autoamplificatory, singlet oxygen-mediated process, which leads to catalase inactivation and reactivation of intercellular apoptosis-inducing signaling. Experimental dissection and quantification of this process is described in this study. When tumor cells were pretreated with H2O2 and nitrite, and then were added to untreated tumor cells, they propaged singlet oxygen mediated catalase inactivation and generation of singlet oxygen to the untreated cell population. This bystander effect allowed to analyze the biochemical requirements of a) induction of the bystander effect-inducing potential, b) transmission of the bystander effect to untreated neighbouring cells, and c) the biochemical consequences of these signaling events. The induction of bystander effect-inducing potential requires the generation of “primary singlet oxygen” through the reactions following the interaction between nitrite and H2O2, followed by local inactivation of a few catalase molecules. This primary effect seems to be very rare, but is efficiently enhanced by the generation of "secondary singlet oxygen" through the interaction between H2O2 and peroxynitrite at the site of inactivated catalase. Transmission of bystander signaling between pretreated and untreated tumor cells depends on the generation of secondary singlet oxygen by the pretreated cells and singlet oxygen-mediated catalase inactivation of the untreated recipient cells. This induces autoamplificatory propagation of secondary singlet oxygen generation in the population. This experimental approach allowed to quantify the efficiencies of primary and secondary singlet oxgen generation after CAP and PAM action, to dissect the system and to study the underlying chemical biology in detail. Our data confirm that CAP and PAM-derived components are merely the trigger for the activation of autoamplificatory mechanisms of tumor cells, whereas the tumor cells efficiently propagate their cell death through their own ROS/RNS signaling potential.

The synergistic effect between hydrogen peroxide and nitrite, two long-lived molecular species from cold atmospheric plasma, triggers tumor cells to induce their own cell death

Nitrite and H2O2 are long-lived species in cold atmospheric plasma and plasma-activated medium. It is known that their synergistic interaction is required for selective apoptosis induction in tumor cells that are treated with plasma-activated medium. This study shows that the interaction between nitrite and H2O2 leads to the formation of peroxynitrite, followed by singlet oxygen generation through the interaction between peroxynitrite and residual H2O2. This primary singlet oxygen causes local inactivation of few catalase molecules on the surface of tumor cells. As a consequence, H2O2 and peroxynitrite that are constantly produced by tumor cells and are usually decomposed by their protective membrane-associated catalase, are surviving at the site of locally inactivated catalase. This leads to the generation of secondary singlet oxygen through the interaction between tumor cell-derived H2O2 and peroxynitrite. This selfsustained process leads to autoamplification of secondary singlet oxygen generation and catalase inactivation. Inactivation of catalase allows the influx of H2O2 through aquaporins, leading to intracellular glutathione depletion and sensitization of the cells for apoptosis induction through lipid peroxidation. It also allows to establish intercellular apoptosis-inducing HOCl signaling, driven by active NOX1 and finalized by lipid peroxidation through hydroxyl radicals that activates the mitochondrial pathway of apoptosis. This experimentally established model is based on a triggering function of CAP and PAM-derived H2O2/nitrite that causes selective cell death in tumor cells based on their own ROS and RNS. This model explains the selectivity of CAP and PAM action towards tumor cells and is in contradiction to previous models that implicated that ROS/RNS from CAP or PAM were sufficient to directly cause cell death of tumor cells.

Signal amplification by tumor cells: Clue to the understanding of the antitumor effects of cold atmospheric plasma and plasma-activated medium

The expression of membrane-associated catalase, SOD, and NADPH oxidase represents a characteristic feature of tumor cells and a novel target for tumor therapy. An analytical approach, based on experimentally determined responses of cells from distinct steps of oncogenesis toward defined reactive oxygen/nitrogen species (ROS/RNS) is presented. It is suggested that singlet oxygen from cold atmospheric plasma (CAP) or plasma-activated medium (PAM) triggers tumor cells to generate high concentrations of secondary singlet oxygen that inactivates their protective catalase. This reactivates intercellular ROS/RNS-dependent apoptosis-inducing signaling and also may lead to aquaporin-mediated influx of extracellular H2O2. Initial targeting of either catalase, SOD or of the FAS receptor by CAP- or PAM-derived singlet oxygen causes the same final effect. The generation of singlet oxygen is based on a complex interaction between H2O2 and peroxy-nitrite. HOCl and peroxynitrite are also potentially involved in the modulation of immunogenic cell death and thus trigger a cytotoxic ${T}$ cell response that acts synergistically with the primary ROS/RNS-related effects induced by CAP or PAM. This approach might be useful to define optimal composition of CAP, to define selective CAP and PAM action, to allow for the establishment of synergistic effects and to link CAP- and PAM-related ROS/RNS effects on apoptosis with immunogenic cell death.

Interaction Between Cap- Derived Singlet Oxygen And Tumor Cell Protective Catalase: Update And Chances

Transformation of cells from various tissues requires NADPH oxidase-dependent generation of extracellular superoxide anions. These drive the proliferation, but also cause the elimination of malignant cells through the HOCl and the NO/peroxynitrite signaling pathways. These intercellular signaling pathways induce apoptosis selectively in malignant cells, due to site-specific concerted interaction of defined reactive oxygen and nitrogen species (ROS/RNS). Tumor progression requires the expression of membrane-associated catalase. This enzyme interferes with HOCl signaling through decomposition of H2O2, and with NO/peroxynitrite signaling through oxidation of NO and decomposition of peroxynitrite. Membrane-associated catalase has been found on all lines of bona fide tumor cells and represents a promising target for novel antitumor strategies. Inactivation of tumor cell-specific membrane-associated catalase reactivates intercellular ROS/RNS-dependent apoptosis-inducing signaling and leads to autocrine apoptotic selfdestruction of tumor cells. Model experiments with defined ROS and RNS led to the conclusion that CAP-derived singlet oxygen might lead to site-specific inactivation of catalase, followed by tumor cell-specific generation of secondary singlet oxygen and further inactivation of catalase. This then allows for subsequent reactivation of intercellular ROS/RNS-dependent apoptosis-inducing signaling [1]. Only on the first sight, this model seemed a) to be in contradiction to the model on the dependence of CAP action from aquaporins [2] and b) to be independent of subsequent immunogenic cell death and activation of a cytotoxic T cell response [3, 4]. The analysis of existing experimental data from several groups and the alignment of site-specific mechanisms, defined by chemical biology and cell biology, allowed to establish an updated and comprehensive model [5] that includes the concepts from references [1-4] in a rational way. This model shows several biochemical amplification loops related to the generation of secondary singlet oxygen, a positive feed-back of HOCl signaling on immunogenic modulation, as well as a feedback loop from activated T cells to catalase inactivation and reactivation of intercellular ROS/RNS signaling. Thereby HOCl seems to play a role as mediator for apoptosis induction and enhancer of immunogenic stimulation. The detailed biochemical analysis of generation of secondary singlet oxygen by tumor cells after initial interaction with exogenous singlet oxygen allowed to predict that an increase in the concentrations of tumor cell-derived extracellular superoxide anions and/or nitric oxide should cause a substantial synergistic effect with exogenous singlet oxygen. Therefore, a hybrid molecule, consisting of a plant-derived NOX stimulator and a plant-derived inhibitor of NO dioxygenase (NOD) (patent application PCT/EP2016/080169) has been synthesized in collaboration with Prof. R. Bruckner and Dr. S. Braukmuller (Organic Chemistry, Freiburg). When applied in vitro at 0.1-0.5 nmoles/liter, this compound caused a remarkable synergistic effect with exogenous singlet oxygen, characterized by a reduction of the optimal dose of singlet oxygen by three log steps. We suggest to evaluate whether this principle can be translated into application of CAP for tumor treatment. If so, the reduction of necessary plasma doses might be useful for further development of microinvasive plasma-mediated tumor therapy

Autoamplificatory singlet oxygen generation sensitizes tumor cells for intercellular apoptosis-inducing signaling

Tumor cells express NADPH oxidase-1 (NOX1) in their membrane and control NOX1-based intercellular reactive oxygen and nitrogen species (ROS/RNS)-dependent apoptosis-inducing signaling through membrane-associated catalase and superoxide dismutase.Treatment: of tumor cells with high concentrations of H2O2, peroxnitrite, HOCl, or increasing the concentration of cell-derived NO causes initial generation of singlet oxygen and local inactivation of membrane-associated catalase. As a result, free peroxynitrite and H2O2 interact and generate secondary singlet oxygen. Inactivation of further catalase molecules by secondary singlet oxygen leads to auto-amplification of singlet oxygen generation and catalase inactivation. This allows reactivation of intercellular ROS/RNS-signaling and selective apoptosis induction in tumor cells.The initial singlet oxygen generation seems to be the critical point in this complex biochemical multistep mechanism. Initial singlet oxygen generation requires the interaction between distinct tumor cell-derived ROS and RNS and may also depend on either the induction of NO synthase expression or NOX1 activation through the FAS receptor. FAS receptor activation can be achieved by singlet oxygen.Autoamplificatory generation of singlet oxygen through the interaction between peroxynitrite and hydrogen peroxide inherits a rich potential for the establishment of synergistic effects that may be instrumental for novel approaches of tumor therapy with high selectivity towards malignant cells.

The Antitumor Effect of Singlet Oxygen.

Tumor cells are protected against intercellular apoptosis-inducing signaling through expression of membrane-associated catalase and superoxide dismutase. Exogenous singlet oxygen derived from activated photosensitizers or from cold atmospheric plasma causes local inactivation of protective catalase which is followed by the generation of secondary extracellular singlet oxygen. This process is specific for tumor cells and is driven by a complex interaction between H2O2 and peroxynitrite. Secondary singlet oxygen has the potential for autoamplification of its generation, resulting in optimal inactivation of protective catalase and reactivation of intercellular apoptosis-inducing signaling. An increase in the endogenous NO concentration also causes inactivation of catalase and autoamplificatory generation of secondary singlet oxygen. This principle is essential for the antitumor activity of secondary plant products, such as cyanidins and other inhibitors of NO dioxygenase. It seems that the action of the established chemotherapeutic taxol and the recently established antitumor effect of certain azoles are based on the same principles.

Ene–Diene Transmissive Cycloaddition Reactions with Singlet Oxygen: The Vinylogous Gem Effect and Its Use for Polyoxyfunctionalization of Dienes

The singlet oxygen reactivities and regioselectivities of the model compounds 1b-d were compared with those of the geminal (gem) selectivity model ethyl tiglate (1a). The kinetic cis effect is k(E)/k(Z) = 5.2 for the tiglate/angelate system 1a/1a' without a change in the high gem regioselectivity. Further conjugation to vinyl groups enabled mode-selective processes, namely, [4 + 2] cycloadditions versus ene reactions. The site-specific effects of methylation on the mode selectivity and the regioselectivity of the ene reaction were studied for dienes 1e-g. A vinylogous gem effect was observed for the γ,δ-dimethylated and α,γ,δ-trimethylated substrates 1h and 1i, respectively. The corresponding phenylated substrates 1j-l showed similar mode selectivity, as monomethylated 1j exhibited exclusively [4 + 2] reactivity while the tandem products 12 and 14 were isolated from the di- and trimethylated substrates 1k and 1l, respectively. The vinylogous gem effect favors the formation of 1,3-dienes from the substrates, and thus, secondary singlet oxygen addition was observed to give hydroperoxy-1,2-dioxenes 19 and 20 in an ene-diene transmissive cycloaddition sequence. These products were reduced to give alcohols (16, 17, and 18) or furans (24 and 25), respectively, or treated with titanium(IV) alkoxides to give the epoxy alcohols 26 and 27. The vinylogous gem effect is rationalized by DFT calculations showing that biradicals are the low-energy intermediates and that no reaction path bifurcations compete.

Singlet Oxygen Oxidation of Isolated and Cellular DNA: Product Formation and Mechanistic Insights

This survey focuses on recent aspects of the singlet oxygen oxidation of the guanine moiety of nucleosides, oligonucleotides, isolated and cellular DNA that has been shown to be the exclusive DNA target for this biologically relevant photogenerated oxidant. A large body of mechanistic data is now available from studies performed on nucleosides in both aprotic solvents and aqueous solutions. A common process to both reaction conditions is the formation of 8-oxo-7,8-dihydroguanine by reduction of 8-hydroperoxyguanine that arises from the rearrangement of initially formed endoperoxide across the 4,8-bond of the purine moiety. However, in organic solvent the hydroperoxide is converted as a major degradation pathway into a dioxirane that subsequently decomposes into a complex pattern of oxidation products. A different reaction that involved the formation of a highly reactive quinonoid intermediate consecutively to the loss of a water molecule from the 8-hydroperoxide has been shown to occur in aqueous solution. Subsequent addition of a water molecule at C5 leads to the generation of a spiroiminodihy-dantoin compound via a rearrangement that involves an acyl shift. However, in both isolated and cellular DNA the latter decomposition pathway is at the best a minor process, because only 8-oxo-7,8-dihydroguanine has been found to be generated. It is interesting to point out that singlet oxygen has been shown to contribute predominantly to the formation of 8-oxo-7,8-dihydroguanine in the DNA of bacterial and human cells upon exposure to UVA radiation. It may be added that the formation of secondary singlet-oxygen oxidation products of 8-oxo-7,8-dihydroguanine, including spiroiminodihydantoin and oxaluric acid that were characterized in nucleosides and oligonucleotide, respectively, have not yet been found in cellular DNA.