p38(MAPK)-regulated induction of p62 and NBR1 after photodynamic therapy promotes autophagic clearance of ubiquitin aggregates and reduces reactive oxygen species levels by supporting Nrf2-antioxidant signaling.

Emerging evidence indicates that oxidative stress instigates the formation of ubiquitin (Ub) aggregates, substrates of autophagy, through a process requiring the ubiquitin binding adaptors p62/SQSTM1 and NBR1. Here, we have investigated the role of p62 and NBR1 in cell survival after hypericin-mediated photodynamic therapy (Hyp-PDT), a procedure known to incite robust reactive oxygen species (ROS)-based endoplasmic reticulum stress and autophagy pathways. We found that Hyp-PDT stimulated the formation of p62- and NBR1-associated Ub aggregates in normal and cancer cells, which were ultimately removed by autophagy, through a mechanism partially regulated by p38(MAPK).

Mitochondria are targets for peroxisome-derived oxidative stress in cultured mammalian cells.

Many cellular processes are driven by spatially and temporally regulated redox-dependent signaling events. Although mounting evidence indicates that organelles such as the endoplasmic reticulum and mitochondria can function as signaling platforms for oxidative stress-regulated pathways, little is known about the role of peroxisomes in these processes. In this study, we employ targeted variants of the genetically encoded photosensitizer KillerRed to gain a better insight into the interplay between peroxisomes and cellular oxidative stress.

Pro-apoptotic signaling induced by photo-oxidative ER stress is amplified by Noxa, not Bim.

Pro-apoptotic signaling instigated by endoplasmic reticulum (ER) stress is tightly governed by the BH3-only proteins like Noxa and Bim, which help trigger apoptosis, in part by inactivating mitochondria protecting proteins like Mcl-1. Bim/Noxa-based pro-apoptotic signaling has been implicated for various ER stressors but not yet for those causing "ER-focused" production of severe oxidative stress. In the present study we found that photo-oxidative (phox)-ER stress induced by hypericin-based photodynamic therapy is associated with activation of PERK (an ER sessile, stress sensor), robust induction of CHOP (a pro-apoptotic transcription factor) and induction of Bim and Noxa (accompanied by an eventual drop in Mcl-1 levels).

Autophagy: shaping the tumor microenvironment and therapeutic response.

Autophagy, the major lysosomal pathway for recycling intracellular components including whole organelles, is emerging as a key process modulating tumorigenesis, tumor-stroma interactions, and cancer therapy. Research over the past decade has highlighted a context-dependent and dynamic role for autophagy in cancer: it is tumor suppressive in the early stages of cancer development, but fuels the growth of established tumors. Likewise, the stimulation of autophagy in response to therapeutics can contextually favor or weaken chemoresistance and antitumor immunity.

Spatiotemporal autophagic degradation of oxidatively damaged organelles after photodynamic stress is amplified by mitochondrial reactive oxygen species.

Although reactive oxygen species (ROS) have been reported to evoke different autophagic pathways, how ROS or their secondary products modulate the selective clearance of oxidatively damaged organelles is less explored. To investigate the signaling role of ROS and the impact of their compartmentalization in autophagy pathways, we used murine fibrosarcoma L929 cells overexpressing different antioxidant enzymes targeted to the cytosol or mitochondria and subjected them to photodynamic (PD) stress with the endoplasmic reticulum (ER)-associated photosensitizer hypericin. We show that following apical ROS-mediated damage to the ER, predominantly cells overexpressing mitochondria-associated glutathione peroxidase 4 (GPX4) and manganese superoxide dismutase (SOD2) displayed attenuated kinetics of autophagosome formation and overall cell death, as detected by computerized time-lapse microscopy.

A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death.

Surface-exposed calreticulin (ecto-CRT) and secreted ATP are crucial damage-associated molecular patterns (DAMPs) for immunogenic apoptosis. Inducers of immunogenic apoptosis rely on an endoplasmic reticulum (ER)-based (reactive oxygen species (ROS)-regulated) pathway for ecto-CRT induction, but the ATP secretion pathway is unknown. We found that after photodynamic therapy (PDT), which generates ROS-mediated ER stress, dying cancer cells undergo immunogenic apoptosis characterized by phenotypic maturation (CD80(high), CD83(high), CD86(high), MHC-II(high)) and functional stimulation (NO(high), IL-10(absent), IL-1β(high)) of dendritic cells as well as induction of a protective antitumour immune response.

5-ALA-PDT induces RIP3-dependent necrosis in glioblastoma.

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are resistant to all current therapies and are associated with a high rate of recurrence. Glioblastoma were previously shown to respond to treatments by 5-aminolevulinic acid (5-ALA)-based photodynamic therapy (PDT) mainly by activating a necrotic type of cell death.

NF-kappaB inhibition improves the sensitivity of human glioblastoma cells to 5-aminolevulinic acid-based photodynamic therapy.

Glioblastoma constitute the most frequent and deadliest brain tumors of astrocytic origin. They are very resistant to all current therapies and are associated with a huge rate of recurrence. In most cases, this type of tumor is characterized by a constitutive activation of the nuclear factor-kappaB (NF-κB).

Phototoxicity of Hoechst 33342 in time-lapse fluorescence microscopy.

Dyes that bind to DNA, such as Hoechst 33342, are commonly used to visualize chromatin in live cells by fluorescence microscopy. A caveat is that the probes themselves should not perturb cellular responses and under normal conditions the dyes are generally non-toxic. However, researchers are increasingly using computerized time-lapse microscopy (CTLM), where cells stained with fluorescent dyes are often imaged frequently over a period of several days, to follow cellular responses in real time.

Autophagy pathways activated in response to PDT contribute to cell resistance against ROS damage.

Reactive oxygen species (ROS) concurrently instigate apoptosis and autophagy pathways, but the link between these processes remains unclear. Because cytotoxic ROS formation is exploited in anticancer therapy, such as in photodynamic therapy (PDT), a better understanding of the complex interplay between autophagy and apoptosis is urgently required. Previously, we reported that ROS generated by PDT with an endoplasmic reticulum (ER)-associated sensitizer leads to loss of ER-Ca(2+) homeostasis, ER stress and apoptosis.

A p38(MAPK)/HIF-1 pathway initiated by UVB irradiation is required to induce Noxa and apoptosis of human keratinocytes.

The signal transduction pathways leading to apoptosis of human keratinocytes responding to UVB irradiation are complex and not completely understood. Previously, we reported that in UVB-irradiated keratinocytes, p38(MAPK) instigates Bcl-2-associated X protein (Bax) activation and mitochondrial apoptosis. However, the molecular mechanism underlying the pro-apoptotic function of p38(MAPK) remained unclear.

Real-time imaging of novel spatial and temporal responses to photodynamic stress.

Cells subjected to various forms of stress have been shown to induce bystander responses in nontargeted cells, thus extending the stress response to a larger population. However, the mechanism(s) of bystander responses remains to be clearly identified, particularly for photodynamic stress. Oxidative stress and cell viability were studied on the spatial and temporal levels after photodynamic targeting of a subpopulation of EMT6 murine mammary cancer cells in a multiwell plate by computerized time-lapse fluorescence microscopy.

Spatial and temporal dynamics of in vitro photodynamic cell killing: extracellular hydrogen peroxide mediates neighbouring cell death.

Photodynamic killing of a cell population is generally considered to result from direct effects that occur in each cell. In some scenarios this may be an over-simplification and the potential for cell-cell signaling processes to contribute to the response of a population to photodynamic stress is addressed in this paper. Photodynamic killing of EMT6 cells in culture was studied in time and space using computerized time-lapse microscopy.

Photochemical production and characterisation of the radical ions of tetraphenylporphycenes.

Both the radical anion and radical cation of 2,7,12,17-tetraphenylporphycene and of its palladium(II) complex have been produced by photochemical methods. Their electronic absorption spectrum and decay kinetics have been characterised by global analysis of the time-absorbance-wavelength matrix. The results provide deeper insight on the role of these radical ion species in the photodynamic activity of tetraphenylporphycenes.

Effect of aza substitution on the photophysical and electrochemical properties of porphycenes: Characterization of the near-IR-absorbing photosensitizers 2,7,12,17-tetrakis(p-substituted phenyl)-3,6,13,16-tetraazaporphycenes.

The need for new photodynamic-therapy photosensitizers has stimulated the search of new families of compounds absorbing strongly in the 700-900 nm range, the region where tissue is most transparent to radiation capable to induce the photodynamic effect. Using computational chemistry techniques, 3,6,13,16-tetraazaporphycenes were previously identified as interesting target candidates. This work reports on the photophysical and electrochemical properties of selected members of this new family of macrocycles.