cytochrome-c-t and temoporfin

Cell death induced by oxidative insult targeted to mitochondrial interior of A431 cells was investigated. For stimulated production of ROS in the inner space of mitochondria, safranin-mediated photodynamic treatment (PDT) was employed. Another photosensitizer, mTHPC, which diffusely localizes to cellular membranes, was used for comparison. Cell response to the oxidative insult in mitochondrial interior was different from the response to the photodamage produced in cellular membranes. Autophagy and apoptotic features of cell death in response to mTHPC-PDT was observed in a wide range of PDT doses. Cell response to the oxidative stress in mitochondrial interior was dose-dependent. Damage up to CD50 did not reveal hallmarks of dead cells. At intermediate damage (CD50), cells manifested enhanced autophagy and reduced population of S-phase, but not apoptosis. Severe damage (beyond CD70) induced apoptosis following release of cytochrome c and caspase activation, in addition to autophagy and cell cycle arrest.

Topics: Apoptosis; Autophagy; Caspase 3; Cell Cycle; Cell Line, Tumor; Cell Survival; Citrate (si)-Synthase; Cyclin-Dependent Kinase Inhibitor p21; Cytochromes c; Humans; Indicators and Reagents; Mesoporphyrins; Mitochondria; Phenazines; Photochemotherapy; Photosensitizing Agents; Reactive Oxygen Species

Photodynamic therapy (PDT) is an approved anticancer treatment modality that eliminates unwanted cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which is selectively taken up by tumor cells. Present study reports the modalities of cell death after photosensitization of human adenocarcinoma HT29 monolayer and spheroid cells with a second generation photosensitizer Foscan. Kinetics of apoptosis and necrosis after Foscan-PDT in monolayer cells determined by flow cytometry using labeling of cleaved poly(ADP-ribose) polymerase (PARP) and staining with propidium iodide (PI) demonstrated that Foscan was not a strong inducer of apoptosis and necrosis was a prevailing mode of cell death. Cytochrome c release (cyt c) and mitochondrial membrane potential (Deltapsim) addressed by flow cytometry technique at different time points post-Foscan-PDT demonstrated that cell photoinactivation was governed by these mitochondrial events. Foscan-loaded HT29 multicell spheroids, subjected to irradiation with different fluence rates and equivalent light doses, displayed much better tumoricidal activity at the lowest fluence rate used. Apoptosis, measured by caspase-3 activation was evidenced only in spheroids irradiated with the lowest fluence rate and moderate fluence inducing 65% of cell death. Application of higher fluence rates for the same level of photocytotoxicity did not result in caspase-3 activation. The observation of the fluence rate-dependent modulation of caspase-3 activity in spheroids offers the possibility of regulating the mechanism of direct cell photodamage and could be of great potential in the clinical context.

Topics: Apoptosis; Blotting, Western; Caspase 3; Caspases; Cell Death; Cell Survival; Colonic Neoplasms; Cytochromes c; Dose-Response Relationship, Drug; Enzyme Activation; Flow Cytometry; HT29 Cells; Humans; Kinetics; Membrane Potentials; Mesoporphyrins; Mitochondria; Necrosis; Photochemotherapy; Photosensitizing Agents; Spheroids, Cellular

Apoptosis induced by photodynamic therapy (PDT) is considered to be an important factor defining the treatment outcome. Nevertheless, the relevance of apoptotic events in overall cell death should be established for every given photosensitizer. The present study addresses the contribution of Foscan-(meta-tetra(hydroxyphenyl)chlorine; mTHPC) photosensitized apoptosis in overall cell death in a model of cultured HT29 adenocarcinoma cells. Early events of cell death were assessed by the evaluation of mitochondrial response to mTHPC-mediated PDT, cytochrome c release and membrane depolarization. Apoptosis was measured through the activity of caspase-3 and the binding of the fluorescent conjugate Ca2+-dependent protein Annexin-V on membrane externalized phosphatidylserine at 2, 4, and 24 h post-PDT. Immediately after mTHPC-PDT, from 28 to 57% cells exhibited cytochrome c release concomitantly with mitochondrial membrane depolarization for light doses inducing more than 90% overall cell death. The maximum of caspase-3 activation (12-fold more than control) was reached 24 h after irradiation at fluence inducing 90% cell death (LD(90)). The corresponding measurement of apoptotic cells (12% of Annexin-V bound cells) confirmed the mild and delayed apoptotic response of HT29 cells to mTHPC-PDT.

Topics: Apoptosis; Caspase 3; Caspases; Colonic Neoplasms; Cytochromes c; Endopeptidases; HT29 Cells; Humans; Membrane Potentials; Mesoporphyrins; Mitochondria; Photochemotherapy; Photosensitizing Agents