benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and temoporfin

Photodynamic therapy (PDT), an approved anticancer treatment, is reported as a potent inducer of programmed cell death (PCD) by both apoptosis and autophagy. The present study investigated the kinetics of both autophagy and caspase activation in MCF-7 cells submitted to mTHPC-PDT upon condition of treatment promoting ER accumulation of mTHPC. Fluence-dependent immediate cytochrome c (cyt C) release followed by caspase-9 and -7 activation at 1 h post-PDT evidenced a mitochondrial oxidative stress triggered by high light doses leading to >90% of cell death. ER oxidative stress was monitored by the induction of the glucose-related protein chaperone GRP78. From 6 h post-PDT, GRP78 induction was accompanied by the conversion of LC3-I into LC3-II, the hallmark of autophagosome formation. The formation of acid vesicles evidenced by fluorescence microscopy was obvious from 22 h post-PDT. Twenty-four hours post-PDT, cyt C release decreased and caspase-9 cleavage disappeared, while the expression of cleaved caspase-7 remained significant. At the same time, the profiles of GRP78, cleaved caspase-7 and LC3-II expression were similar irrespective of light doses. In contrast to an inhibitor of caspase activation Z-VAD-FMK, the use of autophagy inhibitor, Wortmannin, impaired cytotoxicity along with an increase in caspase-7 activation. These results demonstrate a valuable contribution of autophagy to cell death in mTHPC-photosensitized MCF-7 cells.

Topics: Amino Acid Chloromethyl Ketones; Androstadienes; Apoptosis; Autophagy; Caspase Inhibitors; Caspases; Cell Line, Tumor; Cell Survival; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Humans; Kinetics; Mesoporphyrins; Microtubule-Associated Proteins; Mitochondria; Oxidative Stress; Photochemotherapy; Photosensitizing Agents; Poly(ADP-ribose) Polymerases; Protein Kinase Inhibitors; Wortmannin

As many types of cells exposed to photodynamic therapy (PDT) appear to undergo apoptosis, various apoptosis inhibitors have already been used in studies of PDT-induced apoptosis. Although these inhibitors decrease apoptosis, their real effect on the phototoxic efficacy of photosensitisers is unclear. The good phototoxicity of m-THPC was confirmed on murine melanoma B16-A45 cells. Toxicity and phototoxicity studies were then carried out using four apoptosis inhibitors: BAPTA-AM, Forskolin, DSF, and Z.VAD.fmk. Although all inhibitors tested blocked PDT-induced apoptosis, none produced a significant modification of the phototoxic effect of m-THPC on B16 cells. It has been suggested that apoptosis and necrosis share common initiation pathways and that the final outcome is determined by the presence of an active caspase. This implies that apoptosis inhibition reorients cells to necrosis, i.e. those cells sufficiently damaged by PDT appear to be killed, regardless of the mechanism involved.

Topics: Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Cell Survival; Chelating Agents; Colforsin; Cysteine Proteinase Inhibitors; Disulfiram; Dose-Response Relationship, Drug; Egtazic Acid; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Melanoma; Melanoma, Experimental; Mesoporphyrins; Mice; Models, Biological; Photochemotherapy; Tumor Cells, Cultured