title: Regulated forms of cell death are induced by the photodynamic action of the fluorogenic substrate, Hypocrellin B-acetate.
abstract: The addition of chemical groups to a photosensitizer makes it to act as a fluorogenic substrate, increasing its ability to enter the cells. In this work, the cytotoxic efficacy of Hypocrellin B modified by addition of two acetate groups (HypB-Ac) was investigated in HeLa cells. Using transmission electron microscopy, cytochemical and immunocytochemical techniques, and flow cytometry we demonstrated that light irradiation of HypB-Ac-loaded cells resulted in either necrosis or apoptosis, depending on the HypB-Ac concentration. Administration of Hyp-Ac at high concentration (1×10(-)(5) M) resulted in massive necrosis, while at low concentration (2.5×10(-)(7) M) apoptosis along with autophagy were induced. Focusing on cells still exhibiting non-apoptotic features, we provide the evidence of early involvement of different organelles in the photodamage, with the frequent presence of autophagic vacuoles already at very short post-irradiation times (30 min, when ultrastructural apoptotic features are rarely found). These findings suggest that the widespread photodamage rather than the target organelle(s) involved is crucial for inducing either a catastrophic or a regulated form of cell death. Fluorogenic substrates such as HypB-Ac have an increased capability to accumulate in cancer cells compared to the native photosensitizing molecules: this would allow to use lower drug doses in vivo, thus decreasing the risk of systemic cytotoxicity in the absence of irradiation improving the efficacy of photodynamic therapy. The ability of HypB-Ac at very low concentration to induce autophagy and apoptosis would additionally be advantageous for therapeutic application, as the preferential induction of regulated forms of cell death entails the rapid phagocytotic removal of dying cells without affecting the tissue and organ structure.
authors.affiliation: Istituto di Genetica Molecolare del CNR, Pavia, Italy. giada.santin@unipv.it
authors.forename: Giada
authors.initials: G
authors.lastname: Santin
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Bottone
authors.forename: Manuela
authors.initials: M
authors.lastname: Malatesta
authors.forename: Anna Ivana
authors.initials: AI
authors.lastname: Scovassi
authors.forename: Giovanni
authors.initials: G
authors.lastname: Bottiroli
authors.forename: Carlo
authors.initials: C
authors.lastname: Pellicciari
authors.forename: Anna Cleta
authors.initials: AC
authors.lastname: Croce
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001343:Autophagy / Q000187:drug effects; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D008027:Light; D009336:Necrosis / Q000139:chemically induced; D010569:Perylene / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D019800:Phenol; D017319:Photosensitizing Agents / Q000737:chemistry; D011809:Quinones / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D017319:Photosensitizing Agents; D011809:Quinones; C570352:hypocrellin B acetate; D019800:Phenol; D010569:Perylene; C048388:hypocrellin A
keywords: Apoptosis; Autophagy; Electron microscopy; Fluorescence microscopy; Hypocrellin B acetate; Photodynamic therapy