Overview of Cell Death Mechanisms Induced by Rose Bengal Acetate-Photodynamic Therapy

Photodynamic Therapy (PDT) is a non-invasive treatment for different pathologies, cancer included, using three key components: non-toxic light-activated drug (Photosensitizer, PS), visible light, and oxygen. Their interaction triggers photochemical reactions leading to Reactive Oxygen Species (ROS) generation, that mediate cytotoxicity and cell death. In the present paper, the most important findings about the synthetic dye Rose Bengal Acetate (RBAc), an emerging photosensitizer for its efficient induction of cell death, will be reported with the aim to integrate RBAc phototoxicity to novel therapeutic PDT strategies against tumour cells. After its perinuclear intracellular localization, RBAc causes multiple subcellular organelles damage, that is, mitochondria, Endoplasmic Reticulum (ER), lysosomes, and Golgi complex. Indeed, RBAc exerts long-term phototoxicity through activation of both caspase-independent and- dependent apoptotic pathways and autophagic cell death. In particular, this latter cell death type may promote cell demise when apoptotic machinery is defective. The deep knowledge of RBAc photocytotoxicity will allow to better understand its potential photomedicine application in cancer

Comb-assisted cavity ring-down spectroscopy of a buffer-gas-cooled molecular beam

We demonstrate continuous-wave cavity ring-down spectroscopy of a partially hydrodynamic molecular beam emerging from a buffer-gas-cooling source. Specifically, the (ν1 + ν3) vibrational overtone band of acetylene (C2H2) around 1.5 μm is accessed using a narrow-linewidth diode laser stabilized against a GPS-disciplined rubidium clock via an optical frequency comb synthesizer. As an example, the absolute frequency of the R(1) component is measured with a fractional accuracy of ∼1 × 10(-9). Our approach represents the first step towards the extension of more sophisticated cavity-enhanced interrogation schemes, including saturated absorption cavity ring-down or two-photon excitation, to buffer-gas-cooled molecular beams

Immunogenic Cell Death: Can It Be Exploited in PhotoDynamic Therapy for Cancer?

Immunogenic Cell Death (ICD) could represent the keystone in cancer management since tumor cell death induction is crucial as well as the control of cancer cells revival after neoplastic treatment. In this context, the immune system plays a fundamental role. The concept of Damage-Associated Molecular Patterns (DAMPs) has been proposed to explain the immunogenic potential of stressed or dying/dead cells. ICD relies on DAMPs released by or exposed on dying cells. Once released, DAMPs are sensed by immune cells, in particular Dendritic Cells (DCs), acting as activators of Antigen-Presenting Cells (APCs), that in turn stimulate both innate and adaptive immunity. On the other hand, by exposing DAMPs, dying cancer cells change their surface composition, recently indicated as vital for the stimulation of the host immune system and the control of residual ill cells. It is well established that PhotoDynamic Therapy (PDT) for cancer treatment ignites the immune system to elicit a specific antitumor immunity, probably linked to its ability in inducing exposure/release of certain DAMPs, as recently suggested. In the present paper, we discuss the DAMPs associated with PDT and their role in the crossroad between cancer cell death and immunogenicity in PDT

Overview of cell death mechanisms induced by Rose Bengal acetate-photodynamic therapy

Photodynamic Therapy (PDT) is a non-invasive treatment for different pathologies, cancer included, using three key components: non-toxic light-activated drug (Photosensitizer, PS), visible light, and oxygen. Their interaction triggers photochemical reactions leading to Reactive Oxygen Species (ROS) generation, that mediate cytotoxicity and cell death. In the present paper, the most important findings about the synthetic dye Rose Bengal Acetate (RBAc), an emerging photosensitizer for its efficient induction of cell death, will be reported with the aim to integrate RBAc phototoxicity to novel therapeutic PDT strategies against tumour cells. After its perinuclear intracellular localization, RBAc causes multiple subcellular organelles damage, that is, mitochondria, Endoplasmic Reticulum (ER), lysosomes, and Golgi complex. Indeed, RBAc exerts long-term phototoxicity through activation of both caspase-independent and-dependent apoptotic pathways and autophagic cell death. In particular, this latter cell death type may promote cell demise when apoptotic machinery is defective. The deep knowledge of RBAc photocytotoxicity will allow to better understand its potential photomedicine application in cancer

Nanomaterials and Autophagy: New Insights in Cancer Treatment

Autophagy represents a cell’s response to stress. It is an evolutionarily conserved process with diversified roles. Indeed, it controls intracellular homeostasis by degradation and/or recycling intracellular metabolic material, supplies energy, provides nutrients, eliminates cytotoxic materials and damaged proteins and organelles. Moreover, autophagy is involved in several diseases. Recent evidences support a relationship between several classes of nanomaterials and autophagy perturbation, both induction and blockade, in many biological models. In fact, the autophagic mechanism represents a common cellular response to nanomaterials. On the other hand, the dynamic nature of autophagy in cancer biology is an intriguing approach for cancer therapeutics, since during tumour development and therapy, autophagy has been reported to trigger both an early cell survival and a late cell death. The use of nanomaterials in cancer treatment to deliver chemotherapeutic drugs and target tumours is well known. Recently, autophagy modulation mediated by nanomaterials has become an appealing notion in nanomedicine therapeutics, since it can be exploited as adjuvant in chemotherapy or in the development of cancer vaccines or as a potential anti-cancer agent. Herein, we summarize the effects of nanomaterials on autophagic processes in cancer, also considering the therapeutic outcome of synergism between nanomaterials and autophagy to improve existing cancer therapies

Surface exposure of HSP70 and HSP90 in RBAc-PDT-treated HeLa cells.

<p>Kinetic of HSP70 and HSP90 membrane translocation (ecto-HSP70 and ecto-HSP90) was performed by Western blot of electroblotted to nitrocellulose membrane SDS-PAGE of membrane proteins fraction (30 µg protein/lane) of RBAc-PDT treated HeLa cells (10⁻⁵ M RBAc, 1 h, 1.6 J/cm², 90 sec) in the presence of 3-MA (10 mM) and Nec-1 (300 µ) (apoptotic cells) and in the presence of z-VAD-FMK (20 µM) and 3-MA (10 mM) (autophagic cells) at the indicated time intervals. Monoclonal antibodies against HSP70 (70 kDa) and HSP90 (90 kDa) were used. The amount of HSP70 and HSP90 proteins was reported as band intensity ratio (treated/untreated) measured by densitometer analysis. E-cadherin (97 kDa) and β-actin (45 kDa) expression is shown as a control. Actin is absent in the membrane proteins fraction. The data are the mean ± SD of three independent experiments. The values for ecto-HSP70 of the apoptotic cells are always significantly different (p<0.05) with respect to autophagic cells. The values of apoptotic and autophagic cells values are significantly different (p<0.05) with respect to untreated cells for both ecto-HSP70 and ecto-HSP90. One representative Western Blot is shown out of the three independent experiments performed.</p

Involvement of ecto-CRT in phagocytosis of RBAc photosensitized HeLa cells.

<p>Phase contrast micrographs (a, b) and fluorescence micrographs of Hoechst (1 µg/mL) labeled (a’–b’) apoptotic RBAc-PDT-induced (RBAc 10⁻⁵ M 60 min followed by 90 seconds irradiation with green light (1.6 J/cm²) and then by recovery in fresh medium for 12 h, in the presence of 3-MA 10 mM and Nec-1 300) HeLa cells co-incubated with human macrophages. a–b) inhibition of ecto-CRT by polyclonal chicken anti-CRT. Bars = 10 µm.</p

Phagocytosis index and rate, percentage of binding and number of apoptotic and autophagic HeLa cells bound per isolated human macrophages.

<p>Phagocytosis index: number of dead cells internalized per macrophage.</p><p>Phagocytosis rate: number of macrophages internalizing at least one dead cell.</p><p>Percentage of binding: number of macrophages binding at least one dead cell.</p><p>Viable: untreated HeLa cells; RBAc-PDT (12 h rec): apoptotic PDT-induced in the presence of 3-MA (10 mM) and Nec-1 (300 µ) HeLa cells, 12 h of recovery post PDT; RBAc-PDT (8 h rec): autophagic PDT-induced in the presence of z-VAD-FMK (20 µ) and 3-MA (10 mM) HeLa cells, 8 h of recovery post PDT.</p><p>N.D. Not Detected value, corresponding to 0 cells counted.</p><p>RBAc-PDT: HeLa cells incubated with Rose Bengal Acetate for 60 min followed by 90 seconds irradiation with green light (1.6 J/cm²) and then by recovery in fresh medium (8 and 12 h).</p><p>Each value is the average ±SD of 500 cells scored out of three independent experiments. Asterisks show significant (-anti-CRT) values (p<0.05) versus (+anti-CRT) ones.</p

RBAc-PDT induces an early release of HSP70 and HSP90 and a late and passive release of HMGB1 in HeLa cells.

<p>Kinetic of released HSP70 and HSP90 was performed by Western blot of electroblotted to nitrocellulose membrane SDS-PAGE of the conditioned media proteins (30 µg protein/lane) of RBAc-PDT treated HeLa cells (10⁻⁵ M RBAc, 1 h, 1.6 J/cm², 90 sec) in the presence of 3-MA (10 mM) and Nec-1 (300 µ) (apoptotic cells) and in the presence of z-VAD-FMK (20 µM) and 3-MA (10 mM) (autophagic cells) at indicated time intervals. Monoclonal antibodies against HSP70 (70 kDa), HSP90 (90 kDa) and HMGB1 (29 kDa) were used. The amount of HSP70, HSP90 and HMGB1 proteins was reported as band intensity ratio (treated/untreated) measured by densitometer analysis. Actin is absent in the conditioned media proteins. The data are the mean ± SD of three independent experiments. The values (4–12 h) of ecto-HSP70 of apoptotic cells are significantly different (p<0.05) with respect to autophagic cells. HSP90 and HMGB1values of apoptotic cells were always significantly different (p<0.05) with respect to autophagic cells. HSP70, HSP90 and HMGB1 values of apoptotic and autophagic cells were always significantly different (p<0.05) with respect to untreated cells. One representative Western Blot is shown out of the three independent experiments performed.</p

RBAc-PDT induces release of ATP in HeLa cells.

<p>Kinetic of intracellular and extracellular released ATP was performed by ELISA (A) and luciferin-luciferase based (B) assay on the conditioned media of RBAc-PDT treated HeLa cells (10⁻⁵ M RBAc, 1 h, 1.6 J/cm², 90 sec) in the presence of 3-MA (10 mM) and Nec-1 (300 µ) (apoptotic cells) and in the presence of z-VAD-FMK (20 µM) and 3-MA (10 mM) (autophagic cells) at indicated time intervals. The amount of ATP, reported as ng/mL or as concentration (M), is the mean ± SD of three independent experiments. The values of extracellular ATP both of apoptotic and autophagic cells are significantly different (p<0.05) with respect to untreated cells. Extracelluar ATP values of apoptotic cells were always significantly different (p<0.05) with respect to autophagic cells. Intracellular ATP values of apoptoticc cells were always significantly different (p<0.05) with respect to autophagic and untreated cells.</p