Photodynamic Therapy‐Induced Apoptosis of HeLa Cells

Abstract

Photodynamic therapy (PDT), which is a treatment for cancer and certain noncancerous conditions, requires exposure of cells or tissue to a photosensitizing drug followed by irradiation with visible light of the appropriate wavelength. By using Rose Bengal Acetate (RBAc) as the photosensitizer and an innovative green light-emitting diode, we investigated the efficiency with which apoptosis is induced in HeLa cells, focusing our study on mitochondria alteration and cytochrome c release. Indeed, RBAc is a very efficient fluorogenic substrate and easily enters the cells where the original photoactive molecule is restored by specific esterases. HeLa cells after PDT underwent a consistent rate of apoptosis (peaked at 12 h of recovery post-PDT). Necrosis was observed at the longest times of recovery as a result of secondary necrosis. PDT gave rise to a series of shape modifications, mainly referable to apoptotic-related changes (i.e., extensive blebs formation) involving both F-actin and tubulin networks. Soon after PDT, mitochondria lose their potential membranes and release large quantities of cytochrome c.

Sonodynamic Effect of an Anti-Inflammatory Agent – Emodin on Macrophages

“…In a number of papers, deleterious effects of PDT on the cytoskeleton have been documented (Sakharov et al 2003;Panzarini et al 2009;Jung et al 2009;Casas et al 2008). By a similar mechanism, SDT can also affect cytoskeletal proteins.…”

Rose Bengal Acetate PhotoDynamic Therapy (RBAc-PDT) Induces Exposure and Release of Damage-Associated Molecular Patterns (DAMPs) in Human HeLa Cells

“…Co., St Louis, MO, USA) and necroptosis (Necrostatin 1, Nec-1, 300 µM; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) specific inhibitors in order to obtain autophagic and apoptotic cells fractions at 90% purity [30]. The autophagic HeLa cells fraction was obtained by using simultaneously Z-VAD-FMK and Nec-1; apoptotic HeLa cells fraction was obtained by using 3-MA and Nec-1.…”

“…Determination of protein concentration and immunoblotting were performed as described in [30]. Immunoblotting was done with primary antibodies against HSP70 (1 µg/mL), HSP90 (1 µg/mL), CRT (1 µg/mL), ERp57 (20 µg/mL), HMGB1 (1 µg/mL; MBL, Woburn, MA, USA).…”

Rose Bengal Acetate photodynamic therapy-induced autophagy

“…[44][45][46][47] Alternatively, due to the high reactivity of photogenerated ROS it is possible that autophagy is initiated in an attempt to remove organelles damaged by oxidation or to degrade large aggregates of cross-linked proteins, produced by photochemical reactions, which are not removed by the ubiquitin-proteasome system. 48 It is known that RBAc-PDT damages mitochondria as demonstrated by mitochondrial potential loss allowing the release of cytochrome c. 26,27 Examples of specific degradation pathways for damaged mitochondria, a process called mitophagy that often precedes apoptosis and requires caspase inhibition to be appreciated, 49 have been reported for hepatocytes and yeast. 50,51 It is thus likely that autophagy in RBAc-PDT HeLa cells occurs within the first 8 h post-PDT to eliminate damaged mitochondria, while apoptosis is maintained at high level by different caspase-dependent and -independent pathways.…”

“…All together our data confirm the induction of autophagy by the RBAc-PDT; in particular, those reported in Figure 7 indicated that autophagy is concomitantly induced along with apoptosis. Indeed, in previous works we reported that RBAc-PDT induced 35-40% of apoptosis in HeLa cells, mitochondria lost their membrane potential and released cytochrome-c. 26,27 The bulk of autophagic cells occurred quicker than apoptotic cells (8 vs. 12 h) and disappeared faster as well. that accompanies necrosis is prevented also in vivo.…”