Timing the multiple cell death pathways initiated by Rose Bengal acetate photodynamic therapy

Rose Bengal Acetate PhotoDynamic Therapy (RBAc-PDT) induced multiple cell deaths pathways in HeLa cells through ROS generation. The onset of apoptosis, autophagy and of the different apoptotic pathways were timed by determining the levels of caspases, Bcl 2 family, Hsp 70, LC3, Grp78 and phospho-eIF2α proteins. Four different apoptotic pathways plus autophagy were sequentially initiated by RBAc-PDT: intrinsic, extrinsic, caspase 12- dependent and caspase independent as reflected by peaks of the relative caspases, 9, 8, 3 and 12. Autophagy, revealed by the formation of acidic autophagosomes and by increased Light Chain 3-II (LC3BII) expression was conspicuous at 8 h post-PDT. In our system, autophagy had a pro-death role, since its inhibitor, 3-MethylAdenine (3-MA), significantly augmented cell viability. The increase of cleaved caspase 12 was consequent to the increase of Grp78 and phospho-eIF2α proteins, suggesting Endoplasmic Reticulum. Regulation of the intrinsic pathway of apoptosis was under the control of Bcl-2 family (i.e. soon after irradiation Bcl2 decreased, Bax and tBid increased) and of Hsp70 proteins (peak at 12-18h post-PDT). Interestingly, inhibition of one pathway, i.e.caspase-9 (Z-LEHD-FMK), caspase-8 (Z-IETD-FMK), pan-caspases (Z-VAD-FMK), autophagy (3-MA) and necrosis (Nec-1), did not impair the activation of the others, suggesting the independent onset of the different apoptotic and autophagic pathways in a not subordinated fashion.