Esther Buytaert

Role of endoplasmic reticulum depletion and multidomain proapoptotic BAX and BAK proteins in shaping cell death after hypericin-mediated photodynamic therapy

Both the commitment event and the modality of cell death in photodynamic therapy (PDT) remain poorly defined. We report that PDT with endoplasmic reticulum (ER)‐associating hypericin leads to an immediate loss of SERCA2 protein levels, causing disruption of Ca2+ homeostasis and cell death. Protection of SERCA2 protein rescues ER‐Ca2+ levels and prevents cell death, suggesting that SERCA2 photodestruction with consequent incapability of the ER to maintain intracellular Ca2+ homeostasis is causal to cell killing. Apoptosis is rapidly initiated after ER‐Ca2+ depletion and strictly requires the BAX/BAK gateway at the mitochondria. Bax−/− Bak−/− double‐knockout (DKO) cells are protected from apoptosis but undergo autophagy‐associated cell death as revealed by electron microscopy and biochemical analysis. Autophagy inhibitors, but not caspase antagonists, significantly reduce death of DKO cells, suggesting that sustained autophagy is lethal. Thus, following ER photodamage and consequent disruption of Ca2+ homeostasis, BAX and BAK proteins model PDT‐mediated cell killing, which is executed through apoptosis in their presence or via an autophagic pathway in their absence.

Deficiency in Apoptotic Effectors Bax and Bak Reveals an Autophagic Cell Death Pathway Initiated by Photodamage to the Endoplasmic Reticulum

Efficient exploitation of cell death mechanisms for therapeutic purpose requires the identification of the molecular events committing cancer cells to death and the intracellular elements of the pro-death and pro-survival machinery activated in response to the anticancer therapy. Photodynamic therapy (PDT) is a paradigm of anticancer therapy utilizing the generation of reactive oxygen species to kill the cancer cells. In this study we have identified the photodamage to the sarco(endo)plasmic-reticulum Ca2+-ATPase (SERCA) pump and consequent loss in the ER-Ca2+ homeostasis as the most apical molecular events leading to cell death in hypericin-photosensitized cells. Downstream of the ER-Ca2+ emptying, both caspase-dependent and -independent pathways are activated to ensure cell demise. The induction of apoptosis as a cell death modality is dependent on the availability of proapopototic BAX and BAK proteins, which are essential effectors of the mitochondrial outer membrane permeabilization (MOMP) and subsequent caspase activation. In Bax-/-/Bak-/- cells a nonapoptotic pathway dependent on sustained autophagy commits the oxidatively damaged cells to death. These results argue that the decision to die in this paradigm of oxidative stress is taken upstream of BAX-dependent MOMP and that the irreversible photodamage to the ER induced by hypericin-PDT acts as a trigger for an autophagic cell death pathway in apoptosis-deficient cells. Addendum to: Role of Endoplasmic Reticulum Depletion and Multidomain Proapoptotic BAX and BAK Proteins in Shaping Cell Death After Hypericin-Mediated Photodynamic Therapy E. Buytaert, G. Callewaert, N. Hendrickx, L. Scorrano, D. Hartmann, L. Missiaen, J.R. Vandenheede, I. Heirman, J. Grooten and P. Agostinis FASEB J 2006; In press