Determination of cell death type after Hexvix®-PDT in rat tumor bearing bladders
Résumé
We have previously evaluated PDT of rat bladder tumors with the endogenous photosensitizer, protoporphyrin IX (PpIX) induced after intravesical administration of a prodrug Hexvix® or hexyl ester amnolevulinate (hALA). We have observed complete tumor eradication, without damage to underlying tissues with 8 mM hALA-PDT. PDT was performed at 635 nm and a fluence of 20 J/cm² delivered at 100 mW/cm². The aim of this study was to investigate the mechanism of cell destruction. Orthotopic and syngeneic transitional cell tumors were implanted in female Fischer (F344) rats after instillation of AY27 cells (transitional carcinoma cells). Ten days after tumor implantation, 8 mM hALA was instilled intravesically for 1 h, followed by 2 hours resting time and PDT. Immediately and 4 h post PDT, the rats were euthanized for cystectomy and the bladders were fixed. To study the cellular morphology and to determine apoptotic proteins, we have performed transmission electron microscopy and immunohistochemistry of cytochrome C and caspase 3 on section of untreated or PDT treated tumor bladders. Immediately after PDT, all bladders were unaltered as observed by transmission electron microscopy. Four hours post 8 mM HAL-PDT, tumor cells exhibited typical apoptotic features, which consisted in cell shrinking, amorphous pericellular cytoplasm, initiation of chromatin condensation, mitochondrial swelling and damage with crest ruptures but intact plasma membrane. Immunohistochemistry showed cytoplasmic expression of cytochrome C and cleaved caspase 3. Our results demonstrate that hALA mediated PDT induces apoptotic cell death of bladder tumors with cytosolic translocation of cytochrome C followed by caspase 3 cleavage. We have demonstrated that a cytochrome c-mediated caspase-dependent pathway is involved in the apoptotic induction by hALA-PDT. Nevertheless, other pathways such as Apoptosis Inducing Factor (AIF) could also be involved and are currently under investigation.