Abstract
Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated
drugs for the treatment of a variety of pathologic conditions. Human poorly (CNE2)
and moderately differentiated (TW0-1) human nasopharyngeal carcinoma (NPC) cells
undergo rapid apoptosis when treated with PDT sensitized with Hypocrellin A (HA)
and Hypocrellin B (HB). It has been shown that these compounds have a strong photodynamic
effect on tumors and viruses. The initiating events of PDT sensitized HA and HB-induced
apoptosis are poorly defined. In the current study, we sought to determine whether
Fas/FasL upregulation and involvement of mitochondrial events are an early event
in HA and HB-treated PDT induced apoptosis. Loss of mitochondrial transmembrane
potential, release of cytochrome c, involvement of caspases-8 and -3 and the status
caspase-3 specific substrate PARP, were evaluated in PDT treated tumor cells.
Photoactivation of HA and HB enhanced both CD95/CD95L expression and induced CD95-signaling
dependent cell death in all tumor cell lines studied. CD95/ CD95L expression appeared
within 2 h following light activation and appeared to be a primary event in PDT
induced apoptosis. Furthermore, these results indicate that release of mitochondrial
cytochrome c into the cytoplasm is a secondary event following the activation
of initiator caspase-8 preceding caspase-3 activation, cleavage of PARP and DNA
fragmentation. Cytochrome c appeared in the cytosol within 2-3 h post PDT. Cleavage
of PARP was observed at 3-4 h following PDT and caspase-3 specific inhibitor DEVD-CHO
and broad-spectrum caspases inhibitor z-VAD-fmk blocked caspase-3 activation and
PARP cleavage suggesting that caspase-3 plays an important role in HA and HB-induced
apoptosis.