Cell death and growth arrest in response to photodynamic therapy with membrane-bound photosensitizers.

Photodynamic therapy (PDT) is a treatment for cancer and for certain benign conditions that is based on the use of a photosensitizer and light to produce reactive oxygen species in cells. Many of the photosensitizers currently used in PDT localize in different cell compartments such as mitochondria, lysosomes, endoplasmic reticulum and generate cell death by triggering necrosis and/or apoptosis. Efficient cell death is observed when light, oxygen and the photosensitizer are not limiting ("high dose PDT"). When one of these components is limiting ("low dose PDT"), most of the cells do not immediately undergo apoptosis or necrosis but are growth arrested with several transduction pathways activated. This commentary will review the mechanism of apoptosis and growth arrest mediated by two important PDT agents, i.e. pyropheophorbide and hypericin.

Ultraviolet B radiation-induced apoptosis in human keratinocytes: cytosolic activation of procaspase-8 and the role of Bcl-2.

In this study, we show that ultraviolet B radiation (UVB)-induced apoptosis of human keratinocytes involves mainly cytosolic signals with mitochondria playing a central role. Overexpression of Bcl-2 inhibited UVB-induced apoptosis by blocking the early generation of reactive oxygen species, mitochondrial cardiolipin degradation and cytochrome c release, without affecting Fas ligand (FasL)-induced cell death. It also prevented the subsequent activation of procaspase-3 and -8 as well as Bid cleavage in UVB-treated cells. Comparative analysis of UVB and FasL death pathways revealed a differential role and mechanism of caspase activation, with the UVB-induced activation of procaspase-8 only being a bystander cytosolic event rather than a major initiator mechanism, as is the case for the FasL-induced cell death. Our results suggest that Bcl-2 overexpression, by preventing reactive oxygen species production, helps indirectly to maintain the integrity of lysosomal membranes, and therefore inhibits the release of cathepsins, which contribute to the cytosolic activation of procaspase-8 in UVB-irradiated keratinocytes.

Phosphorylation of Bcl-2 in G2/M phase-arrested cells following photodynamic therapy with hypericin involves a CDK1-mediated signal and delays the onset of apoptosis.

The role of Bcl-2 in photodynamic therapy (PDT) is controversial, and some photosensitizers have been shown to induce Bcl-2 degradation with loss of its protective function. Hypericin is a naturally occurring photosensitizer with promising properties for the PDT of cancer. Here we show that, in HeLa cells, photoactivated hypericin does not cause Bcl-2 degradation but induces Bcl-2 phosphorylation in a dose- and time-dependent manner. Bcl-2 phosphorylation is induced by sublethal PDT doses; increasing the photodynamic stress promptly leads to apoptosis, during which Bcl-2 is neither phosphorylated nor degraded. Bcl-2 phosphorylation involves mitochondrial Bcl-2 and correlates with the kinetics of a G(2)/M cell cycle arrest, preceding apoptosis. The co-localization of hypericin with alpha-tubulin and the aberrant mitotic spindles observed following sublethal PDT doses suggest that photodamage to the microtubule network provokes the G(2)/M phase arrest. PDT-induced Bcl-2 phosphorylation is not altered by either the overexpression or inhibition of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH(2)-terminal protein kinase 1 (JNK1) nor by inhibiting the extracellular signal-regulated kinases (ERKs) or protein kinase C. By contrast, Bcl-2 phosphorylation is selectively suppressed by the cyclin-dependent protein kinase (CDK)-inhibitor roscovitine, completely blocked by the protein synthesis inhibitor cycloheximide and enhanced by the overexpression of CDK1, suggesting a role for this pathway. However, in an in vitro kinase assay, active CDK1/cyclin B1 complex failed to phosphorylate immunoprecipitated Bcl-2, suggesting that this protein kinase may not directly modify Bcl-2. Mutation of serine-70 to alanine in Bcl-2 abolishes PDT-induced phosphorylation and restores the caspase-3 activation to the same levels of the vector-transfected cells, indicating that Bcl-2 phosphorylation may be a signal to delay apoptosis in G(2)/M phase-arrested cells.

Hypericin in cancer treatment: more light on the way.

Photodynamic therapy (PDT) has been described as a promising new modality for the treatment of cancer. PDT involves the combination of a photosensitizing agent (photosensitizer), which is preferentially taken up and retained by tumor cells, and visible light of a wavelength matching the absorption spectrum of the drug. Each of these factors is harmless by itself, but when combined they ultimately produce, in the presence of oxygen, cytotoxic products that cause irreversible cellular damage and tumor destruction. Hypericin, a powerful naturally occurring photosensitizer, is found in Hypericum perforatum plants, commonly known as St. John's wort. In recent years increased interest in hypericin as a potential clinical anticancer agent has arisen since several studies established its powerful in vivo and in vitro antineoplastic activity upon irradiation. Investigations of the molecular mechanisms underlying hypericin photocytotoxicity in cancer cells have revealed that this photosensitizer can induce both apoptosis and necrosis in a concentration and light dose-dependent fashion. Moreover, PDT with hypericin results in the activation of multiple pathways that can either promote or counteract the cell death program. This review focuses on the more recent advances in the use of hypericin as a photodynamic agent and discusses the current knowledge on the signaling pathways underlying its photocytotoxic action.