Publication: PDT-mediated apoptosis by hypocrellins and hypericin in tumor cells
Abstract
Photodynamic therapy (PDT) is relatively a new therapeutic procedure used for the management of a variety of solid tumors including skin cancer and is showing promise for the management of many nonmalignant diseases including dermatological disorders. PDT is classically based on photochemical reactions of chromophores of both exogenous and endogenous origin. An increasing body of evidence suggests that apoptosis plays an important role in the biochemical cell death pathway for normal tissue homeostasis, cellular differentiation, development within a multi-cellular organisms and tumors. Apoptosis is a tightly regulated physiological process characterized in part by nuclear condensation and cell shrinkage with preservation of an intact plasma membrane, culminating in the destruction of the nuclear chromatin and the digestion of the genomic DNA, an irreversible event.<br><br>Current developments in understanding the mechanisms of the PDT effect of photosensitizers indicate that a critical factor in the success of the agent is the ability to induce apoptosis in the malignant cell population. PDT with many photosensitizers has shown to induce both apoptosis and necrosis in vitro as well as in vivo. Hypocrellin A (HA), Hypocrellin B (HB) and Hypericin (HY) are lipid-soluble perylquinone derivatives of the genus Hypericum, which in cells bind mostly to the cellular membrane and can be metabolized rapidly in vivo with no toxic properties. It has been reported that HA, HB and HY have a strong photodynamic effect on human immunodeficiency virus type I (HIV-1), and vesicular stomatitis (VSV). The above observations collectively provide a compelling rationale for the development of HA, HB and HY as photosensitizers.<br><br>Although, the differential anti-tumor activities of HY have been well documented on a variety of tumor cell lines, the precise mode of cell death and the intracellular signaling pathway (s) triggered by HY were not well defined. Besides the above, it has been reported that HA can successfully induce cell death in human malignant cell lines. But, hitherto no reports have mentioned cell apoptosis induced by PDT using neither HB nor the precise mode of cell death induced by PDT using HA or HB. Studies on PDT sensitized HA, HB and HY induced apoptosis and their molecular mechanisms are however limited and remains unclear in tumor cells. Therefore, the present study was undertaken to shed some light on the molecular mechanisms and signaling pathways involved in the apoptosis induced by photoactivated HA, HB and HY in poorly and moderately differentiated NPC as well as poorly differentiated colon and bladder cells. To evaluate the susceptibility of tumor cells to apoptosis induced by photoactivated HA, HB and HY, and concomitant changes in the cell size; cell cycle DNA distribution, caspases activation, phosphatidylserine (PS) externalization and chromatin condensation were studied.<br><br>Both NPC and two other tumor cell lines, all respond to PDT by exhibiting morphological and biochemical features consistent with apoptotic cell death, i.e. shrinkage of cell size, appearance of sub-diploid DNA. A loss of membrane phospholipid asymmetry associated with apoptosis was induced in all tumor cell lines as evidenced by the externalization of phosphatidylserine (PS). A dose-dependent increase in caspase-3 protease activity inhibitable by the tetrapeptide inhibitors DEVD-CHO and z-VAD was also observed in all the cell lines. Under apoptotic conditions, Western blot analysis of poly (ADP-ribose) polymerase, a caspase substrate, showed the classical cleavage pattern (116 to 85kDa) associated with apoptosis in photoactivated HA, HB and HY-treated cell lysates. In addition, 85kDa cleaved product was blocked by the tetrapeptide caspases inhibitors such as DEVD-CHO or z-VAD-fmk. Both inhibitors protect tumor cells from apoptosis. Besides, caspase inhibition blocked the externalization of membrane PS, indicating that the loss of membrane phospholipid asymmetry is a downstream event of caspases activation. These results demonstrate that tumor cell death induced by HA, HB and HY is mediated by caspase proteases.<br><br>Using these photosensitizers (HA, HB and HY) as a model system for apoptotic signaling, this study tried to understand the pathways with respect to Fas/FasL system as well as the "cross talk" between caspases and mitochondria in PDT induced apoptosis. Hence, it was sought to determine whether Fas/FasL upregulation and involvement of mitochondrial events were an early event in HA, HB and HY-treated PDT induced apoptosis or not. Loss of mitochondrial transmembrane potential, release of cytochrome c and involvement of caspases-8 and -3 were evaluated in photo-induced HA, HB and HY -treated tumor cells. Photoactivation of HA, HB and HY enhance both CD95/CD95L expression and induce CD95-signalling dependent cell death in all tumor cell lines studied. CD95/CD95L expression appeared within 2 h following light activation and appeared to be an important event in PDT induced apoptosis. Further, these results also indicate that release of mitochondrial cytochrome c into the cytoplasm is a subsequent 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 in HA, HB and HY-treated cells.<br><br>As reactive oxygen species (ROS), particularly hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) has been implicated in drug-induced apoptosis, and the recent observations which highlighted the regulatory role of H<sub>2</sub>O<sub>2</sub> in apoptotic signaling, it was worthwhile to investigate the role of H<sub>2</sub>O<sub>2</sub> in the apoptotic cell death induced by photoactivated HA, HB and HY. The present study also examined whether generation of H<sub>2</sub>O<sub>2</sub> is a critical event for the apoptotic pathway upstream of mitochondrial involvement and caspase-3 protease activation. Generation of H<sub>2</sub>O<sub>2</sub> was commonly detected after photoactivation within hour, and scavenging of H<sub>2</sub>O<sub>2</sub> caused cells to fail to undergo apoptosis. Flow cytometry analysis demonstrated that H<sub>2</sub>O<sub>2</sub> production preceded loss of mitochondrial membrane potential (ΔΨm) in photoactivated cells treated with HA, HB and HY. These results indicate that HA, HB and HY photosensitization induces H<sub>2</sub>O<sub>2</sub> generation, which causes (ΔΨm) and subsequently caspase-3 activation followed by DNA fragmentation, resulting in apoptosis. These findings suggest that H<sub>2</sub>O<sub>2</sub> may function as a primary and common mediator for apoptosis induced by HA, HB and HY.<br><br>Intracellular acidification associated with apoptotic cell death has been reported in many studies, suggesting an execution-phase expression, marking the point of no return in apoptosis, preceding mitochondrial dysfunction and upstream caspases activation. It has also been seen as a requirement for apoptosis. Results of the present study provide evidence to support the fact that intracellular acidification induced by photoactivated HA, HB and HY in CNE2 and TWO-1 cells occurs secondary to H<sub>2</sub>O<sub>2</sub> production. Moreover, kinetic results also showed that acidification of the intracellular milieu may be due to ROS in particular H<sub>2</sub>O<sub>2</sub> (H<sub>2</sub>O<sub>2</sub> generation occurs within 30 min after irradiation) preceding mitochondrial dysfunction and upstream of caspases activation.<br><br>Glutathione (GSH) is a component of the essential antioxidant system that provides animal cells their reducing milieu. GSH not only protects cells against oxidative damage, but also against a variety of toxic compounds of endogenous and exogenous origin. As a parameter of oxidative stress intracellular GSH levels were assessed in this study. At 1h post irradiation, GSH status was checked by flow cytometry analysis. Photoactivated HA, HB and HY-treated cells show that there is a depletion of GSH, the primary mechanism in cells against free radicals as well as other oxidants when compared to drug treated, non-irradiated control cells.<br><br>In conclusion, although the mechanisms for the activation of caspase-3 proteases and DNA fragmentation would depend on each photosensitizer and light, H<sub>2</sub>O<sub>2</sub> production and related mitochondrial down stream events appear to follow general pathways in photoactivated HA, HB and HY induced apoptosis. Further, H<sub>2</sub>O<sub>2</sub> may serve as a major mediator for photo-induced apoptosis in NPC and other cell types. Thus, it is accomplished that photoactivation induces the generation of H<sub>2</sub>O<sub>2</sub> which causes intracellular acidification, GSH depletion and subsequent drop in membrane potential followed by release of cytochrome c and caspases-3 activation, resulting in apoptosis. The present study also identified HA, HB and HY as potent and promising photosensitizers for the treatment of NPC and other tumor cells. These findings suggest that Hypocrellins (A and B) and Hypericin offer photodynamic properties, which in association with red light may render them suitable for further development as potent mediators of photodynamic therapy.<br><br>Thus, it has become clear that multiple pathways are involved in PDT-mediated apoptosis, offering exciting opportunities to take advantage of these pathways in improving PDT treatment protocol.