Cellular Photodestruction Induced by Hypericin in AY-27 Rat Bladder Carcinoma Cells

Abstract

In a recent clinical study we showed that hypericin accumulates selectively in urothelial lesions following intravesical administration of the compound to patients. In the present study the efficacy of hypericin as a photochemotherapeutic tool against urinary bladder carcinoma was investigated using the AY-27 cells (chemically induced rat bladder carcinoma cells). The uptake of hypericin by the cells increased by prolonging the incubation time and increasing the extracellular hypericin concentration. Photodynamic treatment of the cells incubated with 0.8 and 1.6 microM hypericin concentrations resulted in remarkable cytotoxic effects the extent of which depended on the fluence rates. Photoactivation of 1.6 microM hypericin by 0.5, 1.0 or 2.0 mW/cm2 for 15 min resulted in 3, 30 and 95% of the antiproliferative effect, respectively. Increasing the photoactivating light dose from 0.45 to 3.6 J/cm2 resulted in a five-fold increase in hypericin photodynamic activity. Irrespective of the fluence rates and irradiation times incubation of the cells with 10 microM hypericin induced rapid and extensive cell death in all conditions. The type of cell death (apoptosis or necrosis) induced by photoactivated hypericin depended largely on the hypericin concentration and the postirradiation time. At lower hypericin concentrations and shorter postirradiation times apoptosis was the prominent mode of cell death; increasing the hypericin concentration and/or prolonging the postirradiation time resulted in increased necrotic cell death. Cell pretreatment with the singlet oxygen quencher histidine, but not with the free-radical quenchers, significantly protected the cells from photoactivated hypericin-induced apoptosis, at least when a relatively low concentration (1.25 microM) was used. This result suggests the involvement of a Type-II photosensitization process. However, cells treated with higher hypericin concentrations (2.5-5 microM) were inadequately protected by histidine. Since hypericin is thus shown to be a potent and efficient photosensitizer, and since the conditions used were the same as when hypericin is used clinically to locate early-stage urothelial carcinoma lesions, hypericin may well become very important for the photodynamic treatment of superficial bladder carcinoma.

Photosensitizing activity of hypericin and hypericin acetate after topical application on normal mouse skin

“…In contrast to PpIX, hypericin is very photostable 21 and laboratory studies have indicated that, when activated by artificial light, it has enhanced antitumour activity. [22][23][24] Moreover, tests investigating the topical application of hypericin have shown promising results. 13 These results suggest that hypericin may provide a therapeutic benefit when targeted to certain skin diseases.…”

Evidence for Intracellular Aggregation of Hypericin and the Impact on its Photocytotoxicity in PAM 212 Murine Keratinocytes

“…This method has been used in analogous fashion to the extraction of the lipophilic enzymatic product formazan compound from cells, which is part of the standard MTT assay. Another study used 4% sodium dodecyl sulfate in PBS for hypericin extraction (26). In the case of 5 phi' hypericin ( Fig.…”

Cellular Mechanisms and Prospective Applications of Hypericin in Photodynamic Therapy

“…Addition of the singlet oxygen quencher L-histidine was able to prevent the induction of apoptosis by Hyp PDT in AY-27 rat ladder carcinoma cells, whereas necrotic cell death was still feasible at higher PDT doses [114]. These findings point out to the possibility of singlet oxygen having specific signaling functions in the induction of apoptotic cell death (prevented by addition of L-histidine) [64]; however, it is also possible that this substance is able to neutralize singlet oxygen and prevent subsequent ROS formation at concentrations suitable for apoptosis induction, but -over a certain singlet oxygen threshold -fails to prevent severe cellular damage by ROS, resulting in necrosis.…”

“…From that point on, numerous works studied the mechanistic details of apoptosis induced by Hyp PDT using various model systems [85,[112][113][114][115][116][117][118][119][120][121]. A light-or drug dose-dependent transition from apoptosis to necrosis following Hyp PDT was found in human and murine cancer cell lines by the research groups of Agostinis and de Witte [114][115][116]. As discussed in [64] the final intracellular concentration of Hyp and / or the light dose applied most probably account for the different modes of cell death eventually induced.…”