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

Abstract

We have assessed photoinduced toxicity of hypericin in PAM 212 murine keratinocytes and the relationship between concentration, incubation time and light fluence to evaluate the effect of intracellular aggregation at high concentrations. Confocal microscopy was used to establish the subcellular localization of hypericin at 5 and 50 μM and incubation times of 1 and 3 h. From fluorescence uptake time course studies, intracellular hypericin was demonstrated to exist predominantly in the monomeric form for up to 26 h incubation at 5 μM. However, there was a pronounced aggregation effect at 50 μM, with intracellular hypericin fluorescence levels initially showing an increase followed by a decrease with incubation time. This effect was subsequently shown to exert an effect on the phototoxicity of hypericin. On irradiation, the photocytotoxicity for 1 and 7 h incubation with 50 μM hypericin was comparable, whereas using 5 μM the photocytotoxicity showed good correlation with the intracellular fluorescence measurements at 1 and 7 h incubation.

Cytotoxic and Photocytotoxic Effects of Cercosporin on Human Tumor Cell Lines

“…The corresponding ratios for 2 μ m incubation are 1.45 and 3.4. This discrepancy can be attributed to increased intracellular aggregation like we have previously seen for hypericin which is expected to be higher at higher uptakes. Also, as we have shown in our previous work for MCF7 and MDA‐MB‐231 cells , different cell lines have dissimilar tolerances and diverse defense mechanisms to PDT.…”

Melanomas display increased cytoprotection to hypericin‐mediated cytotoxicity through the induction of autophagy

“…Activating it with light, or UV‐A, in the presence of molecular oxygen leads to the generation of reactive oxygen species (ROS) and these ultimately kill the cells (Milanesio et al., 2001; Dolmans et al., 2003). In addition, hypericin accumulates in the endoplasmic reticulum (Agostinis et al., 2002), Golgi (Uzdensky et al., 2001; Theodossiou et al., 2006), lysosomes (Siboni et al., 2002; Theodossiou et al., 2004) and mitochondria (Ali and Olivo, 2002). Although earlier reports suggested that hypericin was excluded from mitochondria, it clearly affects cytochrome c release which induces caspase‐dependent apoptosis and other forms of cell death.…”

Interactive Transport, Subcellular Relocation and Enhanced Phototoxicity of Hypericin Encapsulated in Guanidinylated Liposomes via Molecular Recognition†

“…Following light excitation, HYP exhibits enhanced in vitro cytotoxicity (5–8) attributed to various ROS (7–12). The photocytotoxic action of HYP is highly dependent on its locoregional accumulation within the cell; previous studies on the subcellular localization of HYP indicate a selective affinity to lipid membranes in general (13).…”

“…The photocytotoxic action of HYP is highly dependent on its locoregional accumulation within the cell; previous studies on the subcellular localization of HYP indicate a selective affinity to lipid membranes in general (13). More specifically, HYP has been found to accumulate in the endoplasmic reticulum (ER) and Golgi (14), lysosomes (6,15) and mitochondria (16).…”

“…The main problem in the progression of HYP–PDT to a clinical practice is the lipophilic nature of HYP which tends to form aggregates in aqueous environment. This aggregation significantly diminishes its photosensitizing efficacy (6) and can be overcome either by synthesis of water‐soluble forms of HYP or by encapsulating hydrophobic HYP into appropriate drug‐carriers like liposomes (26).…”