The correlation between hydrophilicity of hypericins and helianthrone: internalization mechanisms, subcellular distribution and photodynamic action in colon carcinoma cells

Abstract

The internalization mechanism and subcellular distribution of hypericin (Hyp), hypericin tetrasulfonic acid (HypS4) and 1,3,4,6-tetrahydroxyhelianthrone (Hel) were studied in murine colon carcinoma CT26 cells, in protein-free medium or in the presence of serum proteins. The correlation between the extent of uptake of the sensitizers by cells that were incubated in the presence of different serum components, and the internalization mechanisms, was studied. The results indicate that sensitizer internalization may be a result of three mechanisms: partitioning, pinocytosis and endocytosis, and as a direct consequence is targeted to specific subcellular sites. While Hyp and Hel, the two lipophilic sensitizers, were localized in the endoplasmic reticulum after protein-free internalization, the hydrophilic HypS4 was localized in the cytoplasmic membrane and in lysosomes. An endolysosomal internalization route was revealed for Hyp and Hel under serum-enriched conditions showing lysosomal localization, as for HypS4. The lysosomal accumulation of Hyp-serum and specifically Hyp-LDL points to an endocytotic mechanism which is supported by its higher uptake parameter in an LDL-enriched medium, compared to the medium with 10% serum. The different uptake parameters of Hyp to cells, with or without serum, reflect the different mechanisms. Smaller differences in the uptake parameter for HypS4 reflect the distinction between partitioning and endocytosis, which, in this case, are both targeted to the lysosomes. The same uptake parameter of Hel to cells incubated in media with or without serum indicates the absence of the endocytotic mechanism. The interrelationship between subcellular targeting and photodynamic treatment was shown for the three sensitizers Hyp was found to be the most efficient sensitizer for PDT under our illumination protocol and it was dependent on internalization and localization sites.

Photochemical internalisation of chemotherapy potentiates killing of multidrug-resistant breast and bladder cancer cells

“…Furthermore, as reported in previous work by colleagues, we showed hypericin partly co-localising with lysotracker green DND-26, a marker known to bind to acidic membranes (eg, lysosomal vesicles) (Theodossiou et al, 2004). This work is supplemented by another study which highlighted that the presence of serum during hypericin incubation favours endocytic uptake (Siboni et al, 2002). We also showed co-localisation between hypericin and MTZ, the latter may be taken up by endocytosis and is retained inpart by lysosomes.…”

Elucidation of the tumoritropic principle of hypericin

“…Alternatively, after collision of the microparticles with interstitial proteins or tumour cell membranes, hypericin can be released from the lipoprotein complex, followed by a lipoprotein-independent diffusion and intracellular uptake of the compound. A similar in vitro cellular uptake of hypericin in serum-free conditions has been documented (Siboni et al, 2002). To investigate which route prevails, lipoproteins were marked with DiOC 18 , a compound that virtually irreversibly associates with lipoproteins.…”

Apoptosis in tumour cells photosensitized with Rose Bengal acetate is induced by multiple organelle photodamage

“…Indeed, the sensitivity of these organelles to photosensitization has previously been reported for other photosensitizers [e.g. hypocrellin B (Miller et al 1997) and its derivatives (Uzdensky et al 2001), hypericin (Siboni et al 2002), toluidine blue (Trembaly et al 2002), temoporWn (Teiten et al 2003)]. The possible role of the ER or the endosome-Golgi compartment in cell death induction through photodamage is supported by the observation that stresses applied to these structures may trigger the apoptotic cascade via the activation of either regulatory proteins or directly acting apoptotic proteases, such as caspase 12 or caspase 2 (Ferri and Kroemer 2001;Boyce and Yuan 2006;Kessel et al 2006;Nakagawa et al 2000).…”