Interaction dynamics of hypericin with low-density lipoproteins and U87-MG cells
Introduction
Hypericin (Hyp) is a natural photosensitizer characterized by high singlet oxygen production (Olivo et al., 2006, Cole et al., 2008, Higuchi et al., 2008, Kacerovská et al., 2008). Its specific accumulation in tumor cells can be used for photo-diagnosis of early epithelial cancer (Sim et al., 2005, Fu et al., 2007, Kah et al., 2008, Ritz et al., 2008a). These properties together with minimal dark toxicity, and high clearance rate from the host body make Hyp a promising agent for PDT of cancer (Falk, 1999, Agostinis et al., 2002, Miskovsky, 2002, Kiesslich et al., 2006).
As expected from its molecular structure (Fig. 1), Hyp is a highly hydrophobic compound that forms non-fluorescent aggregates in aqueous solution. However absorption and fluorescence emission spectra of Hyp monomer can be observed after binding with various biological macromolecules (Das et al., 1999, Falk, 1999, Petrich, 2000, Miskovsky et al., 2001, Agostinis et al., 2002, Miskovsky, 2002, Kiesslich et al., 2006). Formation of reactive oxygen species (ROS) can only be obtained from its monomeric form (Senthil et al., 1992, Guedes and Erikson, 2005, Gbur et al., 2008, Gbur et al., 2009). The very short lifetimes of ROS limit their diffusion through the cells. Consequently, the intracellular biological target and subsequent photo-induced cell death pathways (apoptotic or necrotic) are closely related to the subcellular localization of Hyp as well as to its monomers–aggregates balance. In this view, the intravascular transport of Hyp by LDLs and subsequent cellular uptake processes should be taken in consideration.
LDL is known as the main carrier of cholesterol in the human circulation system. It can be characterized as a spherical particle of 22 nm in diameter having three different regions. The outer surface layer, which consists of phospholipids molecules with a single apoB-100 protein; the core of LDL, which is enriched with triglycerides and cholesterol esters and an interfacial region between these two (Hevonoja et al., 2000). Due to the overexpression of specific receptors in many types of transformed cells (Brown and Goldstein, 1976, Vitols et al., 1992) LDL could play a key role in the targeted delivery of hydrophobic and/or amphiphilic photosensitizers to tumor cells for PDT (Jori, 1996, Reddi, 1997, Konan et al., 2002, Derycke and de Witte, 2004, Sherman et al., 2004). Previous investigations of Hyp interaction with LDL have shown non-specific binding of both its monomer and aggregate forms. Up to ∼30 monomer molecules of Hyp can enter one LDL (Kascakova et al., 2005). Hyp are most likely embedded in the phospholipids shell of LDL close to the apoB-100 protein (Kascakova et al., 2008, Lajos et al., 2008, Mukherjee et al., 2008) and cholesterol has recently been identified as the key determinant of the observed selectivity of Hyp in model membrane systems (Ho et al., 2009). Upon increasing the Hyp concentration, the fluorescence quantum yield is dramatically decreased, suggesting most likely self-quenching of the aggregated form. It was shown that mixing of photosensitizers with LDL before in vivo administration led to a better delivery to target tissues and to an improved photodynamic efficacy (Korbelik, 1992, Korbelik, 1993, Chowdhury et al., 2003). Consistently, we have shown that over expression of LDL receptors leads to an increased accumulation of Hyp within U87 glioma cells (Kascakova et al., 2008). Therefore, the question is; what is the importance of the in vivo plasmatic transport of Hyp by LDL on the cellular uptake process and on its subsequent subcellular distribution. Does it enter cells through the endocytosis pathway of LDL or might the release of Hyp from LDL towards cellular membrane – prior to LDL endocytosis – be the main process? In this view, steady-state and kinetic studies of LDL–Hyp interaction were performed in using the fluorescence properties of Hyp.
Section snippets
Chemicals
Hyp, LDL and LDL-bodipy (LDLb) were all purchased from Gibco-Invitrogen, France.
Cell culture
The U87-MG human glioma cells were grown in Dulbecco's modified Eagle's medium (D-MEM) containing l-glutamine (862 mg L−1), sodium pyruvate (110 mg L−1), glucose (4500 mg L−1), streptomycin (50 μg mL−1), penicillin (50 μg mL−1) and supplemented with 10% fetal bovine serum (FBS) or serum substitute 2% Ultroser G (Pall Corporation, France), in the presence of 5% CO2 humified atmosphere at 37 °C. For all experiment cells were
Dynamic studies of Hyp–LDL interaction
The basic fluorescence spectroscopic study of Hyp interaction with LDL has already been described in our previous studies (Kascakova et al., 2005, Mukherjee et al., 2008). The concentration dependence of Hyp steady-state fluorescence emission in the presence of LDLs was monitored (Kascakova et al., 2005) and result of a similar experiment is given here (Fig. 2). While increasing the number of Hyp molecules per LDL molecule, a saturation effect of the fluorescence intensity is first observed,
Conclusion
In this work, dynamic studies gives clear evidences that the monomer form of Hyp which was already considered as the only photoactive form should also be considered as the only redistributive form while aggregate form should be considered as a monomer delivery delay form. However, the transport of Hyp under aggregate form in overloaded LDL (Hyp/LDL = 200:1) not only allows precluding the toxic photo-oxidation process of LDL prior to cellular uptake but also leads to an higher intracellular
Acknowledgements
This work was supported by the Slovak Research and Development Agency contracts No. APVV-0449-07 and APVV-LPP-0337-06, the Scientific Grant Agency of the Ministry of Education of Slovak Republic (grant VEGA-0164-09). The authors thank the Synchrotron SOLEIL for providing the detection system of the DISCO beamline.
References (46)
- et al.
Hypericin in cancer treatment: more light on the way
Int. J. Biochem. Cell Biol.
(2002) - et al.
Mechanisms in photodynamic therapy: part one—photosensitizers, photochemistry and cellular localization
Photodiagn. Photodyn. Ther.
(2004) - et al.
Liposomes for photodynamic therapy
Adv. Drug Deliv. Rev.
(2004) - et al.
Time resolved luminescence and singlet oxygen formation under illumination of hypericin in acetone
J. Luminescence
(2008) - et al.
Theoretical study of hypericin
J. Photochem. Photobiol. A Chem.
(2005) - et al.
Structure of low density lipoprotein (LDL) particles: basis for understanding molecular changes in modified LDL
Biochem. Biophys. Acta
(2000) - et al.
Lipid-mediated preferential localization of hypericin in lipid membranes
Biochim. Biophys. Acta
(2009) Tumour photosensitizers: approaches to enhance the selectivity and efficacy of photodynamic therapy
J. Photochem. Photobiol. B Biol.
(1996)- et al.
Monitoring of labeled antisense oligonucleotides within living cells by using a multifrequency phase/modulation approach for fluorescence lifetime measurements
J. Mol. Struct.
(2003) - et al.
State of the art in the delivery of photosensitizers for photodynamic therapy
J. Photochem. Photobiol. B Biol.
(2002)
Low density lipoprotein receptor pathway in the delivery of photofrin: how much it is relevant for selective accumulation of the photosensitizers in tumors?
J. Photochem. Photobiol. B Biol.
Signaling pathway in cell death and survival after photodynamic therapy
J. Photochem. Photobiol. B
Role of delivery vehicles for photosensitizers in the photodynamic therapy of tumors
J. Photochem. Photobiol. B Biol.
Hypericin uptake: a prognostic marker for survival in high-grade glioma
J. Clin. Neurosci.
Photosensitization of aqueous model systems by hypericin
Biochim. Biophys. Acta
Bio-distribution and subcellular localization of hypericin and its role in PDT induced apoptosis in cancer cells
Int. J. Oncol.
Receptor mediated control of cholesterol metabolism
Science
Efficacy of antitumoral photodynamic therapy with hypericin: relationship between biodistribution and photodynamic effects in the RIF-1 mouse tumor model
Int. J. Cancer
Release of hydrophobic molecules from polymer micelles into cell membranes revealed by Förster resonance energy transfer imaging
Proc. Natl. Acad. Sci. U.S.A.
Correlation of photosensitizer delivery to lipoproteins and efficacy in tumor and arthritis mouse models: comparison of lipid-based and Pluronic P123 formulations
J. Pharm. Pharm. Sci.
Hypericin-mediated photodynamic therapy of pituitary tumors: preclinical study in a GH4C1 rat tumor model
J. Neurooncol.
Photophysics of hypericin and hypocrellin A in complex with subcellular components: interactions with human serum albumin
Photochem. Photobiol.
From the photosensitizer hypericin to the photoreceptor stenorin: the chemistry of phenanthroperylene quinines
Angew. Chem. Int. Ed. Engl.
Cited by (41)
Imaging of heterogeneity in 3D spheroids of U87MG glioblastoma cells and its implications for photodynamic therapy
2023, Photodiagnosis and Photodynamic TherapyEffective transport of aggregated hypericin encapsulated in SBA-15 nanoporous silica particles for photodynamic therapy of cancer cells
2023, Journal of Photochemistry and Photobiology B: BiologyRedistribution of hydrophobic hypericin from nanoporous particles of SBA-15 silica in vitro, in cells and in vivo
2023, International Journal of PharmaceuticsSearching for combination therapy by clustering methods: Stimulation of PKC in Golgi apparatus combined with hypericin induced PDT
2020, Photodiagnosis and Photodynamic TherapyCitation Excerpt :Application of Gö6976, PMA and hypericin did not result in a significant difference in LDLbodipy uptake by U87 MG. We have previously demonstrated that hypericin can be transported into lysosomes, however, hypericin should be loaded in LDL [39,58,59]. By contrast, 1 h pretreatment of U87 MG cells with rottlerin caused decreasing of LDLbodipy fluorescence.
Encapsulation of anticancer drug curcumin and co-loading with photosensitizer hypericin into lipoproteins investigated by fluorescence resonance energy transfer
2019, International Journal of PharmaceuticsBenefits of hypericin transport and delivery by low- and high-density lipoproteins to cancer cells: From in vitro to ex ovo
2019, Photodiagnosis and Photodynamic TherapyCitation Excerpt :Hyp, in this study, represents a model of a hydrophobic molecule with promising anticancer properties, especially in photodynamic treatment [7,8]. It was demonstrated that only hyp monomers emit fluorescence [57–59]. Other researcher groups, along with ours, observed that a high number of hyp molecules could be loaded in LDL, but this process is accompanied by aggregation and self-quenching of hyp molecules inside LDL [57,59–61].