In vitro study of the photocytotoxicity of some hypericin analogs on different cell lines

In the present study, hypericin analogs with an increased hydrophilic character were synthesized. As chemical modifications alter the lipophilicity/hydrophilicity balance together with the photophysical/chemical background of the molecule the influence of these structural changes on the cellular uptake, retention and subcellular localization in HeLa cells was investigated. Besides, their photocytotoxic effects using three cell lines (HeLa, MCF-7, A431), as well as their plasma protein binding were also assessed. To assess the relative hydrophilic/lipophilic character of hypericin and analogs their retention times were determined on a reversed phase high performance liquid chromatography (C-18) column. The retention time of all the hypericin analogs was < 46 min, except for dibenzyltetramethylhypericin (118 min), while the retention time of hypericin was > 200 min (solvent system: methanol/citrate buffer 30 mM pH 7; 70/30). Hypericin, hexa-, penta- and dibenzyltetramethylhypericin displayed a potent antiproliferative effect at the nanomolar range after photosensitization (3.6 J/cm2). On the contrary, photoactivated tetrasulfonhypericin and fringelite D had no antiproliferative effect on the three cell lines, whereas hypericin polyethylene glycol showed only an intermediate cytotoxic effect on A431 cells. In dark conditions no antiproliferative effect was observed for any photosensitizer. The antiproliferative photo-effect correlated well with the intracellular accumulation as measured using HeLa cells. In general, the photocytotoxic hypericin analogs concentrated to a large extent, while the noncytotoxic compounds were not taken up by the HeLa cells. Furthermore, confocal laser microscopy revealed that all photosensitizers mainly concentrated in the perinuclear region, probably corresponding with Golgi apparatus and the endoplasmic reticulum, except for tetrasulfonhypericin which located at the plasma membrane. In addition, the plasma protein binding studies illustrated that hypericin bind extensively to the low-density lipoproteins, while the other hypericin analogs were mainly bound to heavy proteins (mostly albumin) and to a small extent to low-density lipoproteins.     

Accumulation and photocytotoxicity of hypericin and analogs in two- and three-dimensional cultures of transitional cell carcinoma cells

The aim of this study was to investigate the in vitro cellular accumulation, distribution and photocytotoxic effect of hypericin in two-dimensional (2-D) and three-dimensional (3-D) cultured RT-112 transitional cell carcinoma cells of the bladder. In addition, two iodinated derivatives of hypericin were incorporated to investigate whether these analogs, with their increased lipophilicity and heavy-atom effect, display a different biological behavior and optimized photodynamic effect. The results indicate that hypericin and mono-iodohypericin behave similarly in terms of cellular accumulation, spheroidal distribution and photocytotoxic effect. In contrast, di-iodohypericin concentrated to a higher extent in monolayers and spheroids, but the accumulation was restricted to the outermost part of the spheroid. An inverse correlation therefore seems to exist between the extent of cellular uptake under 2-D conditions and the penetration of the compounds in multicellular systems. Moreover, a less pronounced photocytotoxic effect was observed for di-iodohypericin in both 2-D and 3-D cell culture systems. It can be concluded that iodinated derivatives of hypericin do not show an increased cytotoxic effect upon irradiation in either monolayers or spheroids. Moreover, this study shows that when new photosensitizers are preclinically developed, the use of 3-D cell aggregates is critical for a correct evaluation of their efficacy.     

Hypocrellin A Photosensitization Involves an Intracellular pH Decrease in 3T3 Cells

Abstract— The fluorescent pH probe carboxy-seminaphtorhoda-fluor-1 (C-Snarf-1) has been used for laser microspectrofluorometric assays of intracellular pH in 3T3 mouse fibroblasts treated with hypocrellin A. These results are compared to those previously obtained with the structurally related hydroxylated polycyclic quinone, hypericin (Sureau et al, J. Am. Chem. Soc. 118, 9484-9487, 1996). A mean local intracellular pH drop of 0.6 units has been observed in the presence of 1 μM hypocrellin A after 90 s of exposure to 0.1 μW of laser irradiation at 514.5 nm. The time evolution of the cytoplasm acidification for hypocrellin A-treated cells is faster than that for cells treated by hypericin. Thus, release of protons from an excited state of hypocrellin A appears to be more efficient than that from hypericin. In addition, the pH dependence of the quenching of C-Snarf-1 fluorescence in 3T3 cells under continuous irradiation has been observed. It is shown here that under continuous illumination, a pH decrease is able to induce a modification of the intracellular binding equilibrium of C-Snarf-1 that results in an increase of C-Snarf-1 fluorescence intensity. This latter observation suggests that the protons generated upon the photoexcitation of hypericin or its analogs may be involved in the production of other photoreactive species. Finally, we suggest that, just as for hypericin, this pH drop may be involved in the antiviral and antitumor activity of hypocrellin A.     

The Role of Oxygen in the Antiviral Activity of Hypericin and Hypocrellin

The light-induced antiviral activity of hypericin and hypocrellin in the presence and absence of oxygen was examined under experimental conditions where the effect of oxygen depletion could be quantified. There was a significant reduction of light-induced antiviral activity of hypericin and hypocrellin under hypoxic conditions. Interestingly, antiviral activity of hypocrellin was not observed at low oxygen levels at which hypericin retained measurable virucidal activity. This suggests that additional pathways, such as the generation of protons from excited states of hypericin, may enhance the biological activity of activated oxygen species.     

Identifying the sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) as a potential target for hypericin - a theoretical study

The exact cellular target for the potent anti-cancer agent hypericin has not yet been determined; this thus encourages the application of computational chemistry tools to be employed in order to provide insights that can be employed in further drug development studies. In the present study computational docking and molecular dynamics simulations are applied to investigate possible interactions between hypericin and the Ca(2+) pump SERCA as proposed in the literature. Hypericin was found to bind strongly both in pockets within the transmembrane region and in the cytosolic region of the protein, although the two studied isoforms of SERCA differ slightly in their preferred binding sites. The calculated binding energies for hypericin in the four investigated sites were of the same magnitude as for thapsigargin (TG), the most potent SERCA inhibitor, or in the range between TG and di-tert-butylhydroquinone (BHQ), which is also known to possess inhibitory activity. The hydrophobic character of hypericin indicates that the molecule initially binds in the ER membrane from which it diffuses into the transmembrane region of the protein and to binding pockets therein. The transmembrane TG and BHQ binding pockets provide suitable locations for hypericin as they allow for favourable interactions with the lipid tails that surround these. High binding energies were noted for hypericin in these pockets and are expected to constitute highly possible binding sites due to their accessibility from the ER membrane. Hypericin most likely binds to both isoforms of SERCA and acts as an inhibitor or, under light irradiation, as a singlet oxygen generator that in turn degrades the protein or induces lipid peroxidation.     

Photocytotoxicity of hypericin in normoxic and hypoxic conditions

The normoxic and hypoxic photocytotoxicity of hypericin has been examined on A431 cells as assessed by the Neutral Red method, using cell-culture flasks made of polystyrene and glass, different hypericin concentrations and light fluences. Using polystyrene flasks, lower hypoxic photoactivities of hypericin than those in normoxic conditions are seen under low fluence. In these conditions the hypoxic photocytotoxic effect can be (partially) rescued by increasing the fluence. However, a completely different outcome is observed when using glass flasks, since most of the hypoxic photocytotoxicity is lost under these conditions. The differences can be explained in terms of efficiency of deoxygenation of the medium present in polystyrene or glass flasks. Polystyrene holds large amounts of oxygen that effuses very slowly. Glass, on the other hand, does not cause this inconvenience. Therefore the type of material of the container used to investigate the oxygen dependency of the photobiological activity of photosensitizers dramatically influences the outcome of the hypoxic experiments. Our results unequivocally prove that the cytotoxic effect induced by photoactivated hypericin is completely oxygen dependent. Hence hypericin does not differ from other phototherapeutics used in photodynamic therapy of cancer, since haematoporphyrin derivative and the second-generation photosensitizers used all seem to depend on the presence of oxygen for their antitumour activity.     

Cellular photodestruction induced by hypericin in AY-27 rat bladder carcinoma cells

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.     

Phototoxicity in human retinal pigment epithelial cells promoted by hypericin, a component of St. John's wort

St. John's wort (SJW), an over-the-counter antidepressant, contains hypericin, which absorbs light in the UV and visible ranges. In vivo studies have determined that hypericin is phototoxic to skin and our previous in vitro studies with lens tissues have determined that it is potentially phototoxic to the human lens. To determine if hypericin might also be phototoxic to the human retina, we exposed human retinal pigment epithelial (hRPE) cells to 10(-7) to 10(-5) M hypericin. Fluorescence emission detected from the cells (lambda(ex) = 488 nm; lambda(em) = 505 nm) confirmed hypericin uptake by human RPE. Neither hypericin exposure alone nor visible light exposure alone reduced cell viability. However when irradiated with 0.7 J cm(-2) of visible light (lambda > 400 nm) there was loss of cell viability as measured by MTS and lactate dehydrogenase assays. The presence of hypericin in irradiated hRPE cells significantly changed the redox equilibrium of glutathione and a decrease in the activity of glutathione reductase. Increased lipid peroxidation as measured by the thiobarbituric acid reactive substances assay correlated to hypericin concentration in hRPE cells and visible light radiation. Thus, ingested SJW is potentially phototoxic to the retina and could contribute to retinal or early macular degeneration.     

Liposome binding constants and singlet oxygen quantum yields of hypericin, tetrahydroxy helianthrone and their derivatives: studies in organic solutions and in liposomes

The spectroscopy and photophysics of several hypericin and helianthrone derivatives were studied in methanol and when bound to liposomes. The singlet oxygen quantum yields (phi(delta)) were measured indirectly relative to Rose Bengal and hematoporphyrin IX, employing 9,10-dimethylanthracene as a singlet oxygen trap. Hypericin was found to have a phi(delta) of 0.39+/-0.01 in methanol, and 0.35+/-0.05 in lecithin vesicles, in agreement with literature values. A heavy atom effect was evident upon bromination, resulting in phi(delta) for tetrabromohypericin of 0.72+/-0.02, presumably due to enhanced intersystem crossing. Elimination of the anionic hydroxyls by methylation also enhanced phi(delta) to 0.81+/-0.01. Conversely, addition of anionic sulfate groups drastically reduced phi(delta) resulting in phi(delta)'s of 0.12+/-0.01, 0.052+/-0.003 and 0.40+/-0.01 for hypericin disulfonate, hypericin tetrasulfonate and hexamethyl hypericin tetrasulfonate, respectively. The non-sulfonated helianthrones exhibited low phi(delta)'s in solution. The liposome binding constants, Kb, were measured using a spectroscopic assay. Except for hexamethyl hypericin, all non-sulfonated compounds bound well with Kb's ranging from 15.5+/-0.1 to 48.7+/-3.9 (mg/ml)(-1). None of the tetrasulfonated compounds bound, however the hypericin disulfonate had a Kb of 4.1+/-0.2 (mg/ml)(-1). The phi(delta)'s of the compounds capable of binding were measured and, in the case of the hypericin derivatives, were found not to vary dramatically from those in the free state. Liposome-bound helianthrone and dimethyl tetrahydroxy helianthrone both exhibited high phi(delta)'s, i.e. >0.5. The variations in binding constant and sensitization efficiencies are explained in conjunction with the molecular structure. The relevance of the above data to photodynamic therapy is briefly discussed.     

HYPOCRELLIN, FROM HYPOCRELLA BAMBUASE, IS PHOTOTOXIC TO HUMAN IMMUNODEFICIENCY VIRUS

Abstract Hypocrellin, a photodynamic perylene quinonoid isolated from the Chinese medicinal fungus Hypocrella bambuase , was evaluated for antiviral activity against the human immunodeficiency virus (HIV-1). Hypocrellin was phototoxic to HIV-1, almost as good as the structurally similar plant pigment hypericin, and like hypericin its activity required visible light. In contrast peroxyhypocrellin had little or no effect on the virus.     

Bromohypericins are potent photoactive antiviral agents

Several hypericin derivatives, previously shown to have interesting light-mediated biological activities, were evaluated for antiviral activities against herpes simplex virus and influenza virus. Three brominated hypericins, the dibromo- and tetrabromo-derivatives and the natural compound gymnochrome B were all very active against both viruses, particularly herpes simplex virus, although light was required in all cases for maximum activity. The dibromohypericin was the most potent, under standard assay conditions, gymnochrome B was approximately as active as hypericin itself and tetrabromohypericin significantly less so. Surprisingly, hexamethylhypericin, which is known to have potent anti-protein kinase (PK) C activity, as well as anticell proliferation properties, showed no antiviral activity at all. The compounds were also evaluated in different serum concentrations. All the active compounds were inhibited by increasing concentrations of serum, but to different degrees, such that their relative antiviral potencies changed to some extent. Thus, in summary, there was no correlation between antiviral and anti-PK or anticellular activities, and consequently it is not possible at present to define those structural features of hypericin-type molecules that are required for their various biological activities.     

Toward the molecular flashlight: preparation,properties, and photophysics of a hypericin-luciferin tethered molecule

The synthesis of a molecule containing hypericin and luciferin moieties joined by a tether is reported. The light-induced (in vitro) antiviral activity as well as the photophysical properties of this new compound are measured and compared with those of the parent compounds, hypericin and pseudohypericin. This tethered molecule exhibits excited-state behavior that is very similar to that of its parent compounds and antiviral activity that is identical, within experimental error, to that of its more closely related parent compound, pseudohypericin. The implications for a photodynamic therapy that is independent of external light sources are discussed.     

Photosensitizing and radiosensitizing effects of hypericin on human renal carcinoma cells in vitro

The renal cell carcinoma (RCC) is extremely resistant to chemotherapy and radiotherapy. The prognosis of patients with metastatic RCC still remains poor, the median survival is less than 12 months. Therefore, new therapeutic options are desirable. The aim of this study was to investigate the photosensitizing and radiosensitizing effects of hypericin on human RCC cells in vitro. First the RCC-derived cell lines A498 and ACHN were incubated with different concentrations of hypericin. In vitro uptake and intracellular distribution of hypericin were confirmed by fluorescence microscopy. Subsequently cells were illuminated and irradiated with a dose of 2-8 Gy, respectively. Finally, metabolic activity, apoptosis and clonogenic survival were investigated. Uptake of hypericin was observed for almost all cells. Hypericin treatment combined with illumination led to a 94-97% decrease in metabolic activity and caused apoptosis in nearly 100% of RCC cells. Hypericin enhanced the radiosensitivity of A498 cells in vitro. The clonogenic survival after irradiation was significantly reduced by hypericin treatment. Taken together, the photosensitizing and radiosensitizing effects of hypericin on human RCC cells we found in this investigation could be of clinical relevance, e.g. for radiotherapy and intraoperative photodynamic therapy, respectively.     

Novel methyl helianthrones as photosensitizers: synthesis and biological evaluation

A combination of light, oxygen and a photosensitizer is used to induce death of cancer cells by photodynamic therapy. In this study, we have synthesized several new methyl helianthrone derivatives and compared their phototoxicity with that of hypericin. In contrast to hypericin, methyl helianthrones are soluble in aqueous solutions and have a broad range of light absorbance, which allows the use of polychromatic light. Structural modifications of methyl helianthrone demonstrated that substitution of hydrogen atoms of methyl helianthrone at Positions 2 and 5 with Br atoms or methylation of its phenolic hydroxyls, significantly increases the corresponding singlet oxygen quantum yield and their phototoxicity toward alphaT3-1, M2R and LNCaP cells. The phototoxicity of some of these compounds was similar to that of hypericin. Methyl helianthrones, like hypericin, accumulated mainly in the perinuclear region as evident by confocal microscopy. Irradiation of cells pretreated with methyl helianthrone derivatives generates intracellular reactive oxygen species and lipid free radicals, as shown by a fluorescentic probe and electron paramagnetic resonance methods, respectively. The phototoxicity of these methyl helianthrones as well as their ability to oxidize membrane lipids were significantly decreased on addition of specific Type-II inhibitors, suggesting the involvement of singlet oxygen as the main oxidant.     

LASER PHOTOSENSITIZATION OF CELLS BY HYPERICIN

Abstract— Administering a light dose of 90 J/cm² at 599 nm during incubation with hypericin to a highly differentiated normal epithelial cell line(FRTL–5), derived from Fisher rat thyroid, and to a neoplastic cell line(MPTK–6), derived from the lung metastases of a thyroid carcinoma induced in Fisher rats, produces cell kill at drug doses 1000 times lower than those necessary to cause the same mortality in the dark. The photocytocidal activity of this polycyclic quinone drug on neoplastic cells is superior to that of antitumor anthraquinone drugs, such as daunomycin and mitoxanthrone, and to the photosensitized antiviral activity previously reported for hypericin.     

Photophysics of Hypericin and Hypocrellin A in Complex with Subcellular Components: Interactions with Human Serum Albumin

Time-resolved fluorescence and absorption measurements are performed on hypericin complexed with human serum albumin, HSA (1:4, 1:1 and approximately 5:1 hypericin: HSA complexes). Detailed comparisons with hypocrellin A/HSA complexes (1:4 and 1:1) are made. Our results are consistent with the conclusions of previous studies indicating that hypericin binds to HSA by means of a specific hydrogen-bonded interaction between its carbonyl oxygen and the N1-H of the tryptophan residue in the IIA subdomain of HSA. (They also indicate that some hypericin binds nonspecifically to the surface of the protein.) A single-exponential rotational diffusion time of 31 ns is measured for hypericin bound to HSA, indicating that it is very rigidly held. Energy transfer from the tryptophan residue of HSA to hypericin is very efficient and is characterized by a critical distance of 94 A, from which we estimate a time constant for energy transfer of approximately 3 x 10(-15) s. Although it is tightly bound to HSA, hypericin is still capable of executing excited-state intramolecular proton (or hydrogen atom) transfer in the approximately 5:1 complex, albeit to a lesser extent than when it is free in solution. It appears that the proton transfer process is completely impeded in the 1:1 complex. The implications of these results for hypericin (and hypocrellin A) are discussed in terms of the mechanism of intramolecular excited-state proton transfer, the mode of binding to HSA and the light-induced antiviral and antitumor activity.     

Hypericin-induced photocytotoxicity is connected with G2/M arrest in HT-29 and S-phase arrest in U937 cells

Susceptibility of the HT-29 human colon adenocarcinoma cell line and human myeloid leukemia cell line U937 to hypericin-mediated photocytotoxicity was investigated and compared in this study. Cellular parameters as viability, cell number, metabolic activity and total protein amount were monitored in screening experiments with subsequent cell-cycle analysis and apoptosis detection to determine the cellular response of the different tumor types to various concentrations of photoactivated hypericin. The results show concentration dependence of the photosensitizer's cytotoxicity on the studied cell lines, with higher sensitivity of U937 cells. Whereas the two extreme hypericin concentrations (1 x 10(-9) M and 1 x 10(-6) M) resulted in similar changes in all tested cellular parameters on the two studied cell lines, 1 x 10(-8) M and 1 x 10(-7) M hypericin treatment resulted in different responses of the cell lines in all monitored parameters except for viability. Although leukemic cells proved sensitive to both 1 x 10(-8) M and 1 x 10(-7) M hypericin, significant changes on HT-29 cells were detected only after the 1 x 10(-7) M hypericin concentration. Cell-cycle arrest was related to simultaneously occurring apoptosis in colon cancer. Remarkable is the difference in cell-cycle profile where G2/M arrest in colon cancer cells versus accumulation of leukemic cells in the S phase appears. This suggests that hypericin treatment affecting the cell-cycle machinery of different cancer cells is not universal in effect.     

Skin photosensitization with topical hypericin in hairless mice

Hypericin, a naturally occurring photosensitizer, exhibits interesting in vitro photobiological activities, which suggest that the compound is a potential antipsoriatic agent. In this study, the possibility of hypericin penetrating the skin in photo-active concentrations has been studied. Hypericin is incorporated in either emulsifying ointment supplemented with solketal (hypericin content: 0.05%) or in polyethylene glycol (PEG) ointment (hypericin content: 0.5%) and applied to the skin of hairless mice for 4 h. After removing excess ointment, the mice are then irradiated with different light doses using a 500 W halogen lamp. As a positive control, intraperitoneally (i.p.) administered hypericin (10 and 40 mg/kg) has also been tested. Erythema, desquamation and erosions are demonstrated in the mice treated with hypericin in emulsifying ointment with solketal using a light dose of at least 4.5 J/cm2. In general, these reactions correlate well with those of i.p. administered hypericin (40 mg/kg), indicating that hypericin incorporated in emulsifying ointment with solketal is well absorbed by the skin of the mice. However, for the i.p. administered hypericin (40 mg/kg), we could not evaluate phototoxic reactions in the group of animals that received a light dose of 108 J/cm2, as they all died 12-24 h after irradiation, indicating extreme photosensitization with systemic hypericin at higher light doses. On the contrary, there is no measurable skin photosensitivity induced by hypericin when incorporated in PEG ointment or when 10 mg/kg hypericin is i.p. administered. Our results show that hypericin incorporated in a suitable vehicle can be delivered to the skin in photo-active concentrations. Using a vehicle such as emulsifying ointment with solketal, it will be possible to explore the photo-activity of hypericin in the treatment of psoriasis and other skin diseases.     

Phototoxic effects of Hypericum extract in cultures of human keratinocytes compared with those of psoralen

Extracts of Hypericum perforatum (St. John's wort) are used in the treatment of depression. They contain the plant pigment hypericin and hypericin derivates. These compounds have light-dependent activities. In order to estimate the potential risk of phototoxic skin damage during antidepressive therapy, we investigated the phototoxic activity of hypericin extract using cultures of human keratinocytes and compared it with the effect of the well-known phototoxic agent psoralen. The absorbance spectrum of our Hypericum extract revealed maxima in the whole UV range and in parts of the visible range. We cultivated human keratinocytes in the presence of different Hypericum concentrations and irradiated the cells with 150 mJ/cm2 UVB, 1 J/cm2 UVA or 3 h with a white light of photon flux density 2.6 mumol m-2 s-1. The determination of the bromodeoxyuridine incorporation rate showed a concentration- and light-dependent decrease in DNA synthesis with high hypericin concentrations (> or = 50 micrograms/mL) combined with UVA or visible light radiation. In the case of UVB irradiation a clear phototoxic cell reaction was not detected. We found phototoxic effects even with 10 ng/mL psoralen using UVA with the same study design as in the case of the Hypericum extract. These results confirm the phototoxic activity of Hypericum extract on human keratinocytes. However, the blood levels that are to be expected during antidepressive therapy are presumably too low to induce phototoxic skin reactions.