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1.
J Biol Chem ; 276(37): 34631-6, 2001 Sep 14.
Article in English | MEDLINE | ID: mdl-11457843

ABSTRACT

Natural peptides displaying agonist activity on the orphan G protein-coupled receptor GPR54 were isolated from human placenta. These 54-, 14,- and 13-amino acid peptides, with a common RF-amide C terminus, derive from the product of KiSS-1, a metastasis suppressor gene for melanoma cells, and were therefore designated kisspeptins. They bound with low nanomolar affinities to rat and human GPR54 expressed in Chinese hamster ovary K1 cells and stimulated PIP(2) hydrolysis, Ca(2+) mobilization, arachidonic acid release, ERK1/2 and p38 MAP kinase phosphorylation, and stress fiber formation but inhibited cell proliferation. Human GPR54 was highly expressed in placenta, pituitary, pancreas, and spinal cord, suggesting a role in the regulation of endocrine function. Stimulation of oxytocin secretion after kisspeptin administration to rats confirmed this hypothesis.


Subject(s)
Genes, Tumor Suppressor , Neoplasm Metastasis/genetics , Proteins/genetics , Receptors, Neuropeptide/analysis , Amino Acid Sequence , Animals , CHO Cells , Cricetinae , GTP-Binding Proteins/physiology , Humans , Kisspeptins , Ligands , Molecular Sequence Data , Neoplasm Metastasis/prevention & control , Proteins/isolation & purification , Proteins/metabolism , Rats , Receptors, G-Protein-Coupled , Receptors, Kisspeptin-1 , Receptors, Neuropeptide/metabolism , Tumor Suppressor Proteins
2.
J Photochem Photobiol B ; 56(1): 19-24, 2000 Jun.
Article in English | MEDLINE | ID: mdl-11073312

ABSTRACT

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.


Subject(s)
Cell Hypoxia , Cell Survival/drug effects , Perylene/analogs & derivatives , Radiation-Sensitizing Agents/toxicity , Anthracenes , Cell Survival/radiation effects , Darkness , Glass , Humans , Light , Neutral Red , Perylene/toxicity , Polystyrenes , Skin Neoplasms , Tumor Cells, Cultured
3.
Pharmacol Biochem Behav ; 65(4): 627-33, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10764915

ABSTRACT

Clinical trials have extensively reported the ability of Hypericum perforatum extracts to exert a significant antidepressant activity. Hypericin, the main constituent of H. perforatum extract, is no more regarded as the active principle of the antidepressant activity of the drug. Hence, the question of which constituents are involved in the basic activity of the total extract, is still waiting for an answer. In the present study we focused our attention on the potential anxiolytic activity of H. perforatum total extract, and of some pure components such as protohypericin and a fraction containing hypericin and pseudohypericin. Herein we report that the total extract of H. perforatum increases the locomotor activity in the open field and exerts anxiolytic activity in the light-dark test, whereas the single components did not show any effect. Interestingly, the anxiolytic activity of the total extract was blocked by pretreatment of rats with the benzodiazepine antagonist Flumazenil, hence suggesting an implication of benzodiazepine receptor activation in the anxiolytic effect of H. perforatum extract. Electrophysiological studies, performed to gain more information on the mechanism of action, showed that hypericin reduced the GABA-activated chloride currents, while pseudohypericin did an opposite effect. Furthermore, both hypericin and pseudohypericin inhibited the activation of NMDA receptors.


Subject(s)
Anti-Anxiety Agents/pharmacology , Exploratory Behavior/drug effects , Hypericum/chemistry , Plants, Medicinal , Animals , Anthracenes , Anti-Anxiety Agents/antagonists & inhibitors , Cerebellum/cytology , Cerebellum/drug effects , Electrophysiology , Flumazenil/pharmacology , GABA Modulators/pharmacology , GABA-A Receptor Antagonists , Male , Motor Activity/drug effects , Patch-Clamp Techniques , Perylene/analogs & derivatives , Perylene/isolation & purification , Perylene/pharmacology , Plant Extracts/antagonists & inhibitors , Plant Extracts/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, GABA/drug effects , Receptors, N-Methyl-D-Aspartate/drug effects
4.
Int J Oncol ; 14(4): 759-63, 1999 Apr.
Article in English | MEDLINE | ID: mdl-10087326

ABSTRACT

Hypericin is a natural photo-active pigment produced by plants of the genus Hypericum. The compound exhibits a potent photocytotoxic activity in vitro and in vivo. Using HeLa cells we further investigated whether the photocytotoxic in vitro effect of hypericin is influenced by the cell density. It was demonstrated that hypericin-induced photocytotoxicity in HeLa cells depends significantly on the amount of cells in culture, as low cell density cultures were more responsive to photodynamic therapy (PDT) than confluent or hyperconfluent cell cultures. This confluence dependent resistance (CDR) can be explained in terms of a decrease in hypericin cellular uptake. The phenomenon is not caused by the depletion of hypericin from the medium by high-density cell cultures since the extra-cellular availability of the drug is not altered by the increase in cell density and does not appear to be a limiting factor. Importantly, since confluent or hyperconfluent cell cultures can better mimic the high cell density of the solid tumour, CDR should be taken into consideration whenever resistance of solid tumours to PDT with hypericin is observed.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Count/drug effects , HeLa Cells/drug effects , Perylene/analogs & derivatives , Anthracenes , Antineoplastic Agents/metabolism , Drug Resistance, Neoplasm , Drug Screening Assays, Antitumor , Humans , Perylene/metabolism , Perylene/pharmacology , Photochemistry
5.
Planta Med ; 65(8): 719-22, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10630113

ABSTRACT

In the present study, protohypericin was synthesised in order to compare its intrinsic photocytotoxicity with that of hypericin. The experimental work was performed in specific filtered light conditions that prevented both an unintended photoconversion of protohypericin and photosensitization of the cells. Assessing the photocytotoxicity as a function of irradiation time, it was found that the photocytotoxicity of both compounds converged after a long irradiation time (i.e., 15 min), while the difference between the photocytotoxicities was maximal after a short irradiation time (i.e., 1 min). Since this could not be accounted for by a redistribution of protohypericin during irradiation, and the different irradiation times corresponded to different degrees of photoconversion of protohypericin into hypericin, the results clearly suggest that protohypericin exhibits intrinsically a dramatically lower photoactivity as compared to hypericin.


Subject(s)
Perylene/analogs & derivatives , Photosensitizing Agents/toxicity , Anthracenes , HeLa Cells , Humans , Perylene/pharmacokinetics , Perylene/toxicity , Photosensitizing Agents/pharmacokinetics
6.
J Photochem Photobiol B ; 45(2-3): 87-94, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9868799

ABSTRACT

Pseudohypericin and hypericin, the major photosensitizing constituents of Hypericum perforatum, are believed to cause hypericism. Since hypericin has been proposed as a photosensitizer for photodynamic cancer therapy, the photocytotoxicity of its congener pseudohypericin has been investigated. The presence of foetal calf serum (FCS) or albumin extensively inhibits the photocytotoxic effect of pseudohypericin against A431 tumour cells, and is associated with a large decrease in cellular uptake of the compound. These results suggest that pseudohypericin, in contrast to hypericin, interacts strongly with constituents of FCS, lowering its interaction with cells. Since pseudohypericin is two to three times more abundant in Hypericum than hypericin and the bioavailabilities of pseudohypericin and hypericin after oral administration are similar, these results suggest that hypericin, and not pseudohypericin, is likely to be the constituent responsible for hypericism. Moreover, the dramatic decrease of photosensitizing activity of pseudohypericin in the presence of serum may restrict its applicability in clinical situations.


Subject(s)
Perylene/analogs & derivatives , Photosensitivity Disorders/chemically induced , Anthracenes , Female , Fluorescence , Humans , Molecular Structure , Perylene/chemistry , Perylene/metabolism , Perylene/toxicity , Subcellular Fractions , Tumor Cells, Cultured
7.
Anticancer Res ; 18(2A): 1181-4, 1998.
Article in English | MEDLINE | ID: mdl-9615785

ABSTRACT

BACKGROUND: Three assay models were adopted for assessing the photocytotoxicity of hypericin on A431 cells. The cells were incubated for 1 hour or 24 hours with hypericin to evaluate the importance of the incubation period on the exerted photocytotoxicity. MATERIALS AND METHODS: A neutral red, an antiproliferative and a tetrazolium-reduction (MTT) assay were used for the estimation of cytotoxicity. RESULTS: IC50 values were 296, 321 and > 500 nM after 1 hour, and to 70, 54 and 277 nM after 24 hours, for the neutral red, antiproliferative and MTT assays, respectively. CONCLUSIONS: Our results clearly show that it is imperative to incubate cells for long periods to fully assess the hypericin photocytotoxicity, and that the neutral red assay is as sensitive as the antiproliferative assay but superior to the MTT assay at detecting hypericin cell damage.


Subject(s)
Antineoplastic Agents/pharmacology , Perylene/analogs & derivatives , Photochemotherapy , Radiation-Sensitizing Agents/pharmacology , Anthracenes , Cell Division/drug effects , Cell Line , Humans , Neutral Red , Perylene/pharmacology , Tetrazolium Salts
8.
Photochem Photobiol ; 67(1): 119-25, 1998 Jan.
Article in English | MEDLINE | ID: mdl-9477769

ABSTRACT

The toxicity on three human tumor cell lines (A431, HeLa and MCF7) of five phenanthroperylenequinones (hypericin and derivatives) and two perylenequinones (cercosporin and calphostin C) was investigated after photosensitization (4 J/cm2). Furthermore, the antiproliferative effect on HeLa cells was studied for the phenanthroperylenequinones. Hypericin, 2,5-dibromohypericin, 2,5,9,12-tetrabromohypericin and perylenequinones displayed a potent cytotoxic and antiproliferative effect in the nanomolar range. Hypericin dicarboxylic acid exhibited no photoactivity. In general, the antiproliferative activity correlated well with the photocytotoxicity. However, the nonphotocytotoxic compound hexamethylhypericin showed potent antiproliferative activity in the nanomolar range, probably exerting its action by protein kinase C inhibition. Without light irradiation, no cytotoxic and antiproliferative effect was observed for any photocytotoxic phenanthroperylenequinone compound. Furthermore, confocal laser microscopy revealed that the subcellular localization in A431 cells was similar for the photoactive compounds; the photosensitizers were mainly concentrated in the perinuclear region, probably corresponding with the Golgi apparatus and the endoplasmic reticulum. In addition, the accumulation of the photosensitizers in HeLa cells was investigated. All compounds except hypericin dicarboxylic acid were found to concentrate to a large extent in the cells. The compound 2,5,9,12-tetrabromohypericin seemed intrinsically more effective than hypericin since the intracellular concentration of the bromoderivative was a magnitude of order lower than that of hypericin although both compounds showed similar photobiological activity.


Subject(s)
Antineoplastic Agents/pharmacology , Perylene/analogs & derivatives , Radiation-Sensitizing Agents/pharmacology , Anthracenes , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Cell Division/drug effects , HeLa Cells , Humans , Microscopy, Confocal , Perylene/chemistry , Perylene/metabolism , Perylene/pharmacology , Photochemistry , Radiation-Sensitizing Agents/chemistry , Radiation-Sensitizing Agents/metabolism , Tumor Cells, Cultured
9.
J Photochem Photobiol B ; 38(2-3): 136-42, 1997 Apr.
Article in English | MEDLINE | ID: mdl-9203374

ABSTRACT

The cytotoxic effects of the natural photosensitizing agent hypericin were evaluated. A dramatic difference in the sensitivity of several different human and mouse cell lines towards photoactivated hypericin (4 J cm-2) was demonstrated using a neutral red assay (e.g. A431, IC50 = 0.14 +/- 0.02 microM; HeLa, IC50 = 0.32 +/- 0.05 microM, MCF7, IC50 = 1.84 +/- 0.22 microM). Dark cytotoxicity was absent, even at high hypericin concentration (25 microM). The differential phototoxicity of hypericin did not correlate with the expression of the epidermal growth factor (EGF) receptor nor with the expression of the P170 glycoprotein in the cell. The reduction of the intracellular glutathione content did not enhance further the cytotoxic effects of photoactivated hypericin, as investigated with the A431, HeLa and MCF7 cells. Conversely, using confocal laser microscopy, it was shown that the phototoxicity correlated well with the hypericin cellular uptake.


Subject(s)
Antineoplastic Agents/pharmacology , Perylene/analogs & derivatives , Radiation-Sensitizing Agents/pharmacology , Animals , Anthracenes , Antineoplastic Agents/metabolism , Cell Survival/drug effects , Cell Survival/radiation effects , Humans , Mice , Microscopy, Confocal , Perylene/metabolism , Perylene/pharmacology , Radiation-Sensitizing Agents/metabolism , Tumor Cells, Cultured
10.
Anticancer Res ; 16(4A): 1619-25, 1996.
Article in English | MEDLINE | ID: mdl-8712679

ABSTRACT

The in vivo antitumour activity of the natural photosensitizer hypericin was evaluated. Athymic nude mice xenografted with A431 cells were intraperitoneally administered with different hypericin doses and the tumours were locally irradiated 2 h later with white light (180 J/cm2) using a cold light source. When treatment was started one day after tumour inoculation, a dose-dependent antitumour effect was observed in light-treated animals. Complete inhibition of the tumour growth was achieved with 2.5 mg/kg hypericin. When the efficacy of a single hypericin dose (5 mg/kg) followed by a single light treatment on established tumours (60 mm3) was investigated, an 80% reduction in tumour mass was seen. Furthermore, an accumulation of the photosensitizer in A431 xenografts was observed after local light irradiation. Our results strongly suggest that hypericin holds promise as a new, safe, efficient and selective PDT photosensitizer.


Subject(s)
Antineoplastic Agents/therapeutic use , Carcinoma, Squamous Cell/drug therapy , Perylene/analogs & derivatives , Photochemotherapy , Radiation-Sensitizing Agents/therapeutic use , Animals , Anthracenes , Antineoplastic Agents/pharmacokinetics , Carcinoma, Squamous Cell/pathology , Cell Division/drug effects , Cell Line , Humans , Light , Metabolic Clearance Rate , Mice , Mice, Nude , Perylene/pharmacokinetics , Perylene/therapeutic use , Radiation-Sensitizing Agents/pharmacokinetics , Tissue Distribution , Transplantation, Heterologous , Tumor Cells, Cultured
11.
Biochem Biophys Res Commun ; 220(3): 613-7, 1996 Mar 27.
Article in English | MEDLINE | ID: mdl-8607812

ABSTRACT

The photodynamic inhibitory effect of hypericin and a number of hypericin-derivatives were investigated in vitro using numerous growth-factor regulated protein kinases including receptor-bound (Insulin-R, EGF-R) and non-receptor (Lyn, c-Fgr, CSK, Syk) protein tyrosine kinases as well as Ser/Thr (PK-C, protein kinase CK-2, CK-1) protein kinases. Modification of the hypericin structure altered significantly the specificity of the protein kinase inhibition. In particular, methylation or attachment of long lipophilic chains to both methyl groups of the hypericin molecule strongly enhanced the specificity toward PK-C.


Subject(s)
Enzyme Inhibitors/pharmacology , Growth Substances/pharmacology , Perylene/analogs & derivatives , Photosensitizing Agents/pharmacology , Protein Kinase Inhibitors , Animals , Anthracenes , Enzyme Inhibitors/chemistry , ErbB Receptors/antagonists & inhibitors , Molecular Structure , Perylene/chemistry , Perylene/pharmacology , Photochemotherapy , Photosensitizing Agents/chemistry , Protein Kinases/metabolism , Quinones/pharmacology , Receptor, Insulin/antagonists & inhibitors , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Structure-Activity Relationship
12.
Biochem Pharmacol ; 49(11): 1615-22, 1995 May 26.
Article in English | MEDLINE | ID: mdl-7786302

ABSTRACT

The naphthodianthrone hypericin causes a photosensitized inhibition of protein kinases involved in growth factor signalling pathways. Nanomolar concentrations of hypericin inhibit the protein tyrosine kinase activities (PTK) of the epidermal growth factor receptor and the insulin receptor, while being ineffective towards the cytosolic protein tyrosine kinases Lyn, Fgr, TPK-IIB and CSK. Photosensitized inhibition by hypericin is not restricted to receptor-PTKs since the Ser/Thr protein kinases (protein kinase CK-2, protein kinase C and mitogen-activated kinase) are also extremely sensitive to inhibition (IC50 value for protein kinase CK-2 = 6 nM). A comparison of the hypericin-mediated inhibition of the epidermal growth factor-receptor PTK and protein kinase CK-2 revealed that the inhibition is irreversible, strictly dependent upon irradiation of the enzyme-inhibitor complex with fluorescent light and likely mediated by the formation of radical intermediates (type I mechanism). Although the exact molecular basis for the selectivity of enzyme inhibition by hypericin remains unknown, our results suggest that distantly related protein kinases could still share common reactive domains for the interaction with hypericin.


Subject(s)
Perylene/analogs & derivatives , Photosensitizing Agents/pharmacology , Protein Kinase Inhibitors , Amino Acid Sequence , Anthracenes , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Casein Kinases , Cytosol/enzymology , ErbB Receptors/chemistry , Growth Substances/pharmacology , Kinetics , Light , Molecular Sequence Data , Perylene/pharmacology , Receptor, Insulin/chemistry
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