Photodynamic Effect Of Hypericin Research Articles

Photodynamic effect of hypericin on the microorganisms and primary human fibroblasts

Hypericin (HYP) is a natural photosensitizer considered for the new generation of photodynamic therapy (PDT) drugs. The aim of this study was to evaluate the in vitro bactericidal effect of HYP-PDT on four bacterial species, assessing its photocytotoxicity to primary human fibroblasts to determine possible side effects. Effect of photosensitizer concentration (0.1, 0.3, 0.6, and 1 μg/ml) and light irradiation time (3, 5, 10 min) on photodynamic inactivation of microorganisms and primary human fibroblasts was investigated. A 6.3 log killing was obtained for Staphylococcus aureus (ATCC 25923) treated with 1 μg/ml at 48 J/cm². For this set of PDT parameters, Enterococcus faecalis (ATCC 11700) showed 6.5 log killing, Escherichia coli (ATCC 25922) 6.2 log killing, and Pseudomonas aeruginosa (ATCC 27853) 0.7 log killing. Fibroblasts can be preserved by keeping the HYP concentration below 0.6 μg/ml and the light dose below 48 J/cm². S. aureus, E. faecalis, and E. coli appear to be suitable for treatment with HYP-PDT without significant damage to fibroblasts.

Photodynamic effect of hypericin in primary cultures of human umbilical endothelial cells and glioma cell lines

Hypericin is the most powerful naturally occurring photosensitizer and as such there is renaissant interest in the potentials of this compound for anticancer photodynamic therapy (PDT). The purpose of this study was to investigate the hypericin-mediated photodynamic therapy effects on normal human umbilical endothelial cells (HUVECs) in comparison with cancer human glioma cell lines U-87 MG and U-373 MG, in in vitro conditions. The data suggest that endothelial cells as well as glioma cell lines are sensitive only to photoactivated hypericin. The inhibitory effects of photoactivated hypericin did not differ in endothelial compared with tumor cells in cytotoxicity MTT and DNA fragmentation assays. However, an important difference in sensitivity was found between the above mentioned cell types in migration and metalloproteinases inhibition assays performed as cell function tests. The findings in both function tests were supported by the high sensitivity of endothelial cells in an additional angiogenesis test of tubular formation in vitro.

Photodynamic Effect of Hypericin on the Conformation and Catalytic Activity of Hemoglobin

Hypericin, extracted from H. perforatum, can induce the generation of reactive oxygen species by visible light irradiation, which may consequently induce the conformational change of hemoglobin. We have not only employed UV-vis spectroscopy to observe the changes of UV-vis spectra of the protein, which reveals the conformational changes of the protein, but also employed electrochemical method to obtain its enhanced peroxidase activity. The photodynamic effect of hypericin on the conformation and catalytic activity of the protein has also been proven to be strongly dependent on the irradiation time, the hypericin concentration and the presence of oxygen. This work is beneficial not only to the fabrication of more sensitive hydrogen peroxide biosensor, but also to the guidance of the usage of this medicinal herb molecule, since the conformational change of the protein and the enhanced peroxidase can be easily obtained only by visible light irradiation on hypericin, the process of which is so common to happen.

Photodynamic Therapy of Nonmelanoma Skin Cancer with Topical Hypericum perforatum Extract—A Pilot Study

Hypericin, the photoactive compound of Hypericum perforatum, is probably the most powerful photosensitizer found in nature. This compound has shown high potency in the photodynamic treatment of tumor cells. However, there is only limited knowledge regarding the photodynamic effect of hypericin on nonmelanoma skin cancer cells. The aim of this prospective study was to investigate the efficacy of photodynamic therapy with topical application of an extract of H. perforatum in actinic keratosis, basal cell carcinoma (BCC) and morbus Bowen (carcinoma in situ). The study was carried out on 34 patients--eight with actinic keratoses (AKs), 21 with BCC and five with Bowen's disease. The extract of H. perforatum was applied on the skin lesions under occlusion and that was followed by irradiation with 75 J cm(-2) of red light 2 h later. The treatment was performed weekly for 6 weeks on average. The percentage of complete clinical response was 50% for AKs, 28% in patients with superficial BCC and 40% in patients with Bowen's disease. There was only a partial remission seen in patients with nodular BCCs. A complete disappearance of tumor cells was found in the histologic preparation of 11% of patients with superficial BCCs and 80% in the patients with Bowen's disease. All patients complained of burning and pain sensations during irradiation. Although the results of this first clinical trial could be regarded as disappointing, there are still possibilities for improvement. Better preparation of the lesions, enhancement of hypericin delivery and other types of light exposure procedures could significantly improve the clinical outcomes of this relatively inexpensive treatment modality.

Enhancing the photodynamic effect of hypericin in tumour spheroids by fractionated light delivery in combination with hyperoxygenation

The aim of this study was to explore the hypothesis of oxygen depletion during light irradiation as a possible explanation for the incomplete response seen after hypericin-mediated photodynamic therapy (PDT) under specific conditions. To investigate this, we performed PDT experiments using transitional cell carcinoma spheroids with fractionated light irradiation and hyperoxygenation. After 2-h incubation with 3 different hypericin concentrations, spheroids were irradiated either continuously or with fractionated light delivery. The effect of hyperoxygenation was investigated by bubbling normobaric oxygen in the solution surrounding the spheroids before continuous irradiation or during the dark interval of light fractionation. The PDT efficacy was evaluated with an MTT antiproliferation assay and apoptotic cells were visualized after PDT by DAPI staining. Our results show that fractionated light delivery with dark intervals ranging from 1 to 10 min does not enhance the PDT efficacy in spheroids at all, whereas hyperoxygenation, using appropriate hypericin concentrations and oxygenation intervals, results in a virtually complete malignant cell killing through apoptosis. This study suggests that oxygen depletion is the major source of relative treatment failure in hypericin-mediated PDT with spheroids, which can only be overcome with hyperoxygenation. Therefore, whole bladder wall PDT with hypericin is likely to become a very efficient antitumoural treatment against superficial bladder cancer, on the condition that instillation fluids are hyperoxygenated during light irradiation.

Modulation of Matrix metalloproteinase-1 in nasopharyngeal cancer cells by photoactivation of hypericin

Singlet oxygen (1O2) generated by ultraviolet A irradiation has been reported to induce cytokine-dependent Matrix metalloproteinase-1 (MMP-1) expression in dermal fibroblasts. In the present study, we analyzed the effect of hypericin-based photodynamic therapy (PDT) on MMP-1 expression in two nasopharyngeal cancer (NPC) cell lines and an animal tumor model. MMP-1 protein and mRNA expression were evaluated by Western blot analysis and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) respectively. Photoactivation of hypericin, a polycyclic phenanthroperylenedione, elicited an increase in MMP-1 protein and mRNA expression in well differentiated HK1 and poorly differentiated CNE-2 NPC cells in vitro. Similarly, there was up-regulation of MMP1 mRNA expression in hypericin-PDT-treated NPC/HK1-tumors. To our knowledge, this is the first time that modulation of MMP-1 expression has been demonstrated as a photodynamic effect of hypericin in NPC cells. This has clinical implications as MMP-1 is known to play an essential role in the biological process of matrix remodeling.

Photodynamic effect of hypericin and a water-soluble derivative on isolated crayfish neuron and surrounding glial cells

Hypericin (Hyp) has been proposed as a fluorochrome for fluorescence diagnostics and as a photosensitizer for photodynamic therapy of cancer. However, its insolubility in water is a serious drawback. A novel water-soluble hypericin derivative (Hyp-S) has been constructed, using polyvinylpyrrolidone as a carrier. We used the crayfish stretch receptor, consisting of receptor neuron and satellite glial cells, for comparison of the photodynamic effects of Hyp and Hyp-S. Hyp-S was more toxic in the dark than Hyp and inactivated the neurons at concentrations exceeding 4 μM while Hyp was toxic to the neurons only at the concentrations larger than 20 μM. Electrophysiological investigations revealed polyphasic neuron responses to photosensitization with Hyp as well as with Hyp-S (1 μM concentration, 30 min incubation; irradiation with filtered light from a lamp with an emission maximum near 600 nm and an intensity of 0.2 W/cm 2). In the concentration range 1–4 μM Hyp-S was more phototoxic than Hyp. Fluorescence microscopy showed that both sensitizers were predominately localized in the glial envelope surrounding the neuron. A minor fraction of hypericin was found in the neuron perinuclear area rich in cytoplasm organelles. This suggests the potential application of Hyp and Hyp-S for visualization and selective photodynamic treatment of malignant gliomas.

Oxygen dependence of hypericin-induced phototoxicity to EMT6 mouse mammary carcinoma cells.

The photodynamic effect of hypericin on EMT6 mouse mammary carcinoma cells was investigated in vitro under aerobic and hypoxic conditions. Under aerobic conditions, hypericin-induced photocytotoxicity was dose dependent within a 1-50 microM range. Under hypoxic conditions, cells were resistant to hypericin-induced phototoxicity. In the dark, no cytotoxicity was observed at any hypericin concentration tested either aerobically or hypoxically. Cellular accumulation of hypericin, examined by chemical extraction and spectroscopy, occurred under both hypoxic and aerobic conditions. Fluorescence photomicrographs of cells exposed to hypericin corroborate drug uptake in the plasma membrane and subcellular regions. Our results demonstrate that hypericin cytotoxicity to EMT6 mouse mammary carcinoma cells in vitro is both light and oxygen-dependent. These results suggest that EMT6 cell kill caused by photoactivated hypericin is mediated by an oxygen-dependent mechanism, rather than by a type I oxygen-independent mechanism.

OXYGEN DEPENDENCE OF HYPERICIN‐INDUCED PHOTOTOXICITY TO EMT6 MOUSE MAMMARY CARCINOMA CELLS

AbstractThe photodynamic effect of hypericin on EMT6 mouse mammary carcinoma cells was investigated in vitro under aerobic and hypoxic conditions. Under aerobic conditions, hypericin‐induced photocytotoxicity was dose dependent within a 1–50 μM range. Under hypoxic conditions, cells were resistant to hypericin‐induced phototoxicity. In the dark, no cytotoxicity was observed at any hypericin concentration tested either aerobically or hypoxically. Cellular accumulation of hypericin, examined by chemical extraction and spectroscopy, occurred under both hypoxic and aerobic conditions. Fluorescence photomicrographs of cells exposed to hypericin corroborate drug uptake in the plasma membrane and subcellular regions. Our results demonstrate that hypericin cytotoxicity to EMT6 mouse mammary carcinoma cells in vitro is both light and oxygen‐dependent. These results suggest that EMT6 cell kill caused by photoactivated hypericin is mediated by an oxygen‐dependent mechanism, rather than by a type I oxygen‐independent mechanism.