Lester M. Davids

Photoprotection by honeybush extracts, hesperidin and mangiferin against UVB-induced skin damage in SKH-1 mice

The possible mechanism of photoprotection by polyphenolic extracts of honeybush and the two most abundant polyphenols found in honeybush, hesperidin and mangiferin were determined using a mouse model. Ethanol: acetone soluble extracts and pure honeybush compounds were applied topically to the skin of SKH-1 mice before daily exposures to ultraviolet B (UVB) (180 mJ/cm²) for 10 days. The honeybush extracts reduced signs of sunburn, such as erythema, peeling and hardening of the skin and also significantly (P < 0.05) reduced edema, epidermal hyperplasia and the induction of cyclooxygenase-2 (COX-2), ornithine decarboxylase (ODC), GADD45 and OGG1/2 expression. The fermented honeybush extract significantly (P < 0.05) reduced lipid peroxidation and depletion of the antioxidant enzymes catalase and superoxide dismutase. Hesperidin and mangiferin were less effective. These results show that extracts of honeybush and to some extent, hesperidin and mangiferin, renders protection against UVB-induced skin damage. The mechanisms investigated suggest that honeybush extracts protected the skin via modulation of induced-oxidative damage, inflammation and cell proliferation. Other specific biological properties such as modulation of signaling pathways could also be involved.

Hypericin phototoxicity induces different modes of cell death in melanoma and human skin cells.

Hypericin, the major component of St. Johns Wort, absorbs light in the UV and visible ranges whereupon it becomes phototoxic through the production of reactive oxygen species. Although photodynamic mechanisms (i.e. through endogenous photosensitizers) play a role in UVA phototherapy for the treatment of skin disorders such as eczema and psoriasis, photodynamic therapy employing exogenous photosensitizers are currently being used only for the treatment of certain forms of non-melanoma skin cancers and actinic keratoses. There are few reports however on its use in treating melanomas. This in vitro study analyses the phototoxic effect of UVA (400-315 nm) - activated hypericin in human pigmented and unpigmented melanomas and immortalised keratinocytes and melanocytes. We show that neither hypericin exposure nor UV irradiation alone reduces cell viability. We show that an exposure to 1 microM UVA-activated hypericin does not bring about cell death, while 3 microM activated hypericin induces a necrotic mode of cell death in pigmented melanoma cells and melanocytes and an apoptotic mode of cell death in non-pigmented melanoma cells and keratinocytes. We hypothesis that the necrotic mode of cell death in the pigmented cells is possibly related to the presence of melanin-containing melanosomes in these cells and that the hypericin-induced increase in reactive oxygen species leads to an increase in permeability of melanosomes. This would result in toxic melanin precursors (of an indolic and phenolic nature) leaking into the cytoplasm which in turn leads to cell death. Hypericin localisation in the endoplasmic reticulum in these cells shown by fluorescent microscopy, further support a disruption in cellular processing and induction of cell death. In contrast, this study shows that cells that do not contain melanosomes (non-pigmented melanoma cells and keratinocytes) die by apoptosis. Further, using a mitochondrial-specific fluorescent dye, we show that intracellular accumulation of hypericin induces a mitochondrial-associated caspase-dependent apoptotic mode of cell death. This work suggests that UVA is effective in activating hypericin and that this phototoxicity may be considered as treatment option in some cases of lentigo maligna or lentigo maligna melanoma that are too large for surgical resection.

Melanomas display increased cytoprotection to hypericin-mediated cytotoxicity through the induction of autophagy

Photodynamic therapy (PDT) as a regime for melanoma is of limited success due to factors such as the efficacy of the photosensitizer used, penetration depth and the presence of pigment. We characterised a pigmented and an unpigmented melanoma cell line with respect to their phenotypes. Cell viability was assessed after exposure to hypericin, a UVA‐activated photosensitizer. Exposure to 3 μM activated hypericin induced a cytoprotective (autophagic) response from both cell lines. However, the pigmented cells accumulated a large amount of glycogen in their cytoplasm. We hypothesise that the treatment induces an initial cytoprotective response through autophagy, but with increased stress results in a different mode of cell death in pigmented melanoma cells from unpigmented cells. These results indicate that hypericin‐PDT could be an adjuvant therapy for melanoma.

Kinetic and physical characterisation of recombinant wild-type and mutant human protoporphyrinogen oxidases

The effects of various protoporphyrinogen oxidase (PPOX) mutations responsible for variegate porphyria (VP), the roles of the arginine-59 residue and the glycines in the conserved flavin binding site, in catalysis and/or cofactor binding, were examined. Wild-type recombinant human PPOX and a selection of mutants were generated, expressed, purified and partially characterised. All mutants had reduced PPOX activity to varying degrees. However, the activity data did not correlate with the ability/inability to bind flavin. The positive charge at arginine-59 appears to be directly involved in catalysis and not in flavin-cofactor binding alone. The K(m)s for the arginine-59 mutants suggested a substrate-binding problem. T(1/2) indicated that arginine-59 is required for the integrity of the active site. The dominant alpha-helical content was decreased in the mutants. The degree of alpha-helix did not correlate linearly with T(1/2) nor T(m) values, supporting the suggestion that arginine-59 is important for catalysis at the active site. Examination of the conserved dinucleotide-binding sequence showed that substitution of glycine in codon 14 was less disruptive than substitutions in codons 9 and 11. Ultraviolet melting curves generally showed a two-state transition suggesting formation of a multi-domain structure. All mutants studied were more resistant to thermal denaturation compared to wild type, except for R168C.

St John's Wort (Hypericum perforatum L.) Photomedicine: Hypericin-Photodynamic Therapy Induces Metastatic Melanoma Cell Death

Hypericin, an extract from St Johns Wort (Hypericum perforatum L.), is a promising photosensitizer in the context of clinical photodynamic therapy due to its excellent photosensitizing properties and tumoritropic characteristics. Hypericin-PDT induced cytotoxicity elicits tumor cell death by various mechanisms including apoptosis, necrosis and autophagy-related cell death. However, limited reports on the efficacy of this photomedicine for the treatment of melanoma have been published. Melanoma is a highly aggressive tumor due to its metastasizing potential and resistance to conventional cancer therapies. The aim of this study was to investigate the response mechanisms of melanoma cells to hypericin-PDT in an in vitro tissue culture model. Hypericin was taken up by all melanoma cells and partially co-localized to the endoplasmic reticulum, mitochondria, lysosomes and melanosomes, but not the nucleus. Light activation of hypericin induced a rapid, extensive modification of the tubular mitochondrial network into a beaded appearance, loss of structural details of the endoplasmic reticulum and concomitant loss of hypericin co-localization. Surprisingly the opposite was found for lysosomal-related organelles, suggesting that the melanoma cells may be using these intracellular organelles for hypericin-PDT resistance. In line with this speculation we found an increase in cellular granularity, suggesting an increase in pigmentation levels in response to hypericin-PDT. Pigmentation in melanoma is related to a melanocyte-specific organelle, the melanosome, which has recently been implicated in drug trapping, chemotherapy and hypericin-PDT resistance. However, hypericin-PDT was effective in killing both unpigmented (A375 and 501mel) and pigmented (UCT Mel-1) melanoma cells by specific mechanisms involving the externalization of phosphatidylserines, cell shrinkage and loss of cell membrane integrity. In addition, this treatment resulted in extrinsic (A375) and intrinsic (UCT Mel-1) caspase-dependent apoptotic modes of cell death, as well as a caspase-independent apoptotic mode that did not involve apoptosis-inducing factor (501 mel). Further research is needed to shed more light on these mechanisms.

A rare repigmentation pattern in a vitiligo patient: a clue to an epidermal stem-cell reservoir of melanocytes?

dema, morphoea and fibromatosis. NSF is a rare, acquired, chronic, progressive skin eruption of unknown cause, often affecting patients receiving dialysis for renal failure. It was first described by Cowper et al. as scleromyxoedema-like cutaneous disease in patients on renal-dialysis. Most cases of NSF have been reported in Europe and in the USA, and it was not reported in the Chinese population until July 2007. To our knowledge, our patient is only the second case of NFD in a Chinese patient.

Photodynamic therapy-induced killing is enhanced in depigmented metastatic melanoma cells

The resistance of pigmented human melanomas over their unpigmented counterparts to a number of therapies has suggested that the presence of intracellular melanin plays a role in rendering these cells less susceptible to cell death, probably through the ability of this pigment to act as an intracellular antioxidant, thus neutralizing chemotherapeutic‐induced ROS (reactive oxygen species). PDT (photodynamic therapy) was recently suggested as an attractive, adjunctive therapy owing to its cellular specificity and limited side effects. In the present study, we propose that first depigmenting melanomas with a reversible TYR (tyrosinase) inhibitor such as PTU (phenylthiourea) increases their susceptibility to HYP‐PDT (hypericin‐mediated PDT). Pigmented [UCT Mel‐1 (University of Cape Town melanoma cell line 1)] and unpigmented (A375) melanomas were first characterized with respect to their TYR activities and melanin quantities and then treated with a TYR inhibitor for 48 h. Cell viability assays after treatment with 3 μM HYP‐PDT showed a significant increase in cell death in depigmented melanomas compared with untreated melanomas that returned to the level of untreated melanoma cells on removing the TYR inhibitor. The present study supports the hypothesis that combining the inhibition of melanogenesis with PDT should be explored as a valid therapeutic target for the management of advanced melanoma.

Identification and characterisation of a deletion (537delAT) in the protoporphyrinogen oxidase gene in a South African variegate porphyria family

Variegate porphyria is an autosomal dominant disorder of haem metabolism resulting from a partial decrease in protoporphyrinogen oxidase activity. Variegate porphyria is highly prevalent in South Africa, the result of a founder effect now confirmed genetically as a single point mutation (R59W) which has been described in nearly all South African variegate porphyria patients studied. Only two other mutations (H20P, R168C) have been reported in South Africa. We utilised simultaneous, single‐stranded conformational polymorphism and heteroduplex analysis, and direct sequencing to identify a further mutation; a 2 bp deletion in exon 6 which results in a premature stop codon 11 codons downstream from the mutation and is the first reported deletion in the protoporphyrinogen oxidase gene in a South African family. The familial segregation of this mutation strongly suggests that it is the disease causing mutation for variegate porphyria in this family. This further evidence for allelic heterogeneity limits the utility of tests for the R59W mutation in the diagnosis of variegate porphyria in South Africa. Hum Mutat 12:403–407, 1998.

Depigmentation in melanomas increases the efficacy of hypericin-mediated photodynamic-induced cell death

Melanoma is the main cause of death in skin cancers. Despite combating with early detection, resection and post-operative therapy, melanoma treatment remains unsuccessful and investigations into other forms of adjuvant therapy such as photodynamic therapy (PDT) are prudent. This study proposes that depigmentation i.e. the removal of the free radical scavenging pigment, melanin, in melanotic melanoma cells increases their susceptibility to PDT-induced cell death. Two human melanoma cell lines: one pigmented (Mel-1) and one amelanotic (A(375)) cell lines were used. Kojic acid (KA), a tyrosinase-specific inhibitor, was optimised to 6 μg/ml and shown to quantifiably inhibit melanin synthesis after a 3-day exposure. PDT on these cells resulted in a 3.82 fold increase of intracellular ROS production which correlated to 11% increase in cell death susceptibility compared to untreated controls. Moreover, cells allowed to regain their pigment failed to return to normal even after 72 h thus proving the effectiveness of PDT. Using a DPPH* assay, the results confirmed the scavenging properties of melanin (IC(50) 18.30 μg/ml) proving that this pigment may be one of the reasons for melanoma chemoresistance. Overall this study shows that pigment plays an important role in the efficacy of adjunctive PDT treatment and its removal enhances cell death susceptibility in melanomas.

Mitochondrial targeting of human protoporphyrinogen oxidase

Variegate porphyria is an autosomal dominant disorder of heme metabolism resulting from a deficiency in protoporphyrinogen oxidase, an enzyme located on the inner mitochondrial membrane. This study examined the effect of three South African VP‐causing mutations (H20P, R59W, R168C) on mitochondrial targeting. Only H20P did not target, and of eight protoporphyrinogen oxidase—GFP chimeric fusion proteins created, N‐terminal residues 1–17 were found to be the minimal protoporphyrinogen oxidase sequence required for efficient mitochondrial targeting. Removal of this N‐terminal sequence displayed mitochondrial localization, suggesting internal mitochondrial targeting signals. In addition, six constructs were engineered to assess the effect of charge and helicity on mitochondrial targeting of the protein. Of those engineered, only the PPOX20/H20P‐GFP construct abolished mitochondrial targeting, presumably through disruption of the protoporphyrinogen oxidase α‐helix. Based on our results we propose a mechanism for protoporphyrinogen oxidase targeting to the mitochondrion.