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

Hypericin, an extract from St John''s 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.     

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.     

In vitro clonal analysis of quail cardiac neural crest development

The developmental potentials of cardiac neural crest cells were investigated by in vitro clonal analysis. Five morphologically distinct types of clones were observed: (1) "pigmented" clones contained melanocytes only; (2) "mixed" clones consisted of pigmented and unpigmented cells; (3) "unpigmented dense" clones consisted of flattened, closely aligned unpigmented cells; (4) "unpigmented loose" clones consisted of a few loosely arranged, flattened cells; and (5) "unpigmented large" clones included a large number of small, stellate cells that were highly proliferative. The binding patterns of antibodies against lineage-specific markers showed that cells in the different clones expressed characteristic phenotypes. The following phenotypes were expressed in addition to pigment cells: smooth muscle cells, connective tissue cells, chondrocytes, and cells in the sensory neuron lineage. Mixed clones expressed all five phenotypes. Unpigmented dense clones contained smooth muscle cells, connective tissue cells, chondrocytes, and sensory neurons. Unpigmented loose clones exclusively consisted of smooth muscle cells, whereas unpigmented large clones contained chondrocytes and sensory neuron precursors. Based on these results, the following conclusions can be drawn: (1) Pigmented and unpigmented loose clones are most likely formed by precursors that are committed to the melanogenic and myogenic cell lineages, respectively. (2) Mixed and unpigmented dense clones are derived from pluripotent cells with the capacity to give rise to four or five phenotypes. (3) Unpigmented large clones originate from progenitor cells that appear to have a partially restricted developmental potential, that is, these cells are capable of generating two phenotypes in clonal cultures. Thus, the data indicate that the early migratory cardiac neural crest is a heterogeneous population of cells, consisting of pluripotent cells, cells with a partially restricted developmental potential, and cells committed to a particular cell lineage.     

Molecular characterization of novel melanoma cell lines

We isolated two novel cell lines from different types of sporadic human malignant melanoma: the hmel1 line was obtained from a melanoma skin metastasis and the hmel9 cell line from a primary superficial spreading melanoma. The karyotype and pigmentation parameters were assessed in these cell lines. Cytogenetic analysis in early stages of culture revealed that both cell lines had chromosome instability and simultaneous growth of heteroploid subpopulations. The molecular analysis of some genes involved in melanoma showed that both cell lines harbor BRAF mutations. The unpigmented hmel1 and the pigmented hmel9 lines were found to express the tyrosinase gene. The tyrosine hydroxylase activity was detectable only in hmel9 cells and practically absent in the hmel1 cell line. This activity was found to be correlated with the relative tyrosinase protein amount in both melanoma cell lines. The biological behaviour in the two melanoma cell lines, derived from two different types of melanoma lesions displaying distinct clinical and histopathological features, confirms the heterogeneous characteristics of sporadic melanoma. Similarities and/or differences between cell lines extracted from different melanoma cases could be useful in the future for diagnostic, prognostic and therapeutic purposes.     

The Patchwork Mouse Phenotype: Implication for Melanocyte Replacement in the Hair Follicle

Mice homozygous for the recessive patchwork (pwk) mutation are characterized by a variegated pigment pattern with a mixture of unpigmented and normally pigmented hairs. The pigmented hair bulbs contain functional melanocytes. By contrast, the unpigmented hair bulbs contain no melanocytes. This lack results from the death of melanoblasts in the hair follicle at the end of embryogenesis. Here, we report that melanoblasts and melanocytes are found in the epidermis of pwk/pwk mice. Furthermore, these epidermal pigment cells are able to colonize new hair follicles after skin wounding. Despite the presence of epidermal pigment cells with a colonization potential, a follicle that had produced an unpigmented hair produces a new unpigmented hair during the successive hair growth cycles. This hair color continuity is also true for the pigmented hair follicles. Thus, in normal conditions, the hair acts as an independent functional unit as regards its pigment cells population.     

Lipid-mediated preferential localization of hypericin in lipid membranes

Subcellular localization of a photosensitizer is critical to its therapeutic outcome during photodynamic therapy (PDT). We delineated the distribution of hypericin, a new generation photosensitizer, in model membrane systems to identify the operating principles of its subcellular accumulation. Results from fluorescence microscopy indicated preferential incorporation of hypericin in lipid of giant unilamellar vesicles. Monolayer fluorescence measurements further identified cholesterol as the key determinant for the observed selectivity of hypericin. The emission spectra of hypericin in lipid monolayers varied in a lipid-dependent manner and Stoke's shift behavior suggests that hypericin may form closely packed structure with cholesterol. Overall, our data lead to the conclusion that cholesterol is the major origin of the selectivity for hypericin in membrane systems. A hypothetical model depicting the intracellular and intravascular co-transport of hypericin and cholesterol because of their high affinity is presented.     

Differentiation in mouse melanoma cells: initial reversibility and an on-off stochastic model

Various proposals that a stochastic event, "commitment," is the first and rate-limiting step in mammalian cell differentiation were tested in one cell type, B16C3 mouse melanoma cells. Differentiation (pigment production) was observed in time-lapse films and in cloned single cells. As predicted by all the theories, onset of differentiation was at widely variable times in different cells after stimulation; and selection experiments showed that little of the variability was genetic. Contrary to some theories, differentiation appeared unrelated to cell division. Two properties of the melanoma cells did not fit any of the theories: times of differentiation were highly correlated in sister cells; and differentiation could be reversed in a proportion of cells, which was highest at the lowest levels of pigmentation. Dedifferentiation was associated with cell proliferation, so that most pigmented clones were small and most unpigmented clones large. These findings are accommodated by a model in which functions associated with differentiation can switch on and off, but an inhibition of the off transition builds up in the on state.     

Premature Graying as a Consequence of Compromised Antioxidant Activity in Hair Bulb Melanocytes and Their Precursors

Intricate coordinated mechanisms that govern the synchrony of hair growth and melanin synthesis remain largely unclear. These two events can be uncoupled in prematurely gray hair, probably due to oxidative insults that lead to the death of oxidative stress-sensitive melanocytes. In this study, we examined the gene expression profiles of middle (bulge) and lower (hair bulb) segments that had been micro-dissected from unpigmented and from normally pigmented hair follicles from the same donors using quantitative real-time RT-PCR (qPCR) arrays. We found a significant down-regulation of melanogenesis-related genes (TYR, TYRP1, MITF, PAX3, POMC) in unpigmented hair bulbs and of marker genes typical for melanocyte precursor cells (PAX3, SOX10, DCT) in unpigmented mid-segments compared with their pigmented analogues. qPCR, western blotting and spin trapping assays revealed that catalase protein expression and hydroxyl radical scavenging activities are strongly repressed in unpigmented hair follicles. These data provide the first clear evidence that compromised antioxidant activity in gray hair follicles simultaneously affects mature hair bulb melanocytes and their immature precursor cells in the bulge region.     

The patchwork mouse phenotype: implication for melanocyte replacement in the hair follicle.

Mice homozygous for the recessive patchwork (pwk) mutation are characterized by a variegated pigment pattern with a mixture of unpigmented and normally pigmented hairs. The pigmented hair bulbs contain functional melanocytes. By contrast, the unpigmented hair bulbs contain no melanocytes. This lack results from the death of melanoblasts in the hair follicle at the end of embryogenesis. Here, we report that melanoblasts and melanocytes are found in the epidermis of pwk/pwk mice. Furthermore, these epidermal pigment cells are able to colonize new hair follicles after skin wounding. Despite the presence of epidermal pigment cells with a colonization potential, a follicle that had produced an unpigmented hair produces a new unpigmented hair during the successive hair growth cycles. This hair color continuity is also true for the pigmented hair follicles. Thus, in normal conditions, the hair acts as an independent functional unit as regards its pigment cells population.     

Cytosolic superoxide dismutase activity after photodynamic therapy, intracellular distribution of Photofrin II and hypericin, and P-glycoprotein localization in human colon adenocarcinoma

In photodynamic therapy (PDT), a tumor-selective photosensitizer is administered and then activated by exposure to a light source of applicable wavelength. Multidrug resistance (MDR) is largely caused by the efflux of therapeutics from the tumor cell by means of P-glycoprotein (P-gp), resulting in reduced efficacy of the anticancer therapy. This study deals with photodynamic therapy with Photofrin II (Ph II) and hypericin (Hyp) on sensitive and doxorubicin-resistant colon cancer cell lines. Changes in cytosolic superoxide dismutase (SOD1) activity after PDT and the intracellular accumulation of photosensitizers in sensitive and resistant colon cancer cell lines were examined. The photosensitizers' distributions indicate that Ph II could be a potential substrate for P-gp, in contrast to Hyp. We observed an increase in SOD1 activity after PDT for both photosensitizing agents. The changes in SOD1 activity show that photodynamic action generates oxidative stress in the treated cells. P-gp appears to play a role in the intracellular accumulation of Ph II. Therefore the efficacy of PDT on multidrug-resistant cells depends on the affinity of P-gp to the photosensitizer used. The weaker accumulation of photosensitizing agents enhances the antioxidant response, and this could influence the efficacy of PDT.     

Hypericin in cancer treatment: more light on the way

Photodynamic therapy (PDT) has been described as a promising new modality for the treatment of cancer. PDT involves the combination of a photosensitizing agent (photosensitizer), which is preferentially taken up and retained by tumor cells, and visible light of a wavelength matching the absorption spectrum of the drug. Each of these factors is harmless by itself, but when combined they ultimately produce, in the presence of oxygen, cytotoxic products that cause irreversible cellular damage and tumor destruction. Hypericin, a powerful naturally occurring photosensitizer, is found in Hypericum perforatum plants, commonly known as St. John's wort. In recent years increased interest in hypericin as a potential clinical anticancer agent has arisen since several studies established its powerful in vivo and in vitro antineoplastic activity upon irradiation. Investigations of the molecular mechanisms underlying hypericin photocytotoxicity in cancer cells have revealed that this photosensitizer can induce both apoptosis and necrosis in a concentration and light dose-dependent fashion. Moreover, PDT with hypericin results in the activation of multiple pathways that can either promote or counteract the cell death program. This review focuses on the more recent advances in the use of hypericin as a photodynamic agent and discusses the current knowledge on the signaling pathways underlying its photocytotoxic action.     

Cyclic amines are selective cytotoxic agents for pigmented cells

We have shown that morpholine, a cyclic amine, exerts a selective inhibition of growth on melanocytic pigmented cell lines compared to nonpigmented cells. The ID50 of morpholine for the pigmented B-16 cell line HFH was 1200 micrograms/ml, compared to values greater than 2400 micrograms/ml for baby hamster kidney, Chinese hamster ovary and NP, an unpigmented primate cell line. Two other cyclic amines piperazine and piperidine, were similarly found to be selectively toxic to melanocytes. This selective toxicity could be synergistically enhanced by pretreatment of the cells with theophylline, a stimulator of tyrosinase activity, which indicates that the selective toxicity may be associated with melanin synthesis. Low passage HFH, high passage HFH and Syrian hamster melanoma RPMI 1846 cells that were pretreated with theophylline showed between 13 and 29% greater toxicity compared to controls treated with theophylline or morpholine alone. Unpigmented NP primate cells, Chinese hamster ovary and mouse fibroblast L929 remained unaffected. These cyclic amines join a list of other amines that have also been shown to be melanocytotoxic.     

Clonal analysis of quail neural crest cells: They are pluripotent and differentiate in vitro in the absence of noncrest cells

To determine if neural crest cells are pluripotent and establish whether differentiation occurs in the absence of noncrest cells, a cell culture method was devised in which differentiation could be examined in clones derived from single, isolated neural crest cells. Single neural crest cells, which were isolated before the onset of in vivo migration, gave rise to three types of clones: pigmented, unpigmented, and mixed. Pigmented clones consisted of melanocytes only, whereas some unpigmented cells in mixed and unpigmented clones contained catecholamines, identifying them as adrenergic cells. Extracellular matrix derived from quail somite or chick skin fibroblast cultures stimulated adrenergic differentiation and axon formation. These results demonstrate for the first time the existence of pluripotent quail neural crest cells that give rise to at least two progeny, melanocytes and neuronal cells. They also suggest that continuous direct interactions with noncrest cells are not required for the differentiation of these two cell types. However, components of the extracellular matrix derived from noncrest cells may play an important role in expression of the adrenergic phenotype.     

硫化氢合成酶抑制剂金丝桃素的体内外抗结肠癌作用研究

为研究金丝桃素对硫化氢合成酶活性的影响,探索金丝桃素作为胱硫醚-β-合成酶(CBS)抑制剂的体内外抗结肠癌作用。本研究通过亚甲蓝法测定金丝桃素和氨基氧乙酸(AOAA)对CBS产H2S活性的抑制作用,以HT29人结肠癌细胞和移植HT29细胞的裸鼠肿瘤模型为抗肿瘤研究对象,测定金丝桃素体内外抗肿瘤作用。结果表明,来源于贯叶连翘(Hypericum perforatum L.)的化合物金丝桃素是CBS的高效选择性抑制剂,其IC50为7.9±1.42μmol/L。金丝桃素能显著抑制HT29人结肠癌细胞的增殖,其IC50为22.2±2.25μmol/L。基因沉默实验表明CBS蛋白可能是金丝桃素在细胞内的潜在靶标之一。意外的是,目前普遍使用的CBS抑制剂AOAA抑制HT29细胞增殖作用非常弱,其IC50为152.5±35.68μmol/L。此外,不同剂量的金丝桃素(5~30mg/kg·d)均能显著减少裸鼠肿瘤体积和重量,30mg/kg·d剂量皮下给药2周后,和对照组相比肿瘤重量减少了68%。以上结果表明金丝桃素作为CBS的选择性抑制剂具有显著的抗结肠癌作用。     

Photoinduced antitumour effect of hypericin can be enhanced by fractionated dosing

The in vivo antitumour activity of the natural photosensitizer hypericin was evaluated. C3H/DiSn mice were inoculated with fibrosarcoma G5:1:13 cells. When the tumour reached a volume of 40-80mm3 the mice were intraperitoneally injected with hypericin, either in a single dose (5mg/kg; 1 or 6h before laser irradiation) or two fractionated doses (2.5 mg/kg; 6 and 1 h before irradiation with laser light; 532 nm, 70mW/cm2, 168 J/cm2). All tumours in control groups treated with hypericin alone as well as those irradiated with laser light alone had similar growth rates and none of these tumours regressed spontaneously. Complete remission of tumour in photodynamic therapy (PDT)-treated groups was similar (14-17% single dose vs. 33% fractionated dose), but the fractionated schedule of hypericin dosing was found to be more efficient than the single dose, measured by survival assay (p < 0.05). Our experimental model showed that fractionated administration of hypericin can produce a better therapeutic response than single administration.     

Targeted inhibition of p38alpha MAPK suppresses tumor-associated endothelial cell migration in response to hypericin-based photodynamic therapy

Photodynamic therapy (PDT) is an established anticancer modality and hypericin is a promising photosensitizer for the treatment of bladder tumors. We show that exposure of bladder cancer cells to hypericin PDT leads to a rapid rise in the cytosolic calcium concentration which is followed by the generation of arachidonic acid by phospholipase A2 (PLA2). PLA2 inhibition significantly protects cells from the PDT-induced intrinsic apoptosis and attenuates the activation of p38 MAPK, a survival signal mediating the up-regulation of cyclooxygenase-2 that converts arachidonic acid into prostanoids. Importantly, inhibition of p38alpha MAPK blocks the release of vascular endothelial growth factor and suppresses tumor-promoted endothelial cell migration, a key step in angiogenesis. Hence, targeted inhibition of p38alpha MAPK could be therapeutically beneficial to PDT, since it would prevent COX-2 expression, the inducible release of growth and angiogenic factors by the cancer cells, and cause an increase in the levels of free arachidonic acid, which promotes apoptosis.     

Antioxidant enzymatic systems in pigment tissue of amphibia

Superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase activities in pigmented and unpigmented liver tissues of frog and albino rat, respectively, were studied. Our results show that pigmented tissue is lacking in manganese superoxide dismutase activity and that the main enzymatic activity utilized in the cytosol by pigmented cells to reduce the hydrogen peroxide to water is represented by catalase; on the contrary, for the same reaction, the cells of albino rat liver primarily utilize the glutathione peroxidase activity. Both a low glutathione peroxidase activity and a low glutathione reductase activity were found in pigmented tissue of frog liver when compared with unpigmented tissue of rat liver. In light of our results, we also report a hypothetical interrelationship between melanin and reduced glutathione: We believe that in pigmented cells the melanin could act as a reducing physiological agent replacing the glutathione in the reduction of hydrogen peroxide. This reducing action of melanin could cause a diminished need for GSH and therefore could provoke the low glutathione peroxidase and reductase activities in pigmented tissue.     

Anti-melanoma efficacy of internal radionuclide therapy in relation to melanin target distribution

Targeted internal radionuclide therapy (TRT) could be an efficient, specific way to treat disseminated melanoma. Based on a previous pharmacomodulation study, we selected a quinoxaline-derived molecule (ICF01012) for its melanin specificity and kinetic properties suitable for TRT. Here, we determined the efficacy of [¹³¹I]ICF01012 radiotherapy in vitro and in vivo in relation to melanogenesis using human melanoma models. [¹²⁵I]ICF01012 uptake was first assessed in relation to melanin content. We found that melanin distribution in different models was representative of pathology seen in human tumours: melanin content was high in the extracellular space of SKMel3 tumours, and accumulated primarily in melanophages in M4Beu tumours. Targeted [¹³¹I]ICF01012 radiotherapy had a strong anti-tumoural efficacy in pigmented versus unpigmented tumours, regardless of target distribution and content. This study supports the use of melanin targeting with ¹³¹I-labelled iodoquinoxaline for effective treatment of melanoma.     

Influence of Donor Age on Establishing Well-Differentiated Clonal Cultures from Embryonic Chicken Retinal Pigmented Epithelium

Retinal pigmented epithelia (RPE) isolated from chicken embryos of various developmental stages were dissociated into single cells, and their ability to re-express defferentiated characteristics in clonal culture was investigated. The lighty pigmented, columnar cells isolated from stage 25 to 29 embryos dissociated more easily than the heavily pigmeted, cuboidal cells from embryos of stages 30 to 34. The yield of RPE cells per embryo increased with donor age, paralleling the growth of the epithelium in vivo . However, the potential these cells to attach, to proliferate, and to form typical, welldifferentiated RPE colonies declined with donor age. Cells from stage 25 embryos developed exclusively into large, typical epithelial colonies which expressed all stages of differentiation from flat, unpigmented cells at the margin to cuboidal, pigmented cells in the centre. At the other end of the spectrum, cells from stage 34 embryos frequently formed small, atypical colonies of unpigmented cells, in addition to typical but relatively small colonies. The plating efficiency (calculated on the basis of pigmented colonies formed within 3 weeks) dropped from more than 2% at stage 25 to 0.01% at stage 34.