Amelanotic Melanoma Cells Research Articles

Impact of NAD(P)H:Quinone Oxidoreductase-1 on Pigmentation

We obtained metastasized melanoma tissue from a primary acral lentiginous melanoma (ALM) patient and established a melanoma cell line named primary culture of melanoma cell derived from lymph node (PML)-1. PML-1 cells had a light brown color and decreased the expression of melanogenesis markers, including tyrosinase (TYR), microphthalmia-associated transcription factor, and tyrosinase-related protein-1. To identify genes differentially regulated in PML-1 melanoma cells, we performed DNA microarray and two-dimensional matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses. Among the candidate genes identified, we chose NAD(P)H:quinone oxidoreductase-1 (NQO1) for further study. Reverse transcription-PCR and western blot analyses showed that NQO1 was markedly decreased in PML-1 cells and in several amelanotic melanoma cell lines. To investigate whether NQO1 affects the melanogenesis, we treated the cultured normal human melanocytes (NHMC) and zebrafish with NQO1 inhibitors, ES936 and dicoumarol. Interestingly, melanogenesis was significantly decreased by the addition of NQO1 inhibitors in both NHMC and zebrafish models. In contrast, overexpression of NQO1 using a recombinant adenovirus clearly induced melanogenesis, concomitantly with an increase of TYR protein level. These results suggest that NQO1 is a positive regulator of the pigmentation process.

Photothermal sensitisation and therapeutic properties of a novel far-red absorbing cyanine

A water-soluble disulfonate cyanine was prepared by chemical synthesis and shown to possess photophysical properties which are particularly favourable for the promotion of photothermally sensitised processes, including a very low (<0.1) quantum yield of fluorescence emission and ultra-short (110 to 400 ps) excited state lifetimes, as well as the presence of intense absorption bands at wavelengths longer than 800 nm. This allows the possibility of high-energy irradiation by means of a Ti:sapphire laser operated in a pulse regime. The cyanine was accumulated in comparable amounts by B78H1 amelanotic melanoma cells and HT1080 transformed fibroblasts, however only the B78H1 cells could be extensively damaged by photothermal sensitisation with the cyanine, which was endocellularly distributed as suggested by observations at the optical microscope; the efficiency of the photoprocess could be enhanced by formation of aggregated intracellular cyanine clusters. On the other hand, only a modest photoinactivation of HT1080 cells was induced by photothermal sensitisation, possibly owing to the localization of the cyanine at the periphery of such cells. The cyanine also exhibited a good selectivity of amelanotic melanoma targeting in C57BL/6 mice, bearing the tumour subcutaneously transplanted in the dorsal area: the ratio of cyanine concentration in the melanoma and the surrounding cutaneous districts was as large as 3.8 at 1 h post-injection. The cyanine underwent a fast clearance from the organism, since only traces of the photosensitiser were observed in all the studied tissues at 3 h after i.v. administration. Thus, irradiations were performed at post-injection times shorter than 1 h. Maximum photothermal sensitisation efficiency was obtained at 10 min after injection with a 50% cure rate. Thus, photothermal therapy (PTT) appears to be a very promising and efficient modality of tumour treatment.

Metallo‐naphthalocyanines as photothermal sensitisers for experimental tumours: In Vitro and in vivo studies

Photothermal sensitisation has been recently proposed as a novel approach for the treatment of solid tumours through the development of a therapeutic modality named photothermal therapy (PTT). The technique involves the use of high power pulsed laser irradiation and photosensitising agents with especially short lifetime (in the subnanosecond range) in the electronically excited states. This study aims to investigate the molecular features of the photosensitiser which optimise the photothermal activity. Two octabutoxy-naphthalocyanines centrally coordinated with Pd(II) or Pt(II) ions were prepared by chemical synthesis and tested for their affinity and photothermal sensitisation activity toward a selected tumour cell line, namely B78H1 amelanotic melanoma. Irradiations were performed by using a Ti:sapphire laser operated in a pulsed regime (10 Hz, 30 nanosecond pulses, 120 mJ) at 809 nm (Pt) or 826 nm (Pd). The subcellular distribution pattern of the photosensitiser was also assessed by optical microscopy, while the nature of the photoinduced cell damage was determined by scanning electron microscopy. The results thus obtained provided a basis for subsequent in vivo studies, aimed at defining the phototherapeutic efficiency of the two metallo-naphthalocyanines: the photosensitisers were i.v. injected into C57BL/6 mice bearing a subcutaneously transplanted amelanotic melanoma and at 24 hours post-injection the tumour area was irradiated by the Ti:sapphire laser using the same protocol as above detailed. Both naphthalocyanines exhibited a high affinity for the amelanotic melanoma cells. The subcellular distribution pattern was modulated by the incubation time: after 48 hours incubation with 7.7 microM Pd- and Pt derivatives, the naphthalocyanine appeared to localise in specific sites with a gradual formation of aggregated clusters. Subsequent irradiation of the naphthalocyanine-loaded cells caused an extensive cell death; the photoinduced damage, as observed at the scanning electron microscope, mainly consisted in the formation of large endocellular holes consequent to the loss of cytoplasmic material. This scenario is typical of photothermal sensitisation processes. Lastly, both metallo-naphthalocyanines, and in particular the Pd(II) derivative, promoted an important response by the amelanotic melanoma, when the neoplastic tissue was irradiated by the pulsed Ti:sapphire laser. In certain cases, the photothermal treatment appeared to be curative. In all cases, the in vivo photodamage was confined within the tumour area with no detectable involvement of the perilesional tissues. PTT appears to act very efficiently at least on subcutaneous tumours. The technique can be used either in combination with photodynamic therapy (PDT) or as an alternative to PDT in those cases where the latter modality displays a limited efficacy, such as in the treatment of pigmented or poorly vascularised tumours.

Antiproliferative Activity on Human Cancer Cell Lines after Treatment with Polyphenolic Compounds Isolated from Iris pseudopumila Flowers and Rhizomes

The present study describes the antiproliferative properties of Iris pseudopumila flowers and rhizomes extracts and fourteen constituents isolated from them. The in vitro cytotoxic activity assay against two human cancer cell lines, large lung carcinoma (CORL-23) and amelanotic melanoma (C32), showed that the most antiproliferative extract was the MeOH extract from flowers with a percentage of inhibition of 50.9 at 100 microg/ml against amelanotic melanoma cells. The most antiproliferative compounds against amelanotic melanoma cells were kaempferol-3-O-beta-D-glucopyranoside and irisolidone with a percentage of inhibition of 100 and 96.6, respectively, and against large lung carcinoma cells with a percentage of inhibition of 82.1 and 84.6, respectively. Significant activity on the amelanotic melanoma cell line was also showed by irigenin-7-O-beta-D-glucopyranoside, with a percentage of inhibition of 89.3. The compounds isovitexin and isoorientin-6-O''-beta-D-glucopyranoside showed a selective activity against amelanotic melanoma cells with a percentage of inhibition of 83.2 and 79.8, respectively.

Cholesterol regulates melanogenesis in human epidermal melanocytes and melanoma cells

Cholesterol is important for membrane stability and is the key substrate for the synthesis of steroid hormones and vitamin D. Furthermore, it is a major component of the lipid barrier in the stratum corneum of the human epidermis. Considering that steroid hormone synthesis is taking place in epidermal melanocytes, we tested whether downstream oestrogen receptor/cAMP signalling via MITF/tyrosine hydroxylase/tyrosinase/pigmentation could be possibly modulated by cholesterol. For this purpose, we utilized human primary melanocyte cell cultures and human melanoma cells with different pigmentation capacity applying immunofluorescence, RT-PCR, Western blotting and determination of melanin content. Our in situ and in vitro results demonstrated that melanocytes can synthesize cholesterol via HMG-CoA reductase and transport cholesterol via LDL/Apo-B100/LDLR. Moreover, we show that cholesterol increases melanogenesis in these cells and in human melanoma cells of intermediate pigmentation (FM55) in a time- and dose-dependent manner. Cellular cholesterol levels in melanoma cells with different pigmentation patterns, epidermal melanocytes and keratinocytes do not differ except in the amelanotic (FM3) melanoma cell line. This result is in agreement with decreasing cholesterol content versus increasing pigmentation in melanosomes. Cholesterol induces cAMP in a biphasic manner i.e. after 30 min and later after 6 and 24 h, meanwhile protein expression of oestrogen receptor beta, CREB, MITF, tyrosine hydroxylase and tyrosinase is induced after 72 h. Taken together, we show that human epidermal melanocytes have the capacity of cholesterol signalling via LDL/Apo-B100/LDL receptor and that cholesterol under in vitro conditions increases melanogenesis.

Efficacy of acetaminophen in skin B16-F0 melanoma tumor-bearing C57BL/6 mice

Previously, we reported that acetaminophen (APAP) showed selective toxicity towards melanoma cell lines. In the current study, we investigated further the role of tyrosinase in APAP toxicity in SK-MEL-28 melanoma cells in the presence of a short hairpin RNA (shRNA) plasmid, silencing tyrosinase gene. Results from tyrosinase shRNA experiments showed that APAP led to negligible toxicity in shRNA plasmid-treated cells. It was also found that APAP selectively caused escalation in reactive oxygen species (ROS) formation and intracellular GSH (ICG) depletion in melanocytic human SK-MEL-28 and murine B16-F0 melanoma cells that express functional tyrosinase whereas it lacked significant effects on ROS formation and ICG in amelanotic C32 melanoma cells that do not express functional tyrosinase. These findings suggest that tyrosinase plays a major role in APAP selective induced toxicity in melanocytic melanoma cell lines. Furthermore, the in vivo efficacy and toxicity of APAP in the skin melanoma tumor model in mice was investigated. Mice receiving APAP at 60, 80, 100 and 300 mg/kg/day, day 7 through 13 post melanoma cell inoculation demonstrated tumor size growth inhibition by 7+/-14, 30+/-17, 45+/-11 and 57+/-3%, respectively. Mice receiving APAP day 1 through 13 post melanoma cell inoculation showed tumor size growth inhibition by 11+/-7, 33+/-9, 36+/-20 and 44+/-28%, respectively.

Glycosylated DOTA−α-Melanocyte-Stimulating Hormone Analogues for Melanoma Targeting: Influence of the Site of Glycosylation on in Vivo Biodistribution

alpha-Melanocyte-stimulating hormone (alpha-MSH) is known to bind to the melanocortin receptor 1 (MC1R) which is overexpressed on melanotic and amelanotic melanoma cells. alpha-MSH analogues are potential candidates for specific targeting of melanoma metastases. Several linear and cyclic radiolabeled MSH peptides have been designed and tested in the past, showing both high affinity for the MC1R in vitro and good incorporation in tumor xenografts in vivo. However, considerable kidney reabsorption of the radiopeptides could not be avoided. With the aim to increase the tumor-to-kidney ratio, we synthesized six glycosylated derivatives of NAPamide, an alpha-MSH octapeptide analogue with high tumor selectivity and coupled them to the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). The peptides were evaluated in vitro for MC1R binding and bioactivity and, after labeling with (111)In, for in vitro cellular uptake and in vivo tissue distribution in mice carrying B16F1 melanoma tumors. The glycopeptides showed excellent binding affinities in the low nanomolar to subnanomolar range using both murine and human melanoma cell lines. However, five glycopeptides displayed lower selectivity in vivo than the parent DOTA-NAPamide, because of either a lower tumor uptake or a higher kidney uptake. In particular C-terminal extension of the amide group by a galactosyl moiety increased the kidney retention dramatically. By contrast, an N-terminally positioned galactose residue in DOTA-Gal-NAPamide improved the tumor-to-kidney ratio (4-48 h AUC of 1.34) by a factor of about 1.2 as compared to the parent DOTA-NAPamide (4-48 h AUC of 1.11), thus serving as new lead compound for MC1R-targeting molecules.

Expression of human papilloma virus type 16 E5 protein in amelanotic melanoma cells regulates endo-cellular pH and restores tyrosinase activity

BackgroundMelanin synthesis, the elective trait of melanocytes, is regulated by tyrosinase activity. In tyrosinase-positive amelanotic melanomas this rate limiting enzyme is inactive because of acidic endo-melanosomal pH. The E5 oncogene of the Human Papillomavirus Type 16 is a small transmembrane protein with a weak transforming activity and a role during the early steps of viral infections. E5 has been shown to interact with 16 kDa subunit C of the trans-membrane Vacuolar ATPase proton pump ultimately resulting in its functional suppressions. However, the cellular effects of such an interaction are still under debate. With this work we intended to explore whether the HPV16 E5 oncoprotein does indeed interact with the vacuolar ATPase proton pump once expressed in intact human cells and whether this interaction has functional consequences on cell metabolism and phenotype.MethodsThe expression of the HPV16-E5 oncoproteins was induced in two Tyrosinase-positive amelanotic melanomas (the cell lines FRM and M14) by a retroviral expression construct. Modulation of the intracellular pH was measured with Acridine orange and fluorescence microscopy. Expression of tyrosinase and its activity was followed by RT-PCR, Western Blot and enzyme assay. The anchorage-independence growth and the metabolic activity of E5 expressing cells were also monitored.ResultsWe provide evidence that in the E5 expressing cells interaction between E5 and V-ATPase determines an increase of endo-cellular pH. The cellular alkalinisation in turn leads to the post-translational activation of tyrosinase, melanin synthesis and phenotype modulation. These effects are associated with an increased activation of tyrosine analogue anti-blastic drugs.ConclusionOnce expressed within intact human cells the HPV16-E5 oncoprotein does actually interact with the vacuolar V-ATPase proton pump and this interaction induces a number of functional effects. In amelanotic melanomas these effects can modulate the cell phenotype and can induce a higher sensitivity to tyrosine related anti-blastic drugs.

High-resolution magic angle spinning nuclear magnetic resonance analysis of metabolic changes in melanoma cells after induction of melanogenesis

Melanin synthesis affects melanoma behavior and tumor responsiveness to therapy; therefore, we investigated metabolic changes in melanoma cells after induction of melanogenesis. Amelanotic and melanotic melanoma cells were labeled with 13C precursors and changes in their metabolism was analyzed by high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR). HRMAS NMR demonstrated clear differences in the pattern of metabolic intermediates between amelanotic and melanotic cells. Although the exact nature of the metabolites requires further investigations, our comparative studies clearly show that induction of melanogenesis is associated with changes of glucose and sodium acetate metabolism, demonstrating HRMAS NMR as a powerful and noninvasive technique to investigate such process.

Fas and FasL expression on cells of two transplantable melanoma lines according to their different biological properties.

Fas and FasL interaction induces apoptotic cell death. In immunocompetent cells it plays a crucial role in the effector functions of the cells and in the regulation of host immune response. In tumours (e.g. melanoma), FasL expression possibly counteracts the Fas-positive effector T cells that infiltrate into tumours, and consequently the Fas/FasL interaction can contribute to the escape of tumour cells from the systemic immune response. In this study we examined differences in Fas and FasL expression on cells from the hamster melanotic melanoma line (Ma) and a more aggressive amelanotic melanoma line (Ab). We also tried to find out whether the Fas/FasL expression induces an ability to undergo spontaneous apoptosis in these two transplantable melanoma lines. Our previous studies have shown that cells of the Ma line have a higher ability to undergo spontaneous apoptosis than cells of the Ab line. Isolated transplantable melanoma cells were incubated for 4 and 24 hours and after that time the expression of Fas and FasL was estimated by flow cytometry. The results show that there was no Fas expression, although FasL was detected on both melanoma cell lines. Therefore the data reported by other authors indicate that a lack or a low level of Fas expression and an ectopic expression of FasL on melanoma cells can be an escape mechanism of the tumour, to avoid host immune responses. The content of FasL-positive melanotic melanoma cells was higher than in amelanotic melanoma cells and increased with the prolongation of the incubation time. FasL expression on amelanotic melanoma cells was detected after 24 hours at a level similar to that on melanotic melanoma cells after 4 hours incubation time. FasL expression on melanoma cells can induce apoptosis in cytotoxic T lymphocytes and NK cells which are responsible for tumour cells elimination. The results obtained suggest that the Fas/FasL system does not play any significant role in spontaneous apoptosis of two melanoma cell lines. But these results may indicate the presence of immune privilege of tumour cells with FasL expression.

Biochemical mechanism of acetylsalicylic acid (Aspirin) selective toxicity toward melanoma cell lines

In the current work, we investigated the biochemical toxicity of acetylsalicylic acid (ASA; Aspirin) in human melanoma cell lines using tyrosinase enzyme as a molecular cancer therapeutic target. At 2 h, ASA was oxidized 88% by tyrosinase. Ascorbic acid and NADH, quinone reducing agents, were significantly depleted during the enzymatic oxidation of ASA by tyrosinase to quinone. The 50% inhibitory concentration (48 h) of ASA and salicylic acid toward SK-MEL-28 cells were 100 micromol/l and 5.2 mmol/l, respectively. ASA at 100 micromol/l was selectively toxic toward human melanocytic SK-MEL-28, MeWo, and SK-MEL-5 and murine melanocytic B16-F0 and B16-F10 melanoma cell lines. However, ASA was not significantly toxic to human amelanotic C32 melanoma cell line, which does not express tyrosinase enzyme, and human nonmelanoma BJ, SW-620, Saos, and PC-3 cells. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased ASA toxicity toward SK-MEL-28 cells indicating quinone formation and intracellular GSH depletion played important mechanistic roles in ASA-induced melanoma toxicity. Ascorbic acid, a quinone reducing agent, and GSH, an antioxidant and quinone trap substrate, prevented ASA cell toxicity. Trifluoperazine, inhibitor of permeability transition pore in mitochondria, prevented ASA toxicity. ASA led to significant intracellular GSH depletion in melanocytic SK-MEL-28 melanoma cells but not in amelanotic C32 melanoma cells. ASA also led to significant reactive oxygen species (ROS) formation in melanocytic SK-MEL-28 melanoma cells but not in amelanotic C32 melanoma cells. ROS formation was exacerbated by dicoumarol and 1-bromoheptane in SK-MEL-28. Our investigation suggests that quinone species, intracellular GSH depletion, ROS formation, and mitochondrial toxicity significantly contributed toward ASA selective toxicity in melanocytic SK-MEL-28 melanoma cells.

Antiproliferative effects of essential oils and their major constituents in human renal adenocarcinoma and amelanotic melanoma cells

The purpose of this study was to evaluate cytotoxic activity of Platycladus orientalis, Prangos asperula and Cupressus sempervirens ssp. pyramidalis essential oils and to identify active components involved in inhibition of population growth of human cancer cell lines. Essential oils were obtained by hydrodistillation and were analysed by gas chromatography and gas chromatography coupled to mass spectrometry. Antiproliferative activity was tested on amelanotic melanoma C32 cells and on renal cell adenocarcinoma cells, using the sulphorhodamine B assay. Cupressus sempervirens ssp. pyramidalis leaf oil exerted the highest cytotoxic activity with an IC(50)value of 104.90 microg/mL against C32, followed by activity of P. orientalis and P. asperula on the renal adenocarcinoma cell line (IC(50) of 121.93 and 139.17 microg/mL, respectively). P. orientalis essential oil was also active against amelanotic melanoma with an IC(50) of 330.04 microg/mL. Three identified terpenes, linalool, beta-caryophyllene and alpha-cedrol, were found to be active on both cell lines tested. Our findings provide novel insights into the field of cytotoxic properties of essential oils. This study provided evidence on how cytotoxic activity of the oils is not always related to their major constituents, except for lower activity found in both cell lines for alpha-cedrol. Interestingly, beta-caryophyllene and linalool exhibited comparable IC(50) values to the commercial drug vinblastine on the ACHN cell line. This opens a new field of investigation to discover mechanisms responsible for the observed activity.

In vitro Cytotoxic Activity of Salsola oppositifolia Desf. (Amaranthaceae) in a Panel of Tumour Cell Lines

The aim of the present study was to evaluate for the first time the in vitro cytotoxic activity of fractions and isolated flavonols from Salsola oppositifolia Desf. (Amaranthaceae). The n-hexane fraction demonstrated an effective cytotoxic activity on the large lung carcinoma and amelanotic melanoma cell lines with IC50 values of 19.1 microg/ml and 24.4 microg/ml, respectively. Also the dichloromethane fraction exhibited cytotoxic activity against COR-L23 (IC50 30.4 microg/ml) and C32 (IC50 33.2 microg/ml) cells, while the EtOAc fraction demonstrated a selective cytotoxic activity against MCF-7 cells (IC50 67.9 microg/ml). The major active constituents of this fraction were isorhamnetin-3-O-glucoside (1) and isorhamnetin-3-O-rutinoside (2), which showed an interesting activity against the cell line MCF-7 with IC50 values of 18.2 and 25.2 microg/ml, respectively. Compound 2 exhibited a strong activity against the hormone-dependent prostate carcinoma LNCaP cell line with an IC50 of 20.5 microg/ml. Constituents of S. oppositifolia were identified by GC-MS and NMR analyses.

A Novel Immunohistochemical Marker of Normal and Neoplastic Melanocytes in Formalin-fixed, Paraffin-embedded Tissues of Albino Rats

In albino rats, spontaneous occurrence of melanocytic tumors is rare, with diagnosis difficult. This study evaluated immunoreactivity for PNL2 in normal and neoplastic melanocytes in formalin-fixed and paraffin-embedded tissues of albino rats. The samples consisted of 11 (1.57%) amelanotic melanomas in 700 rats (2 studies), 23 non-melanocytic tumors, and a wide variety of normal tissues. In normal albino rats, PNL2 stained the melanocytes in the iris and choroid of the eyeball and the hair bulb and basal cell layers of the epidermis of the whole body. In amelanotic melanoma, the tumor cells consisted of spindle cells with eosinophilic cytoplasm without melanin granules. PNL2 consistently stained cytoplasm in all amelanotic melanoma cells. In contrast, the nonmelanocytic tumor cells were not labeled. Electron microscopically, neoplastic, and normal melanocytes showed numerous cytoplasmic premelanosomes (stage II melanosome). In conclusion, PNL2 is direct against a fixative- and decalcific-resistant melanocyte-associated antigen, and has high specificity against normal and neoplastic melanocytes of albino rats.

Melanosomal Damage in Normal Human Melanocytes Induced by UVB and Metal Uptake—A Basis for the Pro‐oxidant State of Melanoma†

Melanins are ubiquitous catecholic pigments, formed in organelles called melanosomes within melanocytes, the function of which is to protect skin against harmful effects of UV radiation. Melanosomes within melanoma cells are characteristically abnormal, with fragmented melanin and disrupted membranes. We hypothesize that the disruption of melanosomal melanin might be an early event in the etiology and progression of melanoma, leading to increased oxidative stress and mutation. In this report, we examine the effect of a combination of UV treatment and metal ion exposure on melanosomes within melanocytes, as well as their ability to act as pro-oxidants in ex situ experiments, and assay the effects of this treatment on viability and cell cycle progression. UVB exposure causes morphologic changes of the cells and bleaching of melanosomes in normal melanocytes, both significantly enhanced in Cu(II) and Cd(II)-treated cells, as observed by microscopy. The promoted bleaching by Cu(II) is due to its ability to redox cycle under oxidative conditions, generating reactive oxygen species; verified by the observed enhancement of hydroxyl radical generation when isolated melanosomes were treated with both Cu(II) ions and UVB, as assayed by DNA clipping. Single-dose UVB/Cu treatment does not greatly affect cell viability or cell cycle progression in heavily pigmented cells, but did so in an amelanotic early stage melanoma cell line.

TRP-2 specifically decreases WM35 cell sensitivity to oxidative stress

TRP-2 (dopachrome tautomerase) is a melanogenic enzyme whose expression was recently reported to modulate melanocyte response to different cytotoxic events. Here we studied a possible role of TRP-2 in the oxidative stress response in the amelanotic WM35 melanoma cell line. Cell viability assays showed that TRP-2 overexpression in WM35 cells reduced their sensitivity to oxidative stress. Comet assays linked TRP-2 expression to DNA damage protection, and high-performance liquid chromotography—tandem mass spectrometry experiments showed an increase in intracellular glutathione in TRP-2-overexpressing cells. These effects were specifically reversed when TRP-2 was silenced by RNA interference. Nevertheless, these properties appeared to depend on a particular cell environment because expression of TRP-2 failed to rescue HEK epithelial cells exposed to similar treatments.

The activity of caspases in spontaneous and camptothecin-induced death of melanotic and amelanotic melanoma cell

Loss of pigment in hamster amelanotic melanoma line is accompanied by a faster growth rate, higher tumorigenicity and shorter animal survival time. Thus, the malignancy of melanoma increases during the alteration of melanotic (Ma) into amelanotic (Ab) line. As changes in the ability to undergo a spontaneous or induced apoptosis, and the role of caspases in this process during melanoma progression are not well defined, they were investigated in this work. Our results show that the proportion of spontaneously early apoptotic (caspase+/PI-) cells in the Ab line decreased in comparison to the Ma line. Cytochrome c release into cytosol, and the activation of effector caspases, estimated by PARP degradation clearly showed that during the spontaneous death in the cells from both melanoma lines intrinsic way of apoptosis was activated. Confocal and cytometric flow analyses indicate that camptothecin (CPT) induced apoptosis with caspase activation by the intrinsic way only in the amelanotic melanoma cells, even though cells of the Ma line also underwent CPT-induced apoptosis (the content of TUNEL- positive cells increased). Thus, our results suggest that melanoma progression, associated with a decreased ability to undergo spontaneous apoptosis but an increased susceptibility to CPT-induced apoptosis, relates to different levels of caspase activation; they also show that intrinsic way of apoptotis depends on the phenotype of melanoma cells, being more pronounced in the melanotic melanoma cells. On the other hand, melanotic melanoma cells resistance to camptothecin-induced apoptosis suggests that the melanogenic apparatus or melanin itself may have the protective effect on the ability of the melanoma cells to undergo apoptosis.

Cytotoxic activity of antioxidant constituents from Hypericum triquetrifolium Turra

The Sulforodamine B (SRB) assay was used to test cytotoxicity against four human cancer cell lines and one normal cell line of antioxidant constituents isolated from Hypericum triquetrifolium Turra. Methanolic extract and pure compounds were tested against the large cell lung carcinoma cell line COR-L23, the hepatocellular carcinoma cell line HepG-2, renal cell adenocarcinoma ACHN, the amelanotic melanoma cell line C32 and normal human foetal lung MRC5. The results showed that I3-II8-biapigenin exhibited strong cytotoxic activity (IC50 = 5.73 µg mL−1) showing a certain degree of selectivity against the different cell types.

Selenomethionine inhibits ultraviolet radiation‐induced p53 transactivation

Ultraviolet (UV) radiation damages the cellular DNA of skin cells. In response, wild-type p53 protein accumulates in irradiated cells and the stabilized and transactivated protein can then induce genes involved in cell cycle arrest in G1, or in the initiation of apoptosis. Selenium protects cells from UVB-induced cell death and apoptosis by mechanisms which are unclear, although recent reports suggest that selenium protects against UV-induced cell damage by inducing DNA repair enzymes and transactivating p53. We examined whether selenomethionine could protect human skin cells from UV radiation-induced p53 transactivation, using a pRGCDeltafos-lacZ p53-dependent reporter construct stably transfected in an amelanotic melanoma cell line (Arn-8) which expresses wild-type p53. Cells were pretreated with or without selenomethionine and then irradiated with broadband UVB (approximately 270-350 nm); 0-30 mJ/cm2 from a Phillips TL100 W/12 lamp. The percentage of cells with transcriptionally active p53 increased dose dependently up to 20 mJ/cm2 UVB. Treatment with 50 microM selenomethionine for 24 h both pre- and post-irradiation, significantly diminished p53 activation by 30-43% across the UV dose range (P=0.0085, n=5 independent experiments) and decreased UV-induced p53 protein accumulation as assessed by Western blotting. We conclude that selenomethionine inhibits broad band UVB-induced p53 transactivation and protein accumulation and that this effect correlates with reported protective effects of selenium against UV-induced DNA damage.

Loss of Adhesion in the Circulation Converts Amelanotic Metastatic Melanoma Cells to Melanotic by Inhibition of AKT

Direct injection of murine K-1735 melanoma cells into the subcutis, lung, or brain of syngeneic mice produces amelanotic tumors, whereas intravenous injection into the lateral tail vein or internal carotid artery produces both amelanotic and melanotic foci in the lung and the brain respectively. We hypothesized that loss of adhesion in the circulation may contribute to the melanogenic phenotypes of cells. To test this, we used enforced suspension culture of K-1735 cells by consistent rotating culture of K-1735 cells. We found that the expression of the microphthalmia transcription factor (MITF) and melanin-stimulating hormone receptor (MSHR) were upregulated in cells growing in suspension and were accompanied by inhibitions of AKT and ERK, which were reversed in cells upon regrowth as an adherent monolayer. Inhibition of the AKT pathway was responsible for MITF induction by suspension culture. Stable expression of constitutively active AKT significantly repressed the melanogenesis of K-1735 cells injected via circulation. An amelanotic clone of K-1735 cells was resistant to suspension cultureinduced MITF, although the inhibition of AKT pathway was intact. Collectively, these data suggest that the inhibition of AKT pathway due to loss of adhesion within the circulation renders a subpopulation of K-1735 cells to produce melanin.