Melanocyte Growth Research Articles

Diffuse Cutaneous Melanosis Associated with Malignant Melanoma.

Dear Editor: Diffuse cutaneous melanosis (DCM) is a rare condition affecting a minority of advanced melanoma patients. It is often described in the setting of metastatic disease of the liver and was first defined by the German pathologist Wagner1, who reported on a patient with melanoma arising from a congenital nevus with subsequent gray skin coloration. We report on an otherwise healthy 45-year-old Caucasian woman with a 3-mm-thick nodular melanoma (pT3a) on the right flank. At the time of the first diagnosis (2011), there were no clinical/radiological signs of metastatic disease. After the positive sentinel-lymph-node biopsy (sn1/3), she underwent a complete lymphadenectomy. Two years later and upon negative clinical and radiological follow-up, she complained of intense lower-back pain radiating down to the left leg. Magnetic resonance imaging of the spine showed diffuse bone metastases with pathological fractures and multiple metastases in L5 with penetration of the rear edge and epidural fat tissue with compression of the S1 nerve. A computed tomography-controlled percutaneous aspiration biopsy was performed for routine molecular diagnostics. The melanoma tissue showed the presence of wild-type BRAF. The patient was treated with combination chemotherapy (vindesine/cisplatin) and symptomatic palliative radiotherapy of the spinal cord with five 4-Gy sessions, amounting to a total dose of 20 Gy. After two cycles of chemotherapy, she was hospitalized because of pain exacerbation. Subsequent imaging studies showed disease progression with new liver metastasis and progressive spinal metastasis (M1c) (Fig. 1). High levels of lactate dehydrogenase (1,264 U/L) and S-100 (18.3 µg/L) were detected. Furthermore, she developed diffuse gray hyperpigmentation affecting the entire skin and sclera corresponding to DCM (Fig. 2). No pigment changes of the hair were noted. The patient was treated symptomatically and was transferred to a palliative care unit. Four months after the onset of DCM, she died. Fig. 1 Sagittal magnetic resonance image showing diffuse bone metastases with pathological fractures, metastases in L5 with penetration of the rear edge and epidural fat tissue, and compression of the S1 nerve. Fig. 2 Brown-gray hyperpigmentation of sun-exposed skin areas because of disease progression. (A) Before onset of diffuse cutaneous melanosis (DCM). (B) After onset of DCM. The clinical presentation of DCM consists of diffuse blue-grayish pigmentation of the skin and sclera, with a descending cephalo-caudal progression2. Additional clinical features include melanuria, hair pigment changes, and darkening of the serum or peritoneal fluids. Histologically, in most of the published cases, the presence of dermal pigment, deposition of melanin in histiocytes, and free melanin in dermal connective tissue are reported3. DCM has a poor outcome with a mean time to death of approximately 4~5 months after disease onset4. The pathogenesis of DCM is yet unclear. One current proposed theory is that elevated melanocytic growth factors such as α-melanocyte-stimulating hormone, endothelin-1, and hepatocyte growth factor activate the melanogenesis, proliferation, and motility of normal and malignant melanocytes5. Nevertheless, this hypothesis is contradictory to the fact that no alteration in melanocyte numbers or keratinocyte pigmentation was observed in most cases of DCM. Oncologists and dermato-oncologists should be familiar with DCM and its underlying poor prognosis. Taking into account the anticipated survival of these patients, an interdisciplinary palliative care treatment aimed at providing good individualized symptom control and relief in all areas of the patient's life should be offered in experienced centers.

Abstract 336: IL-6 primes melanoma cells from early stages to statin induced apoptosis

Abstract Introduction: Statins trigger apoptosis in tumour cells in vitro and, particularly, melanoma cells are susceptible to statin induced apoptosis. Growth of normal melanocytes and early stage melanoma can be inhibited by IL-6, whereas metastatic melanoma cells are mostly resistant to the anti-proliferative effects of IL-6. It has been shown that nearly 50% of metastatic melanoma secrete IL-6 into the supernatant. Nevertheless until now the stage dependant effects of IL-6 on melanoma cells are not fully understood. Methods: The secretion of IL-6 by A375, 518A2 human metastatic melanoma cells was compared with WM 35 cells which derive from an early lesion (radial growth phase) and investigated with ELISA. The sensitivity toward statin induced caspase 3 activation was investigated in these cells in the absence and presence of IL-6 or an anti IL-6 receptor antibody (tocilizumab). Apoptosis was also confirmed by FACS analyses for AnnexinV/PI staining and proliferation by cell cycle analysis using PI staining in such treated cells. Phosphorylation of Stat3 and the regulation of pro- (Bax, Bak) and anti-apoptotic proteins (Bcl-2, Bxl-XL) were studied by Western blot. Results: Melanoma cells derived from late stage lesions (A375, 518A2) secrete high amounts of IL-6 in contrast to WM35 cells. IL-6 signalling is intact in A375, 518A2 and WM 35 cells indicated by IL-6 triggered phosphorylation of Stat-3. Most interestingly, the A375 and 518A2 cells show a high sensitivity to simvastatin induced apoptosis with EC50 of 0,52 μM (518A2) and 1,7 μM (A375) in contrast to WM35 cells which are more resistant (EC50 16 μM). Co-treatment with IL-6 leads to an augmentation of the statin induced apoptosis in WM35 cells. Statin induced apoptosis can be mediated by the intrinsic apoptotic pathway which requires the loss of mitochondrial membrane potential. This is often alleviated by a downregulation of antiapoptotic Bcl-2 family members. Interestingly IL-6 treatment led to a marked decrease in the amount of Bcl-2 and Bcl-XL in WM35 cells which may explain the increased activation of apoptosis. Since A375 and 518A2 secrete high amounts of IL-6 we investigated the effects of the anti IL-6 receptor antibody tocilizumab. Conversely interruption of the IL-6 signalling with tocilizumab had no effect on the proliferation of 518A2 and A375 cells. Moreover the simvastatin induced caspase 3 activation was not decreased by blockage of the IL-6 signalling. Conclusion: Taken together our data prove different sensitivities of melanoma cells from various stages to statin induced apoptosis. Moreover, IL-6 facilitates apoptosis in early stage melanoma cells by the down-regulation of anti-apoptotic proteins. Nevertheless, these pro-apoptotic effects of IL-6 are not observed in metastatic melanoma cells indicating a stage specific role of IL-6 in the development and tumorigenesis of melanoma. Citation Format: Christoph Minichsdorfer, Christine Wasinger, Martin Hohenegger. IL-6 primes melanoma cells from early stages to statin induced apoptosis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 336. doi:10.1158/1538-7445.AM2014-336

Regulation of NADPH-dependent Nitric Oxide and reactive oxygen species signalling in endothelial and melanoma cells by a photoactive NADPH analogue

Nitric Oxide (NO) and Reactive oxygen species (ROS) are endogenous regulators of angiogenesis-related events as endothelial cell proliferation and survival, but NO/ROS defect or unbalance contribute to cancers. We recently designed a novel photoactive inhibitor of NO-Synthases (NOS) called NS1, which binds their NADPH site in vitro. Here, we show that NS1 inhibited NO formed in aortic rings. NS1-induced NO decrease led to an inhibition of angiogenesis in a model of VEGF-induced endothelial tubes formation. Beside this effect, NS1 reduced ROS levels in endothelial and melanoma A375 cells and in aorta. In metastatic melanoma cells, NS1 first induced a strong decrease of VEGF and blocked melanoma cell cycle at G2/M. NS1 decreased NOX(4) and ROS levels that could lead to a specific proliferation arrest and cell death. In contrast, NS1 did not perturb melanocytes growth. Altogether, NS1 revealed a possible cross-talk between eNOS- and NOX(4) -associated pathways in melanoma cells via VEGF, Erk and Akt modulation by NS1 that could be targeted to stop proliferation. NS1 thus constitutes a promising tool that modulates NO and redox stresses by targeting and directly inhibiting eNOS and, at least indirectly, NADPH oxidase(s), with great potential to control angiogenesis.

Prevention of hair graying by factors that promote the growth and differentiation of melanocytes.

Epidermal melanocyte precursors migrate into developing hair follicles to form the melanocyte stem cell system required to supply pigmented melanocytes necessary for hair pigmentation in repetitive hair cycles. Hair graying is caused by irreversible defects in the self-renewal and/or development of follicular melanocyte stem cells in the hair follicles. To investigate the mechanism(s) of hair graying during the normal aging process, we established a hair graying model in mice by repeatedly plucking or shaving trunk hairs. We repeatedly plucked or shaved trunk hairs to induce and accelerate the hair graying and counted the gray hairs. By using this functional model of hair graying in mice, we assessed the effects of genes known to affect melanocyte development, such as Kitl, hepatocyte growth factor (HGF) and endotheline 3 (ET3). After increasing the total numbers of cumulative hair cycles by plucking or shaving, we observed a significant increase in the gray hair of C57BL/6 mice. Kitl expression in the skin was the most effective for preventing hair graying and a significant effect was also confirmed for HGF and ET3 expression. The repeated hair plucking or shaving led to hair graying without any genetic lesion. Kitl is a more effective factor for prevention of hair graying than HGF or ET3. Our simple model of hair graying may provide a basic tool for screening the molecules or reagents preventing the progression of hair graying.

Activated Notch1 expression is associated with angiogenesis in cutaneous melanoma.

An early event in melanocytic tumor growth is the upregulation of Notch signaling. When an active form of Notch1 is overexpressed in primary human melanocytes, it increases cell growth, survival and invasive properties, promoting melanoma progression. Recent evidence suggested that tumor initiation and growth are driven by a subset of tumor-initiating cells termed cancer stem cells. Notch1 plays a predominant role in the maintenance of melanoblasts, including melanocyte stem cells, by preventing initiation of apoptosis. Moreover, the importance of Notch1 in the regulation of tumor angiogenesis is supported by growing evidence in various cancers. Nestin has been widely used as a marker for melanocyte stem cells as well as an angiogenic marker to evaluate neovascularity of endothelial cells in tumors. To gain an insight into the impact of Notch1 activation on the maintenance of melanocyte stem cells and angiogenesis in melanoma, the expression levels of activated Notch1 and nestin were analyzed by immunohistochemistry in 114 primary cutaneous melanomas and 35 lymph node metastases. Activated Notch1 and nestin expression was also evaluated in four dysplastic melanocytic nevi. This study provides evidence that activated Notch1 is overexpressed in cutaneous melanoma, in tumor cells as well as in microvessel endothelium, and that it can promote tumor angiogenesis. Indeed, the overexpression of activated Notch1 in both tumor and vascular endothelial cells was significantly associated with microvascular density in melanoma samples. Thus, activated Notch1 inhibitors may provide a therapeutic strategy in the treatment of melanoma by blocking tumor-associated vascularization.

Correlation between fertility drugs use and malignant melanoma incidence: the state of the art.

The relationship between fertility, reproductive hormones, and risk of malignant melanoma has acquired much interest in recent years. Melanocytes are hormonally responsive cells, and some in vitro studies demonstrated that estrogen hormones stimulate the growth of melanocytes. Moreover, estrogen receptors have been identified in melanoma cells, as well as in melanocytic nevi and in normal skin. Some evidences suggest a possible link between fertility treatments and the increased risk of malignant melanoma. This article addresses this association through a scrupulous search of the literature published thus far. The aim of this review is to determine the incidence of malignant melanoma in women treated with fertility drugs and to examine if the exposure to fertility treatments really increases the risk of malignant melanoma. In particular, our analysis focused on the different types of drugs and different treatment schedules used. Finally, this study provides additional insights regarding the long-term relationships between fertility drugs and the risk of malignant melanoma.

A Conditional Zebrafish MITF Mutation Reveals MITF Levels Are Critical for Melanoma Promotion vs. Regression In Vivo

The microphthalmia-associated transcription factor (MITF) is the “master melanocyte transcription factor” with a complex role in melanoma. MITF protein levels vary between and within clinical specimens, and amplifications and gain- and loss-of-function mutations have been identified in melanoma. How MITF functions in melanoma development and the effects of targeting MITF in vivo are unknown because MITF levels have not been directly tested in a genetic animal model. Here, we use a temperature-sensitive mitf zebrafish mutant to conditionally control endogenous MITF activity. We show that low levels of endogenous MITF activity are oncogenic with BRAFV600E to promote melanoma that reflects the pathology of the human disease. Remarkably, abrogating MITF activity in BRAFV600Emitf melanoma leads to dramatic tumor regression marked by melanophage infiltration and increased apoptosis. These studies are significant because they show that targeting MITF activity is a potent antitumor mechanism, but also show that caution is required because low levels of wild-type MITF activity are oncogenic.

Deadliest tumor of oral cavity: A rare case of intra oral malignant melanoma

Malignant melanoma is a rare tumor arising from the uncontrolled growth of melanocytes found in the basal layer of the epithelium. Although cutaneous malignant melanoma is the third most common malignancy of the skin, it accounts only for 3-5% of all dermal malignancies. Primary malignant melanoma of the oral mucosa is extremely rare, accounting for only 0.2-8% of all malignant melanomas. The incidence is slightly higher in males while others report a higher incidence in females. In the oral cavity, 80% of the cases occur in the maxilla with the prevalence of the hard palate or combined with the gingival or alveolar ridge. In this paper, we present a rare case of a 47-year-old female patient who reported with completely asymptomatic, pigmented growth in the maxillary anterior region.

Superoxide dismutase 1 inhibits alpha-melanocyte stimulating hormone and ultraviolet B-induced melanogenesis in murine skin.

BackgroundOver the last decade, the incidence of ultraviolet B (UVB)-related skin problems has increased. Oxidative stress caused by UVB induces the secretion of melanocyte growth and activating factors from keratinocytes, which results in the formation of cutaneous hyperpigmentation. Therefore, increasing the antioxidant abilities of skin cells is thought to be a beneficial strategy for the development of sunscreen agents. Superoxide dismutase 1 (SOD1) is an antioxidant enzyme that is known to exhibit antioxidant properties.ObjectiveThe purpose of this study was to investigate the effect of SOD1 on alpha-melanocyte stimulating hormone (α-MSH) and UVB-induced melanogenesis in B16F10 melanoma cells and HRM-2 melanin-possessing hairless mice.MethodsThe inhibitory effect of SOD1 on tyrosinase activity was evaluated in a cell-free system. Additional experiments were performed using B16F10 melanoma cells to demonstrate the effects of SOD1 in vitro, and HRM-2 melanin-possessing hairless mice were used to evaluate the antimelanogenic effects of SOD1 in vivo.ResultsWe found that SOD1 inhibited melanin production in a dose-dependent manner without causing cytotoxicity in B16F10 melanoma cells. SOD1 did not inhibit tyrosinase activity under cell-free conditions. The results indicate that SOD1 may reduce pigmentation by an indirect, nonenzymatic mechanism. We also found that SOD1 decreased UVB-induced melanogenesis in HRM-2 melanin-possessing hairless mice, as visualized through hematoxylin and eosin staining and Fontana-Masson staining.ConclusionOur results indicate that SOD1 has an inhibitory effect on α-MSH and UVB-induced melanogenesis, indicating that SOD1 may be a promising sunscreen agent.

Melanoma growth and progression after ultraviolet a irradiation: impact of lysosomal exocytosis and cathepsin proteases.

Ultraviolet (UV) irradiation is a risk factor for development of malignant melanoma. UVA-induced lysosomal exocytosis and subsequent cell growth enhancement was studied in malignant melanoma cell lines and human skin melanocytes. UVA irradiation caused plasma membrane damage that was rapidly repaired by calcium-dependent lysosomal exocytosis. Lysosomal content was released into the culture medium directly after irradiation and such conditioned media stimulated the growth of non-irradiated cell cultures. By comparing melanocytes and melanoma cells, it was found that only the melanoma cells spontaneously secreted cathepsins into the surrounding medium. Melanoma cells from a primary tumour showed pronounced invasion ability, which was prevented by addition of inhibitors of cathepsins B, D and L. Proliferation was reduced by cathepsin L inhibition in all melanoma cell lines, but did not affect melano-cyte growth. In conclusion, UVA-induced release of cathepsins outside cells may be an important factor that promotes melanoma growth and progression.

IL-17 and TNF Synergistically Modulate Cytokine Expression while Suppressing Melanogenesis: Potential Relevance to Psoriasis

Inflammation-associated pigmentation changes are extremely common, but the etiology behind this clinical observation remains elusive. Particularly, it is unclear how the myriad of cytokines known to be involved in inflammatory skin processes affect epidermal melanocytes. We sought to determine how IL-17 and TNF influence normal human melanocytes, as these two cytokines have been implicated in various skin diseases. IL-17 and TNF jointly stimulated broad inductions of cytokines , including melanoma mitogens CXCL1 and IL-8. Moreover, IL-17 and TNF synergistically inhibited pigmentation-related signaling and melanin production, and induced keratinocytes production of β-defensin 3, an antagonist for melanocortin-receptor 1. When analyzing psoriasis lesions that are known to over express IL-17 and TNF, we observed an increase in melanocyte number and a simultaneous decrease in pigmentation signaling. Furthermore, therapeutic neutralization of TNF and IL-17 with mAbs results in a rapid recovery of pigment gene expression in psoriasis lesions. These results demonstrate that IL-17 and TNF can impact both the growth and pigment production of melanocytes, which may contribute to the pigmentation changes associated with psoriasis. These findings may allow the development of novel therapeutics for pigmentary disorders and bring new insights into the immune milieu surrounding melanocytes and related neoplasms.

Senescent Fibroblasts in Melanoma Initiation and Progression: An Integrated Theoretical, Experimental, and Clinical Approach

We present an integrated study to understand the key role of senescent fibroblasts in driving melanoma progression. Based on the hybrid cellular automata paradigm, we developed an in silico model of normal skin. The model focuses on key cellular and microenvironmental variables that regulate interactions among keratinocytes, melanocytes, and fibroblasts, key components of the skin. The model recapitulates normal skin structure and is robust enough to withstand physical as well as biochemical perturbations. Furthermore, the model predicted the important role of the skin microenvironment in melanoma initiation and progression. Our in vitro experiments showed that dermal fibroblasts, which are an important source of growth factors in the skin, adopt a secretory phenotype that facilitates cancer cell growth and invasion when they become senescent. Our coculture experiments showed that the senescent fibroblasts promoted the growth of nontumorigenic melanoma cells and enhanced the invasion of advanced melanoma cells. Motivated by these experimental results, we incorporated senescent fibroblasts into our model and showed that senescent fibroblasts transform the skin microenvironment and subsequently change the skin architecture by enhancing the growth and invasion of normal melanocytes. The interaction between senescent fibroblasts and the early-stage melanoma cells leads to melanoma initiation and progression. Of microenvironmental factors that senescent fibroblasts produce, proteases are shown to be one of the key contributing factors that promoted melanoma development from our simulations. Although not a direct validation, we also observed increased proteolytic activity in stromal fields adjacent to melanoma lesions in human histology. This leads us to the conclusion that senescent fibroblasts may create a prooncogenic skin microenvironment that cooperates with mutant melanocytes to drive melanoma initiation and progression and should therefore be considered as a potential future therapeutic target. Interestingly, our simulations to test the effects of a stroma-targeting therapy that negates the influence of proteolytic activity showed that the treatment could be effective in delaying melanoma initiation and progression.

Oncogenic suppression of PHLPP1 in human melanoma

Akt is constitutively activated in up to 70% of human melanomas and has an important role in the pathogenesis of the disease. However, little is known about protein phosphatases that dephosphorylate and thereby inactivate it in melanoma cells. Here we report that suppression of pleckstrin homology domain and leucine-rich repeat Ser/Thr protein phosphatase 1 (PHLPP1) by DNA methylation promotes Akt activation and has an oncogenic role in melanoma. While it is commonly downregulated, overexpression of PHLPP1 reduces Akt activation and inhibits melanoma cell proliferation in vitro, and retards melanoma growth in a xenograft model. In contrast, knockdown of PHLPP1 increases Akt activation, enhances melanoma cell and melanocyte proliferation, and results in anchorage-independent growth of melanocytes. Suppression of PHLPP1 involves blockade of binding of the transcription factor Sp1 to the PHLPP1 promoter. Collectively, these results suggest that suppression of PHLPP1 by DNA methylation contributes to melanoma development and progression.

Primary culture of human face skin melanocytes for the study of hyperpigmentation

Facial epidermal pigmentation and skin tumors can be caused by UV exposure and other physical and chemical irritations. In this report we describe the primary culture of melanocytes from human face skin. The ability to culture these melanocytes will enable their morphological and biological properties to be investigated. Skin specimens were obtained from patients who had undergone lower blepharoplasty procedures. Digestion with neutral protease and trypsin was used to obtain single cell suspensions of epidermal cells. The cells were cultured in M254 medium supplemented with human melanocyte growth solution. Cell morphology was observed using inverted microscopy. Melanocytes were positively identified using both L-DOPA staining and S-100 protein immunohistochemical staining. Immunofluorescence was used to confirm the expression of tyrosinase-related protein-1, a melanocyte-specific protein. The cellular ultrastructure of the melanocytes was observed by transmission electron microscopy. The cultured human melanocytes from face skin were multi-dendritic, and many mature melanosomes were observed. Therefore, using a specific culture medium, melanocytes from face skin can be successfully cultured and made available for further investigations.

Changes of PI3K/AKT/BCL2 signaling proteins in congenital Giant Nevi: melanocytes contribute to their increased survival and integrity

Congenital Giant Nevi (CGN) are rare melanocytic lesions with the potential to regress into malignant melanoma. Simultaneous up-regulation and cooperative interactions of signaling pathways are crucial events in the pathogenesis of melanocytes. Our study aimed to identify changes in the expression and activation of proteins controlling survival and/or apoptosis of the key signaling pathways PI3K/AKT/BCL2 and Wnt/β-catenin of CGN melanocytes. We applied a model of cultured melanocytes from paired CGN and normal appearing skin, and Western blot (WB) analyzed the expression and activation profile of survival and anti-apoptotic proteins of these signaling pathways, growth pattern, cell cycle and apoptosis. WB analysis demonstrated a significant higher expression level of activated AKT and of BCL2 proteins in the CGN melanocytes compared with paired melanocytes from normal appearing skin. A relative increase in the level of GSK3 and FOXO1 proteins, down stream targets of AKT, as well as of pβ-catenin was also detected in the CGN melanocytes compared with the controls. These changes were not affected by growth of the CGN melanocytes in reduced serum (starvation). Both cell populations shared a similar growth pattern, with no significant differences in the proportion of apoptotic cells and in cell cycle fractions. These data demonstrate for the first time, changes in signaling proteins of cultured CGN melanocytes. Further, suggesting that the changes in AKT/BCL2 signaling molecules might mediate growth and anti-apoptosis processes at least in part, thus increasing the survival potential of CGN melanocytes and maintaining their integrity.

Clonal growth of human melanocytes using cell‐free extracellular matrix

Clonal growth of human melanocytes using cell‐free extracellular matrix

Culture of amelanotic melanocytes derived from human fetal hair follicles

Human melanocyte stem cells (MSCs) or melanoblasts are not well-investigated owing to the devoid of suitable culture system. Establishing cell lines of MSCs and/or their progenies from human hair follicles will provide a better opportunity to satisfy clinical needs and to enable a deeper understanding of hair-related diseases. In the present study, we cultured melanocytes derived from human fetal hair follicles, perform immunocytochemistry and Fontana Masson staining on them, and employed atomic force microscopy (AFM) and scanning electron microscopy to observe their subtle morphologies. The results show that the cultured melanocytes have a bipolar or tripolar appearance, which obviously differ from cultured epidermal melanocytes. Compared to cells derived from adult human hair follicles, these cells display a high proliferative capability and exhibit a clonal growth behavior. At the second passage, all these cells were positive for immunocytochemical staining with the NKI/beteb monoclonal antibody and Fontana Masson staining. Under AFM, the cells exhibited rounded, oval, triangular, or quadrangular perikarya, from which two or three dendrites arose. The dendritic arbor was not homogeneous but appeared as spindle-shaped dendritic swellings, knob-like processes, without any filopodia arising from the dendrites or the cell body. Without using a feeder layer, we successfully obtained the clonal growth of melanocytes from human fetal HFs, suggesting that the medium was suitable for the growth of MSCs and their progenies.

Ultrastructural observations of human epidermal melanocytes cultured on polyethylene terephthalate film

ObjectiveTo assess the application of polyethylene terephthalate film as a supporting material of cultured human epidermal melanocytes and to observe the ultrastructure of human epidermal melanocytes in vitro. MethodsHuman epidermal melanocytes were isolated from 7- to 14-years old children foreskins and were cultured in M254 culture medium containing human melanocyte growth supplement. Cultured melanocytes were purified via a differential trypsinization method. Purified melanocytes were cultured on a film and prepared for transmission electron microscopy. ResultsIn comparing with the cellular supporting materials polyvinylidene chloride and cellophane, human epidermal melanocytes only attached and grew on polyethylene terephthalate film. Melanocytes grown on polyethylene terephthalate film maintained an intact shape, and retained special ultrastructures characteristic of melanocytes such as dendrites. ConclusionsPolyethylene terephthalate film proved to be an ideal cellular supporting material for the cultivation of human epidermal melanocytes. Ultrastructure of melanocytes showed melanosomes are transferred both from the tip and middle section of dendrites.

PI(4,5)P2 5-phosphatase A regulates PI3K/Akt signalling and has a tumour suppressive role in human melanoma

Inositol polyphosphate 5-phosphatases can terminate downstream signalling of phosphatidylinositol-3 kinase; however, their biological role in the pathogenesis of cancer is controversial. Here we report that the inositol polyphosphate 5-phosphatase, phosphatidylinositol 4,5-bisphosphate 5-phosphatase, has a tumour suppressive role in melanoma. Although it is commonly downregulated in melanoma, overexpression of phosphatidylinositol 4,5-bisphosphate 5-phosphatase blocks Akt activation, inhibits proliferation and undermines survival of melanoma cells in vitro, and retards melanoma growth in a xenograft model. In contrast, knockdown of phosphatidylinositol 4,5-bisphosphate 5-phosphatase results in increased proliferation and anchorage-independent growth of melanocytes. Although DNA copy number loss is responsible for downregulation of phosphatidylinositol 4,5-bisphosphate 5-phosphatase in a proportion of melanomas, histone hypoacetylation mediated by histone deacetylases HDAC2 and HDAC3 through binding to the transcription factor Sp1 at the PIB5PA gene promoter appears to be another commonly involved mechanism. Collectively, these results establish the tumour suppressive role of phosphatidylinositol 4,5-bisphosphate 5-phosphatase and reveal mechanisms involved in its downregulation in melanoma.

Inhibition of integrins αv/α5-dependent functions in melanoma cells by an ECD-disintegrin acurhagin-C

Acurhagin-C, a Glu–Cys–Asp (ECD)-disintegrin from Agkistrodon acutus venom, has been reported as an endothelial apoptosis inducer, previously. Here we further evaluate its potential applications in cancer therapy. In vitro assays indicated that acurhagin-C not only may influence the cell viability at higher concentration, but also can potently and dose-dependently decrease cell proliferation in murine B16-F10 melanoma. Otherwise, it also had a dose-dependent inhibition on B16-F10 cell adhesion to extracellular matrices, collagen VI, gelatin B and fibronectin, as well as disturbed transendothelial migration of B16-F10 cell. Morphological study found that acurhagin-C dramatically affected B16-F10 cell adhesion to immobilized fibronectin, leading to the formation of multicellular aggregates with rounded shape. Detected by flow cytometry, acurhagin-C was able to induce B16-F10 cell apoptosis and alter cell cycle distribution through its interactions with integrins αv/α5, and thereafter initiation the apoptotic pathways of caspase-8/-9. Furthermore, acurhagin-C could synergistically enhance the anti-proliferative activity of methotrexate in B16-F10 cells and human melanoma SK-MEL-1 cells, without diminishing the growth of human epidermal melanocytes. Taken together, acurhagin-C proved to be a potent inhibitor of integrin-based functions in melanoma cells by activating the complex apoptotic pathways.