Normal Human Epidermal Melanocytes Research Articles

Down-regulation of miRNA-106b inhibits growth of melanoma cells by promoting G1-phase cell cycle arrest and reactivation of p21/WAF1/Cip1 protein.

MiR-106b is overexpressed in various types of cancers and is associated with the regulation of the carcinogenic processes. Using RT-PCR, we have identified overexpression of miRNA-106b in various melanoma cell lines (A375, Hs294t, SK-Mel28, SK-Mel 119, Mel 1241, Mel 1011 and Mel 928) as compared to its expression in normal human epidermal melanocytes (NHEM). The overexpression of miR-106b in melanoma cells (A375, Hs294t) was associated with greater cell proliferation capacity than NHEM. Treatment of A375 and Hs294t cells with anti-miR-106b resulted in inhibition of cell proliferation as well as G1-phase arrest. We determined the effects of grape seed proanthocyanidins (GSPs) on the expression of miRNA-106b and its underlying molecular targets. Treatment of A375 and Hs294t cells with GSPs resulted in suppression of the levels of miRNA-106b, cytotoxicity, G1-phase arrest and reactivation of p21/WAF1/Cip1. Dietary GSPs significantly inhibited growth of A375 melanoma cell tumor xenografts in nude mice, which was associated with reduction in the levels of miRNA-106b, tumor cell proliferation and increases in the levels of p21/WAF1/Cip1 protein. These studies suggest that miRNA-106b plays a crucial role in melanoma growth and that GSPs act as an inhibitor of miR-106b thereby blocking melanoma growth in vitro and in vivo models.

Impact of sparfloxacin on melanogenesis and antioxidant defense system in normal human melanocytes HEMa-LP – An in vitro study

BackgroundFluoroquinolones are a group of broad spectrum bactericidal antibiotics used to treat various infections of urinary and respiratory systems, as well as in ophthalmology and dermatology. This class of antibiotics causes toxic effects directed to pigmented tissues, what introduces a serious limitation to their use. The aim of this work was to examine the impact of sparfloxacin on melanogenesis and the antioxidant defense system in normal human epidermal melanocytes, adult, lightly pigmented (HEMa-LP). MethodsThe effect of sparfloxacin on cell viability was determined by WST-1 assay; melanin content, tyrosinase activity as well as antioxidant enzymes activity were measured spectrophotometrically. ResultsSparfloxacin induced the concentration – dependent loss in melanocytes viability. The value of EC50 was determined to be ∼0.25mM. Sparfloxacin inhibited tyrosinase activity and reduced the melanin content in human melanocytes. To study the antioxidant defense system in melanocytes, the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in cells exposed to sparfloxacin were determined. It was observed that sparfloxacin caused depletion of the antioxidant status of melanocytes. ConclusionsThe observed sparfloxacin-dependent inhibition of melanogenesis and changes of antioxidant enzymes activities in human melanocytes give a new insight into the mechanism of fluoroquinolones toxicity directed to pigmented tissues.

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.

Enhancement of the p38 MAPK and PKA signaling pathways is associated with the pro-melanogenic activity of Interleukin 33 in primary melanocytes

BackgroundInterleukin-33 (IL-33) was recently recognized as a member of the IL-1 cytokine family. The triggers no matter environmental or endogenous that provoke IL-33 cellular release may be associated with inflammation, infection or tissue damage. However, to date, the regulatory role of IL-33 in the control of melanogenesis has not been elucidated. ObjectiveThe present study was designed to investigate the effect of IL-33 on melanogenesis and to explore its underlying mechanisms. MethodsMelanocytes were exposed to IL-33. Then cell viabilities were measured by MTT assay. The improving activities of IL-33 were examined by melanin synthesis, tyrosinase (TYR) activity assay. The expressions of relative proteins were assessed by Western blotting. ResultsIL-33 increased the TYR activity and melanin content in a dosage-dependent manner at concentrations of 1–10ng/ml. Treatment with 10ng/ml of IL-33 enhanced the expression of microphthalmia-associated transcription factor (MITF), TYR, tyrosinase-related protein 1 (TRP-1) and dopachrome tautomerase (DCT) in normal human foreskin-derived epidermal melanocytes (NHEM). Furthermore, IL-33 could remarkably promote the phosphorylation levels of p38 mitogen-activated protein kinases (MAPKs) and cyclic AMP-responsive element-binding protein (CREB). This pro-melanogenic effect could be partially reversed by the pre-treatment with the special p38 MAPK inhibitor, SB203580, and protein kinase A (PKA) inhibitor, H89. ConclusionsOur results collectively indicated that IL-33 improved melanin biosynthesis in NHEM. This function might be attributed to the fact that IL-33 stimulates the phosphorylation of p38 MAPK and CREB, increases the TYR, TRP-1 and DCT expression through MITF, finally resulting in the augment of melanogenesis.

In vitro induction of melanin synthesis and extrusion by tamoxifen

Physical appearance has significant importance psychologically and socially, with skin and hair being of prime relevance. Effective ingredients that modulate melanin synthesis are of growing interest. Tamoxifen, a widely used selective oestrogen receptor modulator, SERM, was described occasionally in medical case reports as causing grey hair repigmentation. This work aimed to study, in vitro, the effect of tamoxifen and 4-hydroxy-tamoxifen, one of its most bioactive derivatives, on melanin production in human melanocytes. Adult normal human epidermal melanocytes (NHEM) were treated with physiological concentrations of tamoxifen and 4-hydroxy-tamoxifen during 72 hours. Cytotoxicity was evaluated by lactate dehydrogenase (LDH) leakage. Total melanin was quantified by spectrophotometry, and cyclic adenosine monophosphate (cAMP) was determined by competitive ELISA. The relative mRNA levels of several genes involved in melanogenesis were investigated by real-time PCR. Under the conditions used, the results showed that tamoxifen and 4-hydroxy-tamoxifen treatments, none of them toxic to NHEM, induced a time-dependent increase in the amount of melanin released to the culture medium. cAMP, one of the major second messenger in signalling pathways important to melanogenesis, was decreased after treatment. The transcript levels of genes coding for catalase, premelanosome protein and melan-A, directly related to skin and hair pigmentation, showed an increased tendency upon tamoxifen and 4-hydroxy-tamoxifen treatment. Induction of catalase gene expression in NHEM points towards a promelanogenic effect mediated by ROS. According to the results, even in such a short treatment period, tamoxifen and 4-hydroxy-tamoxifen promoted melanin extrusion and they seem to act as melanogenesis stimulators at the molecular level. Our data suggest that SERMs might be a new tool for increasing melanogenesis and might be of great interest for topical formulations in cosmetic industry.

Modulation of the pro-inflammatory cytokines and matrix metalloproteinases production in co-cultivated human keratinocytes and melanocytes

The human epidermis exerts immunoregulatory functions through the variety of cytokines and other molecules elaborated by keratinocytes and melanocytes. Their constitutive production is very low; however, considerably increased upon stimulation. In vivo, keratinocytes and melanocytes have a typical exposure in the skin, referred as melanocyte epidermal unit. In the present study we co-cultivated these cells in vitro proposing to elucidate some communication links in close cell-to-cell association. We assessed the amounts of IL-6, IL-8, and matrix metalloproteinases (MMP-2 and MMP-9) in individually and co-cultured cells, exposed or not to UVB radiation. Normal human epidermal keratinocytes and melanocytes were grown in specific media and supplements. Cells were exposed to UVB radiation (100mJ/cm(2)) to create comparable stress to the environmental one. Cytokines were determined with ELISA and confirmed with Western blot and metalloproteinases with gel zimography. Pure cultures of keratinocytes and melanocytes released low amounts of cytokines and metalloproteinases, these secretions being enhanced by UVB irradiation. In co-cultures, the cell-to-cell proximity triggered signals which markedly augmented the cytokines' secretions, whereas metalloproteinases were down-regulated. UVB irradiation did not influence either of these secretions in co-cultures. Concurrently with the highest levels of the pro-inflammatory cytokines, MMP-9 was up-regulated creating pro-inflammatory conditions and premises for changes in cellular survival, differentiation and phenotype. A complex network of interactions occurred between keratinocytes and melanocytes in co-cultures, resulting in modulated pro-inflammatory cytokines and metalloproteinases productions. Therefore, any disturbances in the microenvironmental signaling system and its molecular constituents may result in inflammation or even tumorigenesis in the epidermis.

Abstract LB-209: SIRT3, a mitochondrial sirtuin deacetylase, promotes survival of human melanoma cells.

Abstract Melanoma is one of the most aggressive skin cancers and is often lethal, if not diagnosed early. The existing therapeutic approaches as well as preventive strategies have not been able to effectively manage melanoma. A better understanding of mechanisms of melanoma development and progression may lead to additional means for the management of this deadly neoplasm. Sirtuins are NAD+-dependent protein deacetylases and mono-[ADP-ribosyl]transferases, which have been shown to regulate metabolism, stress responses, and longevity, in model organisms. Mammalian sirtuins, also referred to as class III histone deacetylases, have seven members (SIRT1-7) that differ widely in their localization, activity, and functions. The role of sirtuins in cancer is extremely complex and they have been shown to exhibit dichotomous functions (tumor promoter versus tumor suppressor) depending on cell contexts. SIRT3 is a mitochondrial deacetylase that regulates the activity of enzymes to coordinate global shifts in cellular metabolism. While studies have suggested that the SIRT3 acts as a tumor suppressor in some cancers (such as breast cancer), the tumor promoter function of SIRT3 has also been reported in other cancers (such as oral squamous cell carcinoma). The objective of this study was to determine the role of the SIRT3 in melanoma. Employing a panel of human melanoma cell lines (SK-MEL-28, WM35 and G361) and normal human epidermal melanocytes (NHEMs), we determined the endogenous levels of SIRT3. We found that compared to NHEMs, SIRT3 is significantly upregulated in human melanoma cells, at mRNA as well as protein levels, as shown by quantitative Real-Time PCR and Western blot analysis, respectively. Further, employing a tissue microarray (TMA) analysis, we determined the expression profile of SIRT3 protein in clinical melanoma and normal skin tissues. We found that SIRT3 is overexpressed in human melanoma, when compared to normal skin. Further, we determined the effect of a short hairpin RNA (shRNA)-mediated RNA interference on melanoma cells. Our data demonstrated that shRNA knockdown of SIRT3 resulted in a significant decrease in the growth, viability and clonogenic survival of human melanoma cells. Taken together, our data suggested that i) SIRT3 overexpression could be a contributing factor in melanoma survival, and ii) SIRT3 could serve as a potential target towards developing novel strategies for the management of melanoma. However, further in-depth studies are needed to determine the functional significance of SIRT3 overexpression in melanoma development and progression. Citation Format: Jasmine George, Minakshi Nihal, Chandra K. Singh, Nihal Ahmad. SIRT3, a mitochondrial sirtuin deacetylase, promotes survival of human melanoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-209. doi:10.1158/1538-7445.AM2013-LB-209

Abstract 1662: Dendrogenin A is a newly identified mammalian steroidal alkaloid that induced autophagic cell death in melanoma cells through an LXRbeta-, Nur77- and Nor1-dependent way.

Abstract Dendrogenin A (DDA) is a steroidal alkaloid that we recently discovered in mammalian tissues and normal cells. We did not detect DDA in a panel of melanoma and breast cancer cells, while DDA was present in normal Human Mammary Epithelial Cells and normal Human Epidermal Melanocytes suggesting that a deregulation of DDA metabolism may occur during carcinogenesis. We established that DDA triggered melanoma and breast cancer cells re-differentiation and death in vitro and in vivo, demonstrating its anticancer potency. In the present study, we investigated the molecular mechanisms involved in the cytotoxicity of DDA in melanoma cells. We found that DDA induced a time- and concentration-dependent cytotoxicity with the characteristics of apoptosis in human (SKMEL-28) and mouse (B16F10) melanoma cell lines. DDA induced the impairment of mitochondrial functions and activated the executioner caspase 3. However, caspase inhibitors failed to inhibit cytotoxicity suggesting that other mechanisms were involved. DDA triggered autophagy in melanoma cells and induced an autophagic flux. Genetic and pharmacological inhibition of autophagy inhibited DDA cytotoxicity showing that autophagy was involved in the cytotoxicity, which contrasts with the classical involvement of autophagy in tumor cell survival. In the search for cytotoxic effectors, we found that DDA stimulated the re-expression and re-localization of Nur77 (NR4A1) to the mitochondria, and the re-expression of Nor1 (NR4A3) in melanoma cells. We showed that pharmacological inhibition of the nucleo-cytoplasmic shuttling of Nur77, and the knock-down of Nur77 and Nor1, led to the inhibition of the cytotoxic autophagy. This establishes that Nur77 and Nor1 are involved in the induction of cytotoxic autophagy by DDA. Using a combination of molecular modeling, binding, luciferase gene reporter and transcriptional assays in tumor cells, we showed that DDA was a ligand of LXRα (NR1H3) and LXRβ (NR1H2) and a selective modulator of LXR-dependent gene expression. Interestingly, DDA displayed a specific transcriptional fingerprint compared to prototypical LXR modulators. Finally, we found that LXRβ was required for the stimulation of Nur77 and Nor1 expression by DDA and the induction of a cytotoxic autophagy. Altogether, these data established that the newly identified mammalian steroidal alkaloid DDA is a ligand of LXR that induced cytotoxic autophagy through an LXRβ-,Nur77- and Nor1-dependent mechanism in melanoma cells. Thus, DDA constitutes a promising drug candidate for melanoma treatment through an original mechanism of action. Since Nur77 and Nor1 expression has been found to be repressed in a number of aggressive tumors, DDA may represent an interesting option for the treatment of cancers expressing LXRβ. Citation Format: Gregory Segala, Mathias C. Poirot, Philippe de Medina, Michael Paillasse, Jean-Marc Lobaccaro, Sandrine Silvente-Poirot, Marc Poirot. Dendrogenin A is a newly identified mammalian steroidal alkaloid that induced autophagic cell death in melanoma cells through an LXRbeta-, Nur77- and Nor1-dependent way. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1662. doi:10.1158/1538-7445.AM2013-1662

Interleukin-18 augments growth ability of primary human melanocytes by PTEN inactivation through the AKT/NF-κB pathway

Normal human skin relies on melanocytes to provide photoprotection and thermoregulation by producing melanin. The growth and behavior of melanocytes are controlled by many factors. Interleukin-18 (IL-18) is expressed in both immune and non-immune cells and participates in the adjustment of multitude cellular functions. Nonetheless, the regulative roles of IL-18 in melanogenesis and growth of melanocytes have not been explored. The present study was conducted to investigate the effects of IL-18 on melanocytes and elucidate the underlying mechanisms. We proved that IL-18 increased the tyrosinase activity and melanin content in normal human foreskin-derived epidermal melanocytes (NHEM). Treatment with IL-18 (20ng/ml) enhanced the expression of c-Kit, microphtalmia-associated transcription factor (MITF) and its downstream tyrosinase-related protein 1 (TRP-1), and TRP-2. In addition, IL-18 induced NHEM migration at concentration of 20ng/ml. These results indicated a promotive action of IL-18 on melanogenesis in NHEM. Our data revealed that IL-18 stimulated ERK1/2 and NF-κB activation, improved p-Akt, p70 S6K and anti-apoptotic Bcl-2 levels, and deactivated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in NHEM. Besides, IL-18 increased level of PTEN phosphorylation to protect NHEM from damage induced by H2O2. These results in vitro showed the accommodation of IL-18 in melanocytes growth. Therefore, we suggested an important regulating action of IL-18 to melanogenesis and cell growth ability of skin melanocytes.

CXCR7 mediates SDF1‐induced melanocyte migration

Melanoblasts are derived from the neural crest and migrate to the dermal/epidermal border of skin and hair bulges. Although melanoblast migration during embryogenesis has been well investigated, there are only a few reports regarding the migration of mature melanocytes. Here, we demonstrate that a chemokine, stromal-derived factor-1 (SDF1, also known as CXCL12), and one of its receptor CXCR7 regulate normal human epidermal melanocyte (NHEM) migration. We found that SDF1 induces the directional migration of NHEMs. Interestingly, although both CXCR4 and CXCR7 are expressed in NHEMs, blockade of CXCR4 using a CXCR4-specific neutralizing antibody did not exert any influence on the SDF1-induced migration of NHEMs, whereas blockade of CXCR7 using a CXCR7-specific neutralizing antibody did influence migration. Furthermore, SDF1-induced NHEMs migration exhibited the early hallmark events of CXCR7 signaling associated with MAP kinase activation. It is known that the phosphorylation of ERK through CXCR7 signaling is mediated by β-arrestins. The treatment of NHEMs with SDF1 resulted in the phosphorylation of ERK in a β-arrestin 2-dependent manner. These results suggest that melanocytes may have a unique mechanism of migration via SDF1/CXCR7 signaling that is different from that of other cell types.

Phenytoin induces in vitro melanocyte proliferation

Phenytoin (5,5 diphenylhydantoin), a widely-used anticonvulsant, has been employed both topically and systemically in the treatment of some dermatological disorders. Immunological and non-immunological defects suggests that phenytoin could be therapeutically effective for vitiligo repigmentation. The first objective was to evaluate the effect of phenytoin on cultures of normal human melanocytes and to assess cytotoxicity of the substance using colorimetric methods. The second objective was to select the topical product that releases phenytoin best and fastest, for further clinical studies in vitiligo patients. In the first part of the study, we exposed normal human epidermal melanocyte cultures at 5,5 diphenylhydantoin at different concentrations. Melanocyte proliferation was evaluated using colorimetric assays (MTS assay), by measuring the absorbance at 490 nm with an ELISA plate reader. In the second part of the study, we formulated and obtained three types of phenytoin topical preparations: gel, hydrophilic cream, and ointment, for evaluation of the in vitro phenytoin release. Results The statistical analysis revealed that at low concentrations (0.1, 0.5 μg/ml), phenytoin stimulates growth and cellular proliferation in melanocyte cultures. The in vitro study of drug release indicated that gel formulation gives the highest phenytoin release. Our result indicates that topical applications of low concentrations of phenytoin may have beneficial effects in vitiligo, which justifies its further testing on clinical trials. Our gel formulation occurred to be the most suitable for the clinical studies.

Imatinib inhibits migration of normal human epidermal melanocytes and suppress the phosphorylation of C-kit

To investigate the inhibitory effect of imatinib on the migration of normal human epidermal melanocytes (NHEM), the growth and differentiation of NHEM were measured by MTT assay. Transwell Cell Migration Assay was performed to determine the migration of NHEM following imatinib treatment. The phosphorylation of c-kit in NHEM after imatinib treatment was measured by western blot assay. Imatinib at 0.1 ?M, 1.0 ?M and 5.0 ?M could inhibit the SCF (50 ng/mL) induced NHEM migration and suppress the phosphorylation of c-kit in NHEM. The result concluded that imatinib can inhibit SCF induced NHEM migration and suppress the c-kit phosphorylation in NHEM. This provide theoretical basis for the prevention of post-treatment recurrence of nevus spilus.

Oxidation Levels Differentially Impact Melanocytes: Low versus High Concentration of Hydrogen Peroxide Promotes Melanin Synthesis and Melanosome Transfer

Background: UVB light can generate potentially harmful hydrogen peroxide (H₂O₂) in vivo, but it can also promote the beneficial proliferation and migration of melanocytes. The successful use of UVB monotherapy for treatment of vitiligo suggests that H₂O₂ may have a biphasic effect on melanin synthesis and melanosome transfer. Objective: To study the beneficial role of H₂O₂ on melanogenesis and melanosome transport in living melanocytes and keratinocytes. Methods: A co-culture system model was constructed using the primary human melanocytes and keratinocytes. The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to determine cell proliferation, NaOH was used to determine the melanin content, and real-time PCR was used to determine tyrosinase expression. Western blot was used to determine Rab-27A and protease-activated receptor 2 (PAR-2) expression. Results: This study demonstrated that tyrosinase was activated by low concentrations of H₂O₂ (≤0.3 mM); however, this activity was downregulated by high concentrations of H₂O₂ (>0.3 mM). Activation of high levels of melanin synthesis was induced when cells were treated with low concentrations of H₂O₂ (0.3 mM). Further observation using an in vitro co-culture system of fluorescein (carboxyfluorescein diacetate succinimidyl ester, CFDA-SE)-labeled melanocytes and keratinocytes indicated that melanosome transfer occurred in normal human epidermal melanocytes. Fluorescence microscopy revealed increased melanosome transfer into keratinocytes treated with 0.3 mM H₂O₂ in the co-culture compared to the control. Examination of melanosomes in the keratinocytes by flow cytometry confirmed these results. Furthermore, treatment with H₂O₂ (0.3 mM) upregulated the expression of Rab-27A and PAR-2, significant proteins involved in melanosome transfer, according to Western blot. Conclusion: These results confirmed that low concentration levels of H₂O₂ play a major role in the regulation of human pigmentation by increasing melanin synthesis and melanosome transfer.

Endocannabinoids Stimulate Human Melanogenesis via Type-1 Cannabinoid Receptor

We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), 2-arachidonoylglycerol, the respective target receptors (CB(1), CB(2), and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (∼3-fold over controls at 5 μM) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB(1)-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (∼3- and ∼2-fold over controls at 1 μM). This CB(1)-dependent activity was fully abolished by the selective CB(1) antagonist SR141716 or by RNA interference of the receptor. CB(1) signaling engaged p38 and p42/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB(1) activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor.

Abstract 4722: Targeted inhibition of SIRT1 histone deacetylase by small-molecule inhibitor Tenovin-1 imparts significant anti-proliferative effects in human melanoma cells

Abstract Melanoma, one of the most lethal forms of skin cancer, remains resistant to available treatments. Unfortunately, current treatment options are insufficient for preventing the rapid metastasis and death associated with this disease. Thus, additional target-based approaches are needed for the management of this neoplasm. SIRT1, a member of mammalian sirtuin-family and a class III histone deacetylase has been implicated in several cancers. SIRT1 plays an important role in cell survival, as it is involved in DNA repair and inhibits apoptosis and cellular senescene in response to oxidative stress and DNA damage. It also favors cell survival through its regulation of the FOXO transcription factors and the tumor suppressor gene p53. Previous studies from our laboratory have demonstrated that i) histone deacetylase SIRT1 is expressed at much higher levels in melanoma cells than in normal human epidermal melanocytes, and ii) inhibition of SIRT1 through shRNA-mediated RNA interference or sirtinol-mediated activity inhibition resulted in decreased growth, viability, and clonogenic survival of melanoma cells (Nihal et al; In: AACR Annual Meeting: Proceedings; 2011 Apr 2-6, Orlando, FL Abstract # 1647). Here, in order to determine if SIRT1 is a viable target for melanoma management, we extended our studies to determine the effects of Tenovin-1, which is a more specific and efficient small molecule inhibitor of SIRT1, in multiple melanoma cell lines, representing different stages of disease progression. For this purpose, we employed i) A375 (primary and vertical-growth tumorigenic) and HS294T (metastatic and tumorigenic), both of which are amelanotic cell lines, and ii) G361 (metastatic and tumorigenic) melanin-producing cell line. We determined the effects of Tenovin-1 on the expression levels of SIRT1 protein in melanoma cells. Our data demonstrated that Tenovin-1 treatment resulted in a dose dependent decrease in the level of SIRT1 protein in all the melanoma cell lines tested. Further, we found that Tenovin-1 treatment resulted in a concentration dependent decrease in the growth and viability of melanoma cells, as shown by Trypan blue exclusion and MTT analyses. Furthermore, we found that Tenovin-1 caused an increase in protein level of the tumor suppressor p53 in a concentration-dependent fashion. Taken together, our results suggested that Tenovin-1 reduces melanoma cell growth via inhibition of SIRT1 and subsequent increase in p53 activity. However, additional studies are needed to confirm our results and to further establish the mechanism of how Tenovin-1 mediated SIRT1 inhibition leads to melanoma cell death. This includes the evaluation of other known SIRT1 targets such as the FOXO transcription factors. Finally, in vivo studies are also required to further determine the clinical relevance of our findings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4722. doi:1538-7445.AM2012-4722

Downregulation of cylindromatosis gene, CYLD, confers a growth advantage on malignant melanoma cells while negatively regulating their migration activity

The cylindromatosis gene (CYLD) encodes a deubiquitinase that was initially identified as a tumor suppressor and has recently been investigated in connection with a variety of normal physiological processes. In contrast to its cell-proliferative activity, the effect of CYLD protein on cell migration has been a matter of debate. We investigated the effect of CYLD-siRNA on the migration activity of malignant melanoma cells. Expression of CYLD mRNA/protein was lower in 6 of 8 malignant melanoma cell lines than in 3 sets of primary-cultured normal human epidermal melanocytes. Knockdown of CYLD significantly increased the proliferation activities of two melanoma cell lines (p<0.05), along with BCL3 nuclear translocation followed by CCND1 overexpression. In contrast to the proliferation-related activity, CYLD knockdown significantly decreased the cell migration of all the melanoma cell lines (n=7, p<0.05), and we demonstrated that the mechanism regulating melanoma cell migration was activation of RAC1 through the action of CYLD. Our findings provide new insight into the role of CYLD-induced RAC1 activation in melanoma cell migration.

Nucleus Accumbens-Associated 1 Contributes to Cortactin Deacetylation and Augments the Migration of Melanoma Cells

We investigated the prognostic significance and post-transcriptional acetylation-modification of cortactin (CTTN) via the nucleus accumbens-associated 1 (NACC1)-histone deacetylase 6 (HDAC6) deacetylation system in primary melanomas and melanoma cell lines. Overexpression of CTTN protein was observed in 56 (73%) of 77 stage I-IV melanomas, and was significantly correlated with tumor thickness, lymph node metastasis, distant metastasis, and disease outcome. The patients whose tumors exhibited CTTN overexpression had a poorer outcome than patients without this feature (P=0.028, log-rank test). NACC1 and CTTN proteins, but not HDAC6, were overexpressed in four melanoma cell lines in comparison with a primary culture of normal human epidermal melanocytes. Knockdown of both NACC1 and HDAC6 markedly downregulated the migration activity of all melanoma cell lines (P<0.05), and induced a gain of CTTN protein acetylation status. Confocal microscopy showed that hyperacetylation of CTTN modulated by depletion of both NACC1 and HDAC6 induced disappearance of CTTN protein at the leading edge of migrating cells, resulting in stabilization of the focal adhesion structure and development of actin stress fibers. These data suggest that the acetylation status of CTTN modulated by the NACC1-HDAC6 deacetylation system induces acceleration of melanoma cell migration activity via an actin-dependent cellular process, possibly contributing to aggressive behavior (invasion/metastasis) of the melanoma cells.

CTGF is overexpressed in malignant melanoma and promotes cell invasion and migration

Background:Malignant melanoma cells are known to have altered expression of growth factors compared with normal human melanocytes. These changes most likely favour tumour growth and progression, and influence tumour environment. The induction of transforming growth factor beta1, 2 and 3 as well as BMP4 and BMP7 expression in malignant melanoma has been reported before, whereas the expression of an important modulator of these molecules, connective tissue growth factor (CTGF), has not been investigated in melanomas until now.Methods:Expression of CTGF was analysed in melanoma cell lines and tissue samples by qRT–PCR and immunohistochemistry. To determine the regulation of CTGF expression in malignant melanoma, specific siRNA was used. Additionally, migration, invasion and attachment assays were carried out.Results:We were able to demonstrate that CTGF expression is upregulated in nine melanoma cell lines and in primary and metastatic melanoma in situ. The transcription factor HIF-1α was revealed as a positive regulator for CTGF expression. Melanoma cells, in which CTGF expression is diminished, show a strong reduction of migratory and invasive properties when compared with controls. Further, treatment of normal human epidermal melanocytes with recombinant CTGF leads to an increase of migratory and invasive behaviour of these cells.Conclusion:These results suggest that CTGF promotes melanoma cell invasion and migration and, therefore, has an important role in the progression of malignant melanoma.

1-(2,4-Dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylpheny)propane inhibits melanin synthesis by dual mechanisms

1-(2,4-Dihydroxyphenyl)-3-(2,4-dimethoxy-3-methylpheny)propane (DP) was reported as a novel tyrosinase inhibitor by Nesterov et al. In previous study, we showed that DP is an antioxidant and accelerates the fading of UVB-induced tan in human skin but details of inhibiting mechanism of DP in melanogenesis remain incomplete. To clarify additional mechanisms of DP inhibition of melanogenesis, we studied the effect of DP on tyrosinase processing and degradation. Tyrosinase inhibition was assessed using mushroom and human tyrosinase. The effect of DP on mRNA and protein levels as well as glycosylation and degradation of tyrosinase was examined using normal human epidermal melanocytes (NHEM). DP was 200 times more potent than that of kojic acid in inhibiting mushroom tyrosinase activity. In contrast, DP (IC(50)=200μM) was significantly less effective at inhibiting tyrosinase from NHEM. DP decreased melanin content in cultured NHEM after 7th day (IC(50)=10μM). The IC(50) for DP against human tyrosinase activity was found to be at least 20 times higher than that of melanin synthesis. At a non-cytotoxic concentration DP did not decrease tyrosinase mRNA however protein level decreased by 46% after 48h treatment. DP did not alter the ratio of mature and immature tyrosinase assayed by endo H cleavage. Tyrosinase degradation assays revealed that DP accelerated tyrosinase degradation in NHEM. We found that DP acts through dual mechanisms to reduce melanin synthesis; by inhibition of tyrosinase activity via an anti-oxidant effect, and, more importantly, by the acceleration of tyrosinase degradation.

4-Methylcatechol-induced oxidative stress induces intrinsic apoptotic pathway in metastatic melanoma cells

There has been a steady rise in fatalities associated with thick melanomas (>4 mm). Although understanding of the biology of the disease has improved, effective treatment strategies for patients with advanced metastatic melanoma remain elusive. Therefore, more intensive testing of agents with therapeutic potential are needed to improve survival of patients with metastatic malignant melanoma. We have tested the ability of 4-methylcatechol, a metabolite of quercetin; a naturally occurring compound that is commonly found in a variety of fruits for its potential as an anti-melanoma agent. Our results show that 4-methylcatechol inhibits proliferation of melanoma cells in culture while not affecting the growth of normal human epidermal melanocytes. Further, the ability of metastatic melanoma cells to form colonies on soft agar was also inhibited. 4-Methylcatechol caused the accumulation of cells in G2/M phase of the cell cycle and induced apoptosis. There was an increase in reactive oxygen species following treatment with 4-methylcatechol that led to apoptosis through the intrinsic mitochondrial pathway. Treatment also inhibited cell survival mediated by Akt, a key player in melanoma cell survival. Taken together our results suggest that 4-methylcatechol exhibits cytotoxicity towards metastatic malignant melanoma cells while sparing normal melanocytes and should be tested further as a potential drug candidate for malignant melanoma.