Immortalized Mouse Melanocytes Research Articles

Carvedilol, an Adrenergic Blocker, Suppresses Melanin Synthesis by Inhibiting the cAMP/CREB Signaling Pathway in Human Melanocytes and Ex Vivo Human Skin Culture.

Catecholamines function via G protein-coupled receptors, triggering an increase in intracellular levels of 3′,5′-cyclic adenosine monophosphate (cAMP) in various cells. Catecholamine biosynthesis and the β-adrenergic receptor exist in melanocytes; thus, catecholamines may play critical roles in skin pigmentation. However, their action and mechanisms mediating melanogenesis in human skin have not yet been investigated. Therefore, we examined the potential anti-melanogenetic effect of carvedilol, a nonselective β-blocker with weak α1-blocking activities. Carvedilol reduced melanin content and cellular tyrosinase activity without compromising cellular viability in normal human melanocytes as well as in mel-Ab immortalized mouse melanocytes. Carvedilol downregulated microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2. Carvedilol treatment led to the downregulation of phosphor-cAMP response element-binding protein (CREB). Moreover, the increase in cAMP levels upon treatment with forskolin reversed the anti-melanogenic action of carvedilol. In addition, carvedilol remarkably reduced the melanin index in ultraviolet-irradiated human skin cultures. Taken together, our results indicate that carvedilol effectively suppresses melanogenesis in human melanocytes and ex vivo human skin by inhibiting cAMP/protein kinase A/CREB signaling. The anti-melanogenic effects of carvedilol have potential significance for skin whitening agents.

Abstract 1890: Chloroquine synergizes with MEK1/2 targeted therapy through dual YAP and lysosomal inhibition in GNAQ/11 mutant uveal melanoma

Abstract GNAQ and GNA11 (GNAQ/11) mutations are found in less than 2% of all melanoma, but more than 80% of uveal melanoma. Mutations in these Gα proteins lead to constitutive activation of multiple oncogenic pathways, including MAPK (RAF->MEK1/2->ERK1/2) and YAP signaling. Metastatic uveal melanoma is refractory to all forms of pharmacologic treatment, such as FDA-approved targeted therapies inhibiting MEK1/2 (i.e. trametinib and binimetinib). We show that combining MEK1/2 inhibitors with 4-aminoquinoline antimalarials, chloroquine or hydroxychloroquine, resulted in synergistic and apoptosis-mediated cytotoxicity in GNAQ/11 mutant uveal melanoma cell lines. Interestingly, in contrast to our previous work in pancreatic and other RAS-driven cancers, the lysosomotropic role of chloroquine was not sufficient to promote cytotoxicity with MEK1/2 inhibitors, as neither lysosome inhibition with Bafilomycin A1 nor autophagy-specific and macropinocytosis-specific inhibition yielded enhanced cell death in combination with MEK1/2 inhibition. We then found that chloroquine prevented nuclear localization of the transcriptional coactivator, YAP, suggesting a novel mechanism of chloroquine. YAP inhibition combined with MEK1/2 inhibition enhanced cell death only in the presence of Bafilomycin A1. Gα-specific inhibition (inhibiting YAP and MAPK) combined with Bafilomycin A1 yielded similar results. This implies that the ability of chloroquine to inhibit both YAP signaling and lysosome function is required for promoting cell death in the presence of MEK1/2 inhibition. For in vivostudies, we utilized a hepatic colonization model using luciferized human metastatic uveal melanoma cell lines, OMM2.5 and OMM1. Daily treatment of trametinib with hydroxychloroquine in combination resulted in delayed tumor growth and increased overall survival compared to either treatment as monotherapy or chemotherapy. These findings were also recapitulated in an immunocompetent mouse model in which immortalized mouse melanocytes (Melan-A) with either a GNAQ or GNA11 activating mutation were implanted into syngeneic C57BL/6 mice. Our findings identify a novel mechanism of chloroquine and suggest a potentially effective strategy combining two FDA-approved drugs for the treatment of metastatic uveal melanoma. Citation Format: Amanda Truong, Michael Scherzer, Conan Kinsey, John Michael Sanchez, Jae Hyuk Yoo, Jackson Richards, Donghan Shin, Phaedra Ghazi, Michael Onken, Kendall Blumer, Shannon Odelberg, Martin McMahon. Chloroquine synergizes with MEK1/2 targeted therapy through dual YAP and lysosomal inhibition in GNAQ/11 mutant uveal melanoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1890.

GRM1 to induce M-CSF and CCL2 expression to lead to an upregulation of M2 macrophages in melanoma.

122 Background: GRM1 transgenic mice in an immune competent background (C57BL6) develope melanoma with 100% penetrance (TG-3 mice). Ectopic expression of GRM1 in immortalized mouse melanocytes (MelanA) produce cells (MASS20) that are tumorigenic in both SCID and immune competent C57BL6 mice. We have examined the downstream events of GRM1 signaling that allow transformed cells to evade immune destruction. Methods: Cytokine antibody arrays and Western blot were used to examine differences in cytokine expression between MASS20 cells and parental controls. Next, we cultured C57BL6 bone marrow cells in MASS20 conditioned medium for 24 and 48 hours and determined resultant M2 macrophage levels. Finally, we used oral weekly liposome clodronate (LC) treatment to deplete macrophages in murine models to determine the effect on tumor growth. Results: MASS20 cells produce higher levels of M-CSF and CCL2 as compared with control cells. These two cytokines recruit and induce the polarization of tumor associated macrophages (TAM) to increase the M2 fraction resulting in accelerated tumor progression. Culturing C57BL6 bone marrow cells in MASS20 conditioned medium increased expression of the M2 macrophage marker arginase1 at both time points. Weekly oral LC treatment to C57BL6 mice greatly inhibited the growth of MASS20 tumors as compared to growth in control animals. Single-cell suspensions of spleen and tumor cells from these animals were subjected to flow cytometry and showed that LC treatment depleted macrophages by 90% in the spleen, and increased the percentage of cd8+T and cd335+ NK cells in tumors from treated mice as compared to controls. Finally, we treated TG-3 transgenic mice with weekly oral LC and found that treated animals still developed tumors, but progression was greatly slowed, and survival greatly prolonged, compared to untreated animals. Within 1 week of stopping LC in the treated animals there was rapid growth of the tumors. These findings suggest a link between GRM1-signal transduction and TAM-induced suppression of tumor-immunity in melanoma. Conclusions: We conclude that GRM1-induced suppression of tumor immunity occurs through induction of M2 macrophages in the tumor microenvironment.

The transcription factor TBX2 regulates melanogenesis in melanocytes by repressing Oca2.

The T-box transcription factor TBX2 is known for its role as a critical regulator of melanoma cell proliferation, but its role in regulating melanogenesis has not been widely studied. Here we use a series of experiments to show in primary and immortalized mouse melanocytes that TBX2 acts as regulator of melanogenesis by repressing the expression of the gene encoding the melanosomal protein OCA2. We find that α-MSH or forskolin, both of which stimulate melanogenesis, also reduce TBX2 expression, and that specific knockdown of TBX2 increases melanogenesis. This effect primarily involves an increase in Oca2 expression as the combined knockdown of both Tbx2 and Oca2 interferes with the Tbx2 knockdown-mediated increase in melanogenesis. Standard chromatin immunoprecipitation and reporter assays suggest that TBX2 represses Oca2 at least in part directly. Hence, the results suggest that TBX2 may act as a nexus linking cell proliferation and melanogenesis.

Proton pump inhibitors decrease melanogenesis in melanocytes.

Proton pump inhibitors (PPIs) are widely used as inhibitors of gastric juice secretion for treatment of gastroesophageal reflux disease. However, there are no previous studies of the effects on melanogenesis resulting from PPI treatments. Therefore, the aim of the present study was to investigate the effects of PPIs on melanogenesis in melan-a cells derived from immortalized mouse melanocytes. Tyrosinase activity and copper-chelating activity were measured spectrophotometrically. In addition, the melanin content and viability of melan-a cells treated with PPIs were assessed and the mRNA levels of melanogenesis-associated genes were measured by reverse transcription-polymerase chain reaction. Treatment with rabeprazole, but not the other PPIs tested, resulted in strong, dose-dependent inhibition of mushroom tyrosinase (TYR). By contrast, each of the PPIs tested exhibited copper-chelating activity. Treatment of melan-a cells with 100 µM concentrations of the PPIs resulted in significantly reduced melanin synthesis and reduced expression of several melanogenesis-associated genes, including TYR, TYR-related protein-1 (TRP-1) and TRP-2, and microphthalmia-associated transcription factor, but did not result in cytotoxic effects. These results suggest that PPIs inhibit melanin biosynthesis in melan-a cells via the downregulation of melanogenesis-associated genes. Furthermore, the findings indicate that PPIs in general could be utilized as skin-whitening agents and/or as biomaterial for treating hyperpigmentation disorders.

Abstract A23: Epigenetic Cis-regulatory interactions in HIF1a-activated melanocytes

Abstract Melanoma tumors are heterogeneous lesions that progress through distinct cellular phenotypes exhibiting either highly proliferative or invasive cell state gene expression signatures (SOX10+ vs. TGFB+ pathways, respectively) as disease progression occurs. It is appreciated that even within a single tumor, individual cells respond to hypoxic conditions through Hypoxia Inducible Factor 1a (HIF1a) activation, leading to gene expression that promotes invasive cell properties. Regulation of gene expression integrates the binding of intrinsic cell lineage factors and general transcription machinery to key cis-regulatory DNA sequences that are epigenetically defined. In order to identify the cis-regulatory sequences that are utilized in a cell's response to HIF1a activation, we have established an integrated regulatory dataset from melan-Ink4a-ARF-/- immortalized mouse melanocytes. This dataset includes Dnase1 HS regions, enhancer regions marked by H3K4me1 and EP300 co-binding, ChIP-Seq for the transcription factors HIF1a and SOX10, and RNA-SEQ datasets under proliferative and HIF1a-activated growth conditions. We are evaluating HIF1a binding regions in order to identify the cis-regulatory sequences utilized to regulate gene expression, identify the key factors engaged at these sites, and assess the role that DNA variation has in modulating factor binding and gene transcription in the switch from proliferative to hypoxic cell signaling conditions. By identifying the epigenetic cis-regulatory interactions that occur as a result of HIF1a activation, we hypothesize that we will highlight pathways important for melanoma progression that may be relevant to future therapeutics. Citation Format: Stacie Loftus, Julie Cronin, Temesgen Fufa, Andrew McCallion, Gregory Crawford, William Pavan. Epigenetic Cis-regulatory interactions in HIF1a-activated melanocytes. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A23.

Cornus officinalis Methanol Extract Upregulates Melanogenesis in Melan-a Cells.

Cornus officinalis is widely distributed in Korea, and its fruit has been used to make as herbal drug for traditional medicine in Korea, Japan, and China because of its tonic, analgesic, and diuretic properties. However, the effects of C. officinalis methanol extract (COME) on melanogenesis remain poorly understood. We evaluated the melanogenic capability of COME in melan-a cells, which are immortalized mouse melanocytes. COME increased melanin synthesis in a dose-dependent manner. Treatment with 12.5 μg/mL of COME significantly increased melanin content by 36.1% (p < 0.001) to a level even higher than that (31.6%) of 3-isobutyl-1-methyl-xanthine, a well-known pigmentation agent. COME also upregulated tyrosinase activity and its messenger RNA and protein expression. In addition, COME upregulated the expression of tyrosinase-related proteins 1 and 2 and microphthalmia-associated transcription factor-M messenger RNA expression. These results imply that COME may be appropriate for development as a natural product to treat hair graying.

Cellular Anti-Melanogenic Effects of a Euryale ferox Seed Extract Ethyl Acetate Fraction via the Lysosomal Degradation Machinery.

The aim of this study was to investigate the effect of ethyl acetate fraction of Euryale ferox seed extracts (Efse-EA) on melanogenesis in immortalized mouse melanocyte cell line, melan-a. Efse-EA showed strong dose-dependent mushroom tyrosinase inhibitory activity. Treatment of melan-a cells with 30 μg/mL Efse-EA produced strong inhibition of cellular tyrosinase and melanin synthesis. Efse-EA significantly reduced the levels of melanogenesis-related proteins, such as tyrosinase, tyrosinase-related proteins 1 and 2, and microphthalmia-associated transcription factor. Because Efse-EA treatment reduced tyrosinase protein levels without changing its mRNA expression, we investigated whether this decrease was related to proteasomal or lysosomal degradation of tyrosinase. We found that chloroquine, a lysosomal proteolysis inhibitor, almost completely abolished both the down-regulation of tyrosinase and the inhibition of melanin synthesis induced by Efse-EA. These results suggested that Efse-EA may contribute to the inhibition of melanogenesis by altering lysosomal degradation of tyrosinase, and that this extract may provide a new cosmetic skin-whitening agent.

Metabotropic glutamate receptor 1 mediates melanocyte transformation via transactivation of insulin‐like growth factor 1 receptor

Our laboratory previously described the oncogenic properties of metabotropic glutamate receptor 1 (mGluR1) in melanocytes. mGluR1 transformed immortalized mouse melanocytes in vitro and induced vigorous tumor formation in vivo. Subsequently, we observed the activation of PI3K/AKT in mGluR1-mediated melanocytic tumorigenesis in vivo. In particular, we identified AKT2 being the predominant isoform contributing to the activation of AKT. Suppression of Grm1 or AKT2 using an inducible Tet-R siRNA system resulted in a 60 or 30% reduction, respectively, in in vivo tumorigenesis. We show that simultaneous downregulation of Grm1 plus AKT2 results in a reduction of approximately 80% in tumor volumes, suggesting that both mGluR1 and AKT2 contribute to the tumorigenic phenotype in vivo. The discrepancy between the mild in vitro transformation characteristics and the aggressive in vivo tumorigenic phenotypes of these stable mGluR1-melanocytic clones led us to investigate the possible involvement of other growth factors. Here, we highlight a potential crosstalk network between mGluR1 and tyrosine kinase, insulin-like growth factor 1 receptor (IGF-1R).

Regression of melasma with platelet-rich plasma treatment.

Dear Editor: Platelet-rich plasma (PRP) treatment is performed via the autologous injection of high concentration of platelets in a small volume of plasma1. A 27-year-old woman presented at our dermato-cosmetology department for skin rejuvenation, as she had epidermal hyperpigmentation over the cheeks, perioral region, and forehead for about 5 years. PRP treatment was initiated and her face was injected with autologous PRP prepared by using RegenKit® (Regen Lab., Le Montsur-Lausanne, Switzerland). Before the treatment sessions, 8 ml of blood was collected from the patient into a special tube containing a separation gel and an anticoagulant. The tube was then centrifuged for 8 minutes at 3,500 rpm, and PRP was obtained from the upper part of the buffy coat. A 32-G needle was used for superficial microinjections via the mesotherapy technique, and the injections were administered in to the papillary dermis (1.5~2.0 mm deep). Approximately 1.5 ml of PRP was injected in to the dermis of the face at each session with 15-day intervals. At the end of the third session of PRP treatment, >80% reduction in epidermal hyperpigmentation was observed (Fig. 1, ​,2).2). We did not provide any other treatment or post treatment care besides prescribing the use of a sun-screen product. There has been no recurrence of melasma for 6 months now. Fig. 1 Hyperpigmentation over the cheeks, perioral region, and forehead. Fig. 2 Significant regression in epidermal hyperpigmentation. PRP is commonly used in dermatology and plastic surgery, especially for treating chronic wounds, ulcers, and burns. In recent years, PRP has also started to be used in the field of cosmetology1. Volumetric filling, skin rejuvenation, acne scars, and alopecia are the main targets of PRP application in cosmetology1. The most important contents of platelets are contained in the α-granules. There are >30 bioactive substances in these granules2. Some of the bioactive substances present in the α-granules include platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β1, 2, epidermal growth factor, and mitogenic growth factors such as platelet-derived angiogenesis factor and fibrinogen3. To our knowledge, only TGF-β1 has been investigated about its relation with melanogenesis. Kim et al.4 investigated the effects of TGF-β1 on melanogenesis by using a spontaneously immortalized mouse melanocyte cell line, and asserted that TGF-β1 significantly inhibits melanin synthesis in a concentration-dependent manner. They declared that TGF-β1 decreases melanogenesis via delayed extracellular signal-regulated kinase activation. The pigmentary improvement that occurs with PRP treatment may be associated with the increase in skin volume. PDGF has a significant role in blood vessel formation and in the synthesis of collagen and components of the extracellular matrix, including hyaluronic acid. Hyaluronic acid has been shown to increase skin tone and volume, resulting in providing a more 'glowing skin'5. Although, it is not possible to reach a definitive conclusion, we consider the regression of melasma after PRP treatment as an interesting finding. Controlled clinical trials are needed to confirm this preliminary observation.

ARP101 inhibits α-MSH-stimulated melanogenesis by regulation of autophagy in melanocytes

Autophagy is a cooperative process between autophagosomes and lysosomes that degrades cellular organelles. Although autophagy regulates the turnover of cellular components, its role in melanogenesis is not clearly established. Previously, we reported that ARP101 induces autophagy in various cancer cells. Here, we show that ARP101 inhibits melanogenesis by regulation of autophagy. ARP101 inhibited α-MSH-stimulated melanin synthesis and suppressed the expression of tyrosinase and TRP1 in immortalized mouse melanocytes. ARP101 also induced autophagy in melanocytes. Knockdown of ATG5 reduced both anti-melanogenic activity and autophagy mediated by ARP101 in α-MSH treated melanocytes. Electron microscopy analysis further revealed that autophagosomes engulf melanin or melanosome in α-MSH and ARP101-treated cells. Collectively, our results suggest that ARP101 inhibits α-MSH-stimulated melanogenesis through the activation of autophagy in melanocytes.

Okadaic Acid Suppresses Melanogenesis &lt;i&gt;via&lt;/i&gt; Proteasomal Degradation of Tyrosinase

Okadaic acid is a C38 fatty acid derivative that is known to specifically inhibit the activity of protein phosphatase 2A (PP2A). Previously, we reported that inhibition of PP2A by okadaic acid elicited extracellular signal-regulated kinase (ERK) activation, and that PP2A may be involved in melanogenesis. However, the effects of okadaic acid on melanogenesis have not been completely evaluated. In the present study, we investigated the molecular mechanisms involved in okadaic acid modulation of melanin synthesis in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab. Treatment with okadaic acid inhibited melanin production in a dose-dependent manner. Moreover, okadaic acid led to a decrease in tyrosinase protein levels without altering mRNA expression. Therefore, we investigated whether the decreased level of tyrosinase by okadaic acid was related to proteasomal degradation of tyrosinase. We found that MG132, a proteasome inhibitor, almost completely abolished both the downregulation of tyrosinase levels and the inhibition of melanin synthesis by okadaic acid. Taken together, our data indicate that okadaic acid inhibits melanin synthesis via proteasomal degradation of tyrosinase.

Expression of cytosolic NADP+-dependent isocitrate dehydrogenase in melanocytes and its role as an antioxidant

Cytosolic NADP(+)-dependent ICDH (IDPc) has an antioxidant effect as a supplier of NADPH to the cytosol, which is needed for the production of glutathione. To evaluate the expression of IDPc in melanocytes and to elucidate its role as an antioxidant. The knock-down of IDPc expression in immortalized mouse melanocyte cell lines (melan-a) was performed using the short interfering RNA (siRNA)-targeted gene silencing method. After confirming the silencing of IDPc expression with mRNA and protein levels, viability, apoptosis and necrosis, as well as ROS production in IDPc-silenced melanocytes were monitored under conditions of oxidative stress and non-stress. Also, the ratio of oxidized glutathione to total glutathione was examined, and whether the addition of glutathione recovered cell viability, decreased by oxidant stress, was checked. The expression of IDPc in both primary human melanocytes and melan-a cells was confirmed by Western blot and RT-PCR. The silencing of IDPc expression by transfecting IDPc siRNA in melan-a cells was observed by Western blotting and real-time RT-PCR. IDPc knock-down cells showed significantly decreased cell viability and an increased number of cells under apoptosis and necrosis. IDPc siRNA-treated melanocytes demonstrated a higher intensity of DCFDA after the addition of H(2)O(2) compared with scrambled siRNA-treated melanocytes, and a lower ratio of reduced glutathione to oxidized glutathione were observed in IDPc siRNA transfected melanocytes. In addition, the addition of glutathione recovered cell viability, which was previously decreased after incubation with H(2)O(2). This study suggests that decreased IDPc expression renders melanocytes more vulnerable to oxidative stress, and IDPc plays an important antioxidant function in melanocytes.

Abstract 3587: XL184: c-Met inhibition is effective in a mouse xenograft model of metastatic uveal melanoma

Abstract Oncogenic mutation of GNAQ is an early event in uveal melanoma development. Identification of critical downstream effectors of GNAQ could allow for targeted therapy of this deadly form of melanoma. We identified c-Met upregulation as a downstream effect of GNAQ Q209L mutation by analyzing expression changes in immortalized mouse melanocytes transduced with GNAQ Q209L. By Western blot, we established that c-Met protein expression is increased in human uveal melanoma cell lines that harbor a GNAQ activating mutation compared to lines with wild-type GNAQ. XL184 is a small-molecule tyrosine kinase inhibitor that has activity against c-Met. In vitro assays of XL184 demonstrated preferential activity against human uveal melanoma cell lines (OMM1.3 and Mel202) with GNAQ activating mutations. Intravenous injection of 1×106 OMM1.3 cells into immunocompromised NSG mice results in the development of multiple liver tumor nodules after 6-8 weeks, recapitulating liver tropism (over 80% of patients with metastatic uveal melanoma have liver metastases). We treated mice starting four weeks after OMM1.3 intravenous injection with XL184. After four weeks of treatment, the treated mice demonstrated decreased liver tumor size with central tumor necrosis on histologic examination. The activity of XL184 is likely through its inhibition of c-Met signaling as we found little expression of the other targets of XL184 in OMM1.3. In conclusion, XL184 demonstrates activity in a mouse xenograft model of metastatic uveal melanoma, and has potential to be active against hepatic metastases of uveal melanoma, the major cause of mortality in this rare cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3587. doi:10.1158/1538-7445.AM2011-3587

The regulatory mechanism of melanogenesis by FTY720, a sphingolipid analogue

We previously reported that sphingosine-1-phosphate (S1P) decreases melanin synthesis via extracellular signal-regulated protein kinase (ERK) activation and microphthalmia-associated transcription factor (MITF) degradation. Although FTY720 is an S1P structural analogue, the effects of FTY720 on melanogenesis are not completely understood. Thus, we investigated the influence of FTY720 on melanin synthesis in a spontaneously immortalized mouse melanocyte cell line (Mel-Ab). FTY720 inhibited melanin synthesis in a concentration-dependent manner. Further, FTY720 has a different signal transduction mechanism to regulate melanogenesis from the S1P-induced signalling pathway. Our results showed that FTY720 down-regulated MITF and tyrosinase expression without ERK activation. MITF, the master regulator of pigmentation, is a target for the Wnt signalling pathway, including glycogen synthase kinase 3β (GSK3β) and β-catenin. Thus, the influence of FTY720 on GSK3β and β-catenin was further investigated. Decreased MITF and tyrosinase were associated with a reduction of β-catenin protein and mRNA levels. Decreased β-catenin expression by FTY720 may down-regulate expression of MITF, which finally reduces melanin synthesis.

Abstract 5076: Dissection of RAS downstream pathways in melanomagenesis

Abstract Cutaneous malignant melanoma is considered one of the most deadly human cancers, based upon both its penchant for metastatic spread and its typical resistance to currently available therapy. Long known to harbor oncogenic NRAS mutations, melanomas were more recently reported to be frequent bearers of activating mutations in BRAF, one of the effectors situated downstream of wildtype NRAS. RAS gene products (HRas, KRas and NRas) are 21kd G-proteins that serve as molecular switches converting cell-surface kinase activation events to nuclear events, thus influencing cell behavior. The major downstream effectors of Ras are the Rafs (ARaf, BRaf and CRaf), phosphatidylinositol 3-kinase (PI3K) and the Ral guanine exchange factors (RalGEF). NRAS and BRAF mutations are rarely found in the same melanoma, suggesting that they may possess important overlapping oncogenic activities. Here we compare and contrast the oncogenic roles of the three major NRas downstream effectors, Raf, PI3K and RalGEF, using genetically engineered Arf-deficient immortalized mouse melanocytes as a model system. Activation of each of the three major downstream pathways of NRas has overt phenotypic effects on immortalized mouse melanocytes, many distinct. Our data indicate a prominent role for BRaf and PI3K in melanocyte senescence and invasiveness, respectively. More surprisingly, we found that constitutively activated RalGEF stimulated melanocyte invasiveness and anchorage-independent growth almost as impressively as NRasQ61K. The suppressive effects of expression of a dominant negative Ral corroborated the role of the RalGEF-Ral pathway in anchorage-independent growth. Although no single downstream pathway could recapitulate all of the consequences of oncogenic NRas expression, melanocytes expressing constitutively activated mutants of all three NRas downstream factors (RLF-CAAX plus p110α-CAAX plus BRafV600E) achieved soft agar colony forming efficiency that was fully comparable to those expressing NRasQ61K alone. In conclusion, our data suggested a distinct function for all three NRas downstream pathways - Raf, PI3K and Ral in melanocyte senescence, invasiveness and transformation, respectively. Importantly, our finding that constitutive RalGEF activation confers malignant phenotypes in melanocytes, particularly anchorage-independent growth, indicates that this often overlooked pathway should be more carefully evaluated as a possible therapeutic target. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5076.

Hypopigmentary Action of Dihydropyranocoumarin D2, a Decursin Derivative, as a MITF-Degrading Agent

In this study, the decursin derivative dihydropyranocoumarin D2 (1) was selected for its effects on melanogenesis using a spontaneously immortalized mouse melanocyte cell line (Mel-Ab). The results showed that 1 effectively inhibited melanin synthesis in a concentration-dependent manner, but that it did not inhibit tyrosinase in a cell-free system. In addition, the changes in ERK, Akt, and microphthalmia-associated transcription factor (MITF) in response to treatment with 1 were assessed. The results revealed that ERK was dramatically up-regulated and MITF was down-regulated in response to treatment with 1, but that Akt was unchanged. Therefore, the effects of 1 on melanogenesis were examined in the absence or presence of PD98059 (a specific inhibitor of the ERK pathway). PD98059 restored hypopigmentation and the down-regulation of MITF induced by 1. Finally, MITF down-regulation by 1 was clearly restored by both chloroquine, a lysosomal proteolysis inhibitor, and MG132, a proteasome inhibitor.

The hypopigmentary action of KI-063 (a new tyrosinase inhibitor) combined with terrein

Resorcinol derivatives are known to inhibit melanin synthesis. In this study, resorcinol derivatives were synthesized and screened for their activity on melanogenesis. KI-063 (a tyrosinase inhibitor) was examined for its effects on melanogenesis using a spontaneously immortalized mouse melanocyte cell line (Mel-Ab). In a cell-free system, KI-063 directly inhibited tyrosinase, the rate-limiting melanogenic enzyme. Moreover, in a cell system, it inhibited melanin synthesis in a concentration-dependent manner. In addition, KI-063 inhibited the activity of cellular tyrosinase. Thus, this study examined the effects of a combination of KI-063 with terrein, an agent that down-regulates microphthalmia-associated transcription factor. The data suggest that KI-063 has an additive effect in combination with terrein. Thus, the suppression of tyrosinase activity by KI-063 and the inhibition of tyrosinase production by terrein appear to be an optimal combination for skin whitening.

Dissection of RAS downstream pathways in melanomagenesis: a role for Ral in transformation

Cutaneous malignant melanoma is considered one of the most deadly human cancers, based on both its penchant for metastatic spread and its typical resistance to currently available therapy. Long known to harbor oncogenic NRAS mutations, melanomas were more recently reported to be frequent bearers of activating mutations in BRAF, one of the effectors situated downstream of wild-type NRAS. NRAS and BRAF mutations are rarely found in the same melanoma, suggesting that they may possess important overlapping oncogenic activities. Here, we compare and contrast the oncogenic roles of the three major NRas downstream effectors, Raf, phosphatidylinositol 3-kinase (PI3K) and Ral guanine exchange factor (RalGEF), using genetically engineered Arf-deficient immortalized mouse melanocytes as a model system. Although no single downstream pathway could recapitulate all of the consequences of oncogenic NRas expression, our data indicate a prominent role for BRaf and PI3K in melanocyte senescence and invasiveness, respectively. More surprisingly, we discovered that constitutive RalGEF activation had a major impact on several malignant phenotypes, particularly anchorage-independent growth, indicating that this often overlooked pathway should be more carefully evaluated as a possible therapeutic target.

Silymarin inhibits melanin synthesis in melanocyte cells

Abstract Objectives The aim was to search for inhibitors of melanogenesis from natural resources. Methods The inhibitory effect of silymarin on melanogenesis in a spontaneously immortalized mouse melanocyte cell line, Mel-Ab, was studied. Key findings Silymarin significantly prevented melanin production in a dose-dependent manner with an IC50 value (concentration producing 50% maximal inhibition) of 28.2 μg/ml, without effects on cell viability. Also, silymarin inhibited l-DOPA oxidation activity of tyrosinase, the rate-limiting melanogenic enzyme, in cell based-systems but it did not directly affect cell-free tyrosinase activity. Furthermore, Western blot analysis indicated that silymarin decreased the expression of tyrosinase protein. Conclusions This study suggests that the depigmenting effect of silymarin might be attributable to inhibition of tyrosinase expression and that silymarin may be useful as a natural skin-lightening agent.