Peroxiredoxin-2 represses NRAS-mutated melanoma cells invasion by modulating EMT markers.

The second most common mutation in melanoma occurs in NRAS oncogene, being a more aggressive disease that has no effective approved treatment. Besides, cellular plasticity limits better outcomes of the advanced and therapy-resistant patients. Peroxiredoxins (PRDXs) control cellular processes through direct hydrogen peroxide oxidation or by redox-relaying processes. Here, we demonstrated that PRDX2 could act as a modulator of multiple EMT markers in NRAS-mutated melanomas. PRDX2 knockdown lead to phenotypic changes towards invasion in human reconstructed skin and the treatment with a PRDX mimetic (gliotoxin), decreased migration in PRDX2-deficient cells. We also confirmed the favorable clinical outcome of patients expressing PRDX2 in a large primary melanoma cohort. This study contributes to our knowledge about genes involved in phenotype switching and opens a new perspective for PRDX2 as a biomarker and target in NRAS-mutated melanomas.

Modeling Melanoma Heterogeneity In Vitro: Redox, Resistance and Pigmentation Profiles.

Microenvironment and transcriptional plasticity generate subpopulations within the tumor, and the use of BRAF inhibitors (BRAFis) contributes to the rise and selection of resistant clones. We stochastically isolated subpopulations (C1, C2, and C3) from naïve melanoma and found that the clones demonstrated distinct morphology, phenotypic, and functional profiles: C1 was less proliferative, more migratory and invasive, less sensitive to BRAFis, less dependent on OXPHOS, more sensitive to oxidative stress, and less pigmented; C2 was more proliferative, less migratory and invasive, more sensitive to BRAFis, less sensitive to oxidative stress, and more pigmented; and C3 was less proliferative, more migratory and invasive, less sensitive to BRAFis, more dependent on OXPHOS, more sensitive to oxidative stress, and more pigmented. Hydrogen peroxide plays a central role in oxidative stress and cell signaling, and PRDXs are one of its main consumers. The intrinsically resistant C1 and C3 clones had lower MITF, PGC-1α, and PRDX1 expression, while C1 had higher AXL and decreased pigmentation markers, linking PRDX1 to clonal heterogeneity and resistance. PRDX2 is depleted in acquired BRAFi-resistant cells and acts as a redox sensor. Our results illustrate that decreased pigmentation markers are related to therapy resistance and decreased antioxidant defense.

2-Methoxyestradiol-3,17-O,O-bis-sulfamate (STX140) Inhibits Proliferation and Invasion via Senescence Pathway Induction in Human BRAFi-Resistant Melanoma Cells.

The endogenous estradiol derivative 2-Methoxyestradiol (2-ME) has shown good and wide anticancer activity but suffers from poor oral bioavailability and extensive metabolic conjugation. However, its sulfamoylated derivative, 2-methoxyestradiol-3,17-O,O-bis-sulfamate (STX140), has superior potential as a therapeutic agent, acts by disrupting microtubule polymerization, leading to cell cycle arrest and apoptosis in cancer cells and possesses much better pharmaceutical properties. This study investigated the antiproliferative and anti-invasive activities of STX140 in both SKMEL-28 naïve melanoma (SKMEL28-P) cells and resistant melanoma cells (SKMEL-28R). STX140 inhibited cell proliferation in the nanomolar range while having a less pronounced effect on human melanocytes. Additionally, STX140 induced cell cycle arrest in the G2/M phase and sub-G1, reduced migration, and clonogenic potential in monolayer models, and inhibited invasion in a 3D human skin model with melanoma cells. Furthermore, STX140 induced senescence features in melanoma and activated the senescence machinery by upregulating the expression of senescence genes and proteins related to senescence signaling. These findings suggest that STX140 may hold potential as a therapeutic agent for melanoma treatment.

Epidermal melanocytes metabolize extracellular nucleotides by purinergic enzymes.

The human epidermal melanocyte (hEM) are melanin-producing cells that provide skin pigmentation and protection against ultraviolet radiation. Although purinergic signaling is involved in skin biology and pathology, the presence of NTPDase members, as well as the rate of nucleotides degradation by melanocytes were not described yet. Therefore, in this study, we analyzed the expression of ectonucleotidases in hEM derived from discarded foreskin of male patients. The expression of purinergic enzymes was confirmed by mRNA and flow cytometry. Among the ectonucleotidases, ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1) and ecto-5´-nucleotidase were the ectoenzymes with higher expressions. The hydrolysis rate for ATP, ADP, and AMP was low in comparison to other primary cells already investigated. The amount of ATP in the culture medium was increased after a scratch wound and decreased to basal levels in 48 h, while the NTPDase1 and P2X7 expressions increased. Therefore, it is possible to suggest that after cell injury, the ATP released by hEM into the extracellular space will be hydrolyzed by ectonucleotidases as the NTPDase1 that will control the levels of nucleotides in the skin micro-environment.

Mutually exclusive genetic interactions and gene essentiality shape the genomic landscape of primary melanoma.

Melanoma is a heterogenous malignancy with an unpredictable clinical course. Most patients who present in the clinic are diagnosed with primary melanoma, yet large-scale sequencing efforts have focused primarily on metastatic disease. In this study we sequence-profiled 524 American Joint Committee on Cancer Stage I-III primary tumours. Our analysis of these data reveals recurrent driver mutations, mutually exclusive genetic interactions, where two genes were never or rarely co-mutated, and an absence of co-occurring genetic events. Further, we intersected copy number calls from our primary melanoma data with whole-genome CRISPR screening data to identify the transcription factor interferon regulatory factor 4 (IRF4) as a melanoma-associated dependency. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

3-Bromopyruvate Suppresses the Malignant Phenotype of Vemurafenib-Resistant Melanoma Cells.

(1) BRAF mutations are associated with high mortality and are a substantial factor in therapeutic decisions. Therapies targeting BRAF-mutated tumors, such as vemurafenib (PLX), have significantly improved the overall survival of melanoma patients. However, patient relapse and low response rates remain challenging, even with contemporary therapeutic alternatives. Highly proliferative tumors often rely on glycolysis to sustain their aggressive phenotype. 3-bromopyruvate (3BP) is a promising glycolysis inhibitor reported to mitigate resistance in tumors. This study aimed to evaluate the potential of 3BP as an antineoplastic agent for PLX-resistant melanoma treatment. (2) The effect of 3BP alone or in combination with PLX on viability, proliferation, colony formation, cell death, migration, invasion, epithelial-mesenchymal marker and metabolic protein expression, extracellular glucose and lactate, and reactive species were evaluated in two PLX-resistant melanoma cell lines. (3) 3BP treatment, which was more effective as monotherapy than combined with PLX, disturbed the metabolic and epithelial-mesenchymal profile of PLX-resistant cells, impairing their proliferation, migration, and invasion and triggering cell death. (4) 3BP monotherapy is a potent metabolic-disrupting agent against PLX-resistant melanomas, supporting the suppression of the malignant phenotype in this type of neoplasia.

Redox-Related Proteins in Melanoma Progression.

Melanoma is the most aggressive type of skin cancer. Despite the available therapies, the minimum residual disease is still refractory. Reactive oxygen and nitrogen species (ROS and RNS) play a dual role in melanoma, where redox imbalance is involved from initiation to metastasis and resistance. Redox proteins modulate the disease by controlling ROS/RNS levels in immune response, proliferation, invasion, and relapse. Chemotherapeutics such as BRAF and MEK inhibitors promote oxidative stress, but high ROS/RNS amounts with a robust antioxidant system allow cells to be adaptive and cooperate to non-toxic levels. These proteins could act as biomarkers and possible targets. By understanding the complex mechanisms involved in adaptation and searching for new targets to make cells more susceptible to treatment, the disease might be overcome. Therefore, exploring the role of redox-sensitive proteins and the modulation of redox homeostasis may provide clues to new therapies. This study analyzes information obtained from a public cohort of melanoma patients about the expression of redox-generating and detoxifying proteins in melanoma during the disease stages, genetic alterations, and overall patient survival status. According to our analysis, 66% of the isoforms presented differential expression on melanoma progression: NOS2, SOD1, NOX4, PRX3, PXDN and GPX1 are increased during melanoma progression, while CAT, GPX3, TXNIP, and PRX2 are decreased. Besides, the stage of the disease could influence the result as well. The levels of PRX1, PRX5 and PRX6 can be increased or decreased depending on the stage. We showed that all analyzed isoforms presented some genetic alteration on the gene, most of them (78%) for increased mRNA expression. Interestingly, 34% of all melanoma patients showed genetic alterations on TRX1, most for decreased mRNA expression. Additionally, 15% of the isoforms showed a significant reduction in overall patient survival status for an altered group (PRX3, PRX5, TR2, and GR) and the unaltered group (NOX4). Although no such specific antioxidant therapy is approved for melanoma yet, inhibitors or mimetics of these redox-sensitive proteins have achieved very promising results. We foresee that forthcoming investigations on the modulation of these proteins will bring significant advances for cancer therapy.

Upregulation of the novel lncRNA U731166 is associated with migration, invasion and vemurafenib resistance in melanoma.

Our previous work using a melanoma progression model composed of melanocytic cells (melanocytes, primary and metastatic melanoma samples) demonstrated various deregulated genes, including a few known lncRNAs. Further analysis was conducted to discover novel lncRNAs associated with melanoma, and candidates were prioritized for their potential association with invasiveness or other metastasis-related processes. In this sense, we found the intergenic lncRNA U73166 (ENSG00000230454) and decided to explore its effects in melanoma. For that, we silenced the lncRNA U73166 expression using shRNAs in a melanoma cell line. Next, we experimentally investigated its functions and found that migration and invasion had significantly decreased in knockdown cells, indicating an essential association of lncRNA U73166 for cancer processes. Additionally, using naïve and vemurafenib-resistant cell lines and data from a patient before and after resistance, we found that vemurafenib-resistant samples had a higher expression of lncRNA U73166. Also, we retrieved data from the literature that indicates lncRNA U73166 may act as a mediator of RNA processing and cell invasion, probably inducing a more aggressive phenotype. Therefore, our results suggest a relevant role of lncRNA U73166 in metastasis development. We also pointed herein the lncRNA U73166 as a new possible biomarker or target to help overcome clinical vemurafenib resistance.

Quercetin increases mitochondrial proteins (VDAC and SDH) and downmodulates AXL and PIM-1 tyrosine kinase receptors in NRAS melanoma cells.

Melanoma is a type of skin cancer with low survival rates after it has metastasized. In order to find molecular differences that could represent targets of quercetin in anti-melanoma activity, we have chosen SKMEL-103 and SKMEL-28 melanoma cells and human melanocytes as models. Firstly, we observed that quercetin was able in reducing SKMEL-103 cell viability, but not in SKMEL-28. Besides that, quercetin treatment caused inhibition of AXL in both cell lines, but upregulation of PIM-1 in SKMEL-28 and downregulation in SKMEL-103. Moreover, HIF-1 alpha expression decreased in both cell lines. Interestingly, quercetin was more effective against SKMEL-103 than kinases inhibitors, such as Imatinib, Temsirolimus, U0126, and Erlotinib. Interestingly, we observed that while the levels of succinate dehydrogenase and voltage-dependent anion channel increased in SKMEL-103, both proteins were downregulated in SKMEL-28 after quercetin's treatment. Furthermore, AKT, AXL, PIM-1, ABL kinases were much more active and chaperones HSP90, HSP70 and GAPDH were highly expressed in SKMEL-103 cells in comparison with melanocytes. Our findings indicate, for the first time, that the efficacy of quercetin to kill melanoma cells depends on its ability in inhibiting tyrosine kinase and upregulating mitochondrial proteins, at least when SKMEL-103 and SKMEL-28 cells response were compared.

Kynurenine inhibits melanogenesis in human melanocyte-keratinocyte co-cultures and in a reconstructed 3D skin model.

Kynurenine (KYN), the most abundant metabolite of tryptophan, is classically associated with immune tolerance and tumor immune escape. In the last years, KYN is in the spotlight in other biological processes. Here, we showed that KYN inhibited tyrosinase expression and melanin content in primary human melanocyte and keratinocyte co-cultures. Furthermore, KYN decreased melanosome content in a 3D human skin reconstruction model. In these experiments, we used tyrosine + NH4 Cl to induce pigmentation. We compared the inhibitory effect of KYN on melanogenesis with the already known inhibitory effect promoted by IFN-γ. Since increased KYN production depends on the IFN-γ-inducible enzyme indoleamine-2,3-dioxygenase (IDO), we propose that part of the effect of IFN-γ on melanogenesis involves KYN production. From that, we tested if, during melanogenesis, changes in tryptophan metabolism would occur. For this purpose, we measured tryptophan, KYN and downstream products along with pigmentation. There were no significant changes in Trp metabolism, except for the high consumption of kynurenic acid. Our data identify the skin as a potential target for the action of KYN relevant for skin physiology and pigmentation. The results are discussed concerning the high production of KYN in skin inflammatory disorders and cancer.

Mesenchymal stem cells express epidermal markers in an in vitro reconstructed human skin model.

Introduction: In skin traumas, such as burns, epidermal homeostasis is affected, often requiring clinical approaches. Different therapeutic strategies can be used including transplantation, besides the use of synthetic or natural materials with allogeneic cells. In this context, tissue engineering is an essential tool for skin regeneration, and using mesenchymal stem cells (MSC) from the umbilical cord appears to be a promising strategy in regenerative medicine due to its renewal and differentiation potential and hypo immunogenicity. We evaluated the transdifferentiation of MSC from umbilical cord into keratinocytes in three-dimensional (3D) in vitro skin models, using dermal equivalents composed by type I collagen with dermal fibroblasts and a commercial porcine skin decellularized matrix, both cultured at air-liquid interface (ALI). Methods: The expression of epidermal proteins cytokeratins (CK) 5, 14 and 10, involucrin and filaggrin was investigated by real-time PCR and immunofluorescence, in addition to the activity of epidermal kallikreins (KLK) on the hydrolysis of fluorogenic substrates. Results and discussion: The cultivation of MSCs with differentiation medium on these dermal supports resulted in organotypic cultures characterized by the expression of the epidermal markers CK5, CK14, CK10 and involucrin, mainly on the 7th day of culture, and filaggrin at 10th day in ALI. Also, there was a 3-fold increase in the KLK activity in the epidermal equivalents composed by MSC induced to differentiate into keratinocytes compared to the control (MSC cultivated in the proliferation medium). Specifically, the use of collagen and fibroblasts resulted in a more organized MSC-based organotypic culture in comparison to the decellularized matrix. Despite the non-typical epithelium structure formed by MSC onto dermal equivalents, the expression of important epidermal markers in addition to the paracrine effects of these cells in skin may indicate its potential use to produce skin-based substitutes.

Physicochemical characterization and cosmetic applications of Passiflora nitida Kunth leaf extract

Abstract Passiflora nitida Kunth, an Amazonian Passiflora species, is little studied, although the specie's high biological potential. Herein the plant's pharmacognostic characterization, extract production, antioxidant potential evaluation, and application of this extract in cosmetic products is reported. The physical chemical parameters analyzed were particle size by sieve analysis, loss through drying, extractive yield, total ash content, laser granulometry, specific surface area and pore diameter (SBET), differential scanning calorimetry, thermogravimetry (TG), and wave dispersive X-Ray fluorescence (WDXRF). Total phenol/flavonoid content, LC-MS/MS analysis, DPPH and ABTS antioxidant radical assays, cytotoxicity, melanin, and tyrosinase inhibition in melanocytes test provided evidence to determine the content of the major constituent. P. nitida dry extract provided a fine powder with mesopores determined by SBET, with the TG curve showing five stages of mass loss. The antioxidant potential ranged between 23.5-31.5 mg∙mL-1 and tyrosinase inhibition between 400-654 µg∙mL-1. The species presented an antimelanogenic effect and an inhibitory activity of cellular tyrosinase (26.6%) at 25 µg/mL. The LC-MS/MS analysis of the spray-dried extract displayed the main and minor phenolic compounds constituting this sample. The results indicate that P. nitida extract has promising features for the development of cosmetic formulations

TOP1 modulation during melanoma progression and in adaptative resistance to BRAF and MEK inhibitors.

In melanomas, therapy resistance can arise due to a combination of genetic, epigenetic and phenotypic mechanisms. Due to its crucial role in DNA supercoil relaxation, TOP1 is often considered an essential chemotherapeutic target in cancer. However, how TOP1 expression and activity might differ in therapy sensitive versus resistant cell types is unknown. Here we show that TOP1 expression is increased in metastatic melanoma and correlates with an invasive gene expression signature. More specifically, TOP1 expression is highest in cells with the lowest expression of MITF, a key regulator of melanoma biology. Notably, TOP1 and DNA Single-Strand Break Repair genes are downregulated in BRAFi- and BRAFi/MEKi-resistant cells and TOP1 inhibition decreases invasion markers only in BRAFi/MEKi-resistant cells. Thus, we show three different phenotypes related to TOP1 levels: i) non-malignant cells with low TOP1 levels; ii) metastatic cells with high TOP1 levels and high invasiveness; and iii) BRAFi- and BRAFi/MEKi-resistant cells with low TOP1 levels and high invasiveness. Together, these results highlight the potential role of TOP1 in melanoma progression and resistance.

Skin Equivalent Models: Protocols for In Vitro Reconstruction for Dermal Toxicity Evaluation.

This chapter presents the protocols for developing of skin equivalents (SE) and reconstructed human epidermis (RHE) models for dermal toxicity evaluation as an alternative method to animal use in research. It provides a detailed protocol for the in vitro reconstruction of human skin from primary keratinocytes, melanocytes, and fibroblasts obtained from foreskin biopsies, including the procedures for reconstruction of a stratified epidermis on a polyester membrane. SE and RHE developed through these methods have been proven suitable not only for dermal toxicity studies, but also for investigating of pathological conditions in the skin, such as diabetes and invasion of melanoma.

Organotypic Models in Drug Development "Tumor Models and Cancer Systems Biology for the Investigation of Anticancer Drugs and Resistance Development".

The landscape of cancer treatment has improved over the past decades, aiming to reduce systemic toxicity and enhance compatibility with the quality of life of the patient. However, at the therapeutic level, metastatic cancer remains hugely challenging, based on the almost inevitable emergence of therapy resistance. A small subpopulation of cells able to survive drug treatment termed the minimal residual disease may either harbor resistance-associated mutations or be phenotypically resistant, allowing them to regrow and become the dominant population in the therapy-resistant tumor. Characterization of the profile of minimal residual disease represents the key to the identification of resistance drivers that underpin cancer evolution. Therapeutic regimens must, therefore, be dynamic and tailored to take into account the emergence of resistance as tumors evolve within a complex microenvironment in vivo. This requires the adoption of new technologies based on the culture of cancer cells in ways that more accurately reflect the intratumor microenvironment, and their analysis using omics and system-based technologies to enable a new era in the diagnostics, classification, and treatment of many cancer types by applying the concept "from the cell plate to the patient." In this chapter, we will present and discuss 3D model building and use, and provide comprehensive information on new genomic techniques that are increasing our understanding of drug action and the emergence of resistance.

Survivin modulation in the antimelanoma activity of prodiginines.

Melanoma is a type of skin cancer with an elevated incidence of metastasis and chemoresistance. Such features hamper treatment success of these neoplasms, demanding the search for new therapeutic options. Using a two-step resin-based approach, we recently demonstrated that cytotoxic prodiginines bind to the inhibitor of apoptosis protein, survivin. Herein, we explore the role of survivin in melanoma and whether its modulation is related to the antimelanoma properties of three cytotoxic prodiginines (prodigiosin, cyclononylprodigiosin, and nonylprodigiosin) isolated from marine bacteria. In melanoma patients and cell lines, survivin is overexpressed, and higher levels negatively impact survival. All three prodiginines caused a decrease in cell growth with reduced cytotoxicity after 24 h compared to 72 h treatment, suggesting that low concentrations promote cytostatic effects in SK-Mel-19 (BRAF mutant) and SK-Mel-28 (BRAF mutant), but not in SK-Mel-147 (NRAS mutant). An increase in G1 population was observed after 24 h treatment with prodigiosin and cyclononylprodigiosin in SK-Mel-19. Further studies indicate that prodigiosin induced apoptosis and DNA damage, as detected by increased caspase-3 cleavage and histone H2AX phosphorylation, further arguing for the downregulation of survivin. Computer simulations suggest that prodigiosin and cyclononylprodigiosin bind to the BIR domain of survivin. Moreover, knockdown of survivin increased long-term toxicity of prodigiosin, as observed by reduced clonogenic capacity, but did not alter short-term cytotoxicity. In summary, prodiginine treatment provoked cytostatic rather than cytotoxic effects, cell cycle arrest at G0/G1 phase, induction of apoptosis and DNA damage, downregulation of survivin, and decreased clonogenic capacity in survivin knockdown cells.

Targeting mitochondria in melanoma: Interplay between MAPK signaling pathway and mitochondrial dynamics.

Melanoma is a malignant proliferative disease originated in melanocytes, characterized by high metastatic activity and by the activation of oncogenes, such as B-RAF (40-60% of cases). Recent studies have shown that vemurafenib (a MAPK inhibitor) promoted disturbance of mitochondrial bioenergetics, although underlying mechanisms are not fully comprehended. Here we showed that MAPK inhibition by vemurafenib in B-RAFV600E-mutated human melanoma culminated in the inhibition of DRP1 phosphorylation, associated to a large mitochondrial network remodeling to the hyperfused phenotype, and increased oxidative phosphorylation capacity. Such alterations may be associated to melanoma resistance to vemurafenib, since the impairment of oxidative phosphorylation increased the vemurafenib cytotoxicity. These results point to the potential of mitochondrial dynamics as a targetable pathway in melanoma.

Indoleamine 2,3-dioxygenase in melanoma progression and BRAF inhibitor resistance.

Indoleamine 2,3-dioxygenase (IDO) is associated with the progression of many types of tumors, including melanoma. However, there is limited information about IDO modulation on tumor cell itself and the effect of BRAF inhibitor (BRAFi) treatment and resistance. Herein, IDO expression was analyzed in different stages of melanoma development and progression linked to BRAFi resistance. IDO expression was increased in primary and metastatic melanomas from patients' biopsies, especially in the immune cells infiltrate. Using a bioinformatics approach, we also identified an increase in the IDO mRNA in the vertical growth and metastatic phases of melanoma. Using in silico analyses, we found that IDO mRNA was increased in BRAFi resistance. In an in vitro model, IDO expression and activity induced by interferon-gamma (IFNγ) in sensitive melanoma cells was decreased by BRAFi treatment. However, cells that became resistant to BRAFi presented random IDO expression levels. Also, we identified that treatment with the IDO inhibitor, 1-methyltryptophan (1-MT), was able to reduce clonogenicity for parental and BRAFi-resistant cells. In conclusion, our results support the hypothesis that the decreased IDO expression in tumor cells is one of the many additional outcomes contributing to the therapeutic effects of BRAFi. Still, the IDO production changeability by the BRAFi-resistant cells reiterates the complexity of the response arising from resistance, making it not possible, at this stage, to associate IDO expression in tumor cells with resistance. On the other hand, the maintenance of 1-MT off-target effect endorses its use as an adjuvant treatment of melanoma that has become BRAFi-resistant.

Air Particulate Matter Induces Skin Barrier Dysfunction and Water Transport Alteration on a Reconstructed Human Epidermis Model.

Knowing the damage that particulate matter (PM) can cause in skin is important for tightly controlling the release of air pollutants and preventing more serious diseases. This study investigates if such alterations are present in reconstructed human epidermis exposed to coarse air PM. Exposure of reconstructed human epidermis to increasing concentrations (2.2, 8.9, and 17.9 µg/cm2) of standard urban PM over time led to decreased cell viability at 48 hours. The barrier function was shown to be compromised by 24 hours of exposure to high doses (17.9 µg/cm2). Morphological alterations included cytoplasm vacuolization and partial loss of epidermal stratification. Cytokeratin 10, involucrin, loricrin, and filaggrin protein levels were significantly decreased. We confirmed an inflammatory process by IL-1α release and found a significant increase in AQP3 expression. We also demonstrated changes in NOTCH1 and AhR expression of epidermis treated with coarse air PM. The use of hydrogen peroxide altered AQP3 and NOTCH1 expression, and the use of N-acetyl-L-cysteine altered NOTCH1 expression, suggesting that this is a redox-dependent process. These results demonstrate that coarse air PM induces dose-dependent inflammatory response and alterations in protein markers of differentiation and water transport in the epidermis that could ultimately compromise the structural integrity of the skin, promoting or exacerbating various skin diseases.

In vivo antitumoral effect of 4-nerolidylcatechol (4-NC) in NRAS-mutant human melanoma.

NRAS-mutations arise in 15-20% of all melanomas and are associated with aggressive disease and poor prognosis. Besides, the treatment for NRAS-mutant melanoma are not very efficient and is currently limited to immune checkpoints inhibitors or aggressive chemotherapy. 4-nerolidylcathecol (4-NC), a natural product extracted from Pothomorphe umbellata, induces apoptosis in melanoma cells by ROS production, DNA damage and increased p53 expression, in addition to inhibiting invasion in reconstructed skin. Moreover, 4-NC showed cytotoxicity in BRAF/MEKi-resistant and naive melanoma cells by Endoplasmic Reticulum (ER) stress induction in vitro. We evaluated the in vivo efficacy and the systemic toxicity of 4-NC in a NRAS-mutant melanoma model. 4-NC was able to significantly suppress tumor growth 4-fold compared to controls. Cleaved PARP and p53 expression were increased indicating cell death. As a proof of concept, MMP-2 and MMP-14 gene expression were decreased, demonstrating a possible role of 4-NC in melanoma invasion inhibition. Toxicological analysis indicated minor changes in the liver and bone marrow, but this toxicity was very mild when compared to other proteasome inhibitors and ER stress inductors already described. Our data indicate that 4-NC can counteract melanoma growth in vivo with minor adverse effects, suggesting further investigation as a potential NRAS-mutant melanoma treatment.