Effect of 460 nm blue light PBM on human MeWo melanoma cells.

Photobiomodulation (PBM) using 460 nm blue light has been shown to have an inhibitory effect on skin cancer cells. In this study, we used a continuous LED light source with a wavelength of 460 nm and designed various combinations of power density (ranging from 6.4 to 25.6 mW) and dose (ranging from 0.96 to 30.72 J/cm2) to conduct treatment experiments on MeWo cells to investigate the effects of blue light on MeWo melanoma cells. We are focusing on cell viability, cytotoxicity, mitochondrial function, oxidative stress, and apoptosis. We found that blue light inhibits these melanoma cells through oxidative stress and DNA damage, and this inhibition intensifies at higher irradiance levels. Although the cells initially attempt to resist the stress induced by the treatment, they eventually undergo apoptosis over time. These findings contribute to understanding melanoma's molecular response to blue light PBM, lay the groundwork for future clinical applications.

Ibrutinib Modulates Proliferation, Migration, Mitochondrial Homeostasis, and Apoptosis in Melanoma Cells.

Ibrutinib, a tyrosine kinase inhibitor with a broad spectrum of action, has been successfully explored to treat hematological and solid cancers. Herein, we investigated the anti-cancer effect of Ibrutinib on melanoma cell lines. Cytotoxicity was evaluated using the MTT assay. Apoptosis, mitochondrial membrane potential, reactive oxygen species (ROS) production, cell proliferation, and cell cycle stages were determined by flow cytometry. LDH release and Caspase 3/7 activity were determined by colorimetric and luminescent assays, respectively. Cell migration was evaluated by wound scratch assay. Gene expression was determined by real-time PCR. Gene Ontology (GO) enrichment analysis of melanoma clinical samples was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID). MTT assays showed that Ibrutinib is toxic for MeWo, SK-MEL-28, and WM164 cells. The annexin V/PI staining, Caspase 3/7 activity, and LDH release in MeWo cells revealed that apoptosis is the primary mechanism of death caused by Ibrutinib. Corroborating such observation, we identified that Ibrutinib treatment impairs the mitochondrial membrane potential of such cells and significantly increases the transcriptional levels of the pro-apoptotic factors ATM, HRK, BAX, BAK, CASP3, and CASP8. Furthermore, Ibrutinib showed antimetastatic potential by inhibiting the migration of MeWo cells. Finally, we performed a functional enrichment analysis and identified that the differential expression of Ibrutinib-target molecules is associated with enrichment of apoptosis and necrosis pathways in melanoma samples. Taken together, our results clearly suggest that Ibrutinib can be successfully explored as an effective therapeutic approach for melanomas.

Molecular and functional anticancer effects of GLP/G9a inhibition by UNC0646 in MeWo melanoma cells.

In recent years, histone methyltransferases (HMTs) have emerged as important therapeutic targets in cancer due to their oncogenic role. Herein, we used the GLP/G9a inhibitor UNC0646 to assess whether the inhibition of such HMTs could induce cell death in MeWo melanoma cells. Furthermore, we investigated the cellular and molecular mechanisms involved in the observed cell death events. Finally, we performed a functional genomics analysis of 480 melanoma samples to characterize G9a/GLP involvement in melanoma. Interestingly, after UNC0646 treatment, MeWo cells underwent apoptosis, followed by loss of mitochondrial membrane potential and the generation of reactive oxygen species (ROS). Furthermore, MeWo cells treated with UNC0646 showed cell cycle arrest and inhibition of proliferation. At the molecular level, UNC0646 treatment increased the transcriptional levels of CDK1 and BAX, and decreased BCL-2 mRNA levels. Finally, we performed a functional enrichment analysis, which demonstrated that dozens of biological pathways were enriched in melanoma samples according to GLP and G9a expression, including apoptosis and necrosis. Taken together, our data show that inhibition of GLP/G9a using UNC0646 exerts anticancer effects on melanoma cells by controlling their proliferation and inducing apoptosis.

Adrenomedullin Secreted by Melanoma Cells Promotes Melanoma Tumor Growth through Angiogenesis and Lymphangiogenesis.

INTRODUCTION: Metastatic melanoma is an aggressive tumor and can constitute a real therapeutic challenge despite the significant progress achieved with targeted therapies and immunotherapies, thus highlighting the need for the identification of new therapeutic targets. Adrenomedullin (AM) is a peptide with significant expression in multiple types of tumors and is multifunctional. AM impacts angiogenesis and tumor growth and binds to calcitonin receptor-like receptor/receptor activity-modifying protein 2 or 3 (CLR/RAMP2; CLR/RAMP3). METHODS: In vitro and in vivo studies were performed to determine the functional role of AM in melanoma growth and tumor-associated angiogenesis and lymphangiogenesis. RESULTS: In this study, AM and AM receptors were immunohistochemically localized in the tumoral compartment of melanoma tissue, suggesting that the AM system plays a role in melanoma growth. We used A375, SK-MEL-28, and MeWo cells, for which we demonstrate an expression of AM and its receptors; hypoxia induces the expression of AM in melanoma cells. The proliferation of A375 and SK-MEL-28 cells is decreased by anti-AM antibody (αAM) and anti-AMR antibodies (αAMR), supporting the fact that AM may function as a potent autocrine/paracrine growth factor for melanoma cells. Furthermore, migration and invasion of melanoma cells increased after treatment with AM and decreased after treatment with αAMR, thus indicating that melanoma cells are regulated by AM. Systemic administration of αAMR reduced neovascularization of in vivo Matrigel plugs containing melanoma cells, as demonstrated by reduced numbers of vessel structures, which suggests that AM is one of the melanoma cells-derived factors responsible for endothelial cell-like and pericyte recruitment in the construction of neovascularization. In vivo, αAMR therapy blocked angiogenesis and lymphangiogenesis and decreased proliferation in MeWo xenografts, thereby resulting in tumor regression. Histological examination of αAMR-treated tumors showed evidence of the disruption of tumor vascularity, with depletion of vascular endothelial cells and a significant decrease in lymphatic endothelial cells. CONCLUSIONS: The expression of AM by melanoma cells promotes tumor growth and neovascularization by supplying/amplifying signals for neoangiogenesis and lymphangiogenesis.

Cornelian Cherry (Cornus mas L.) Extracts Exert Cytotoxicity in Two Selected Melanoma Cell Lines-A Factorial Analysis of Time-Dependent Alterations in Values Obtained with SRB and MTT Assays.

Despite the fact that phytochemicals of Cornaceae species have long been discussed as possible auxiliary agents in contemporary treatment, the insights on their properties remain relatively scarce. This study focuses on Cornus mas L. (Cornelian cherry), the extracts of which are reported to exert a pleiotropic effect shown in both in vivo and in vitro studies. This study aimed to explore the cytotoxic effect of extracts from fruits of red (Cornus mas L. 'Podolski') and yellow (Cornus mas L. 'Yantarnyi' and 'Flava') Cornelian cherries on two melanoma cell lines (A375 and MeWo). The extracts were characterized in the context of the concentration of bioactive compounds of antioxidative properties. Cytotoxicity was investigated with the use of the following two assays: SRB and MTT. An additional, alternative protocol for the SRB assay was used in this study so as to account for possible bias. Cytotoxicity was assessed as a difference in the whole time series of cell viability, instead of analyzing differences in raw values (often found in the literature). Both extracts from Cornus mas L. induced cytotoxicity in both A375 and MeWo cell lines, although the response of these cells was different. Moreover, based on this study, there is no evidence for claiming a different magnitude of cytotoxicity between these two extracts.

Hybrid Theranostic Cubosomes for Efficient NIR-Induced Photodynamic Therapy.

In recent years, lipid bicontinuous cubic liquid-crystalline nanoparticles known as cubosomes have been under investigation because of their favorable properties as drug nanocarriers useful for anticancer treatments. Herein, we present organic/inorganic hybrid, theranostic cubosomes stabilized in water with a shell of alternate layers of chitosan, single strand DNA (model genetic material for potential gene therapy), and folic acid-chitosan conjugate (the outmost layer), coencapsulating up-converting Er3+ and Yb3+ codoped NaYF4 nanoparticles and daunorubicin. The latter acts as a chemotherapeutic drug of photosensitizing activity, while up-converting nanoparticles serve as energy harvester and diagnostic agent. Cellular uptake and NIR-induced photodynamic therapy were evaluated in vitro against human skin melanoma (MeWo) and ovarian (SKOV-3) cancer cells. Results evidenced the preferential uptake of the theranostic cubosomes in SKOV-3 cells in comparison to uptake in MeWo cells, and this effect was enhanced by the folic acid functionalization of the cubosomes surface. Nanocarriers coloaded with the hybrid fluorophores exhibited a superior NIR-induced photodynamic activity, also confirmed by the improved mitochondrial activity and the most affecting f-actin fibers of cytoskeleton. Similar results, but with higher photocytotoxicity, were detected when folic acid-functionalized cubosomes were incubated with SKOV-3 cells. Taken on the whole, these results prove these hybrid cubosomes are good candidates for the photodynamic treatment of tumor lesions.

Exoenzyme C3 transferase lowers actin cytoskeleton dynamics, genomic stability and survival of malignant melanoma cells under UV-light stress.

Actin cytoskeleton remodeling is the major motor of cytoskeleton dynamics driving tumor cell adhesion, migration and invasion. The typical RhoA, RhoB and RhoC GTPases are the main regulators of actin cytoskeleton dynamics. The C3 exoenzyme transferase from Clostridium botulinum is a toxin that causes the specific ADP-ribosylation of Rho-like proteins, leading to its inactivation. Here, we examine what effects the Rho GTPase inhibition and the consequent actin cytoskeleton instability would have on the emergence of DNA damage and on the recovery of genomic stability of malignant melanoma cells, as well as on their survival. Therefore, the MeWo cell line, here assumed as a melanoma cell line model for the expression of genes involved in the regulation of the actin cytoskeleton, was transiently transfected with the C3 toxin and subsequently exposed to UV-radiation. Phalloidin staining of the stress fibers revealed that actin cytoskeleton integrity was strongly disrupted by the C3 toxin in association with reduced melanoma cells survival, and further enhanced the deleterious effects of UV light. MeWo cells with actin cytoskeleton previously perturbed by the C3 toxin still showed higher levels and accumulation of UV-damaged DNA (strand breaks and cyclobutane pyrimidine dimers, CPDs). The interplay between reduced cell survival and impaired DNA repair upon actin cytoskeleton disruption can be explained by constitutive ERK1/2 activation and an inefficient phosphorylation of DDR proteins (γH2AX, CHK1 and p53) caused by C3 toxin treatment. Altogether, these results support the general idea that actin network help to protect the genome of human cells from damage caused by UV light through unknown molecular mechanisms that tie the cytoskeleton to processes of genomic stability maintenance.

Monitoring of tumor burden in vivo by optical imaging in a xenograft SCID mouse model: evaluation of two fluorescent proteins of the GFP-superfamily.

BACKGROUND: Mouse models of human-malignant-melanoma (MM) are important tools to study tumor dynamics. The enhanced green fluorescent protein (EGFP) is widely used in molecular imaging approaches, together with optical scanners, and fluorescence imaging. PURPOSE: Currently, there are no data available as to whether other fluorescent proteins are more suitable. The goal of this preclinical study was to analyze two fluorescent proteins of the GFP superfamily under real-time in vivo conditions using fluorescence reflectance imaging (FRI). MATERIAL AND METHODS: The human melanoma cell line MeWo was stable transfected with one plasmid: pEGFP-C1 or pDsRed1-N1. We investigated two severe combined immunodeficiency (SCID)-mice groups: A (solid xenografts) and B (xenografts as metastases). After three weeks, the animals were weekly imaged by FRI. Afterwards the mice were euthanized and metastases were imaged in situ: to quantify the cutis-dependent reduction of emitted light, we compared signal intensities obtained by metastases in vivo with signal intensities obtained by in situ liver parenchyma preparations. RESULTS: More than 90% of cells were stable transfected. EGFP-/DsRed-xenograft tumors had identical growth kinetics. In vivo the emitted light by DsRed tumors/metastases was much brighter than by EGFP. DsRed metastases were earlier (3 vs. 5 weeks) and much more sensitive detectable than EGFP metastases. Cutis-dependent reduction of emitted light was greater in EGFP than in DsRed mice (tenfold). Autofluorescence of DsRed was lower than of EGFP. CONCLUSION: We established an in vivo xenograft mouse model (DsRed-MeWo) that is reliable, reproducible, and superior to the EGFP model as a preclinical tool to study innovative therapies by FRI under real-time in vivo conditions.

Polymer-free cubosomes for simultaneous bioimaging and photodynamic action of photosensitizers in melanoma skin cancer cells.

We designed novel polymer-free cubic bicontinuous liquid crystalline dispersions (cubosomes) using monoolein as molecular building block, phospholipids as stabilizers, propylene glycol as hydrotrope. Their kinetic stability was evaluated by analysing the backscattering profiles upon ageing, and the most stable formulation was chosen as potential photosensitizers delivery vehicle for photodynamic therapy (PDT) of human skin melanoma cells. Morphological and topological features of such formulation alternatively loaded with Chlorin e6 or meso-Tetraphenylporphine-Mn(III) chloride photosensitizing dyes were investigated by cryo-TEM, DLS, and SAXS. Bioimaging studies demonstrated that Me45 and MeWo cell lines effectively internalized these cubosomes formulations. Particularly, photodynamic activity experiments proved both the very low cytotoxicity of the cubosomes formulation loaded with Chlorin e6 dye in the "dark" condition, and its significant cytotoxic effect after photoirradiation. The toxic effect recorded when the photosensitizer was encapsulated within the cubosomes was shown to be one order of magnitude higher than that caused by the free photosensitizer. This is the first report of biocompatible polymer-free cubosomes for potential application in both PDT and bioimaging of skin malignant melanoma.

Loss of DUSP3 activity radiosensitizes human tumor cell lines via attenuation of DNA repair pathways.

BACKGROUND: Radiotherapy causes the regression of many human tumors by increasing DNA damage, and the novel molecular mechanisms underlying the genomic instability leading to cancer progression and metastasis must be elucidated. Atypical dual-specificity phosphatase 3 (DUSP3) has been shown to down-regulate mitogen-activated protein kinases (MAPKs) to control the proliferation and apoptosis of human cancer cells. We have recently identified novel molecular targets of DUSP3 that function in DNA damage response and repair; however, whether DUSP3 affects these processes remains unknown. METHODS: Tumor cell lines in which DUSP3 activity was suppressed by pharmacological inhibitors or a targeted siRNA were exposed to gamma radiation, and proliferation, survival, DNA strand breaks and recombination repair pathways were sequentially analyzed. RESULTS: The combination of reduced DUSP3 activity and gamma irradiation resulted in decreased cellular proliferation and survival and increased cellular senescence compared with the effects of radiation exposure alone. Gamma radiation-induced DNA damage was increased by the loss of DUSP3 activity and correlated with increased levels of phospho-H2AX protein and numbers of ionizing radiation-induced γ-H2AX foci, which were reflected in diminished efficiencies of homologous recombination (HR) and non-homologous end-joining (NHEJ) repair. Similar results were obtained in ATM-deficient cells, in which reduced DUSP3 activity increased radiosensitivity, independent of increased MAPK phosphorylation. CONCLUSION: The loss of DUSP3 activity markedly increases gamma radiation-induced DNA strand breaks, suggesting a potential novel role for DUSP3 in DNA repair. GENERAL SIGNIFICANCE: The radioresistance of tumor cells is effectively reduced by a combination of approaches through the inhibition of DUSPs.

[Homogeneous and heterogeneous 3D melanoma models in vitro].

Growth of malignant tumors occurs in three-dimensional space and depends on a presence of stromal component, which performs critical functions of tumor cell protection and growth support. Therefore, development and analysis of tumor models in 3D cell cultures in vitro, including co-culture systems, presents a significant interest. In this study, the results of 3D culturing of two human melanoma cell lines using the hanging drop method, with or without human mesenchymal stem cells (MSCs), are presented. Melanoma lines were shown to behave differently in 3D cultures; in particular, Mel Cher melanoma cells have the ability to form uniform spheroids within 24 h, whereas MeWo cells under similar conditions failed to form spheroids even after 2 days of culture. However, co-culturing of melanoma cells with MSCs resulted in formation of compact 3D cell spheroids in both cases. Visualization of MeWo cells and MSCs in the mixed spheroids using fluorescent dyes revealed certain clustering of melanoma cells. The observed properties of melanoma cells in homogeneous and heterogeneous spheroids may be used in the complex analysis of results of testing of antimelanoma chemotherapy drugs and evaluation of their therapeutic properties.

Non-toxic melanin production inhibitors from Garcinia livingstonei (Clusiaceae).

ETHNOPHARMACOLOGICAL RELEVANCE: The stem bark of Garcinia livingstonei is used traditionally as a skin lightening agent. AIM OF THE STUDY: To isolate and identify compounds responsible for the observed skin lightening activity of Garcinia livingstonei and to evaluate their cytotoxicity. MATERIALS AND METHODS: Constituents of the stem bark and fruits of Garcinia livingstonei were isolated using chromatographic techniques and structures were determined using 1D and 2D NMR and MS analysis. MeWo cells were used to evaluate the cytotoxicity and impact on melanin levels of extracts and compounds isolated, in vitro. RESULTS: Twelve known compounds, morelloflavone (1), morelloflavone-7″-sulphate (2), guttiferone A (3), sargaol (4), isojacareubin (5), 6-deoxyisojacareubin (6) and in addition to the common triterpenoids, betulin, betulin aldehyde, lupeol, lupenone, euphol and stigmasterol were isolated in this investigation. Morelloflavone, morelloflavone-7″-sulphate and sargaol, were found to be considerably less cytotoxic and more effective as skin lightening agents than hydroquinone. CONCLUSIONS: A range of compounds was isolated from the stem bark and fruit of Garcinia livingstonei. Although the bark extract contained the cytotoxic guttiferone A, it was found to be less toxic than hydroquinone, and morelloflavone, the 7″-sulphate derivative and sargaol show potential for development as depigmentation/skin lightening agents.