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This study aimed to synthesize four new semisynthetic derivatives of natural oleanolic acid (OA) and, based on an analysis of their cytotoxic and anti-proliferative effects against human MeWo and A375 melanoma cell lines, select those with anti-cancer potential. We also screened the treatment time with the concentration of all four derivatives. We synthesized oxime 2 and performed its acylation with carboxylic acids into new derivatives 3a, 3b, 3c and 3d according to the methods previously described. Colorimetric MTT and SRB assays were used to measure the anti-proliferative and cytotoxic activity of OA and its derivatives 3a, 3b, 3c and 3d against melanoma cells. Selected concentrations of OA, the derivatives, and different time periods of incubation were used in the study. The data were analyzed statistically. The present results revealed the possible anti-proliferative and cytotoxic potential of two selected OA derivatives 3a and 3b, on A375 and MeWo melanoma cells, especially at concentrations of 50 μM and 100 μM at 48 h of incubation (p < 0.05). Further studies will be necessary to analyze the proapoptotic and anti-cancer activities of 3a and 3b against skin and other cancer cells. The bromoacetoxyimine derivative (3b) of OA morpholide turned out to be the most effective against the tested cancer cells.

Clinical outcomes of melanoma patients pointed out a gender disparity that supports a correlation between sex hormone activity on estrogen receptors (ER) and melanoma development and progression. Here, we found that the epithelial-to-mesenchymal transition (EMT) of melanoma cells induced by extracellular acidosis, which is a crucial hallmark of solid cancers, correlates with the expression of ERβ, the most representative ER on melanoma cells. Extracellular acidosis induces an enhanced expression of ERβ in female cells and EMT markers remain unchanged, while extracellular acidosis did not induce the expression of ERβ in male cells and EMT was strongly promoted. An inverse relationship between ERβ expression and EMT markers in melanoma cells of different sex exposed to extracellular acidosis was revealed by two different technical approaches: florescence-activated cell sorting of high ERβ expressing cell subpopulations and ERβ receptor silencing. Finally, we found that ERβ regulates EMT through NF-κB activation. These results demonstrate that extracellular acidosis drives a differential ERβ regulation in male and female melanoma cells and that this gender disparity might open new perspectives for personalized therapeutic approaches.

Background: Aegle marmelos commonly known as Bael is a herbal plant. Itis from a family called Rutaceae. It has many medicinal uses: anti-diarrheal, anti- microbial, anti-viral, anti-cancer, chemo-preventive, Ulcer healing and many others. Aim: To study the effect of Aegle marmelos hydroethanolic leaf extract on expression of antiapoptotic markers in human melanoma cells. Objective: The present study investigated the effect of Aegle marmelos hydroethanolic leaf extract on expression of antiapoptotic markers in human melanoma cells. Materials and Methods: DMSO and MTT chemicals were purchased from Sigma chemical Pvt Ltd. Trypsin EDTA, FBS, RPMI 1640 medium and PBS, Real time PCR kit was purchased from Canada. Human melanoma cell line (A375) was purchased from NCCS, Pune, India. Results: The data was analysed statistically by ANOVA and Duncan’s multiple range test with a computer based software (Graph Pad Prism version 5). The percentage of cell viability decreases with the increase in dosage of Aegle marmelos leaf extract. Conclusion: The study concluded that Aegle marmelos hydro ethanolic leaf extract a novel and innovative herbal drug has a significant effect on the expression of antiapoptotic markers in human melanoma cell lines.

Onconase (ONC) is a monomeric amphibian "pancreatic-type" RNase endowed with remarkable anticancer activity. ONC spontaneously forms traces of a dimer (ONC-D) in solution, while larger amounts can be formed when ONC is lyophilized from mildly acidic solutions. Here, we report the crystal structure of ONC-D and analyze its catalytic and antitumor activities in comparison to ONC. ONC-D forms via the three-dimensional swapping of the N-terminal α-helix between two monomers, but it displays a significantly different quaternary structure from that previously modeled [Fagagnini A et al., 2017, Biochem J 474, 3767-81], and based on the crystal structure of the RNase A N-terminal swapped dimer. ONC-D presents a variable quaternary assembly deriving from a variable open interface, while it retains a catalytic activity that is similar to that of ONC. Notably, ONC-D displays antitumor activity against two human melanoma cell lines, although it exerts a slightly lower cytostatic effect than the monomer. The inhibition of melanoma cell proliferation by ONC or ONC-D is associated with the reduction of the expression of the anti-apoptotic B cell lymphoma 2 (Bcl2), as well as of the total expression and phosphorylation of the Signal Transducer and Activator of Transcription (STAT)-3. Phosphorylation is inhibited in both STAT3 Tyr705 and Ser727 key-residues, as well as in its upstream tyrosine-kinase Src. Consequently, both ONC species should exert their anti-cancer action by inhibiting the pro-tumor pleiotropic STAT3 effects deriving either by its phospho-tyrosine activation or by its non-canonical signaling pathways. Both ONC species, indeed, increase the portion of A375 cells undergoing apoptotic cell death. This study expands the variety of RNase domain-swapped dimeric structures, underlining the unpredictability of the open interface arrangement upon domain swapping. Structural data also offer valuable insights to analyze the differences in the measured ONC or ONC-D biological activities.

Background: Malignant melanomas frequently metastasize to the brain, but metastases in the cerebellum are underrepresented compared with metastases in the cerebrum. Methods: We established animal models by injecting intracardially in athymic nude fox1nu mice two human melanoma cell lines, originating from a cerebral metastasis (HM19) and a cerebellar metastasis (HM86). Results: Using magnetic resonance imaging (MRI), metastases were first detected after a mean of 34.5 days. Mean survival time was 59.6 days for the mice in the HM86 group and significantly shorter (43.7 days) for HM19-injected animals (p < 0.001). In the HM86 group, the first detectable metastasis was located in the cerebellum in 15/55 (29%) mice compared with none in the HM19 group (p < 0.001). At sacrifice, cerebellar metastases were found in 34/55 (63%) HM86- injected mice compared with 1/53 (2%) in the HM19-injected (p < 0.001) mice. At that time, all mice in both groups had detectable metastases in the cerebrum. Comparing macroscopic and histologic appearances of the brain metastases with their clinical counterparts, the cell line-based tumors had kept their original morphologic characteristics. Conclusions: The present work demonstrates that human brain-metastatic melanoma cells injected intracardially in mice had retained inherent characteristics also in reproducing interaction with subtle microenvironmental brain tissue compartment-specific features. The models offer new possibilities for investigating tumor- and host-associated factors involved in determining tissue specificity of brain metastasis.

The aim of this study was to evaluate the Porophyllum ruderale effects on the liver in murine melanoma model B16-F10 and to determine the antioxidant potential. The plant’s aerial parts of the were collected in Campo Grande, MS, Brazil, dried, extracted with ethanol, ultrasonic bath and static maceration. The hydroalcoholic formulation at 100 and 200 mg/kg were submitted to antioxidant activity (DPPH) and hepatoprotective effect. Thirty-eight black mice with a mean weight of 30 g were distributed in 4 experimental groups: negative control, positive control (doxorubicin), P. ruderale at 100 mg/kg and P. ruderale at 200 mg/kg. All the animals received subcutaneous injection of 5x105 B16F10 cells on the 1st day of the experiment and were treated until the 14th day. Although a significant antioxidant activity was verified for the two concentrations analyzed, they were not sufficient to minimize the melanoma effects toxic in the mice liver as observed by histological changes and that leads us to conclude that the hydroalcoholic extract of aerial parts of P. ruderale, in the concentrations used, possesses antioxidant properties, good performance in inhibiting the proliferation of human melanoma cells, but it has no hepatoprotective effect in the melanoma cutaneous treatment. Keywords: Cutaneous Melanoma. Asteraceae. Hepatoprotective Effect. Phenolic Compounds. DPPH. Resumo O objetivo deste estudo foi avaliar os efeitos de Porophyllum ruderale no fígado em melanoma murino modelo B16-F10 e determinar o potencial antioxidante. As folhas da planta foram coletadas em Campo Grande, MS, Brasil, secas, extraídas com etanol, banho ultrassônico e maceração estática. A formulação hidroalcoólica de 100 e 200 mg/kg foi submetida à atividade antioxidante (DPPH) e ao efeito hepatoprotetor. Trinta e oito camundongos pretos com peso médio de 30 g foram distribuídos em 4 grupos experimentais: controle negativo, controle positivo (doxorrubicina), P. ruderale na concentração de 100 mg/kg e P. ruderale na concentração de 200 mg/kg. Todos os animais receberam injeção subcutânea de 5x105 células B16F10 no 1º dia de experimento até o 14º. Embora tenha sido verificada atividade antioxidante significativa para as duas concentrações analisadas, elas não foram suficientes para minimizar os efeitos tóxicos do melanoma no fígado dos camundongos, como observado nas alterações histológicas, o que nos leva a concluir que o extrato hidroalcoólico das folhas de P. ruderale, nas concentrações utilizadas, possui propriedades antioxidantes, bom desempenho na inibição da proliferação de células de melanoma humano, mas não apresenta efeito hepatoprotetor no tratamento do melanoma cutâneo. Palavras-chave: Melanoma Cutâneo. Asteraceae. Efeito Hepatoprotetor. Compostos Fenólicos. DPPH.

Brazil is a country that presents a vast biodiversity, where we find the Caatinga region, represented by plants of the Cordiaceae family, which are employed by local communities as an alternative for the treatment of some diseases, as is the case with the species Varronia dardani and V. leucocephala, whose infusions are used for the relief of gastric pain, menstrual colic, and arthritis. Therefore, the aim of this work was to contribute to the knowledge about these species, by studying their phytochemical and biological properties. To this end, the plant material was collected in the locations of Serra Branca-PB and Maturéia-PB and submitted to extraction processes. The crude ethanolic extract of V. dardani (CEE-VD) was submitted to the determination of total phenolics and to a quantification of rosmarinic acid, considered one of the chemotaxonomic markers of the Cordiaceae family. A screening was performed with CEE-VD to evaluate its spasmolytic activity in various organs. In addition to this biological activity, the antiproliferative activity of the extracts and fractions of all species was evaluated against human melanoma cells (A2058). Using NMR techniques, the compounds isolated from the study species were identified. The screening concerning the activity in smooth muscle, showed that CEE-VD showed a non-selective activity in the different tissues and for the different agonists tested, showing highest potency in the uterus of rats. From the ethyl acetate fraction of the CEE-VD it was possible the isolation and identification of five flavonoids, being three aglycones (isosakuranetin, naringenin, and rhamnocitrin) and two glycosylated (astragalin and isoquercitrin), besides rosmarinic acid. Regarding the cytotoxic test, the isopropanolic fraction of the extract of roots from V. dardani proved to be more active, and from this fraction the compound vardanone was isolated, the first hydroanthraquinone reported in V. dardani species. This compound, was used for a chemoinformatics study, where its set of isomers was analyzed for activity in the A2058 cell line and some interactions between amino acid residues were observed, compared to the positive controls dacarbazine and vemurafenib, drugs used in current melanoma therapy. The second most active species in the cytotoxic tests was V. leucocephala, from which it was possible to isolate a saikosaponin, already isolated previously in the species. Thus, the chemical and biological results, obtained in this work, contributed to the knowledge about the genus Varronia, furthermore, the study revealed new possibilities for research on these species involving the search for new secondary metabolites, mainly of the class of quinones. RÉSUMÉ. Le Brésil est un pays qui présente une vaste biodiversité, en particulier dans la région de la Caatinga, dans le Nordeste. Dans ce biome poussent des plantes de la famille des Cordiaceae, qui sont utilisées par les communautés locales pour le traitement de certaines maladies. C’est notamment le cas des espèces Varronia dardani et V. leucocephala, dont les infusions sont utilisées pour soulager les douleurs gastriques, les coliques menstruelles et l'arthrite. L'objectif de ce travail était de contribuer à la connaissance de ces espèces, en étudiant leurs propriétés phytochimiques et biologiques. Le matériel végétal a été collecté à Serra Branca-PB et Maturéia-PB et soumis à des processus d'extraction. L'extrait éthanolique brut de V. dardani (EEBVD) a été soumis à la détermination des composés phénoliques totaux et à la quantification de l'acide rosmarinique, considéré comme l'un des marqueurs chimiotaxonomiques de la famille des Cordiaceae. Un criblage a été effectué avec le EEB-VD pour évaluer son activité spasmolytique dans différents organes. L'activité antiproliférative des extraits et fractions de ces espèces a également été évaluée contre des cellules de mélanome humain (A2058). En utilisant des techniques de RMN, les composés isolés des espèces étudiées ont été identifiés. Le screening concernant l'activité dans le muscle lisse, a montré que l’extrait EEB-VD présente une activité spasmolytique non sélective dans différents tissus et pour différents agonistes testés, avec une puissance maximale sur l’utérus des rats. À partir de la fraction acétate d'éthyle du EEB-VD, il a été possible d'isoler et d'identifier cinq flavonoïdes, dont trois aglycones (isosakuranetine, naringenine et rhamnocitrine) et deux glycosylés (astragaline et isoquercitrine), en plus de l'acide rosmarinique. En ce qui concerne le test d’activité antiproliféartif sur cellules de mélanomes, la fraction isopropanolique de l'extrait de racines de V. dardani s'est révélée la plus active, et c'est à partir de cette fraction qu'a été isolé une nouvelle molécule, la vardanone, la première hydroanthraquinone rapportée dans l'espèce V. dardani. Ce composé a été évalué dans une étude chimio-informatique, dans laquelle l'activité de son groupe d'isomères sur la lignée cellulaire A2058 a été analysée, par rapport aux témoins positifs que sont la dacarbazine et le vemurafenib, des médicaments utilisés dans le schéma thérapeutique actuel du mélanome. La deuxième espèce la plus active dans les tests d’inhibition de prolifération était V. leucocephala, à partir de laquelle il a été possible d'isoler une saikosaponine, déjà isolée précédemment dans l'espèce. Ainsi, les résultats chimiques et biologiques, obtenus dans ce travail, ont contribué à la connaissance du genre Varronia, de plus, l'étude a révélé de nouvelles possibilités de recherche sur ces espèces grâce à la découverte de nouveaux métabolites secondaires, principalement de la classe des quinones. Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES O Brasil é um país que possui uma vasta biodiversidade, onde encontramos a região da Caatinga, representada por vegetais da família Cordiaceae, os quais são empregados pelas comunidades locais como uma alternativa para o tratamento de algumas doenças, como é o caso das espécies Varronia dardani e V. leucocephala, cujas infusões são utilizadas para o alívio das dores gástricas, cólica menstrual e artrite. Sendo assim, buscou-se contribuir para o conhecimento sobre essas espécies, mediante o estudo fitoquímico e biológico das mesmas. Para isso, o material vegetal foi coletado nos municípios de Serra Branca-PB e Maturéia-PB e submetidos a processos de extração. O extrato etanólico bruto de V. dardani (EEB-VD) foi submetido à determinação de fenólicos totais e à uma quantificação do ácido rosmarínico, considerado um dos marcadores quimiotaxonômicos da família Cordiaceae. Foi realizada uma triagem com o EEB-VD para avaliar a sua atividade espasmolítica em diversos órgãos. Além dessa atividade biológica, foi avaliada a atividade antiproliferativa dos extratos e frações de todas as espécies frente às células de melanoma humano (A2058). Através de técnicas de RMN foram identificadas as substâncias isoladas das espécies do estudo. A triagem relativa à atividade no músculo liso, mostrou que o EEB-VD apresentou uma atividade não seletiva nos diferentes tecidos e para os diferentes agonistas testados, apresentando maior potência no útero de ratos. Da fração acetato de etila do EEB-VD foi possível o isolamento e identificação de cinco flavonoides, sendo três agliconas (isosakuranetina, naringenina e ramnocitrina) e dois glicosilados (astragalina e isoquercitrina), além do ácido rosmarínico. Com relação ao teste citotóxico, a fração isopropanólica do extrato das raízes de V. dardani se mostrou mais ativa, e desta fração foi isolada a vardanona, primeira hidroantraquinona relatada na espécie V. dardani. O composto VD-R-I, foi utilizado para um estudo de quimioinformática, onde o conjunto de isômeros dessa substância foi analisado quanto à atividade na linhagem celular A2058 e foram observadas algumas interações entre resíduos de aminoácidos, comparando com o controle positivo dacarbazina e vemurafenibe, drogas utilizadas na terapia atual do melanoma. A segunda espécie mais ativa nos testes citotóxicos foi a V. leucocephala, de onde foi possível isolar uma saikosaponina, já isolada ateriormente na espécie. Logo, os resultados químicos e biológicos, obtidos nesse trabalho, contribuíram para o conhecimento sobre o gênero Varronia, ainda assim, o estudo revelou novas possibilidades de pesquisas nessas espécies envolvendo a busca de novos metabólitos secundários, principalmente da classe das quinonas.

Cancer cells migrating in confined microenvironments exhibit plasticity of migration modes. Confinement of contractile cells in a nonadhesive environment drives “leader bleb–based migration” (LBBM), morphologically characterized by a long bleb that points in the direction of movement separated from a cell body by a contractile neck. Although cells undergoing LBBM have been visualized within tumors, the organization of organelles and actin regulatory proteins mediating LBBM is unknown. We analyzed the localization of fluorescent organelle-specific markers and actin-associated proteins in human melanoma and osteosarcoma cells undergoing LBBM. We found that organelles from the endolysosomal, secretory, and metabolic systems as well as the vimentin and microtubule cytoskeletons localized primarily in the cell body, with some endoplasmic reticulum, microtubules, and mitochondria extending into the leader bleb. Overexpression of fluorescently tagged actin regulatory proteins showed that actin assembly factors localized toward the leader bleb tip, contractility regulators and cross-linkers in the cell body cortex and neck, and cross-linkers additionally throughout the leader bleb. Quantitative analysis showed that excess filamin-A and fascin-1 increased migration speed and persistence, while their depletion by small interfering RNA indicates a requirement in promoting cortical tension and pressure to drive LBBM. This indicates a critical role of specific actin crosslinkers in LBBM.

In this study, the compound 4-(2-chlorobenzyl)-1-(5-fluoro-2-hydroxy-3-((4-methylpiperidin -1-yl)methyl)phenyl)-[1,2,4]triazolo[4,3-a]quinazolin-5(4H)-one (1) was synthesized for the first time, and its structure was characterized by 1H NMR, 13C NMR, MS and FT-IR spectra. Single crystals of compound 1 were grown in acetonitrile and the structure was confirmed by X-ray diffraction. The DFT-optimized structure was consistent with that determined by X-ray diffraction. Geometrical parameter, molecular electrostatic potential and frontier molecular orbital analyses were performed using the DFT/B3LYP/6-311G (2d, p) method. The vibrational assignment was based on the characteristic vibrational absorption band of compound 1. The theoretical and experimental 13C NMR chemical shifts exhibited good correlation. Molecular docking suggests favorable interactions between compound 1 and SHP2 protein. The SHP2 enzyme inhibition rate of compound 1 was 12.40% at 10 μM. The antitumor activity of 1 was better than that of the reference compound in human hepatoma cells SMMC7721 (IC50 = 757 µM) and human melanoma cells A375 (IC50 = 70.19 µM).

Acid ceramidase (AC) is a lysosomal hydrolase encoded by the ASAH1 gene, which cleaves ceramides into sphingosine and fatty acid. AC is expressed at high levels in most human melanoma cell lines and may confer resistance against chemotherapeutic agents. One such agent, doxorubicin, was shown to increase ceramide levels in melanoma cells. Ceramides contribute to the regulation of autophagy and apoptosis. Here we investigated the impact of AC ablation via CRISPR-Cas9 gene editing on the response of A375 melanoma cells to doxorubicin. We found that doxorubicin activates the autophagic response in wild-type A375 cells, which effectively resist apoptotic cell death. In striking contrast, doxorubicin fails to stimulate autophagy in A375 AC-null cells, which rapidly undergo apoptosis when exposed to the drug. The present work highlights changes that affect melanoma cells during incubation with doxorubicin, in A375 melanoma cells lacking AC. We found that the remarkable reduction in recovery rate after doxorubicin treatment is strictly associated with the impairment of autophagy, that forces the AC-inhibited cells into apoptotic path.

A recent study by Oliveira et al. provides, in unprecedented detail, novel insights into the relationship between tumorreactivity and functional states of CD8 T cells in metastatic melanoma.

Cold atmospheric plasma (CAP) is a novel technology which is widely used in the biomedical field and has developed quickly over the past few years, especially in cancer therapy. Compared to traditional anti-cancer approaches, CAP treatment shows a selective anti-cancer mode which makes CAP a promising anti-cancer treatment modality. However, the underlying mechanism of this selective cytotoxicity is not fully elaborated. In this study, we demonstrated that A375 human melanoma cells were more sensitive to CAP treatment than HaCaT cells and compared the differences in metabolites of these two cell lines after CAP treatment. Using ultra-high performance liquid tandem chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS) approach, we identified 361 changes (metabolites) in HaCaT cells and 1531 changes in A375 cells. Further research using KEGG pathway analysis found that purine metabolism and Pantothenate and CoA biosynthesis were highly correlated with changes of HaCaT cells and A375 cells after CAP treatment. Our results shed light on the mechanism of selective cytotoxicity in the metabolites' aspect.

Objective: This work aimed to establish and formulate the gossypinproniosomal gel drug delivery system. Methods: Gossypin-loaded proniosomal gels (GPG) was prepared using specific non-ionic surfactants (Spans), followed by particle size (PS), entrapment efficiency (percent EE), in vitro, ex-vivo drug release, and in vitro efficacy of Gossypin against human melanoma cells (A-375). Results: The results showed that the percentage EE for the GPG is appropriate (81.3 %–95.5 %) and they are Nano-sized (189.3–912.0 nm) and the gels diffusion provided the desired sustaining effect for GPG-F7 formulation (75.5 percent). The GPG reported cell viability of 14.9±2.3 percent compared with 16.1±1.1 percent for free Gossypin at the maximum dose of 100 μg/ml for A-375 human melanoma cells after 24 hr incubation time. No major changes were seen in the percentage EE, PS of GPG after storage for 90d, in the physical stability report. Conclusion: The results obtained suggest that the proniosomal drug delivery system can enhance the flux to the skin and achieve the ideal Gossypin sustainability effect. Consequently, the use of proniosomal gel may be advantageous with regard to the topical delivery of Gossypin for melanoma treatment management.

We present the novel ReactomeGSA resource for comparative pathway analyses of multi-omics datasets. ReactomeGSA is accessible through Reactome's web interface and the novel ReactomeGSA R Bioconductor package with explicit support for scRNA-seq data. We showcase ReactomeGSA's functionality by characterizing the role of B cells in anti-tumour immunity. Combining multi-omics data of five TCGA studies reveals marked opposing effects of B cells in different cancers. This showcases how ReactomeGSA can quickly derive novel biomedical insights by integrating large multi-omics datasets., Graphical Abstract Highlights ReactomeGSA is a novel tool for multi-species, multi-omics pathway analysis. Its quantitative pathway analysis methods offer high statistical power. Combining data of five TCGA studies shows B cells have opposing effects in cancers. ReactomeGSA reveals differences in key pathways between transcript- and protein-level., Pathway analyses are key methods to analyze 'omics experiments. Nevertheless, integrating data from different 'omics technologies and different species still requires considerable bioinformatics knowledge. Here we present the novel ReactomeGSA resource for comparative pathway analyses of multi-omics datasets. ReactomeGSA can be used through Reactome's existing web interface and the novel ReactomeGSA R Bioconductor package with explicit support for scRNA-seq data. Data from different species is automatically mapped to a common pathway space. Public data from ExpressionAtlas and Single Cell ExpressionAtlas can be directly integrated in the analysis. ReactomeGSA greatly reduces the technical barrier for multi-omics, cross-species, comparative pathway analyses. We used ReactomeGSA to characterize the role of B cells in anti-tumor immunity. We compared B cell rich and poor human cancer samples from five of the Cancer Genome Atlas (TCGA) transcriptomics and two of the Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteomics studies. B cell-rich lung adenocarcinoma samples lacked the otherwise present activation through NFkappaB. This may be linked to the presence of a specific subset of tumor associated IgG+ plasma cells that lack NFkappaB activation in scRNA-seq data from human melanoma. This showcases how ReactomeGSA can derive novel biomedical insights by integrating large multi-omics datasets.

Introduction.The molecular basis of radio- and photodynamic therapy (PDT), the mechanism of action of a number of antitumor chemotherapy drugs is oxidative stress (OS). The enzyme hemoxygenase-I (НO-1), a molecular marker of OS, is a key participant in the system of protection and adaptation of tumor cells under stress.Objective.To find out whether the sensitivity of human melanoma tumor cells to OS depends on the basal and modulator-induced levels of НO-1 expressionMaterial and methods.Human melanoma cell lines were used in the study. The expression of mRNA НO-1 in cells was studied by real-time RT-PCR, the reactive oxygen species content in cells – by flow cytometry and the cytotoxicity of drugs – by MTT assay.Results.According to our data, human melanoma cells have different basal levels of HO-1 transcription: high (3.0–3.5 o. u.) in lines MelIL, MelP, medium (1.5 o. u.) in lines MeWo, MelZ, MelIbr and low (0.5 o. e.) – MelMe, A375).There is no direct correlation between the level of basal cell expression of HO-1 and their sensitivity to the OS inducer – Н2О2. The hemin-induced increase in HO-1 expression in cells is accompanied by doubled resistance to Н2О2. It was found that HO-1 repression in the presence of apigenin was registered in melanoma cells with different basal levels, but sensitization to Н2О2 (2–4 times) was observed only for cells with medium (MeWo) and low (A375) levels of basal HO-1 expression. It was found that the decrease in basal expression of HO-1 induced by apigenin is accompanied by an increase in the reactive oxygen species content in cells.Conclusions.The results of our research allow us to recommend natural flavon apigenin, a modulator of HO-1 expression, for inclusion in the chemotherapy and PDT regimens to increase the effectiveness of human melanoma treatment.

A new Sr(II)-based metal organic framework (MOF) formulated as [Sr(HTATB)(H2O)2](DMF)4 (1) has been prepared by reaction of Sr(NO3)2 with the rigid tricarboxylic acid linker 4,4′,4″-s-triazine-2, 4...

Human malignant melanoma exhibits imbalances in redox status, leading to activation of many redox-sensitive signaling pathways. APE/Ref-1 is a multifunctional protein that serves as a redox chaperone that regulates many nuclear transcription factors and is an important mechanism in cancer cell survival of oxidative stress. Previous studies showed that APE/Ref-1 is a potential druggable target for melanoma therapy. In this study, we synthesized a novel APE/Ref-1 inhibitor, bis-cinnamoyl-1,12-dodecamethylenediamine (2). In a xenograft mouse model, compound 2 treatment (5 mg/kg) significantly inhibited tumor growth compared to the control group, with no significant systemic toxicity observed. We further synthesized compound 2 analogs to determine the structure-activity relationship based on their anti-melanoma activities. Among those, 4-hydroxyphenyl derivative (11) exhibited potent anti-melanoma activities and improved water solubility compared to its parental compound 2. The IC50 of compound 11 was found to be less than 0.1 μM. Compared to other known APE/Ref-1 inhibitors, compound 11 exhibited increased potency in inhibiting melanoma proliferation. As determined by luciferase reporter analyses, compound 2 was shown to effectively inhibit H2O2-activated AP-1 transcription activities. Targeting APE/Ref-1-mediated signaling using pharmaceutical inhibitors is a novel and effective strategy for melanoma treatment with potentially high impact.

Given the known pro-oxidant status of tumour cells, the development of anti-proliferative strategies focuses on products with both anti- and pro-oxidant properties that can enhance antitumour drug cytotoxicity. We used a C. zeylanicum essential oil (CINN-EO) and assessed its effect on a human metastatic melanoma cell line (M14). Human PBMCs and MDMs from healthy donors were used as normal control cells. CINN-EO induced cell growth inhibition, cell cycle perturbation, ROS and Fe(II) increases, and mitochondrial membrane depolarization. To assess whether CINN-EO could affect the stress response, we analysed iron metabolism and stress response gene expression. CINN-EO increased HMOX1, FTH1, SLC7A11, DGKK, and GSR expression but repressed OXR1, SOD3, Tf, and TfR1 expression. HMOX1, Fe(II), and ROS increases are associated with ferroptosis, which can be reversed by SnPPIX, an HMOX1 inhibitor. Indeed, our data demonstrated that SnPPIX significantly attenuated the inhibition of cell proliferation, suggesting that the inhibition of cell proliferation induced by CINN-EO could be related to ferroptosis. Concurrent treatment with CINN-EO enhanced the anti-melanoma effect of two conventional antineoplastic drugs: the mitochondria-targeting tamoxifen and the anti-BRAF dabrafenib. We demonstrate that CINN-EO-mediated induction of an incomplete stress response specifically in cancer cells affects the proliferation of melanoma cells and can enhance drug cytotoxicity.

We developed a human melanoma model using the HT168-M1 cell line to induce IFN-α2 resistance in vitro (HT168-M1res), which was proven to be maintained in vivo in SCID mice. Comparing the mRNA profile of in vitro cultured HT168-M1res cells to its sensitive counterpart, we found 79 differentially expressed genes (DEGs). We found that only a 13-gene core of the DEGs was stable in vitro and only a 4-gene core was stable in vivo. Using an in silico cohort of IFN-treated melanoma tissues, we validated a differentially expressed 9-gene core of the DEGs. Furthermore, using an in silico cohort of immune checkpoint inhibitor (ICI)-treated melanoma tissues, we tested the predictive power of the DEGs for the response rate. Analysis of the top four upregulated and top four downregulated genes of the DEGs identified WFDC1, EFNA3, DDX10, and PTBP1 as predictive genes, and analysis of the “stable” genes of DEGs for predictive potential of ICI response revealed another 13 genes, out of which CDCA4, SOX4, DEK, and HSPA1B were identified as IFN-regulated genes. Interestingly, the IFN treatment associated genes and the ICI-therapy predictive genes overlapped by three genes: WFDC1, BCAN, and MT2A, suggesting a connection between the two biological processes.

In vitro tumour spheroid experiments have been used to study avascular tumour growth and drug design for the last 50 years. Unlike simpler two-dimensional cell cultures, tumour spheroids exhibit heterogeneity within the growing population of cells that is thought to be related to spatial and temporal differences in nutrient availability. The recent development of real-time fluorescent cell cycle imaging allows us to identify the position and cell cycle status of individual cells within the growing population, giving rise to the notion of a four-dimensional (4D) tumour spheroid. In this work we develop the first stochastic individual-based model (IBM) of a 4D tumour spheroid and show that IBM simulation data qualitatively and quantitatively compare very well with experimental data from a suite of 4D tumour spheroid experiments performed with a primary human melanoma cell line. The IBM provides quantitative information about nutrient availability within the spheroid, which is important because it is very difficult to measure these data in standard tumour spheroid experiments. Software required to implement the IBM is available on GitHub, https://github.com/ProfMJSimpson/4DFUCCI.

Photodynamic therapy (PDT) is a treatment which uses light-activated compounds to produce reactive oxygen species, leading to membrane damage and cell death. Multicellular cancer spheroids are a preferable alternative for PDT evaluation in comparison to monolayer cell cultures due to their ability to better mimic in vivo avascular tumour characteristics such as hypoxia and cell-cell interactions, low cost, and ease of production. However, inconsistent growth kinetics and drug responsiveness causes poor experimental reproducibility and limits their usefulness. Herein, we used image analysis to establish a link between human melanoma C8161 spheroid morphology and drug responsiveness. Spheroids were pre-selected based on sphericity, area, and diameter, reducing variation in experimental groups before treatment. Spheroid morphology after PDT was analyzed using AnaSP and ReViSP, MATLAB-based open-source software, obtaining nine different parameters. Spheroids displayed a linear response between biological assays and morphology, with area (R2 = 0.7219) and volume (R2 = 0.6138) showing the best fit. Sphericity, convexity, and solidity were confirmed as poor standalone indicators of spheroid viability. Our results indicate spheroid morphometric parameters can be used to accurately screen inefficient treatment combinations of novel compounds.

Despite recent therapeutic progress, advanced melanoma remains lethal for many patients. The composition of the immune tumor microenvironment (TME) has decisive impacts on therapy response and disease outcome. High dimensional analyses of patient samples can reveal the composition and heterogeneity of the immune TME. In particular, macrophages are known for their cancer-supportive role, but the underlying mechanisms are incompletely understood, and experimental in vivo systems are needed to test the functional properties of these cells. We characterized a humanized mouse model, reconstituted with a human immune system and a human melanoma, in which: (1) human macrophages support metastatic spread of the tumor; and (2) tumor-infiltrating macrophages have a specific transcriptional signature that faithfully represents the transcriptome of macrophages from patient melanoma samples and is associated with shorter survival. This model complements patient sample analyses, enabling the elucidation of fundamental principles in melanoma biology, and the development and evaluation of candidate therapies.

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazoliumbromide (MTT) assay is a widely used assessment method for the determination of anticancerogenic effects of active compounds including plant secondary metabolites. Recently, some important plant active ingredients have been widely investigated for anticancerogenic properties on melanoma cancer lines which are the most lethal type of skin cancer. Although some methods including DNA assay, 3H-thymidine incorporation and flow cytometry have been recommended for counting cells in the culture, MTT is one of the most frequent method and therefore, MTT assay needs to be optimized for melanoma cell lines. In this study, the MTT analytical procedure for determination of cell viability of human melanoma cell line (SK-Mel-30) was divided into nine steps and various parameters in each step (reagent amount, incubation time, centrifugation, solvent type, waiting time before spectrophotometric analysis and spectrophotometric parameters) were optimized. Optimum amount of MTT reagent and incubation time after MTT addition were determined as 10 µL and 4 h for 96 well plate, respectively. Various solvents were evaluated for solubility effectiveness of the formed crystals and DMSO was found to be the best solvent to dissolve the crystals. Waiting time before spectrophotometric reading and Uv-vis spectrums were also evaluated. At the end of the study a flowchart, presented the best analytical conditions, was constructed. Obtained findings can be used for the determination of anticancerogenic properties of plant ingredients.

Seven previously undescribed withanolides, namely physaminilide A–G (1–7), and two artificial withanolides (8–9), along with 10 known analogues (10–19) were isolated from Physalis minima. The structures were established by spectroscopic analysis, including NMR and electronic circular dichroism (ECD) data. Cytotoxicity of all the isolates was evaluated against A375 human melanoma cells. Compounds 2, 5, 8, 10, 11 and 15 exhibited significant cytotoxic activities with IC50 values in the range of 1.2–9.4 μM.

—Molecular heterogeneity in seven human metastatic melanoma cells lines is revealed using a double immunofluorescent assay by flow cytometry. It is evaluated that quite a small population of cells (up to 10%) expressed only vimentin, the main marker of melanoma. Only epithelial cytokeratins expression is revealed in a significantly greater number of cells (from 25 to 50%). The dominant molecular phenotype (50–80% cells expressed both markers), the coexpression of vimentin and cytokeratins, is identified for the first time. The authors assume that the latter phenotype of melanoma cells may be a molecular marker of the tumor’s aggressiveness and metastatic potential.

The use of selective BRAF inhibitors (BRAFi) has produced remarkable outcomes for patients with advanced cutaneous melanoma harboring a BRAFV600E mutation. Unfortunately, the majority of patients eventually develop drug-resistant disease. We employed a genetic screening approach to identify gain-of-function mechanisms of BRAFi resistance in two independent melanoma cell lines. Our screens identified both known and unappreciated drivers of BRAFi resistance, including multiple members of the DBL family. Mechanistic studies identified a DBL/RAC1/PAK signaling axis capable of driving resistance to both current and next-generation BRAFis. However, we show that the SRC inhibitor, saracatinib, can block the DBL-driven resistance. Our work highlights the utility of our straightforward genetic screening method in identifying new drug combinations to combat acquired BRAFi resistance. Significance: A simple, rapid, and flexible genetic screening approach identifies genes that drive resistance to MAPK inhibitors when overexpressed in human melanoma cells.

Immune-cell infiltration is associated with improved survival in melanoma. Human melanoma metastases may be grouped into immunotypes representing patterns of immune-cell infiltration: A (sparse), B (perivascular cuffing), and C (diffuse). Immunotypes have not been defined for murine melanomas, but may provide opportunities to understand mechanism-driving immunotype differences. We performed immunohistochemistry with immune-cell enumeration, immunotyping, and vascular density scoring in genetically engineered (Braf/Pten and Braf/Pten/β-catenin) and transplantable (B16-F1, B16-OVA, and B16-AAD) murine melanomas. The transplantable tumors were grown in subcutaneous (s.c.) or intraperitoneal (i.p.) locations. Braf/Pten and Braf/Pten/β-catenin tumors had low immune-cell densities, defining them as Immunotype A, as did B16-F1 tumors. B16-OVA (s.c. and i.p.) and B16-AAD s.c. tumors were Immunotype B, while B16-AAD i.p. tumors were primarily Immunotype C. Interestingly, the i.p. location was characterized by higher immune-cell counts in B16-OVA tumors, with counts that trended higher for B16-F1 and B16-AAD. The i.p. location was also characterized by higher vascularity in B16-F1 and B16-AAD tumors. These findings demonstrate that spontaneously mutated neoantigens in B16 melanomas were insufficient to induce robust intratumoral immune-cell infiltrates, but instead were Immunotype A tumors. The addition of model neoantigens (OVA or AAD) to B16 enhanced infiltration, but this most often resulted in Immunotype B. We find that tumor location may be an important element in enabling Immunotype C tumors. In aggregate, these data suggest important roles both for the antigen type and for the tumor location in defining immunotypes.

Ethylhexyl dimethyl para-aminobenzoic acid (PABA) is an oily yellow liquid derivative of water-soluble PABA commonly used in sunscreen. Ethylhexyl dimethyl PABA is widely used as an ingredient in many cosmetics at an average concentration of 1.25% (0.5-2.0%) in Korea. Previous studies, including those involving animals, have demonstrated that ethylhexyl dimethyl PABA is toxic to the following four organs: testis, epididymis, spleen, and liver. In addition, experiments using human keratinocytes found that ethylhexyl dimethyl PABA inhibits cell growth and DNA synthesis at low concentrations, and halted the cell cycle of MM96L cells (human melanoma cell line) at the G1 phase. Despite limited clinical data in humans, many studies have confirmed increased mutagenicity of ethylhexyl dimethyl PABA following exposure to sunlight, which suggests that this molecule is likely to contribute to onset of sun-induced cancer despite protecting the skin through absorption of UVB. For risk assessment, the no observed adverse effect level (NOAEL) chosen was 100 mg/kg bw/day in a 4 weeks oral toxicity study. Systemic exposure dosage (SED) was 0.588 mg/kg bw/day for maximum use of ethylhexyl dimethyl PABA in cosmetics. Based on the risk assessment and exposure scenarios conducted in this study, the margin of safety (MOS) was calculated to be 180.18 for a sunscreen containing 8% ethylhexyl dimethyl PABA, which is the maximum level allowed by the relevant domestic authorities.

Background Malignant melanomas frequently metastasize to the brain, but metastases in the cerebellum are underrepresented compared with metastases in the cerebrum. Methods We established animal models by injecting intracardially in athymic nude fox1nu mice two human melanoma cell lines, originating from a cerebral metastasis (HM19) and a cerebellar metastasis (HM86). Results Using magnetic resonance imaging (MRI), metastases were first detected after a mean of 34.5 days. Mean survival time was 59.6 days for the mice in the HM86 group and significantly shorter (43.7 days) for HM19‐injected animals (p, We present two new models that offer novel possibilities for investigating tumor‐ and host‐associated factors involved in malignant melanoma brain metastasis in general and tissue‐specific metastasis in particular. Two cell lines derived from a cerebral and a cerebellar human‐brain metastasis gave rise to two completely different patterns of spread, where 62.9% of animals injected with the cell line derived from a cerebellar human metastasis developed cerebellar metastases compared with 2% in the group injected with the cerebral cell line.

Activated antigen-experienced B cells play an unexpected complex role in anti-tumor immunity in human melanoma patients. However, correlative studies between B cell infiltration and tumor progression are limited by the lack of distinction between functional B cell subtypes. In this study, we examined a series of 59 primary and metastatic human cutaneous melanoma specimens with B cell infiltration. Using seven-color multiplex immunohistochemistry and automated tissue imaging and analysis, we analyzed the spatiotemporal dynamics of three major antigen-experienced B cell subpopulations expressing lymphotoxin alpha (LTA/TNFSF1) or interleukin-10 (IL-10) outside tertiary lymphoid structures. The expression of both LTA and IL-10 was not restricted to a particular B cell subtype. In primary melanomas, these cells were predominantly found at the invasive tumor-stroma front and, in metastatic melanomas, they were also found in the intratumoral stroma. In primary melanomas, decreased densities of LTA+ memory-like and, to a lesser extent, activated B cells were associated with metastasis. Compared with metastatic primary tumors, B cell infiltrates in melanoma metastases were enriched in both LTA+ memory-like and LTA+ activated B cells, but not in any of the IL-10+ B cell subpopulations. Melanoma disease progression shows distinct dynamics of functional B cell subpopulations, with the regulation of LTA+ B cell numbers being more significant than IL-10+ B cell subpopulations.

Sanghuangporus vaninii, also called ‘Sanghuang’ mushroom in Chinese, has various medicinal uses, but its effects on human melanoma cells have not been reported. The present study investigated the inhibitory ability and potential anticancer mechanism of the aqueous extracts of S. vaninii (SH). The results revealed that SH inhibited the proliferation of A375 human melanoma cells in a dose-dependent manner, and flow cytometry analysis suggested that SH induced A375 cell cycle arrest at S phase and apoptosis. Reverse transcription-quantitative PCR, western blotting and immunofluorescence analyses indicated that SH induced S-phase arrest by upregulating p21 expression, and p21 inhibited the expression of cyclin-cyclin-dependent kinases complexes at both the RNA and protein levels. In addition, SH induced apoptosis of A375 cells by inhibiting the expression levels of the anti-apoptosis gene Bcl-2. Therefore, the results suggested that SH may be a potential candidate for the treatment of human melanoma, thus providing new ideas for developing drugs that target melanoma.

There is increasing evidence that tertiary lymphoid structures (TLS) control not only local adaptive B cell responses at melanoma tumor sites but also the cellular composition and function of other immune cells. In human melanoma, however, a comprehensive analysis of TLS phenotypes, density and spatial distribution at different disease stages is lacking. Here we used 7-color multiplex immunostaining of whole tissue sections from 103 human melanoma samples to characterize TLS phenotypes along the expression of established TLS-defining molecular and cellular components. TLS density and spatial distribution were determined by referring TLS counts to the tissue area within defined intra- and extratumoral perimeters around the invasive tumor front. We show that only a subgroup of primary human melanomas contains TLS. These TLS rarely formed germinal centers and mostly located intratumorally within 1 mm distance to the invasive tumor front. In contrast, melanoma metastases had a significantly increased density of secondary follicular TLS. They appeared preferentially in stromal areas within an extratumoral 1 mm distance to the invasive tumor front and their density varied over time and site of metastasis. Interestingly, secondary follicular TLS in melanoma often lacked BCL6+ lymphatic cells and canonical germinal center polarity with the formation of dark and light zone areas. Our work provides an integrated qualitative, quantitative and spatial analysis of TLS in human melanoma and shows disease progression- and site-associated changes in TLS phenotypes, density and spatial distribution. The frequent absence of canonical germinal center polarity in melanoma TLS highlights the induction of TLS maturation as a potential additive to future immunotherapy studies. Given the variable evaluation strategies used in previous TLS studies of human tumors, an important asset of this study is the standardized quantitative evaluation approach that provides a high degree of reproducibility.

The development and progression of melanoma tumors is associated with angiogenesis, manifesting as changes in vessel density, morphology, and architecture that may extend through the entire skin depth. Three-dimensional imaging of vascular characteristics in skin lesions could allow diagnostic insights not available to the conventional visual inspection. Raster-scan optoacoustic mesoscopy (RSOM) has emerged as a unique modality to image microvasculature through the entire skin depth with resolutions of tens of micrometers, offering new possibilities to assess angiogenetic processes. However, current RSOM implementations are slow, exacerbating motion artifacts and reducing image quality, particularly when imaging melanoma lesions that often appear on the upper torso where breathing motion is strongest. To visualize for the first time melanoma vasculature in humans, in high-resolution, we accelerated RSOM scanning using an illumination scheme that is co-axial with a high-sensitivity ultrasound detector path, yielding 15 second single-breath-hold scans that minimize motion artifacts. Applied to 10 melanomas and 10 benign nevi in humans, we demonstrate visualization of microvasculature with performance never before shown in vivo. We show marked differences between malignant and benign lesions, supporting the possibility to use vasculature as a biomarker for lesion characterization. The study points to promising clinical potential for Fast-RSOM (FRSOM) as a three dimensional visualization method that can enable the complete assessment of microvascular parameters of melanoma and improve diagnostics.

Background: The role of tumor-associated B cells in human cancer is only starting to emerge. B cells typically undergo a series of developmental changes in phenotype and function, however, data on the composition of the B cell population in human melanoma are largely absent including changes during tumor progression and their potential clinical significance. Methods: In this study, we compared the number and distribution of six major B cell and antibody secreting cell subpopulations outside tertiary lymphoid structures in whole tumor sections of 154 human cutaneous melanoma samples (53 primary tumors without subsequent metastasis, 44 primary tumors with metastasis, 57 metastatic samples) obtained by seven color multiplex immunohistochemistry and automated tissue imaging and analysis. Results: In primary melanomas, we observed the highest numbers for plasmablast-like, memory-like, and activated B cell subtypes. These cells showed a patchy, predominant paratumoral distribution at the invasive tumor-stroma margin. Plasma cell-like cells were hardly detected, germinal center- and transitional/regulatory-like B cells not at all. Of the major clinicopathologic prognostic factors for primary melanomas, metastasis was associated with decreased memory-like B cell numbers and a higher age associated with higher plasmablast-like cell numbers. When we compared the composition of B cell subpopulations in primary melanomas and metastatic samples, we found a significantly higher proportion of plasma cell-like cells at distant metastatic sites and a higher proportion of memory-like B cells at locoregional than distant metastatic sites. Both cell types were detected mainly in the para- and intratumoral stroma. Conclusion: These data provide a first comprehensive and comparative spatiotemporal analysis of major B cell and antibody secreting cell subpopulations in human melanoma and describe metastasis-, tumor stage-, and age-associated dynamics, an important premise for B cell-related biomarker and therapy studies.

Background: The role of tumor-associated B cells in human cancer is only starting to emerge. B cells typically undergo a series of developmental changes in phenotype and function, however, data on the composition of the B cell population in human melanoma are largely absent including changes during tumor progression and their potential clinical significance.Methods: In this study, we compared the number and distribution of six major B cell and antibody secreting cell subpopulations outside tertiary lymphoid structures in whole tumor sections of 154 human cutaneous melanoma samples (53 primary tumors without subsequent metastasis, 44 primary tumors with metastasis, 57 metastatic samples) obtained by seven color multiplex immunohistochemistry and automated tissue imaging and analysis.Results: In primary melanomas, we observed the highest numbers for plasmablast-like, memory-like, and activated B cell subtypes. These cells showed a patchy, predominant paratumoral distribution at the invasive tumor-stroma margin. Plasma cell-like cells were hardly detected, germinal center- and transitional/regulatory-like B cells not at all. Of the major clinicopathologic prognostic factors for primary melanomas, metastasis was associated with decreased memory-like B cell numbers and a higher age associated with higher plasmablast-like cell numbers. When we compared the composition of B cell subpopulations in primary melanomas and metastatic samples, we found a significantly higher proportion of plasma cell-like cells at distant metastatic sites and a higher proportion of memory-like B cells at locoregional than distant metastatic sites. Both cell types were detected mainly in the para- and intratumoral stroma.Conclusion: These data provide a first comprehensive and comparative spatiotemporal analysis of major B cell and antibody secreting cell subpopulations in human melanoma and describe metastasis-, tumor stage-, and age-associated dynamics, an important premise for B cell-related biomarker and therapy studies.

Spatial transcriptomics and multiplexed imaging are complementary methods for studying tissue biology and disease. Recently developed spatial transcriptomic methods use fresh-frozen specimens but most diagnostic specimens, clinical trials, and tissue archives rely on formaldehyde-fixed tissue. Here we describe the Pick-Seq method for deep spatial transcriptional profiling of fixed tissue. Pick-Seq is a form of micro-region sequencing in which small regions of tissue, containing 5-20 cells, are mechanically isolated on a microscope and then sequenced. We demonstrate the use of Pick-Seq with several different fixed and frozen human specimens. Application of Pick-Seq to a human melanoma with complex histology reveals significant differences in transcriptional programs associated with tumor invasion, proliferation, and immuno-editing. Parallel imaging confirms changes in immuno-phenotypes and cancer cell states. This work demonstrates the ability of Pick-Seq to generate deep spatial transcriptomic data from fixed and archival tissue with multiplexed imaging in parallel.

Photochemo and photodynamic therapies are minimally invasive approaches for the treatment of cancers and powerful weapons for competing bacterial resistance to antibiotics. Synthetic and naturally occurring quinazolinones are considered privileged anticancer and antibacterial agents, with several of them to have emerged as commercially available drugs. In the present study, applying a single-step green microwave irradiation mediated protocol we have synthesized eleven quinazolinon-4(3H)-ones, from cheap readily available anthranilic acids, in very good yields and purity. These products were irradiated in the presence of pBR322 plasmid DNA under UVB, UVA and visible light. Four of the compounds proved to be very effective DNA photocleavers, at low concentrations, being time and concentration dependent as well as pH independent. Participation of reactive oxygen species was related to the substitution of quinazolinone derivatives. 6-Nitro-quinazolinone in combination with UVA irradiation was found to be in vitro photodestructive for three cell lines; glioblastoma (U87MG and T98G) and mainly melanoma (A-375). Thus, certain appropriately substituted quinazolinones may serve as new lead photosensitizers for the development of promising biotechnological applications and as novel photochemo and photodynamic therapeutics.

Plasma medicine is a new field focusing on biomedical and clinical applications of cold physical plasmas, including their anticancer effects. Cold plasmas can be applied directly or indirectly as plasma activated liquids (PAL). The effect of plasma activated cell growth medium (PAM) and plasma activated phosphate buffered saline (PAPBS) were tested using a plasma pen generating streamer corona discharge in ambient air, on different cancer cell lines (melanoma A375, glioblastoma LN229 and pancreatic cancer MiaPaCa-2) and normal cells (human dermal fibroblasts HDFa). The viability reduction and apoptosis induction were detected in all cancer cells after incubation in PAL. In melanoma cells we focused on detailed insights to the apoptotic pathways. The anticancer effects depend on the plasma treatment time or PAL concentration. The first 30 minutes of incubation in PAL were enough to start processes leading to the cell death. In fibroblasts, no apoptosis induction was observed, only PAPBS, activated for longer time, slightly decreased their viability. Anticancer effects of PAM and PAPBS on cancer cells showed selectivity compared to normal fibroblasts, depended on correctly chosen activation time and PAL concentration. This selectivity, supported by optimum ratio of hydrogen peroxide and nitrites in PAL, is very promising for potential clinical applications.

Melanoma is the most prevalent type of skin cancer with high mortality rates. This study demonstrates the

Early differential diagnosis between malignant and benign tumors and their underlying intrinsic differences are the most critical issues for life-threatening cancers. To study whether human acral melanomas, deadly cancers that occur on non-hair-bearing skin, have distinct origins that underlie their invasive capability, we develop fate-tracing technologies of melanocyte stem cells in sweat glands (glandular McSCs) and in melanoma models in mice and compare the cellular dynamics with human melanoma. Herein, we report that glandular McSCs self-renew to expand their migratory progeny in response to genotoxic stress and trauma to generate invasive melanomas in mice that mimic human acral melanomas. The analysis of melanocytic lesions in human volar skin reveals that genetically unstable McSCs expand in sweat glands and in the surrounding epidermis in melanomas but not in nevi. The detection of such cell spreading dynamics provides an innovative method for an early differential diagnosis of acral melanomas from nevi.

The classic model for development of a human therapeutic involves initial chemical/molecular selection or design, in vitro testing, and frequently molecular optimization. The Food and Drug Administration preclinical testing for safety for eventual human trials usually requests two animal species to be evaluated (one rodent and one non-rodent). A major reason cited for the inefficiency of drug development is the relatively low power of current rodent models to predict therapeutic efficacy in humans. A review of the scientific literature demonstrates numerous examples in which successful murine studies did not predict success in humans. A widely distributed and updated volume of fascicles of Animal Models of Human Disease was published by the Armed Forces Institute of Pathology in 1979. Naturally occurring melanoma in dogs has continued to be studied as a complementary model for human melanoma treatment. In 2016, researchers at the University of Colorado School of Medicine and Colorado State University College of Veterinary Medicine collaborated on a canine study using photodynamic therapy to treat lung cancer via bronchoscopy.

The synthesis and characterization of potent 7-substituted-2-(4-substitutedphenyl)imidazo[2,1-b][1,3]benzothiazoles as effective radiosensitizers and anticarcinogenic compound is reported. The activity is determined against human liver cancer Hep G2 cell line, two parental melanoma cell lines (human melanoma cell line, A375C and mouse melanoma cell line, A375C). The compounds 7-sulfonamide-2-(4-fluorophenyl)imidazo[2,1-b][1,3]benzothiazole (3f, IC50 = 0.097 μM) and 7-sulfonamide-2-(4-methylphenyl)imidazo[2,1-b][1,3]benzothiazole (3g, IC50 = 0.114 μM) exhibited considerable in vitro anticancer activity against Hep G2 cell line while 7-bromo-2-(4-fluorophenyl)imidazo[2,1-b][1,3]benzothiazole showed effectiveness against both parental melanoma cell lines (3c, IC50 = 0.04 and 0.052 μM). Further the active molecules 3f, 3g and 3h are tested for radiosensitizing activity over Hep G2 cell line at 4 Gy, with the comet formed at 0.054 μM . Similarly, 3c tested against two parental melanoma cell lines, it has proven to be more potent when combined with 2 Gy ϒ-radiation. The present study reveals that presence of sulfonamide in combination with methoxy substitution enhanced DNA fragmentation and there by acting as effective derivative for Hepatocellular carcinoma.

A 67-year-old female with a history of colon cancer underwent colonoscopy. An 8 mm semi-pedunculated, friable, and ulcerated lesion of the ascending colon was removed completely using a hot snare. Immunohistochemical staining showed strong positivity for transcription factor binding to IGHM enhancer 3 (TFE-3) and was partially positive for Human Melanoma Black (HMB-45), consistent with a diagnosis of perivascular epithelioid cell tumor (PEComa). The patient underwent endoscopic submucosal dissection of the residual lesion in the ascending colon without complications. Here, we discuss the clinical and histopathologic characterizations that helped guide the diagnosis and management of this exceedingly rare entity.

While much of the research concerning factors associated with responses to immunotherapies focuses on the contributions of conventional peptide-specific T cells, the role of unconventional T cells, such as mucosalassociated invariant T (MAIT) cells, in human melanoma remains largely unknown. MAIT cells are innate-like T cells expressing a semi-invariant T cell receptor restricted to the non-classical MHC class I molecule MR1 presenting vitamin metabolites derived from bacteria. In this prospective clinical study, we sought to characterize MAIT cells in melanoma patients and determine their association with clinical outcomes. We identified tumor-infiltrating MAIT cells in melanomas across metastatic sites and found that the number of circulating MAIT cells is reduced in melanoma patients. However, circulating MAIT cell frequency is restored by anti-PD1 treatment in responding patients, correlating with treatment responses in which patients with high frequencies of MAIT cells exhibited improved overall survival. These data provide evidence for leveraging MAIT cells and their functions as novel targets for future therapies.

The purpose of this study was to figure out the dysregulation of miR-942-5p in melanoma and its role in melanoma pathogenesis.Quantitative real-time PCR (qRT-PCR) assay was used to determine the change of RNA expression. Protein expression was examined by Western blotting. miRNA target was validated through TargetScan and luciferase assay. Cell migration and invasion were detected by wound healing and transwell assay, respectively.Results of qRT-PCR manifested miR-942-5p were upregulated in melanoma cell. High expression of miR-942-5p in melanoma patients presented a poor prognosis. Upregulation of miR-942-5p accelerated cell proliferation, migration, and invasion in melanoma cells. Cell apoptosis was inhibited by miR-942-5p mimics. Suppression of miR-942-5p by its inhibitor showed the opposite effects in melanoma cells. TargetScan and luciferase assay showed that miR-942-5p directly targeted to the 3'-untranslated region (3'-UTR) of DKK3. Overexpression of DKK3 inhibited GSK-3β phosphorylation and reduced the expression of β-catenin in both cytoplasm and nucleus, which were induced by miR-942-5p mimics leading to the activation of Wnt/β-catenin pathway.Upregulation of miR-942-5p was observed in melanoma cells and tissues and significantly associated with a poor prognosis. Though targeting 3'-UTR of DKK3, miR-942-5p could activate Wnt/β-catenin pathway, resulting in melanoma cell proliferation, migration, and invasion, which promoted the development of melanoma. These results showed that miR-942-5p might be a diagnosis and prognosis biomarker in melanoma.

The goal of this study is to explore the effects of columbamine in melanoma cells and the signaling pathway involved.Human melanoma cell line A375 cells were used in this study. Cell proliferative ability was detected by MTT assay and clone formation assay. Cell migration and invasion were measured by wound healing assay and transwell assay, respectively. Protein expression was examined by Western blotting.Columbamine reduced cell proliferative ability and the number of clone spots in A375 cells. Western blotting results demonstrated that expression of cleaved caspase 3, an activated cell death protease, was upregulated by 20 and 50 µM of columbamine. Wound healing results showed that the scratch width was wider in cell treated with 20 and 50 µM of columbamine than that in cell treated with 0 and 10 µM of columbamine. Phosphorylation of STAT3 and expression of HSP90 was also repressed by columbamine in a concentration-dependent manner. Overexpression of HSP90 attenuated the inhibition of cell proliferation, migration and invasion induced by columbamine.Columbamine inhibited melanoma cell proliferation, migration, and invasion in A375 cells through inactivation of STAT3, which is mediated by HSP90.