Investigation of antimicrobial and anti-cancer activity of thermally sensitive SnO2 nanostructures with green-synthesized cauliflower morphology at ambient weather conditions.

A tin oxide (SnO2) nanostructure was prepared using Matricaria recutita leaf extract to investigate its anticancer activity against SK-MEL-28 cells. The tetragonal crystal structure of tin oxide nanoparticles with an average crystal size of 27 nm was confirmed by X-ray diffraction (XRD) analysis. The tetragonal crystal structure of the tin oxide nanoparticles, with an average crystallite size of 27 nm, was confirmed by XRD an absorbance peak at 365 nm was identified by UV-visible spectroscopy analysis as belonging to the bio-mediated synthesis of SnO2 nanoparticles. The SnO2 NPs are capped and stabilized with diverse functional groups derived from bioactive molecules, including aldehydes, benzene rings, amines, alcohols, and carbonyl stretch protein molecules. Fourier transform infrared spectroscopy (FTIR) analysis validated the presence of these capping and stabilizing chemical bonds. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) studies revealed the cauliflower-shaped morphology of the SnO2 nanoparticles with an average particle size of 28 nm. The antimicrobial activity of both prepared and encapsulated samples confirmed their biological activities. Furthermore, both prepared and encapsulated tin oxide samples exhibited excellent anticancer activity against SK-MEL-28 human cancer cells. The present study introduces a reliable and uncomplicated approach to produce SnO2 nanoparticles and demonstrates their effectiveness in various applications, including cancer therapy, drug administration, and disinfectant.

TumFlow: An AI Model for Predicting New Anticancer Molecules.

Melanoma is the fifth most common cancer in the United States. Conventional drug discovery methods are inherently time-consuming and costly, which imposes significant limitations. However, the advent of Artificial Intelligence (AI) has opened up new possibilities for simulating and evaluating numerous drug candidates, thereby mitigating the requisite time and resources. In this context, normalizing flow models by employing machine learning techniques to create new molecular structures holds promise for accelerating the discovery of effective anticancer therapies. This manuscript introduces TumFlow, a novel AI model designed to generate new molecular entities with potential therapeutic value in cancer treatment. It has been trained on the NCI-60 dataset, encompassing thousands of molecules tested across 60 tumour cell lines, with an emphasis on the melanoma SK-MEL-28 cell line. The model successfully generated new molecules with predicted improved efficacy in inhibiting tumour growth while being synthetically feasible. This represents a significant advancement over conventional generative models, which often produce molecules that are challenging or impossible to synthesize. Furthermore, TumFlow has also been utilized to optimize molecules known for their efficacy in clinical melanoma treatments. This led to the creation of novel molecules with a predicted enhanced likelihood of effectiveness against melanoma, currently undocumented on PubChem.

α-Chitosan and ß-Oligochitosan Mixtures-Based Formula for In Vitro Assessment of Melanocyte Cells Response.

Chitosan is a natural polymer with numerous biomedical applications. The cellular activity of chitosan has been studied in various types of cancer, including melanoma, and indicates that these molecules can open new perspectives on antiproliferative action and anticancer therapy. This study analyzes how different chitosan conformations, such as α-chitosan (CH) or ß-oligochitosan (CO), with various degrees of deacetylation (DDA) and molar mass (MM), both in different concentrations and in CH-CO mixtures, influence the cellular processes of SK-MEL-28 melanocytes, to estimate the reactivity of these cells to the applied treatments. The in vitro evaluation was carried out, aiming at the cellular metabolism (MTT assay), cellular morphology, and chitinase-like glycoprotein YKL-40 expression. The in vitro effect of the CH-CO mixture application on melanocytes is obvious at low concentrations of α-chitosan/ß-oligochitosan (1:2 ratio), with the cell's response supporting the hypothesis that ß-oligo-chitosan amplifies the effect. This oligochitosan mixture, favored by the ß conformation and its small size, penetrates faster into the cells, being more reactive when interacting with some cellular components. Morphological effects expressed by the loss of cell adhesion and the depletion of YKL-40 synthesis are significant responses of melanocytes. ß-oligochitosan (1.5 kDa) induces an extension of cytophysiological effects and limits the cell viability compared to α-chitosan (400-900 kDa). Statistical analysis using multivariate techniques showed differences between the CH samples and CH-CO mixtures.

Structure and Metabolically Oriented Efficacy of Fucoidan from Brown Alga Sargassum muticum in the Model of Colony Formation of Melanoma and Breast Cancer Cells.

This work reports the detailed structure of fucoidan from Sargassum miticum (2SmF2) and its ability to potentiate the inhibitory effect of glycolysis inhibitor 2-deoxy-d-glucose (2-DG). 2SmF2 was shown to be sulfated and acetylated galactofucan containing a main chain of alternating residues of 1,3- and 1,4-linked α-l-fucopyranose, fucose fragments with monotonous 1,3- and 1,4-type linkages (DP up to 3), α-d-Gal-(1→3)-α-L-Fuc disaccharides, and 1,3,4- and 1,2,4-linked fucose branching points. The sulfate groups were found at positions 2 and 4 of fucose and galactose residues. 2SmF2 (up to 800 µg/mL) and 2-DG (up to 8 mM) were not cytotoxic against MDA-MB-231 and SK-MEL-28 as determined by MTS assay. In the soft agar-based model of cancer cell colony formation, fucoidan exhibited weak inhibitory activity at the concentration of 400 µg/mL. However, in combination with low non-cytotoxic concentrations of 2-DG (0.5 or 2 mM), 2SmF2 could effectively inhibit the colony formation of SK-MEL-28 and MDA-MB-231 cells and decreased the number of colonies by more than 50% compared to control at the concentration of 200 µg/mL. Our findings reveal the metabolically oriented effect of fucoidan in combination with a glycolysis inhibitor that may be beneficial for a therapy for aggressive cancers.

The Combined Metabolically Oriented Effect of Fucoidan from the Brown Alga Saccharina cichorioides and Its Carboxymethylated Derivative with 2-Deoxy-D-Glucose on Human Melanoma Cells.

Melanoma is the most aggressive and treatment-resistant form of skin cancer. It is phenotypically characterized by aerobic glycolysis that provides higher proliferative rates and resistance to cell death. The glycolysis regulation in melanoma cells by means of effective metabolic modifiers represents a promising therapeutic opportunity. This work aimed to assess the metabolically oriented effect and mechanism of action of fucoidan from the brown alga Saccharina cichorioides (ScF) and its carboxymethylated derivative (ScFCM) in combination with 2-deoxy-D-glucose (2-DG) on the proliferation and colony formation of human melanoma cell lines SK-MEL-28, SK-MEL-5, and RPMI-7951. The metabolic profile of melanoma cells was determined by the glucose uptake and Lactate-GloTM assays. The effect of 2-DG, ScF, ScFCM, and their combination on the proliferation, colony formation, and activity of glycolytic enzymes was assessed by the MTS, soft agar, and Western blot methods, respectively. When applied separately, 2-DG (IC50 at 72 h = 8.7 mM), ScF (IC50 at 72 h > 800 µg/mL), and ScFCM (IC50 at 72 h = 573.9 µg/mL) inhibited the proliferation and colony formation of SK-MEL-28 cells to varying degrees. ScF or ScFCM enhanced the inhibiting effect of 2-DG at low, non-toxic concentrations via the downregulation of Glut 1, Hexokinase II, PKM2, LDHA, and pyruvate dehydrogenase activities. The obtained results emphasize the potential of the use of 2-DG in combination with algal fucoidan or its derivative as metabolic modifiers for inhibition of melanoma SK-MEL-28 cell proliferation.

In vitro anti-melanoma activity of imiquimod in ultradeformable nanovesicles.

BACKGROUND: The wide spectrum of antitumoral mechanisms of imiquimod (IMQ), made it a good candidate for topical therapy of melanoma. However, physicochemical properties make IMQ formulation a difficult task. Solubility and skin penetration of IMQ are increased when loaded into ultradeformable nanovesicles. OBJECTIVE: Survey the in vitro anti-melanoma activity of IMQ loaded into two types of ultradeformable nanovesicles: archaeosomes (UDA-IMQ) (containing sn-2,3 ether-linked phytanyl saturated archaeolipids extracted from Halorubrum tebenquichense) and liposomes lacking archaeolipids (UDL-IMQ). METHODS: We prepared and structurally characterized UDA-IMQ and UDL-IMQ. Cytotoxicity was determined on human melanoma cells (SK-Mel-28) and keratinocytes (HaCaT cells) by MTT assay and LDH release. The cellular uptake was determined by flow cytometry. Apoptosis/necrosis induction was determined by fluorescence microscopy after double staining with YO-PRO-1® and propidium iodide. RESULTS: Neither IMQ nor IMQ-nanovesicles reduced the viability of HaCaT cells; but UDL-IMQ (371 nm, -24 mV ζ potential, 31 µg IMQ/mg lipids) and UDA-IMQ (216 nm, -32 mV ζ potential, 61 µg IMQ/mg lipids) showed time and concentration-dependent cytotoxicity on SK-Mel-28 that resulted between 4 and 33 folds higher than free IMQ, respectively. While both UDA-IMQ and UDL-IMQ retained 60% of IMQ against dilution, UDA-IMQ uptaken by SK-Mel-28 cells was nine-fold higher than UDL-IMQ. UDL-IMQ induced early apoptosis, but UDA-IMQ induced both apoptosis and necrosis on SK-Mel-28 cells. CONCLUSIONS: UDA-IMQ was innocuous to keratinocytes but was highly uptaken and induced apoptosis and necrosis on melanoma cells, being a candidate for future investigations as adjuvant topical anti-melanoma therapy.

Dipotassium Glycyrrhizinate on Melanoma Cell Line: Inhibition of Cerebral Metastases Formation by Targeting NF-kB Genes-Mediating MicroRNA-4443 and MicroRNA-3620-Dipotassium Glycyrrhizinate Effect on Melanoma.

Glycyrrhizic acid (GA), a natural compound isolated from licorice (Glycyrrhiza glabra), has exhibited anti-inflammatory and anti-tumor effects in vitro. Dipotassium glycyrrhizinate (DPG), a dipotassium salt of GA, also has shown an anti-tumor effect on glioblastoma cell lines, U87MG and T98G. The study investigated the DPG effects in the melanoma cell line (SK-MEL-28). MTT assay demonstrated that the viability of the cells was significantly decreased in a time- and dose-dependent manner after DPG (IC50 = 36 mM; 24 h). DNA fragmentation suggested that DPG (IC50) induced cellular apoptosis, which was confirmed by a significant number of TUNEL-positive cells (p-value = 0.048) and by PARP-1 [0.55 vs. 1.02 arbitrary units (AUs), p-value = 0.001], BAX (1.91 vs. 1.05 AUs, p-value = 0.09), and BCL-2 (0.51 vs. 1.07 AUs, p-value = 0.0018) mRNA compared to control cells. The proliferation and wound-healing assays showed an anti-proliferative effect on DPG-IC50-treated cells, also indicating an inhibitory effect on cell migration (p-values < 0.001). Moreover, it was observed that DPG promoted a 100% reduction in melanospheres formation (p-value = 0.008). Our previous microRNAs (miRs) global analysis has revealed that DPG might increase miR-4443 and miR-3620 expression levels. Thus, qPCR showed that after DPG treatment, SK-MEL-28 cells presented significantly high miR-4443 (1.77 vs. 1.04 AUs, p-value = 0.02) and miR-3620 (2.30 vs. 1.00 AUs, p-value = 0.01) expression compared to control cells, which are predicted to target the NF-kB, CD209 and TNC genes, respectively. Both genes are responsible for cell attachment and migration, and qPCR revealed significantly decreased CD209 (1.01 vs. 0.54 AUs, p-value = 0.018) and TNC (1.00 vs. 0.31 AUs, p-value = 2.38 × 10−6) mRNA expression levels after DPG compared to untreated cells. Furthermore, the migration of SK-MEL-28 cells stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) was attenuated by adding DPG by wound-healing assay (48 h: p-value = 0.004; 72 h: p-value = 7.0 × 10−4). In addition, the MMP-9 expression level was inhibited by DPG in melanoma cells stimulated by TPA and compared to TPA-treated cells (3.56 vs. 0.99 AUs, p-value = 0.0016) after 24 h of treatment. Our results suggested that DPG has an apoptotic, anti-proliferative, and anti-migratory effect on SK-MEL-28 cells. DPG was also able to inhibit cancer stem-like cells that may cause cerebral tumor formation.

Cancer microcell initiation and determination.

BACKGROUND: Cancer remains one of the leading causes of death worldwide, despite the possibilities to detect early onset of the most common cancer types. The search for the optimal therapy is complicated by the cancer diversity within tumors and the unsynchronized development of cancerous cells. Therefore, it is necessary to characterize cancer cell populations after treatment has been applied, because cancer recurrence is not rare. In our research, we concentrated on small cancer cell subpopulation (microcells) that has a potential to be cancer resistance source. Previously made experiments has shown that these cells in small numbers form in specific circumstances after anticancer treatment. METHODS: In experiments described in this research, the anticancer agents' paclitaxel and doxorubicin were used to stimulate the induction of microcells in fibroblast, cervix adenocarcinoma, and melanoma cell lines. Mainly for the formation of microcells in melanoma cells. The drug-stimulated cells were then characterized in terms of their formation efficiency, morphology, and metabolic activity. RESULTS: We observed the development of cancer microcells and green fluorescent protein (GFP) transfection efficiency after stress. In the time-lapse experiment, we observed microcell formation through a renewal process and GFP expression in the microcells. Additionally, the microcells were viable after anticancer treatment, as indicated by the nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) enzyme activity assay results. Taken together, these findings indicate that cancer microcells are viable and capable of resisting the stress induced by anticancer drugs, and these cells are prone to chemical substance uptake from the environment. CONCLUSION: Microcells are not only common to a specific cancer type, but can be found in any tumor type. This study could help to understand cancer emergence and recurrence. The appearance of microcells in the studied cancer cell population could be an indicator of the individual anticancer therapy effectiveness and patient survival.

Integrin αvß3-Akt signalling plays a role in radioresistance of melanoma.

Melanoma is a deadly type of skin cancer that is particularly difficult to treat owing to its resistance to radiation therapy. Here, we attempted to determine the key proteins responsible for melanoma radioresistance, with the aim of improving disease response to radiation therapy. Two melanoma cell lines, SK-Mel5 and SK-Mel28, with different radiosensitivities were analysed via RNA-Seq (Quant-Seq) and target proteins with higher abundance in the more radioresistant cell line, SK-Mel28, identified. Among these proteins, integrin αvß3, a well-known molecule in cell adhesion, was selected for analysis. Treatment of SK-Mel28 cells with cilengitide, an integrin αvß3 inhibitor, as well as γ-irradiation resulted in more significant cell death than γ-irradiation alone. In addition, Akt, a downstream signal transducer of integrin αvß3, showed high basic activation in SK-Mel28 and was significantly decreased upon co-treatment with cilengitide and γ-irradiation. MK-2206, an Akt inhibitor, exerted similar effects on the SK-Mel28 cell line following γ-irradiation. Our results collectively demonstrate that the integrin αvß3-Akt signalling pathway contributes to radioresistance in SK-Mel28 cells, which may be manipulated to improve therapeutic options for melanoma.

A novel spheroid-based co-culture model mimics loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced selection of ABCB5-expressing cells.

BACKGROUND: Different 3D-cell culture approaches with varying degrees of complexity have been developed to serve as melanoma models for drug testing or mechanistic studies. While these 3D-culture initiatives are already often superior to classical 2D approaches, they are either composed of only melanoma cells or they are so complex that the behavior of individual cell types is hard to understand, and often they are difficult to establish and expensive. METHODS: This study used low-attachment based generation of spheroids composed of up to three cell types. Characterization of cells and spheroids involved cryosectioning, immunofluorescence, FACS, and quantitative analyses. Statistical evaluation used one-way ANOVA with post-hoc Tukey test or Student's t-test. RESULTS: The tri-culture model allowed to track cellular behavior in a cell-type specific manner and recapitulated different characteristics of early melanoma stages. Cells arranged into a collagen-IV rich fibroblast core, a ring of keratinocytes, and groups of highly proliferating melanoma cells on the outside. Regularly, some melanoma cells were also found to invade the fibroblast core. In the absence of melanoma cells, the keratinocyte ring stratified into central basal-like and peripheral, more differentiated cells. Conversely, keratinocyte differentiation was clearly reduced upon addition of melanoma cells. Treatment with the cytostatic drug, docetaxel, restored keratinocyte differentiation and induced apoptosis of external melanoma cells. Remaining intact external melanoma cells showed a significantly increased amount of ABCB5-immunoreactivity. CONCLUSIONS: In the present work, a novel, simple spheroid-based melanoma tri-culture model composed of fibroblasts, keratinocytes, and melanoma cells was described. This model mimicked features observed in early melanoma stages, including loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced increase of ABCB5 expression in external melanoma cells.

Extraction, isolation and in vitro evaluation of affinisine from Tabernaemontana catharinensis in human melanoma cells.

Plant compounds have been identified as new drug prototypes. In this line, this work aimed to isolate the indole alkaloid affinisine from Tabernaemontana catharinensis and test its antitumor activity. The alkaloid was isolated by silica gel open column chromatography from the ethanolic extract of the stem of T. catharinensis. Afterwards, this molecule was characterized by high-resolution mass spectrometry and nuclear magnetic resonance. In the next step, the cytotoxicity of the compound was tested against human melanoma cell lines (A375, WM1366 and SK-MEL-28) and a normal skin cell line (CCD-1059Sk) using a MTT (3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells treated with affinisine were evaluated by flow cytometry to analyze apoptosis and the induction of cell cycle arrest, to evaluate the dead mechanism. The metabolite was isolated in a 0.2% yield relative to the extract. Cytotoxic activity of the molecule was observed at 48 h, resulting in considerable growth inhibition rates in melanoma cells, especially in WM1366, which had the lowest IC50 (32.86 ± 2.54 µg/mL). The apoptosis rate was lower in A375 (56.66 and 86.71% with 57 and 65 µg/mL, respectively). Moreover, affinisine was able to significantly induce cell cycle arrest in different phases in the A375 and WM1366 cell lines. However, in SK-MEL-28 cells, cycle arrest was not observed. In summary, this compound significantly decreased the viability of tumor cells in a dose- and time-dependent manner for all evaluated lineages, reduced cell viability by the apoptosis mechanism and presented prominent activities of cell cycle arrest. In this way, the use of antineoplastic agents is among the most widely used therapeutic measures for the control and treatment of cancer. Affinisine is a promising prototype in the search for new drugs to treat cancer.

Efficient ferrocifen anticancer drug and Bcl-2 gene therapy using lipid nanocapsules on human melanoma xenograft in mouse.

Metastatic melanoma has been described as a highly aggressive cancer with low sensibility to chemotherapeutic agents. New types of drug, such as metal-based drugs (ferrocifens) have emerged and could represent an alternative for melanoma treatment since they show interesting anticancer potential. Furthermore, molecular analysis has evidenced the role of apoptosis in the low sensibility of melanomas and especially of the key regulator, Bcl-2. The objective of this study was to combine two strategies in the same lipid nanocapsules (LNCs): i) gene therapy to modulate anti-apoptotic proteins by the use of Bcl-2 siRNA, and ii) ferrocifens as a new type of anticancer agent. The efficient gene silencing with LNCs was verified by the specific extinction of Bcl-2 in melanoma cells. The cellular toxicity of ferrocifens (ferrociphenol (FcDiOH) or Ansa-FcDiOH) was demonstrated, showing higher efficacy than dacarbazine. Interestingly, the association of siBcl-2 LNCs with Ansa-FcDiOH demonstrated a significant effect on melanoma cell viability. Moreover, the co-encapsulation of siRNA and ferrocifens was successfully performed into LNCs for animal experiments. A reduction of tumor volume and mass was proved after siBcl-2 LNC treatment and Ansa-FcDiOH LNC treatment, individually (around 25%). Finally, the association of both components into the same LNCs increased the reduction of tumor volume to about 50% compared to the control group. In conclusion, LNCs appeared to provide a promising tool for the co-encapsulation of a metal-based drug and siRNA.

Development of gummy bear supplements and in vitro exploration of antioxidant and antiproliferative potential of Nuciferine.

BACKGROUND: Nuciferine's extensive therapeutic potential, including its robust antioxidant properties, is explored in response to the growing consumer preference for value-added organic foods. OBJECTIVE: This study focuses on the formulation of gummy bear supplements fortified with nuciferine from Nelumbonucifera. The research highlights nuciferine's ability to combat oxidative stress induced by reactive oxygen species (ROS) and examines its application in maintaining basal ROS levels during oxidative stress conditions in skin melanoma cells. METHODS: Characterization of extracted nuciferine through FTIR and UV-vis spectroscopy ensures product quality, while sensory evaluation compares honey and sugar as natural sweeteners for optimal flavor and consumer preference. SK-Mel-28 cellular ROS levels were measured using 2',7' -dichlorofluorescin diacetate dye before and after nuciferine treatment. SK-Mel-28 cell viability and dose response of nuciferine treatment was assessed using MTT assay. RESULTS: Nuciferine shows potent inhibition of SK-Mel-28 cell proliferation with an IC50 of 39.31 ± 5.280 µg/ml and showed no cytotoxicity in normal L6 skeletal muscle cells. This study compares the sensory properties of honey and sugar based gummy bear formulations. CONCLUSION: This project aims to create a high-quality, health-promoting dietary supplement that aligns with the evolving trends in organic nutrition and antioxidant supplementation.

Altered regulatory T cell-mediated Natural Killer cells suppression may lead to generalized vitiligo.

Generalized vitiligo (GV) is characterized by white patches due to autoimmune loss of melanocytes. Regulatory T cells (Tregs) maintain immune homeostasis, while NK cells eliminate pathogens and tumors. Increased NK cell frequency and reduced Treg frequency and suppressive capacity are observed in vitiligo patients. However, studies assessing Treg-mediated suppression of NK cell functions in GV are lacking. Therefore, our study aimed to assess in vitro Treg-mediated suppression of NK cells function over K562 and SK-Mel-28 cells in 31 GV patients and 30 controls using the BrdU-cell proliferation assay. We found decreased Treg-mediated suppression of NK cell function in GV patients (p = 0.0289). Moreover, increased NK cell-mediated K562 and SK-Mel-28 cells' suppression was observed in GV patients (p = 0.0207,p = 0.0419). Disease activity-based analysis, suggested reduced Treg-mediated suppression of NK cell function and increased NK cell function in active vitiligo patients (p = 0.03,p = 0.0436). Interestingly, age-based analysis suggested decreased Treg-mediated suppression of NK cell function in 1-20 and 21-40 years age groups compared to 41-60 years age group of GV patients (p = 0.005,p = 0.0380). Overall, our study, for the first time, suggests that decreased Treg-mediated suppression of NK cells may lead to increased destruction of melanocytes in GV, and this knowledge may help in developing effective therapeutics based on Tregs and NK cells for GV.

Genotoxicity and apoptotic effect of silver(I) complexes with mixed-ligands of thiosemicarbazones and diphenyl(p-tolyl)phosphine on malignant melanoma cells, SK-MEL-28.

Complexes of coinage metals can potentially be used as alternatives to platinum-based chemotherapeutic drugs. Silver is a coinage metal that can potentially improve the spectrum of efficacy in various cancers treatment, such as malignant melanoma. Melanoma is the most aggressive form of skin cancer that is often diagnosed in young and middle-aged adults. Silver has high reactivity with skin proteins and can be developed as a malignant melanoma treatment modality. Therefore, this study aims to identify the anti-proliferative and genotoxic effects of silver(I) complexes with mixed-ligands of thiosemicarbazones and diphenyl(p-tolyl)phosphine ligands in the human melanoma SK-MEL-28 cell line. The anti-proliferative effects of a series of silver(I) complex compounds labelled as OHBT, DOHBT, BrOHBT, OHMBT, and BrOHMBT were evaluated on SK-MEL-28 cells by using the Sulforhodamine B assay. Then, DNA damage analysis was performed in a time-dependent manner (30 min, 1 h and 4 h) by using alkaline comet assay to investigate the genotoxicity of OHBT and BrOHMBT at their respective IC50 values. The mode of cell death was studied using Annexin V-FITC/PI flow cytometry assay. Our current findings demonstrated that all silver(I) complex compounds showed good anti-proliferative activity. The IC50 values of OHBT, DOHBT, BrOHBT, OHMBT, and BrOHMBT were 2.38 ± 0.3 µM, 2.70 ± 0.17 µM, 1.34 ± 0.22 µM, 2.82 ± 0.45 µM, and 0.64 ± 0.04 µM respectively. Then, DNA damage analysis showed that OHBT and BrOHMBT could induce DNA strand breaks in a time-dependent manner, with OHBT being more prominent than BrOHMBT. This effect was accompanied by apoptosis induction in SK-MEL-28, as evaluated using Annexin V-FITC/PI assay. In conclusion, silver(I) complexes with mixed-ligands of thiosemicarbazones and diphenyl(p-tolyl)phosphine exerted anti-proliferative activities by inhibiting cancer cell growth, inducing significant DNA damage and ultimately resulting in 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.

Dexamethasone Promotes a Stem-Like Phenotype in Human Melanoma Cells via Tryptophan 2,3 Dioxygenase.

In addition to its well-established immunosuppressive actions, tryptophan 2,3-dioxygenase (TDO) appears to elicit direct effects on tumor cell function. Although TDO has been associated with cancer stemness, its involvement in melanoma stem cell biology remains largely unknown. Since we showed that by upregulating TDO, dexamethasone (dex) promotes proliferation and migration of SK-Mel-28 human melanoma cells, we sought to investigate dex effects on melanoma spherogenesis and stemness, and whether these events are mediated by TDO. We demonstrate here that dex significantly upregulates TDO in A375, a more aggressive melanoma cell line, confirming that dex effects are not limited to SK-Mel-28 cells. Moreover, dex stimulates spherogenesis of both cell lines, which is mediated by TDO, evident by its suppression with 680C91, a TDO inhibitor. The formed melanospheres appear to be enriched with embryonic stem cell marker mRNAs, the expression of which is potentiated by dex. Expression of cancer stem cell markers (CD133, CD44, ganglioside GD2) was significantly increased in A375 spheres, as detected by flow cytometry. Taken together, our results suggest that TDO could represent a promising target in the management of melanoma and that dex, routinely used as a co-medication also in advanced melanoma, may stimulate melanoma cell function/tumor-supporting properties, a rather debilitating and undesired side effect.

Exploring the anticancer and antioxidant properties of Vicia faba L. pods extracts, a promising source of nutraceuticals.

Background: Pulse crops are considered the major sources of proteins, dietary fiber, micronutrients, and bioactive phytochemicals. Among the numerous pulse crops, broad beans (Vicia faba L.) have received particular attention due to their nutraceutical, functional and economic importance. Our attention was mainly focused on the broad bean pods (VFs), which are the primary by-product of the domestic and industrial processing of broad beans and an attractive source of valuable ingredients. Methods: In order to investigate the VFs properties, the flours from broad beans of three different harvest periods were extracted with acetone, methanol and 70% aqueous ethanol and the dried extracts were analyzed, qualitatively and quantitatively, and tested for their antioxidant through DPPH and ABTS assay and anticancer activities using the MTT assay and immunofluorescence analysis. Results: The VF extracts demonstrated a good in vitro radical scavenging activity from the first stage of collection of all the V. faba L. extracts. Additionally, the extracts were tested for their cytotoxicity against a panel of cancer and normal cells and the outcomes indicated the ethanol extract as the most active against the melanoma cell line Sk-Mel-28, without affecting the viability of the normal cells. Finally, we found out that the ethanol extract interfered with the microtubules organization, leading to the cancer cells death by apoptosis.

22ß-hydroxytingenone induces apoptosis and suppresses invasiveness of melanoma cells by inhibiting MMP-9 activity and MAPK signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: 22ß-hydroxytingenone (22-HTG) is a quinonemethide triterpene isolated from Salacia impressifolia (Miers) A. C. Smith (family Celastraceae), which has been used in traditional medicine to treat a variety of diseases, including dengue, renal infections, rheumatism and cancer. However, the anticancer effects of 22-HTG and the underlying molecular mechanisms in melanoma cells have not yet been elucidated. AIM OF THE STUDY: The present study investigated apoptosis induction and antimetastatic potencial of 22-HTG in SK-MEL-28 human melanoma cells. MATERIALS AND METHODS: First, the in vitro cytotoxic activity of 22-HTG in cultured cancer cells was evaluated. Then, cell viability was determined using the trypan blue assay in melanoma cells (SK-MEL-28), which was followed by cell cycle, annexin V-FITC/propidium iodide assays (Annexin/PI), as well as assays to evaluate mitochondrial membrane potential, production of reactive oxygen species (ROS) using flow cytometry. Fluorescence microscopy using acridine orange/ethidium bromide (AO/BE) staining was also performed. RT-qPCR was carried out to evaluate the expression of BRAF, NRAS, and KRAS genes. The anti-invasiveness potential of 22-HTG was evaluated in a three-dimensional (3D) model of reconstructed human skin. RESULTS: 22-HTG reduced viability of SK-MEL-28 cells and caused morphological changes, as cell shrinkage, chromatin condensation, and nuclear fragmentation. Furthermore, 22-HTG caused apoptosis, which was demonstrated by increased staining with AO/BE and Annexin/PI. The apoptosis may have been caused by mitochondrial instability without the involvement of ROS production. The expression of BRAF, NRAS, and KRAS, which are important biomarkers in melanoma development, was reduced by the 22-HTG treatment. In the reconstructed skin model, 22-HTG was able to decrease the invasion capacity of melanoma cells in the dermis. CONCLUSIONS: Our data indicate that 22-HTG has anti-tumorigenic properties against melanoma cells through the induction of cell cycle arrest, apoptosis and inhibition of invasiveness potential, as observed in the 3D model. As such, the results provide new insights for future work on the utilization of 22-HTG in malignant melanoma treatment.

Raman spectral imaging of 13C2H15N-labeled α-synuclein amyloid fibrils in cells.

Parkinson's disease is characterized by the intracellular accumulation of α-synuclein (α-syn) amyloid fibrils, which are insoluble, ß-sheet-rich protein aggregates. Raman spectroscopy is a powerful technique that reports on intrinsic molecular vibrations such as the coupled vibrational modes of the polypeptide backbone, yielding secondary structural information. However, in order to apply this method in cells, spectroscopically unique frequencies are necessary to resolve proteins of interest from the cellular proteome. Here, we report the use of 13C2H15N-labeled α-syn to study the localization of preformed fibrils fed to cells. Isotopic labeling shifts the amide-I (13CO) band away from endogenous 12CO vibrations, permitting secondary structural analysis of internalized α-syn fibrils. Similarly, 13C2H stretches move to lower energies in the "cellular quiet" region, where there is negligible biological spectral interference. This combination of well-resolved, distinct vibrations allows Raman spectral imaging of α-syn fibrils across a cell, which provides conformational information with spatial context.