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.

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.

Fucoidan from brown algae Fucus evanescens potentiates the anti-proliferative efficacy of asterosaponins from starfish Asteropsis carinifera in 2D and 3D models of melanoma cells.

This study was aimed to determine the efficacy of combination of fucoidan from the brown algae Fucus evanescens (FeF) or its derivatives with thornasteroside A (ThA) or asteropsiside A (AsA) from the starfish Asteropsis carinifera in combating human melanoma cells. In vitro MTS and soft agar methods were performed to determine effect of FeF, its derivatives, ThA, AsA or their combination on proliferation and colony formation of SK-MEL-28 cells in 2D and 3D culture. Desulfation of FeF, but not deacetylation, led decreasing of its Mw and anti-proliferative activity. The combinatorial effect of FeF with ThA and AsA depended on the sequences of treatment by compounds. There was additive anticancer effect of FeF with ThA or AsA during simultaneous treatment of cells. ThA and AsA were not active against SK-MEL-28 cells after their pre-treatment with FeF. Potential synergism of action was identified only when SK-MEL-28 cells were pre-treated with ThA and AsA and then by FeF. This process going through the regulation of MEK1/2/ERK1/2/MSK1 pathway and expression of the cell cycle proteins as determined by Western Blot. Thus, the combination of fucoidan with the asterosaponins opens up the prospects for the development of effective combined chemotherapeutic methods for melanoma treatment.

Selective cytotoxicity of PAMAM G5 core--PAMAM G2.5 shell tecto-dendrimers on melanoma cells.

BACKGROUND: The controlled introduction of covalent linkages between dendrimer building blocks leads to polymers of higher architectural order known as tecto-dendrimers. Because of the few simple steps involved in their synthesis, tecto-dendrimers could expand the portfolio of structures beyond commercial dendrimers, due to the absence of synthetic drawbacks (large number of reaction steps, excessive monomer loading, and lengthy chromatographic separations) and structural constraints of high-generation dendrimers (reduction of good monodispersity and ideal dendritic construction due to de Gennes dense-packing phenomenon). However, the biomedical uses of tecto-dendrimers remain unexplored. In this work, after synthesizing saturated shell core-shell tecto-dendrimers using amine-terminated polyamidoamine (PAMAM) generation 5 (G5) as core and carboxyl-terminated PAMAM G2.5 as shell (G5G2.5 tecto-dendrimers), we surveyed for the first time the main features of their interaction with epithelial cells. METHODS: Structural characterization of G5G2.5 was performed by polyacrylamide gel electrophoresis, matrix-assisted laser desorption time-of-flight mass spectrometry, and microscopic techniques; their hydrodynamic size and Z-potential was also determined. Cellular uptake by human epidermal keratinocytes, colon adenocarcinoma, and epidermal melanoma (SK-Mel-28) cells was determined by flow cytometry. Cytotoxicity was determined by mitochondrial activity, lactate dehydrogenase release, glutathione depletion, and apoptosis/necrosis measurement. RESULTS: The resultant 60%-67% saturated shell, 87,000-dalton G5G2.5 (mean molecular weight) interacted with cells in a significantly different fashion in comparison to their building blocks and to its closest counterpart, PAMAM G6.5. After being actively taken up by epithelial cells, G5G2.5 caused cytotoxicity only on SK-Mel-28 cells, including depletion of intracellular glutathione and fast necrosis that was manifested above 5 µM G5G2.5. It cannot be discounted that traces of LiCl within G5G2.5 were involved in such deleterious effects. CONCLUSION: These preliminary results suggest that at concentrations that do not damage healthy keratinocytes, G5G2.5 could display antimelanoma activity.