Anti-Melanogenic and Anti-Inflammatory Effects of 2'-Hydroxy-4',6'-dimethoxychalcone in B16F10 and RAW264.7 Cells.

Chalcone is a type of flavonoid compound that is widely biosynthesized in plants. Studies have shown that consuming flavonoids from fruits and vegetables or applying individual ingredients reduces the risk of skin disease. However, the effects of chalcone on melanogenesis and inflammation have not been fully investigated. The aim of this study was to evaluate the anti-melanogenic and anti-inflammatory effects of 2'-hydroxy-3,4'-dimethoxychalcone (3,4'-DMC), 2'-hydroxy-4,4'-dimethoxychalcone (4,4'-DMC), 2'-hydroxy-3',4'-dimethoxychalcone (3',4'-DMC), and 2'-hydroxy-4',6'-dimethoxychalcone (4',6'-DMC). Among the derivatives of 2'-hydroxy-4'-methoxychalcone, 4',6'-DMC demonstrated the most potent melanogenesis-inhibitory and anti-inflammatory effects. As evidenced by various biological assays, 4',6'-DMC showed no cytotoxicity and notably decreased the expression of tyrosinase, tyrosinase-related protein (TRP)-1, and TRP-2 enzymes. Furthermore, it reduced cellular melanin content and intracellular tyrosinase activity in B16F10 melanoma cells by downregulating microphthalmia-associated transcription factor (MITF), cAMP-dependent protein kinase (PKA), cAMP response element-binding protein (CREB), p38, c-Jun N-terminal kinase (JNK), ß-catenin, glycogen synthase kinase-3ß (GSK3ß), and protein kinase B (AKT) proteins, while upregulating extracellular signal-regulated kinase (ERK) and p-ß-catenin. Additionally, treatment with 4',6'-DMC significantly mitigated the lipopolysaccharide (LPS)-induced expression of NO, PGE2, inflammatory cytokines, COX-2, and iNOS proteins. Overall, 4',6'-DMC treatment notably alleviated LPS-induced damage by reducing nuclear factor kappa B (NF-κB), p38, JNK protein levels, and NF-kB/p65 nuclear translocation. Finally, the topical applicability of 4',6'-DMC was evaluated in a preliminary human skin irritation test and no adverse effects were found. These findings suggest that 4',6'-DMC may offer new possibilities for use as functional ingredients in cosmeceuticals and ointments.

CD24 flags anastasis in melanoma cells.

Anastasis is a phenomenon observed in cancer cells, where cells that have initiated apoptosis are able to recover and survive. This molecular event is increasingly recognized as a potential contributor to cancer metastasis, facilitating the survival and migration of tumor cells. Nevertheless, the identification of a specific surface marker for detecting cancer cells in anastasis remained elusive. Here we report our observation that the cell surface expression of CD24 is preferentially enriched in a non-adherent FSClowSSChigh melanoma subpopulation, which is generally considered a non-viable population in cultivated melanoma cell lines. More than 90% of non-adherent FSClowSSChighCD24+ve metastatic melanoma cells exhibited bonafide features of apoptosis on the cell surface and in the nucleus, marking apoptotic or seemingly apoptotic subpopulations of the in vitro cultivated metastatic melanoma cell lines. Unexpectedly, however, the CD24+ve subpopulation, despite being apoptotic, showed evidence of metabolic activity and exhibited proliferative capacities, including anchorage-independent growth, when inoculated in soft agarose growth medium. These findings indicate that apoptotic FSClowSSChighCD24+ve melanoma subpopulations are capable of reversing the progression of apoptosis. We report CD24 as the first novel cell surface marker for anastasis in melanoma cells.

CD27 signaling inhibits tumor growth and metastasis via CD8 + T cell-independent mechanisms in the B16-F10 melanoma model.

CD27 belongs to the tumor necrosis factor receptor superfamily and acts as a co-stimulatory molecule, modulating T and B cell responses. CD27 stimulation enhances T cell survival and effector functions, thus providing opportunities to develop therapeutic strategies. The current study aims to investigate the role of endogenous CD27 signaling in tumor growth and metastasis. CD8 + T cell-specific CD27 knockout (CD8Cre-CD27fl) mice were developed, while global CD27 knockout (KO) mice were also used in our studies. Flow cytometry analyses confirmed that CD27 was deleted specifically from CD8 + T cells without affecting CD4 + T cells, B cells, and HSPCs in the CD8Cre-CD27fl mice, while CD27 was deleted from all cell types in global CD27 KO mice. Tumor growth and metastasis studies were performed by injecting B16-F10 melanoma cells subcutaneously (right flank) or intravenously into the mice. We have found that global CD27 KO mice succumbed to significantly accelerated tumor growth compared to WT controls. In addition, global CD27 KO mice showed a significantly higher burden of metastatic tumor nests in the lungs compared to WT controls. However, there was no significant difference in tumor growth curves, survival, metastatic tumor nest counts between the CD8Cre-CD27fl mice and WT controls. These results suggest that endogenous CD27 signaling inhibits tumor growth and metastasis via CD8 + T cell-independent mechanisms in this commonly used melanoma model, presumably through stimulating antitumor activities of other types of immune cells.

Enhanced Therapeutic Efficacy Against Melanoma through Exosomal Delivery of Hesperidin.

Melanoma, characterized as the most aggressive and metastatic form of skin cancer, currently has limited treatment options, predominantly chemotherapy and radiation therapy. However, the drawbacks associated with parenterally administered chemotherapy underscore the urgent need for alternative compounds to combat melanoma effectively. Hesperidin (HES), a flavonoid present in various citrus fruits, exhibits promising anticancer activity. Nevertheless, the clinical utility of HES is hindered by challenges such as poor water solubility, a short half-life, and low oral bioavailability. In response to these limitations, we introduced a novel approach by formulating HES-loaded exosomes (Exo-HES). Isolation of exosomes was achieved through the ultracentrifugation method, and HES was efficiently loaded using the sonication method. The resulting formulations displayed a desirable particle size (∼106 nm) and exhibited a spherical morphology, as confirmed by scanning electron and atomic force microscopy. In vitro studies conducted on B16F10 cell lines demonstrated higher cytotoxicity of Exo-HES compared to free HES, supported by enhanced cellular uptake validated through coumarin-6-loaded exosomes. This superior cytotoxicity was further evidenced by DNA fragmentation, increased generation of free radicals (ROS), loss of mitochondrial membrane potential, and effective inhibition of colony formation. The antimetastatic properties of Exo-HES were confirmed through wound healing and transwell migration assays. Oral pharmacokinetics studies revealed a remarkable increase of approximately 2.5 times in oral bioavailability and half-life of HES when loaded into exosomes. Subsequent in vivo experiments utilizing a B16F10-induced melanoma model in Swiss mice established that Exo-HES exhibited superior anticancer activity compared to HES after oral administration. Importantly, no biochemical, hematological, or histological toxicities were observed in tumor-bearing mice treated with Exo-HES. These findings suggest that exosomes loaded with HES represent a promising nanocarrier strategy to enhance the therapeutic effectiveness of hesperidin in melanoma treatment.

In Silico Identification and Molecular Mechanism of Novel Tyrosinase Inhibitory Peptides Derived from Nacre of Pinctada martensii.

Pearl and nacre powders have been valuable traditional Chinese medicines with whitening properties for thousands of years. We utilized a high-temperature and high-pressure method along with compound enzyme digestion to prepare the enzymatic hydrolysates of nacre powder of Pinctada martensii (NP-PMH). The peptides were identified using LC-MS/MS and screened through molecular docking and molecular dynamics simulations. The interactions between peptides and tyrosinase were elucidated through enzyme kinetics, circular dichroism spectropolarimetry, and isothermal titration calorimetry. Additionally, their inhibitory effects on B16F10 cells were explored. The results showed that a tyrosinase-inhibitory peptide (Ala-His-Tyr-Tyr-Asp, AHYYD) was identified, which inhibited tyrosinase with an IC50 value of 2.012 ± 0.088 mM. The results of the in vitro interactions showed that AHYYD exhibited a mixed-type inhibition of tyrosinase and also led to a more compact enzyme structure. The binding reactions of AHYYD with tyrosinase were spontaneous, leading to the formation of a new set of binding sites on the tyrosinase. The B16F10 cell-whitening assay revealed that AHYYD could reduce the melanin content of the cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that AHYYD could be widely used as a tyrosinase inhibitor in whitening foods and pharmaceuticals.

In Vitro In Silico Screening Strategy and Mechanism of Novel Tyrosinase Inhibitory Peptides from Nacre of Hyriopsis cumingii.

For thousands of years, pearl and nacre powders have been important traditional Chinese medicines known for their skin whitening effects. To prepare the enzymatic hydrolysates of Hyriopsis cumingii nacre powder (NP-HCH), complex enzymatic hydrolysis by pineapple protease and of neutral protease was carried out after the powder was pre-treated with a high-temperature and high-pressure method. The peptides were identified using LC-MS/MS and picked out through molecular docking and molecular dynamics simulations. Subsequently, the tyrosinase inhibitory and antioxidant properties of novel tyrosinase inhibitory peptides were investigated in vitro. In addition, the enzymatic activity of tyrosinase in B16F10 cells as well as melanin content and antioxidant enzyme levels were also examined. The results showed that a tyosinase inhibitory peptide (Tyr-Pro-Asn-Pro-Tyr, YPNPY) with an efficient IC50 value of 0.545 ± 0.028 mM was identified. The in vitro interaction results showed that YPNPY is a reversible competitive inhibitor of tyrosinase, suggesting that it binds to the free enzyme. The B16F10 cell whitening test revealed that YPNPY can reduce the melanin content of B16F10 cells by directly inhibiting the activity of intracellular tyrosinase. Additionally, it indirectly affects melanin production by acting as an antioxidant. These results suggest that YPNPY could be widely used as a tyrosinase inhibitor in whitening foods and drugs.

Antioxidant and Skin-Whitening Efficacy of a Novel Decapeptide (DP, KGYSSYICDK) Derived from Fish By-Products.

The skin is vulnerable to damage from ultraviolet rays and oxidative stress, which can lead to aging and pigmentation issues. This study investigates the antioxidant and whitening efficacy of a decapeptide (DP, KGYSSYICDK) derived from marine fish by-products and evaluates its potential as a new skin-whitening agent. DP demonstrated high antioxidant activity, showing comparable or superior performance to Vitamin C (Vit. C) in ferric reducing antioxidant power (FRAP) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging assays. In hydrogen peroxide (H2O2)-treated HaCaT cells, DP increased cell viability and reduced reactive oxygen species (ROS) generation. Furthermore, DP inhibited tyrosinase activity and decreased melanin production in α-melanocyte stimulating hormone (α-MSH)-induced B16F10 melanoma cells in a dose-dependent manner. Reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that DP reduces the mRNA expression of MITF, tyrosinase, and MC1R, thus suppressing melanin production. DP exhibits strong binding interactions with multiple amino acid residues of tyrosinase, indicating potent inhibitory effects on the enzyme. These results suggest that DP possesses significant antioxidant and whitening properties, highlighting its potential as a skin-whitening agent. Future research should focus on optimizing DP's structure and exploring structure-activity relationships.

Comparative Analysis of Olive-Derived Phenolic Compounds' Pro-Melanogenesis Effects on B16F10 Cells and Epidermal Human Melanocytes.

Olive leaf contains plenty of phenolic compounds, among which oleuropein (OP) is the main component and belongs to the group of secoiridoids. Additionally, phenolic compounds such as oleocanthal (OL) and oleacein (OC), which share a structural similarity with OP and two aldehyde groups, are also present in olive leaves. These compounds have been studied for several health benefits, such as anti-cancer and antioxidant effects. However, their impact on the skin remains unknown. Therefore, this study aims to compare the effects of these three compounds on melanogenesis using B16F10 cells and human epidermal cells. Thousands of gene expressions were measured by global gene expression profiling with B16F10 cells. We found that glutaraldehyde compounds derived from olive leaves have a potential effect on the activation of the melanogenesis pathway and inducing differentiation in B16F10 cells. Accordingly, the pro-melanogenesis effect was investigated by means of melanin quantification, mRNA, and protein expression using human epidermal melanocytes (HEM). This study suggests that secoiridoid and its derivates have an impact on skin protection by promoting melanin production in both human and mouse cell lines.

Maclura cochinchinensis (Lour.) Corner Heartwood Extracts Containing Resveratrol and Oxyresveratrol Inhibit Melanogenesis in B16F10 Melanoma Cells.

This study aimed to isolate and purify resveratrol and oxyresveratrol from the heartwoods of Maclura cochinchinensis, and to evaluate their inhibitory effects on melanogenesis in B16F10 murine melanoma cells. A methanol maceration process yielded a crude extract comprising 24.86% of the initial mass, which was subsequently analyzed through HPTLC, HPLC, and LC-MS/MS. These analyses revealed the presence of resveratrol and oxyresveratrol at concentrations of 4.32 mg/g and 33.6 mg/g in the extract, respectively. Initial purification employing food-grade silica gel column chromatography separated the extract into two fractions: FA, exhibiting potent inhibition of both tyrosinase activity and melanogenesis, and FM, showing no such inhibitory activity. Further purification processes led to the isolation of fractions Y11 and Gn12 with enhanced concentrations of resveratrol (94.9 and 110.21 mg/g, respectively) and fractions Gn15 and Gn16 with elevated levels of oxyresveratrol (321.93 and 274.59 mg/g, respectively), all of which significantly reduced melanin synthesis. These outcomes affirm the substantial presence of resveratrol and oxyresveratrol in the heartwood of M. cochinchinensis, indicating their promising role as natural agents for skin lightening.

The Anti-Melanogenic Effects of Ganodermanontriol from the Medicinal Mushroom Ganoderma lucidum through the Regulation of the CREB and MAPK Signaling Pathways in B16F10 Cells.

Ganoderma lucidum, a member of the Basidiomycetes family, is attracting attention for its medicinal potential due to its biological activity and the presence of numerous bioactive compounds. Although it is known that extracts of this mushroom inhibit melanin production, there are few reports on a single substance associated with this effect. In this study, we identified ganodermanontriol (GT), a novel compound from G. lucidum, that effectively inhibited melanin biosynthesis in B16F10 cells. GT inhibits melanin production by suppressing the expression of cellular tyrosinase proteins and microphthalmia-related transcription factor (MITF). Furthermore, GT affects the phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) and mitogen-activated protein kinase (MAPK) signaling molecules, which are involved in melanogenesis in B16F10 cells. Finally, the biosynthesis of GT and other substances by G. lucidum was evaluated using HPLC analysis. Thus, this study revealed the mechanism by which GT in G. lucidum inhibits melanin production in B16F10 cells, and these findings will contribute to promoting the potential use of this mushroom in the future.

Polysaccharide extracted from the Sargassum fusiforme induces cell cycle arrest and apoptosis of B16F10 melanoma cells through the PI3K/AKT pathway.

BACKGROUND: SARGASSUM FUSIFORME: (S. fusiforme) is a brown alga that has been utilized as a medicine for a long time. Polysaccharides extracted from S. fusiforme demonstrate antitumor activities. METHODS: The impact of S. fusiforme polysaccharides (SFPS 191,212) on the proliferation, apoptosis, and cell cycle kinetics of B16F10 murine melanoma cells were thoroughly investigated in this work. The anticancer activities of the SFPS 191,212 compounds were assayed in the B16F10 cells at both transcriptional and translational levels. RESULTS: The compound exhibited concentration-dependent effects. Moreover, SPFS 191,212 increased the numbers of apoptotic cells and arrested the cell cycle in the S phase of the quantitative real-time PCR. From western blotting, it was verified that the SFPS 191,212 treatment improved the expression of Bax, Caspase-9, and Caspase-3 genes and proteins, while it reduced phosphatidylinositol 3 kinase and Bcl-2 genes and proteins, suggesting the involvement of mitochondria. CONCLUSION: Overall, SFPS 191,212 can be further explored as a potential functional food or adjuvant agent for the prevention or treatment of melanoma.

Antimelanoma effect of manool in 2D cell cultures and reconstructed human skin models.

Melanoma is the most aggressive and lethal type of skin cancer, characterized by therapeutic resistance. In this context, the present study aimed to investigate the cytotoxic potential of manool, a diterpene from Salvia officinalis L., in human (A375) and murine (B16F10) melanoma cell lines. The analysis of cytotoxicity using the XTT assay showed the lowest IC50 after 48 h of treatment with the manool, being 17.6 and 18.2 µg/ml for A375 and B16F10, respectively. A selective antiproliferative effect of manool was observed on the A375 cells based on the colony formation assay, showing an IC50 equivalent to 5.6 µg/ml. The manool treatments led to 43.5% inhibition of the A375 cell migration at a concentration of 5.0 µg/ml. However, it did not affect cell migration in the B16F10 cells. Cell cycle analysis revealed that the manool interfered in the cell cycle of the A375 cells, blocking the G2/M phase. No changes in the cell cycle were observed in the B16F10 cells. Interestingly, manool did not induce apoptosis in the A375 cells, but apoptosis was observed after treatment of the B16F10 cells. Additionally, manool showed an antimelanoma effect in a reconstructed human skin model. Furthermore, in silico studies, showed that manool is stabilized in the active sites of the tubulin dimer with comparable energy concerning taxol, indicating that both structures can inhibit the proliferation of cancer cells. Altogether, it is concluded that manool, through the modulation of the cell cycle, presents a selective antiproliferative activity and a potential antimelanoma effect.

Comparison of two methods for tumour-targeting peptide modification of liposomes.

Liposomes decorated with tumour-targeting cell-penetrating peptides can enhance specific drug delivery at the tumour site. The TR peptide, c(RGDfK)-AGYLLGHINLHHLAHL(Aib)HHIL, is pH-sensitive and actively targets tumour cells that overexpress integrin receptor αvß3, such as B16F10 melanoma cells. Liposomes can be modified with the TR peptide by two different methods: utilization of the cysteine residue on TR to link DSPE-PEG2000-Mal contained in the liposome formula (LIPTR) or decoration of TR with a C18 stearyl chain (C18-TR) for direct insertion into the liposomal phospholipid bilayer through electrostatic and hydrophobic interactions (LIPC18-TR). We found that both TR and C18-TR effectively reversed the surface charge of the liposomes when the systems encountered the low pH of the tumour microenvironment, but LIPC18-TR exhibited a greater increase in the charge, which led to higher cellular uptake efficiency. Correspondingly, the IC50 values of PTX-LIPTR and PTX-LIPC18-TR in B16F10 cells in vitro were 2.1-fold and 2.5-fold lower than that of the unmodified PTX-loaded liposomes (PTX-LIP), respectively, in an acidic microenvironment (pH 6.3). In B16F10 tumour-bearing mice, intravenous administration of PTX-LIPTR and PTX-LIPC18-TR (8 mg/kg PTX every other day for a total of 4 injections) caused tumour reduction ratios of 39.4% and 56.1%, respectively, compared to 20.8% after PTX-LIP administration. Thus, we demonstrated that TR peptide modification could improve the antitumour efficiency of liposomal delivery systems, with C18-TR presenting significantly better results. After investigating different modification methods, our data show that selecting an adequate method is vital even when the same molecule is used for decoration.

Quercetin 3-O-Galactoside Isolated from Limonium tetragonum Inhibits Melanogenesis by Regulating PKA/MITF Signaling and ERK Activation.

Quercetin 3-O-galactoside (Q3G) is a common dietary flavanol that has been shown to possess several bioactivities, including anti-melanogenesis. However, how Q3G exerts its anti-melanogenic effect has not been studied. The current study, therefore aimed to investigate the anti-melanogenesis potential of Q3G and elucidate the underlying action mechanism in α-melanocyte-stimulating hormone (α-MSH)-induced hyperpigmentation model of B16F10 murine melanoma cells. Results showed that α-MSH stimulation significantly increased tyrosinase (TYR) and melanin production, which were significantly downregulated by Q3G treatment. The treatment with Q3G suppressed the transcriptional and protein expressions of melanogenesis-related enzymes TYR, tyrosinase related protein-1 (TRP-1), and TRP-2, along with the melanogenic transcription factor microphthalmia-associated transcription factor (MITF) in B16F10 cells. It was shown that Q3G downregulated MITF expression and suppressed its transcriptional activity by inhibiting the cAMP-dependent protein kinase A (PKA)-mediated activation of CREB and GSK3ß. In addition, MAPK-regulated MITF activation signaling was also involved in the inhibition of melanin production by Q3G. The results suggest that the anti-melanogenic properties of Q3G rationalize further studies in vivo to confirm its action mechanism and consequent utilization as a cosmetic ingredient against hyperpigmentation.

Syringetin Promotes Melanogenesis in B16F10 Cells.

Syringetin, an active compound present in red grapes, jambolan fruits, Lysimachia congestiflora, and Vaccinium ashei, is a dimethyl myricetin derivative which contains free hydroxyl groups at the C-2' and C-4' positions in ring B. Recent studies have revealed that syringetin possesses multiple pharmacological properties, such as antitumor, hepatoprotective, antidiabetic, antioxidative, and cytoprotective activities. To date, there has been no attempt to test the action of syringetin on melanogenesis. In addition, the molecular mechanism for the melanogenic effects of syringetin remains largely unknown. In this study, we investigated the effect of syringetin on melanogenesis in a murine melanoma cell line from a C57BL/6J mouse, B16F10. Our results showed that syringetin markedly stimulated melanin production and tyrosinase activity in a concentration-dependent manner in B16F10 cells. We also found that syringetin increased MITF, tyrosinase, TRP-1, and TRP-2 protein expression. Moreover, syringetin inhibited ERK and PI3K/Akt phosphorylation by stimulating p38, JNK, PKA phosphorylation levels, subsequently stimulating MITF and TRP upregulation, resulting in the activation of melanin synthesis. Furthermore, we observed that syringetin activated phosphorylation of GSK3ß and ß-catenin and reduced the protein level of ß-catenin, suggesting that syringetin stimulates melanogenesis through the GSK3ß/ß-catenin signal pathway. Finally, a primary skin irritation test was conducted on the upper backs of 31 healthy volunteers to determine the irritation or sensitization potential of syringetin for topical application. The results of the test indicated that syringetin did not cause any adverse effects on the skin. Taken together, our findings indicated that syringetin may be an effective pigmentation stimulator for use in cosmetics and in the medical treatment of hypopigmentation disorders.

The Effects of 2'-Hydroxy-3,6'-Dimethoxychalcone on Melanogenesis and Inflammation.

In this study, we demonstrated that 2'-hydroxy-3,6'-dimethoxychalcone (3,6'-DMC) alleviated α-MSH-induced melanogenesis and lipopolysaccharides (LPS)-induced inflammation in mouse B16F10 and RAW 264.7 cells. In vitro analysis results showed that the melanin content and intracellular tyrosinase activity were significantly decreased by 3,6'-DMC, without cytotoxicity, via decreases in tyrosinase and the tyrosinase-related protein 1 (TRP-1) and TRP-2 melanogenic proteins, as well as the downregulation of microphthalmia-associated transcription factor (MITF) expression through the upregulation of the phosphorylation of extracellular-signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K)/Akt, and glycogen synthase kinase-3ß (GSK-3ß)/catenin, and downregulation of the phosphorylation of p38, c-Jun N-terminal kinase (JNK), and protein kinase A (PKA). Furthermore, we investigated the effect of 3,6'-DMC on macrophage RAW264.7 cells with LPS stimulation. 3,6'-DMC significantly inhibited LPS-stimulated nitric oxide production. 3,6'-DMC also suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 on the protein level. In addition, 3,6'-DMC decreased the production of the tumor necrosis factor-α and interleukin-6. Successively, our mechanistic studies revealed that 3,6'-DMC also suppressed the LPS-induced phosphorylation of the inhibitor of IκBα, p38MAPK, ERK, and JNK. The Western blot assay results showed that 3,6'-DMC suppresses LPS-induced p65 translocation from cytosol to the nucleus. Finally, the topical applicability of 3,6'-DMC was tested through primary skin irritation, and it was found that 3,6'-DMC, at 5 and 10 µM concentrations, did not cause any adverse effects. Therefore, 3,6'-DMC may provide a potential candidate for preventing and treating melanogenic and inflammatory skin diseases.

Comparative Study of Metastasis Suppression Effects of Extracellular Vesicles Derived from Anaplastic Cell Lines, Nanog-Overexpressing Melanoma, and Induced Pluripotent Stem Cells.

Previous studies have demonstrated that extracellular vesicles (EVs) derived from an anaplastic mouse melanoma cell line made using Nanog overexpression of F10 (Nanog+F10) suppressed the metastasis of Nanog+F10. Here, an induced pluripotent stem (iPS) cell line was focused as a more anaplastic cell line, potentially producing EVs with higher metastasis-suppressive effects. The EVs were introduced into the tail vein nine times before introducing Nanog+F10 cells. Two weeks later, the liver and lung were resected and metastatic colonies were quantified. The involvement of macrophages (invasion inhibiting ability, phagocytic activity) and cytotoxic T cells (cytotoxicity) was evaluated using J774.1 and CTLL-2 cell lines. iPS EVs showed similar level effects to Nanog+F10 EVs in every item relevant to metastasis suppression. Differential expression analysis of miRNAs in EVs and functional network database analysis revealed that dominant regulatory miRNAs were predicted. The candidate hub genes most highly associated with the metastasis suppression mechanism were predicted as six genes, including Trp53 and Hif1a, for Nanog+F10 EVs and ten genes, including Ins1 and Kitl, for iPS EVs. Regarding the mechanism, Nanog+F10 EVs and iPS EVs were very different. This suggests synergistic effect when used together as metastasis preventive vaccine.

NLRC5-CIITA Fusion Protein as an Effective Inducer of MHC-I Expression and Antitumor Immunity.

Aggressive tumors evade cytotoxic T lymphocytes by suppressing MHC class-I (MHC-I) expression that also compromises tumor responsiveness to immunotherapy. MHC-I defects strongly correlate to defective expression of NLRC5, the transcriptional activator of MHC-I and antigen processing genes. In poorly immunogenic B16 melanoma cells, restoring NLRC5 expression induces MHC-I and elicits antitumor immunity, raising the possibility of using NLRC5 for tumor immunotherapy. As the clinical application of NLRC5 is constrained by its large size, we examined whether a smaller NLRC5-CIITA fusion protein, dubbed NLRC5-superactivator (NLRC5-SA) as it retains the ability to induce MHC-I, could be used for tumor growth control. We show that stable NLRC5-SA expression in mouse and human cancer cells upregulates MHC-I expression. B16 melanoma and EL4 lymphoma tumors expressing NLRC5-SA are controlled as efficiently as those expressing full-length NLRC5 (NLRC5-FL). Comparison of MHC-I-associated peptides (MAPs) eluted from EL4 cells expressing NLRC5-FL or NLRC5-SA and analyzed by mass spectrometry revealed that both NLRC5 constructs expanded the MAP repertoire, which showed considerable overlap but also included a substantial proportion of distinct peptides. Thus, we propose that NLRC5-SA, with its ability to increase tumor immunogenicity and promote tumor growth control, could overcome the limitations of NLRC5-FL for translational immunotherapy applications.

A 7-Hydroxy 4-Methylcoumarin Enhances Melanogenesis in B16-F10 Melanoma Cells.

The objectives of this study were to investigate the melanogenetic potentials of the naturally occurring 7-hydroxy coumarin derivatives 7-hydroxy 5,6-dimethoxycoumarin (7H-5,6DM), 7-hydroxy 6,8-dimethoxycoumarin (7H-6,8DM), 7-hydroxy 6-methoxycoumarin (7H-6M), and 7-hydroxy 4-methylcoumarin (7H-4M) in the melanogenic cells model for murine B16F10 melanoma cells. The initial results indicated that melanin production and intracellular tyrosinase activity were significantly stimulated by 7H-4M but not by 7H-5,6DM, 7H-6,8DM, or 7H-6M. Therefore, our present study further investigated the melanogenic effects of 7H-4M in B16-F10 cells, as well as its mechanisms of action. In a concentration-dependent manner, 7H-4M increased intracellular tyrosinase activity, leading to the accumulation of melanin without affecting the viability of B16-F10 cells. Our study further investigated the effects of 7H-4M on melanogenesis, including its ability to promote tyrosinase activity, increase melanin content, and activate molecular signaling pathways. The results indicate that 7H-4M effectively stimulated tyrosinase activity and significantly increased the expression of melanin synthesis-associated proteins, such as microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP1), and TRP2. Based on our findings, we can conclude that 7H-4M has the ability to activate the melanogenesis process through the upregulation of cAMP-dependent protein kinase (PKA) and the cAMP response element-binding protein (CREB). Additionally, our study showed that 7H-4M induced melanogenic effects by downregulating the extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthesis kinase-3ß (GSK-3ß) cascades, while upregulating the JNK and p38 signaling pathways. Finally, the potential of using 7H-4M in topical applications was tested through primary human skin irritation tests. During these tests, no adverse reactions were induced by 7H-4M. In summary, our results indicate that 7H-4M regulates melanogenesis through various signaling pathways such as GSK3ß/ß-catenin, AKT, PKA/CREB, and MAPK. These findings suggest that 7H-4M has the potential to prevent the development of pigmentation diseases.

The Cytotoxic Activity of Dammarane-Type Triterpenoids Isolated from the Stem Bark of Aglaia cucullata (Meliaceae).

The Aglaia genus, a member of the Meliaceae family, is generally recognized to include a number of secondary metabolite compounds with diverse structures and biological activities, including triterpenoids. Among the members of this genus, Aglaia cucullata has been reported to have unique properties and thrives exclusively in mangrove ecosystems. This plant is also known to contain various metabolites, such as flavaglines, bisamides, and diterpenoids, but there are limited reports on the isolation of triterpenoid compounds from its stem bark. Therefore, this research attempted to isolate and elucidate seven triterpenoids belonging to dammarane-type (1-7) from the stem bark of Aglaia cucullata. The isolated compounds included 20S,24S-epoxy-3α,25-dihydroxy-dammarane (1), dammaradienone (2), 20S-hydroxy-dammar-24-en-3-on (3), eichlerianic acid (4), (20S,24RS)-23,24-epoxy-24-methoxy-25,26,27-tris-nor dammar-3-one (5), 3α-acetyl-cabraleahydroxy lactone (6), and 3α-acetyl-20S,24S-epoxy-3α,25-dihydroxydammarane (7). Employing spectroscopic techniques, the chemical structures of the triterpenoids were identified using FTIR, NMR, and HRESITOF-MS. The cytotoxic activity of compounds 1-7 was tested with the PrestoBlue cell viability reagent against MCF-7 breast cancer, B16-F10 melanoma, and CV-1 normal kidney fibroblast cell lines. The results displayed that compound 5 had the highest level of bioactivity compared to the others. Furthermore, the IC50 values obtained were more than 100 µM, indicating the low potential of natural dammarane-type triterpenoids as anticancer agents. These findings provided opportunities for further studies aiming to increase their cytotoxic activities through semi-synthetic methods.