In vitro effects and mathematical modelling of CTCE-9908 (a chemokine receptor 4 antagonist) on melanoma cell survival.

CTCE-9908, a CXC chemokine receptor 4 (CXCR4) antagonist, prevents CXCR4 phosphorylation and inhibits the interaction with chemokine ligand 12 (CXCL12) and downstream signalling pathways associated with metastasis. This study evaluated the in vitro effects of CTCE-9908 on B16 F10 melanoma cells with the use of mathematical modelling. Crystal violet staining was used to construct a mathematical model of CTCE-9908 B16 F10 (melanoma) and RAW 264.7 (non-cancerous macrophage) cell lines on cell viability to predict the half-maximal inhibitory concentration (IC50). Morphological changes were assessed using transmission electron microscopy. Flow cytometry was used to assess changes in cell cycle distribution, apoptosis via caspase-3, cell survival via extracellular signal-regulated kinase1/2 activation, CXCR4 activation and CXCL12 expression. Mathematical modelling predicted IC50 values from 0 to 100 h. At IC50, similar cytotoxicity between the two cell lines and ultrastructural morphological changes indicative of cell death were observed. At a concentration 10 times lower than IC50, CTCE-9908 induced inhibition of cell survival (p = 0.0133) in B16 F10 cells but did not affect caspase-3 or cell cycle distribution in either cell line. This study predicts CTCE-9908 IC50 values at various time points using mathematical modelling, revealing cytotoxicity in melanoma and non-cancerous cells. CTCE-9908 significantly inhibited melanoma cell survival at a concentration 10 times lower than the IC50 in B16 F10 cells but not RAW 264.7 cells. However, CTCE-9908 did not affect CXCR4 phosphorylation, apoptosis,\ or cell cycle distribution in either cell line.

Epigallocatechin gallate protects against fat and muscle atrophy in B16BL6 melanoma-bearing mice on a high-fat diet.

AIMS: Epidemiological evidence indicates that there is a substantial association between body mass index (BMI) and at least ten forms of cancer, including melanoma, and BMI imbalance contributes to the poor survival rate of cancer patients before and after therapy. Nevertheless, few pharmacological studies on models of obesity and cancer have been reported. In this study, we administered epigallocatechin gallate (EGCG) to B16BL6 tumor-bearing mice that received a high-fat diet (HFD) to examine its impact. METHODS: B16BL6 tumor-bearing mice were fed a HFD. Body weight and food intake were documented every week. We conducted a Western blot analysis to examine the protein levels in the tumor, gastrocnemius (GAS), and tibialis anterior (TA) muscles, as well as the inguinal and epididymal white adipose tissues (iWAT and eWAT). KEY FINDINGS: EGCG has been shown to have anti-cancer effects equivalent to those of cisplatin, a chemotherapy drug. Furthermore, EGCG protected against the loss of epidydimal white adipose tissue by regulating protein levels of lipolysis factors of adipose triglyceride lipase and hormone-sensitive lipase as well as WAT browning factors of uncoupling protein 1, as opposed to cisplatin. EGCG was shown to reduce the protein levels of muscular atrophy factors of muscle RING-finger protein-1, whereas cisplatin did not contribute to rescuing the atrophy of TA and GAS muscles. CONCLUSION: Taken together, our findings indicate that EGCG has a preventive effect against cachexia symptoms and has anti-cancer effects similar to those of cisplatin in tumor-bearing mice fed a high-fat diet.

The Expression of Markers of Acute Kidney Injury Kim1 and NGAL after Administration of High Doses of Lithium Carbonate in Mice with Engrafted Skin Melanoma B16.

The expression of marker proteins of acute kidney injury after administration of high doses of lithium carbonate was assessed to evaluate the possibility of lithium use in neutron capture therapy. In mice with implanted skin melanoma B16, the expression of Kim1 (kidney injury molecule 1) and NGAL (neutrophil gelatinase-associated lipocalin) proteins in the kidneys was evaluated immunohistochemically 15, 30, 90, 180 min, and 7 days after peroral administration of lithium carbonate at single doses of 300 and 400 mg/kg. An increase in the expression of the studied proteins was found in 30 and 90 min after administration of 400 mg/kg lithium carbonate, however, 7 days after the drug administration, the expression returned to the level observed in the control group. It can be suggested that single administration of lithium carbonate in the studied doses effective for lithium neutron capture therapy will not significantly affect the renal function.

Combining large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers for promoting protective melanosome autophagy via the PI3K/Akt/mTOR signalling pathway for the treatment of melasma.

Melasma is a common condition of hyperpigmented facial skin. Picosecond lasers are reported to be effective for the treatment of melasma. We aimed to identify the most effective therapeutic mode and elucidate the potential molecular mechanisms of picosecond lasers for the treatment of melasma. Female Kunming mice with melasma-like conditions were treated using four different picosecond laser modes. Concurrently, in vitro experiments were conducted to assess changes in melanin and autophagy in mouse melanoma B16-F10 cells treated with these laser modes. Changes in melanin in mouse skin were detected via Fontana-Masson staining, and melanin particles were evaluated in B16-F10 cells. Real-time polymerase chain reaction and western blotting were used to analyse the expression levels of melanosome and autophagy-related messenger ribonucleic acid (mRNA) and proteins. A combination of large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers resulted insignificant decreases in melanin as well as in mRNA and protein expression of melanin-synthesizing enzymes (TYR, TRP-1 and MITF). This combination also led to increased expression of the autophagy-related proteins, Beclin1 and ATG5, with a marked decrease in p62 expression. Intervention with the PI3K activator, 740 Y-P, increased TYR, TRP-1, MITF, p-PI3K, p-AKT, p-mTOR and p62 expression but decreased the expression of LC3, ATG5 and Beclin1. A combination of large-spot low-fluence 1064-nm and fractional 1064-nm picosecond lasers proved more effective and safer. It inhibits melanin production, downregulates the PI3K/AKT/mTOR pathway, enhances melanocyte autophagy and accelerates melanin metabolism, thereby reducing melanin content.

Ribosomal S6 kinase 2-forkhead box protein O4 signaling pathway plays an essential role in melanogenesis.

Although previous studies have examined the signaling pathway involved in melanogenesis through which ultraviolet (UV) or α-melanocyte-stimulating hormones (α-MSH) stimuli act as key inducers to produce melanin at the stratum basal layer of the epidermis, the signaling pathway regulating melanogenesis is still controversial. This study reports that α-MSH, not UVA and UVB, acted as a major stimulus of melanogenesis in B16F10 melanoma cells. Signaling pathway analysis using gene knockdown technology and chemical inhibitors, the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase 2 (RSK2) played an important role in melanogenesis. Unexpectedly, LY294002, a PI3K inhibitor, increased melanogenesis without UV or α-MSH stimulation, suggesting that the PI3K/AKT signaling pathway may not be a major signaling pathway for melanogenesis. Chemical inhibition of the MEKs/ERKs/RSK2 signaling pathway using U0126 or BI-D1870 suppressed melanogenesis by stimulation of UVA or α-MSH stimulation, or both. In particular, the genetic depletion of RSK2 or constitutive active (CA)-RSK2 overexpression showed that RSK2 plays a key role in melanogenesis. Interestingly, forkhead box protein O4 (FOXO4) was phosphorylated by RSK2, resulting in the increase of FOXO4's transactivation activity. Notably, the FOXO4 mutant harboring serine-to-alanine replacement at the phosphorylation sites totally abrogated the transactivation activity and reduced melanin production, indicating that RSK2-mediated FOXO4 activity plays a key role in melanogenesis. Furthermore, kaempferol, a flavonoid inhibiting the RSK2 activity, suppressed melanogenesis. In addition, FOXO4-wt overexpression showed that FOXO4 enhance melanin synthesis. Overall, the RSK2-FOXO4 signaling pathway plays a key role in modulating melanogenesis.

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.

Osteocytes support bone metastasis of melanoma cells by CXCL5.

Bone metastasis is a common complication of certain cancers such as melanoma. The spreading of cancer cells into the bone is supported by changes in the bone marrow environment. The specific role of osteocytes in this process is yet to be defined. By RNA-seq and chemokines screening we show that osteocytes release the chemokine CXCL5 when they are exposed to melanoma cells. Osteocytes-mediated CXCL5 secretion enhanced the migratory and invasive behaviour of melanoma cells. When the expression of the CXCL5 receptor, CXCR2, was down-regulated in melanoma cells in vitro, we observed a significant decrease in melanoma cell migration in response to osteocytes. Furthermore, melanoma cells with down-regulated CXCR2 expression showed less bone metastasis and less bone loss in the bone metastasis model in vivo. Furthermore, when simultaneously down-regulating CXCL5 in osteocytes and CXCR2 in melanoma cells, melanoma progression was abrogated in vivo. In summary, these data suggest a significant role of osteocytes in bone metastasis of melanoma, which is mediated through the CXCL5-CXCR2 pathway.

Melanin Inhibitory Effect of Tuber himalayense Isolated in Incheon, Korea.

There has been a growing interest in skin beauty and antimelanogenic products. Melanogenesis is the process of melanin synthesis whereby melanocytes are activated by UV light or hormone stimulation to produce melanin. Melanogenesis is mediated by several enzymes, such as tyrosinase (TYR), microphthalmia-associated transcription factor (MITF), tyrosinase-related protein-1 (TRP-1), and TRP-2. In this study, we investigated the effect of Tuber himalayense extract on melanin synthesis in α-melanocyte-stimulating hormone (α-MSH)-treated B16F10 melanoma cells. We confirmed that T. himalayense extract was not toxic to α-MSH-treated B16F10 melanoma cells and exhibited a significant inhibitory effect on melanin synthesis at concentrations of 25, 50, and 100 µg/ml. Additionally, the T. himalayense extract inhibited melanin, TRP-1, TRP-2, tyrosinase, and MITF, which are enzymes involved in melanin synthesis, in a concentration-dependent manner. Furthermore, T. himalayense extract inhibited the mitogen-activated protein kinase (MAPK) pathways, such as extracellular signal-regulated kinase-1/2 (ERK), c-Jun N-terminal kinase (JNK), and p38. Therefore, we hypothesized that various components of T. himalayense extract affect multiple factors involved in melanogenesis in B16F10 cells. Our results indicate that T. himalayense extract could potentially be used as a new material for preparing whitening cosmetics.

Screening of Key Components for Melanogenesis Inhibition of Polygonum cuspidatum Extract Based on the Spectrum-Effect Relationship and Molecular Docking.

Polygonum cuspidatum (PC) extract has been listed in the "Catalog of Used Cosmetic Ingredients (2021 Edition)", which can inhibit melanogenesis, thus exerting a whitening effect, and has been widely used in cosmetics. However, there are currently no quality standards for PC extract used in cosmetics, and the bioactive components associated with anti-melanogenesis remain unclear. In view of this, the present study was the first to investigate the spectrum-effect relationship between fingerprints of PC extract and melanogenesis inhibition. Ten batches of PC extract fingerprints were established by HPLC. Pearson's correlation analysis, gray correlation analysis (GRA) and orthogonal partial least squares regression analysis (OPLSR) were used to screen out resveratrol, emodin and physcion as the main whitening active ingredients using the inhibition of tyrosinase in B16F10 cells as the pharmacological index. Then, the melanogenesis inhibitory effects of the above three components were verified by tyrosinase inhibition and a melanin content assay in B16F10 cells. The interaction between small molecules and proteins was investigated by the molecular docking method, and it was confirmed by quantitative real-time PCR (qRT-PCR) that resveratrol, emodin and physcion significantly down-regulated the transcript levels of melanogenesis-related factors. In conclusion, this study established a general model combining HPLC fingerprinting and melanogenesis inhibition and also analyzed the spectrum-effect relationship of PC extract, which provided theoretical support for the quality control of PC extract in whitening cosmetics.

Inhibition of α-melanocyte-stimulating hormone-induced melanogenesis and molecular mechanisms by polyphenol-enriched fraction of Tagetes erecta L. flower.

BACKGROUND: The pursuit for safe and efficacious skin-whitening agents has prompted a dedicated exploration of plant-derived compounds. Notably, Tagetes erecta L. flowers have been used as a medicinal extract and possessed in vitro mushroom tyrosinase activity. However, whether polyphenol-enriched fraction extracted from T. erecta L. flowers (TE) regulates melanogenesis within cellular and animal models has not yet been investigated. PURPOSE: This study aimed to investigate the effect of TE as a prospective inhibitor of melanogenesis. METHODS: Through advanced UPLC-QTof/MS analysis, the components of TE were analyzed. Anti-melanogenic effects of TE were evaluated in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells by measuring cell viability assay, extracellular and intracellular melanin biosynthesis, cyclic adenosine monophosphate (cAMP) production, and melanogenesis-related gene and protein expression. Zebrafish larvae were employed for in vivo studies, assessing both heart rate and melanogenesis. Furthermore, molecular docking analyses were employed to predict the interaction between TE components and the melanocortin 1 receptor (MC1R). Direct binding activity of TE components to MC1R was compared with [Nle4, d-Phe7]-MSH (NDP-MSH). RESULTS: TE was found to contain significant phenolic compounds such as patulitrin, quercetagetin, kaempferol, patuletin, and isorhamnetin. This study revealed that TE effectively inhibits melanin biosynthesis in both in vitro and in vivo models. This inhibition was attributed to interference of TE with the cAMP-cAMP response element-binding protein (CREB)-microphthalmia-associated transcription factor (MITF)-tyrosinase pathway, which plays a pivotal role in regulating melanogenesis. Importantly, TE exhibited the remarkable ability to curtail α-MSH-induced melanogenesis in zebrafish larvae without impacting heart rates. Molecular docking analyses predicted that the components of TE possibly interact with the melanocortin 1 receptor, suggesting their role as potential inhibitors of melanin biosynthesis. However, through the direct binding activity compared with NDP-MSH, any TE components did not directly bind to MC1R, suggesting that TE inhibits α-MSH-induced melanogenesis by inhibiting the cAMP-mediated intracellular signaling pathway. The assessment of anti-melanogenic activity, conducted both in vitro and in vivo, revealed that patulitrin and patuletin exhibited significant inhibitory effects on melanin formation, highlighting their potency as major contributors. DISCUSSION: This investigation demonstrated the considerable potential of TE as a natural remedy endowed with remarkable anti-melanogenic properties. The demonstrated capacity of TE to attenuate melanin production by modulating the cAMP-CREB-MITF-tyrosinase pathway underscores its central role in management of disorders associated with excessive pigmentation. Importantly, the implications of these findings extend to the cosmetics industry, where TE emerges as a prospective and valuable ingredient for the formulation of skin-whitening products. The elucidated interactions between TE components and MC1R not only provide insight into a potential mechanism of action but also elevate the significance of this study. In summary, this study not only contributes to our comprehension of pigmentation-related conditions but also firmly establishes TE as a secure and natural strategy for the regulation of melanin production. The innovative aspects of TE propel it into the forefront of potential interventions, marking a noteworthy advancement in the pursuit of effective and safe solutions for pigmentation disorders.

Arctigenin Induces Apoptosis in Melanoma Cells by Reducing the Expression of BCL-2 and VEGF.

BACKGROUND: To investigate the biological effects of arctigenin on B16-F10 melanoma cells in vitro and to explore its mechanism. METHODS: B16-F10 melanoma cells in vitro were treated with the blank control solution and arctigenin solution of different concentrations, respectively. Cell proliferation and apoptosis were analyzed using the CCK-8 assay and cell loss assay, and the effect of arctigenin on melanoma cell proliferation was evaluated. Western blot was used to analyze the expression of BCL-2 protein and vascular endothelial growth factor (VEGF) in the cells of different groups and to explore the mechanism of action of arctigenin. RESULTS: The proliferation rate of B16-F10 melanoma cells treated with arctigenin solutions was significantly lower than that of the blank control group (P < .05), and the proliferation rate decreased with increasing concentration of arctigenin. The apoptosis rate of B16-F10 melanoma cells treated with arctigenin solutions was significantly higher than that of the blank control group (P < .05), and the apoptosis rate increased with increasing concentration of arctigenin. The expression levels of BCL-2 and VEGF in B16-F10 melanoma cells treated with arctigenin solutions were significantly lower than those in the blank control group (P < .05), and the expression levels decreased as the concentration of arctigenin increased. CONCLUSIONS: Arctigenin can inhibit the proliferation and promote the apoptosis of melanoma cells, and the mechanism may be associated with decreasing the expression of BCL-2 and VEGF in melanoma cells.

Anti-melanogenic effect of exosomes derived from human dermal fibroblasts (BJ-5ta-Ex) in C57BL/6 mice and B16F10 melanoma cells.

Exosomes are involved in intercellular communication by transferring cargo between cells and altering the specific functions of the target cells. Recent studies have demonstrated the therapeutic effects of exosomes in several skin diseases. However, understanding of the effects of exosomes on anti-pigmentation is limited. Therefore, we investigated whether BJ-5ta exosomes (BJ-5ta-Ex) derived from human foreskin fibroblasts regulate melanogenesis and delineated the underlying mechanism. Interestingly, treatment with BJ-5ta-Ex induced decreased melanin content, tyrosinase (TYR) activity, and expression of melanogenesis-related genes, including microphthalmia-related transcription factor (MITF), TYR, tyrosinase-related protein-1 (TRP1), and tyrosinase-related protein-2 (TRP2). In addition, BJ-5ta-Ex downregulated the cAMP/PKA and GSK-3ß/ß-catenin signaling pathways and upregulated the MAPK/ERK signaling pathway. Notably, treatment with BJ-5ta-Ex inhibited α-melanocyte-stimulating hormone-induced melanosome transport and decreased the expression of key proteins involved in melanosome transport, namely, rab27a and melanophilin (MLPH). To further confirm the depigmenting effects of BJ-5ta-Ex, we conducted experiments using a three-dimensional reconstituted human full skin model and ultraviolet B (UVB)-irradiated mouse model. Treatment with BJ-5ta-Ex improved tissue brightness and reduced the distribution of melanosomes. In UVB-irradiated mouse ears, BJ-5ta-Ex reduced the number of active melanocytes and melanin granules. These results demonstrate that BJ-5ta-Ex can be useful for the clinical treatment of hyperpigmentation disorders.

Astaxanthin induces autophagy and apoptosis in murine melanoma B16F10-Nex2 cells and exhibits antitumor activity in vivo.

Countless efforts have been made to prevent and suppress the formation and spread of melanoma. Natural astaxanthin (AST; extracted from the alga Haematococcus pluvialis) showed an antitumor effect on various cancer cell lines due to its interaction with the cell membrane. This study aimed to characterize the antitumor effect of AST against B16F10-Nex2 murine melanoma cells using cell viability assay and evaluate its mechanism of action using electron microscopy, western blotting analysis, terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, and mitochondrial membrane potential determination. Astaxanthin exhibited a significant cytotoxic effect in murine melanoma cells with features of apoptosis and autophagy. Astaxanthin also decreased cell migration and invasion in vitro assays at subtoxic concentrations. In addition, assays were conducted in metastatic cancer models in mice where AST significantly decreased the development of pulmonary nodules. In conclusion, AST has cytotoxic effect in melanoma cells and inhibits cell migration and invasion, indicating a promising use in cancer treatment.

Integrative pharmacology reveals the mechanisms of Erzhi pills, A traditional Chinese formulation, stimulating melanogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: Erzhi pills (EZP), a traditional Chinese medicine formula prescribed for the treatment of vitiligo, has shown promising efficacy. However, the oral bioactive components and mechanisms underlying the promotion of melanogenesis by EZP remain unclear. AIM OF THE STUDY: This study aimed to investigate the pharmacological basis and mechanism of EZP in promoting melanogenesis. MATERIALS AND METHODS: UHPLC-TOF-MS analysis was used to identify absorbed phytochemicals in serum after oral administration of EZP. Network pharmacology methods were used to predict potential targets and pathways involved in the melanogenic activity of EZP, resulting in the construction of a "compound-target-pathway" network. Zebrafish and B16F10 cells were used to evaluate the effects of EZP on tyrosinase activity and melanin content. Western blot and ELISA analyses were used to validate the effects of EZP on melanogenesis-related proteins, including MITF, TYR, CREB, p-CREB, and cAMP. RESULTS: UHPLC-TOF-MS analysis identified 36 compounds derived from EZP in serum samples. Network pharmacology predictions revealed 89 target proteins associated with the identified compounds and closely related to vitiligo. GO and KEGG analyses indicated the involvement of the cAMP/PKA signaling pathway in the promotion of melanogenesis by EZP. Experimental results showed that EZP increased tyrosinase activity and melanin content in zebrafish and B16F10 cells without inducing toxicity. Western blot and ELISA results suggested that the melanogenic effect of EZP may be related to the activation of the cAMP/PKA signaling pathway. These results confirm the feasibility of combining serum pharmacological and network pharmacological approaches. CONCLUSIONS: EZP have the potential to increase tyrosinase activity and melanin content in zebrafish and cells possibly through activation of the cAMP/PKA pathway.

Dietary flavonoid myricetin 3-O-galactoside suppresses α-melanocyte stimulating hormone-induced melanogenesis in B16F10 melanoma cells by regulating PKA and ERK1/2 activation.

Melanogenesis is the process where skin pigment melanin is produced through tyrosinase activity. Overproduction of melanin causes skin disorders such as freckles, spots, and hyperpigmentation. Myricetin 3-O-galactoside (M3G) is a dietary flavonoid with reported bioactivities. M3G was isolated from Limonium tetragonum and its anti-melanogenic properties were investigated in α-melanocyte stimulating hormone-stimulated B16F10 melanoma cells. The in vitro anti-melanogenic capacity of M3G was confirmed by inhibited tyrosinase and melanin production. M3G-mediated suppression of melanogenic proteins, tyrosinase, microphthalmia-associated transcription factor (MITF), and tyrosinase-related proteins (TRP)-1 and TRP-2, were confirmed by mRNA and protein levels, analyzed by RT-qPCR and Western blot, respectively. Furthermore, M3G suppressed Wnt signaling through the inhibition of PKA phosphorylation. M3G also suppressed the consequent phosphorylation of CREB and nuclear levels of MITF. Analysis of MAPK activation further revealed that M3G increased the activation of ERK1/2 while p38 and JNK activation remained unaffected. Results showed that M3G suppressed melanogenesis in B16F10 cells by decreasing tyrosinase production and therefore inhibiting melanin formation. A possible action mechanism was the suppression of CREB activation and upregulation of ERK phosphorylation which might cause the decreased nuclear levels of MITF. In conclusion, M3G was suggested to be a potential nutraceutical with anti-melanogenic properties.

The spread of interferon-γ in melanomas is highly spatially confined, driving nongenetic variability in tumor cells.

Interferon-γ (IFNγ) is a critical antitumor cytokine that has varied effects on different cell types. The global effect of IFNγ in the tumor depends on which cells it acts upon and the spatial extent of its spread. Reported measurements of IFNγ spread vary dramatically in different contexts, ranging from nearest-neighbor signaling to perfusion throughout the entire tumor. Here, we apply theoretical considerations to experiments both in vitro and in vivo to study the spread of IFNγ in melanomas. We observe spatially confined niches of IFNγ signaling in 3-D mouse melanoma cultures and human tumors that generate cellular heterogeneity in gene expression and alter the susceptibility of affected cells to T cell killing. Widespread IFNγ signaling only occurs when niches overlap due to high local densities of IFNγ-producing T cells. We measured length scales of ~30 to 40 µm for IFNγ spread in B16 mouse melanoma cultures and human primary cutaneous melanoma. Our results are consistent with IFNγ spread being governed by a simple diffusion-consumption model and offer insight into how the spatial organization of T cells contributes to intratumor heterogeneity in inflammatory signaling, gene expression, and immune-mediated clearance. Solid tumors are often viewed as collections of diverse cellular "neighborhoods": Our work provides a general explanation for such nongenetic cellular variability due to confinement in the spread of immune mediators.

Ginsenoside Re inhibits melanogenesis and melanoma growth by downregulating microphthalmia-associated transcription factor.

Panax ginseng, also known as Korean ginseng, is a traditional remedy widely used in Asian countries. Its major active compounds are ginsenosides, specifically triterpenoid saponins. Among them, one notable ginsenoside called Re has shown various biological effects, including anti-cancer and anti-inflammatory properties. However, the potential beneficial effects of Re on melanogenesis and skin cancer remain poorly understood. To investigate this, we conducted a comprehensive study using biochemical assays, cell-based models, a zebrafish pigment formation model, and a tumor xenograft model. Our results revealed that Re effectively inhibited melanin biosynthesis in a dose-dependent manner by competitively inhibiting the activity of tyrosinase, an enzyme involved in melanin production. Moreover, Re significantly reduced the mRNA expression levels of microphthalmia-associated transcription factor (MITF), a key regulator of melanin biosynthesis and melanoma growth. Furthermore, Re decreased the protein expression of MITF and its target genes, including tyrosinase, TRP-1, and TRP-2, through a partially ubiquitin-dependent proteasomal degradation mechanism, mediated by the AKT and ERK signaling pathways. These findings indicate that Re exerts its hypopigmentary effects by directly inhibiting tyrosinase activity and suppressing its expression via MITF. Additionally, Re demonstrated inhibitory effects on skin melanoma growth and induced tumor vascular normalization in our in vivo experiments. This study represents the first evidence of Re-mediated inhibition of melanogenesis and skin melanoma, shedding light on the underlying mechanisms. These promising preclinical findings warrant further investigation to determine the suitability of Re as a natural agent for treating hyperpigmentation disorders and skin cancer.

6-Methylcoumarin Promotes Melanogenesis through the PKA/CREB, MAPK, AKT/PI3K, and GSK3ß/ß-Catenin Signaling Pathways.

We investigated the effects of four coumarin derivatives, namely, 6-methylcoumarin, 7-methylcoumarin, 4-hydroxy-6-methylcoumarin, and 4-hydroxy-7-methylcoumarin, which have similar structures on melanogenesis in a murine melanoma cell line from a C57BL/6J mouse called B16F10. Our results showed that only 6-methylcoumarin significantly increased the melanin synthesis in a concentration-dependent manner. In addition, the tyrosinase, TRP-1, TRP-2, and MITF protein levels were found to significantly increase in response to 6-methylcoumarin in a concentration-dependent manner. To elucidate the molecular mechanism whereby 6-methylcoumarin-induced melanogenesis influences the melanogenesis-related protein expression and melanogenesis-regulating protein activation, we further assessed the B16F10 cells. The inhibition of the ERK, Akt, and CREB phosphorylation, and conversely, the increased p38, JNK, and PKA phosphorylation activated the melanin synthesis via MITF upregulation, which ultimately led to increased melanin synthesis. Accordingly, 6-methylcoumarin increased the p38, JNK, and PKA phosphorylation in the B16F10 cells, whereas it decreased the phosphorylated ERK, Akt, and CREB expressions. In addition, the 6-methylcoumarin activated GSK3ß and ß-catenin phosphorylation and reduced the ß-catenin protein level. These results suggest that 6-methylcoumarin stimulates melanogenesis through the GSK3ß/ß-catenin signal pathway, thereby affecting the pigmentation process. Finally, we tested the safety of 6-methylcoumarin for topical applications using a primary human skin irritation test on the normal skin of 31 healthy volunteers. We found that 6-methylcoumarin did not cause any adverse effects at concentrations of 125 and 250 µM. Our findings indicate that 6-methylcoumarin may be an effective pigmentation stimulator for use in cosmetics and the medical treatment of photoprotection and hypopigmentation disorders.

Silver Carp (Hypophthalmichthys molitrix) Scale Collagen Peptides-1 (SCPs1) Inhibit Melanogenesis through Downregulation of the cAMP-CREB Signaling Pathway.

The mechanism of silver carp scale collagen peptides (SCPs1) on melanogenesis and its mechanism of action were examined in mouse melanoma cells (B16). The cell viability and effects of SCPs1 on intracellular tyrosinase (TYR) activity and melanin, reactive oxygen species (ROS), glutathione (GSH) and cyclic adenosine monophosphate (cAMP) content were examined. The regulatory mechanism of SCPs1 on the cAMP response element-binding protein (CREB) signaling pathway was analyzed. The cell viability of the SCPs1 group was >80% (0.01-1 mg/mL) and the inhibitory rate of SCPs1 on B16 cell melanin increased in a dose-dependent manner. The highest inhibitory rate of SCPs1 on melanin content reaching 80.24%. SCPs1 significantly increased the GSH content and decreased the tyrosinase activity, as well as the content of ROS and cAMP. Western blot analysis showed that SCPs1 significantly inhibited melanocortin-1 receptor (MC1R) expression and CREB phosphorylation in the cAMP-CREB signaling pathway, leading to downregulation of microphthalmia-associated transcription factor (MITF) and the expression of TYR, TYR-related protein-1 (TRP-1) and TRP-2. SCPs1 also inhibited the expression of MC1R, MITF, TYR, TRP-1 and TRP-2 at the transcriptional level. Taken together, SCPs1 inhibited melanin synthesis through the downregulation of the cAMP-CREB signaling pathway. Fish-derived collagen peptides could potentially be applied in skin whitening products.

Enzyme-treated caviar extract ameliorates melanogenesis in UVB-induced SKH-1 hairless mice.

This study investigated anti-melanogenesis effects of enzyme-treated caviar extract (CV) in murine melanoma B16F10 cells and SKH-1 hairless mice. To induce melanin production in vitro and in vivo studies, B16F10 cells were treated with 3-Isobutyl-1-methylxanthine (IBMX), and SKH-1 hairless mice were irradiated with UVB, respectively. The expression of melnogenesis-related factors and signaling molecules were analyzed by ELISA and western blotting. 50, 100 and 200 µg/mL of CV significantly decreased the melanin contents and the activities of tyrosinase, nitric oxide, glutathione, and cAMP, melanogenesis factor, in B16F10 cells treated IBMX. In addition, CV significantly suppressed the expression of melanogenesis proteins such as pPKA, pCREB, MITF, TRP-1and TRP-2. Similarly, results of oral administration of CV (20, 50 and 100 mg/kg) for 8 weeks in UVB-Induced SKH-1 hairless mice, the expression of melanogenesis-related factor tyrosinase, nitric oxide, and cAMP and protein expression of pPKA, pCREBa, MITF, TRP-1and TRP-2 was significantly reduced. In particular, 100 mg/kg of CV exhibited an excellent effect similar to control group. Therefore, we suggest the possibility of developing CV as a food supplement having skin whitening effects by ameliorating melanogenesis.