Melanin-Ce6-loaded polydopamine nanoparticles-based enhanced phototherapy for B16 melanoma cancer cells.

Melanoma is one of the most aggressive and lethal types of cancer owing to its metastatic propensity and chemoresistance property. An alternative therapeutic option is photodynamic and photothermal therapies (PDT/PTT), which employ near-infrared (NIR) light to generate heat and reactive oxygen species (ROS). As per previous reports, Melanin (Mel), and its synthetic analogs (i.e. polydopamine nanoparticles) can induce NIR light-mediated heat energy, thereby selectively targeting and ameliorating cancer cells. Similarly, chlorin e6 (Ce6) also has high ROS generation ability and antitumor activity against various types of cancer. Based on this tenet, In the current study, we have encapsulated Mel-Ce6 in a polydopamine (PDA) nanocarrier (MCP NPs) synthesized by the oxidation polymerization method. The hydrodynamic diameter of the synthesized spherical MCP NPs was 139 ± 10 nm. The MCP NPs, upon irradiation with NIR 690 nm laser for 6 min, showed photothermal efficacy of more than 50 °C. Moreover, the red fluorescence in the MCP NPs due to Ce6 can be leveraged for diagnostic purposes. Further, the MCP NPs exhibited considerable biocompatibility with the L929 cell line and exerted nearly 70% ROS-mediated cytotoxicity on the B16 melanoma cell line after the laser irradiation. Thus, the prepared MCP NPs could be a promising theranostic agent for treating the B16 melanoma cancer.

The potential of Paeonia lactiflora pall seeds oil as a pure natural cosmetics raw material: In Vitro findings.

BACKGROUND: As a traditional Chinese herbal medicine, Paeonia lactiflora Pall is rich in various active ingredients such as polysaccharides and total flavonoids while having ornamental value. It has potential application value in the development of food and cosmetics. OBJECTIVE: To study the in vitro efficacy of Paeonia lactiflora Pall seeds oil. METHODS: Firstly, the levels of linolenic acid and linoleic acid in Paeonia lactiflora Pall seeds oil were quantified using gas chromatography. The impact of Paeonia lactiflora Pall seeds oil on the proliferation rate of B16F10 cells was assessed through the CCK-8 method, while the melanin content of B16F10 cells was determined using the sodium hydroxide lysis method. The inhibitory effects of Paeonia lactiflora Pall seeds oil on elastase, collagenase and hyaluronidase were evaluated by biochemical techniques in vitro. Lastly, the hen's egg chorioallantoic membrane test (HET-CAM) was conducted to confirm the absence of eye irritation caused by Paeonia lactiflora Pall seeds oil. RESULTS: Paeonia lactiflora Pall seeds oil within a certain volume concentration range (0.5%-4%) had no effect on the proliferation of B16F10 cells. Paeonia lactiflora Pall seeds oil showed significant inhibition of elastase, collagenase and hyaluronidase. Notably, the highest concentration tested, 4% Paeonia lactiflora Pall seed oil, yielded the most pronounced outcomes without causing any irritation. CONCLUSION: A certain concentration of Paeonia lactiflora Pall seeds oil has a significant effect on decreasing the melanin content in B16F10 cells and inhibiting the activities of elastase, collagenase, and hyaluronidase, which can provide a reference for the development of pure natural cosmetics raw materials.

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.

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.

The whitening effect of cuscutin responsible for traditional use of Bergenia purpurascens.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots and rhizomes of Bergenia purpurascens (Hook. f. et Thomson) Engl., was used as a sunscreen to protect against ultraviolet rays in Tibet of China historically, but its skin whitening constituents and pharmacological effects of this plant remained unknown. AIM OF THE STUDY: To investigate the anti-melanogenesis effect of B. purpurascens in vitro and in vivo, and then explore the preliminary mechanism. MATERIALS AND METHODS: An ultraviolet B (UVB)-induced skin injury model of mice was used to verify the ameliorative effect of B. purpurascens extract (BPE) on ultraviolet damage. Then, alpha-melanocyte stimulating hormone (α-MSH)-induced murine melanoma cell line (B16F10) melanin generation model was further adopted to approval the effects of BPE and its bioactive compound, cuscutin, in vitro. Moreover, α-MSH stimulated melanogenesis model in zebrafish was employed to confirm the anti-pigmentation effect of cuscutin. Then, proteins expressions associated with melanin production were observed using western blotting assay to explore preliminary mechanism. RESULTS: BPE inhibited UVB-induced mice injury and restored skin barrier function observably in vivo. BPE and cuscutin suppressed the overproduction of melanin in α-MSH induced B16F10 significantly, in which cuscutin exhibited better effect than well-known whitening agent α-arbutin at same 10 µg/mL concentration. Moreover, the pigmentation of zebrafish embryo was decreased by cuscutin. Finally, cuscutin showed significant downregulation of expressions of tyrosinase (TYR) and tyrosinase related protein-1 (TRP-1), TRP-2 and microphthalmia-associated transcription factor (MITF) in the melanogenic signaling pathway. CONCLUSION: B. purpurascens extract and its major bioactive constituent, cuscutin, showed potent anti-melanogenesis and skin-whitening effect by targeting TYR and TRP-2 proteins for the first time, which supported its traditional use.

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.

Natural Products Induce Different Anti-Tumor Immune Responses in Murine Models of 4T1 Mammary Carcinoma and B16-F10 Melanoma.

Natural products obtained from Petiveria alliacea (Anamu-SC) and Caesalpinia spinosa (P2Et) have been used for cancer treatment, but the mechanisms by which they exert their antitumor activity appear to be different. In the present work, we show that the Anamu-SC extract reduces tumor growth in the 4T1 murine mammary carcinoma model but not in the B16-F10 melanoma model, unlike the standardized P2Et extract. Both extracts decreased the levels of interleukin-10 (IL-10) in the B16-F10 model, but only P2Et increased the levels of tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ). Likewise, co-treatment of P2Et and doxorubicin (Dox) significantly reduced tumor size by 70% compared to the control group, but co-treatment of Anamu-SC with Dox had no additive effect. Analysis of intratumoral immune infiltrates showed that Anamu-SC decreased CD4+ T cell frequency more than P2Et but increased CD8+ T cell frequency more significantly. Both extracts reduced intratumoral monocytic myeloid-derived suppressor-like cell (M-MDSC-LC) migration, but the effect was lost when co-treated with doxorubicin. The use of P2Et alone or in co-treatment with Anamu-SC reduced the frequency of regulatory T cells and increased the CD8+/Treg ratio. In addition, Anamu-SC reduced glucose consumption in tumor cells, but this apparently has no effect on IFNγ- and TNFα-producing T cells, although it did reduce the frequency of IL-2-producing T cells. The efficacy of these herbal preparations is increasingly clear, as is the specificity conditioned by tumor heterogeneity as well as the different chemical complexity of each preparation. Although these results contribute to the understanding of specificity and its future benefits, they also underline the fact that the development of each of these standardized extracts called polymolecular drugs must follow a rigorous path to elucidate their biological activity.

67-kDa laminin receptor mediates oolonghomobisflavan B-induced cell growth inhibition in melanoma.

BACKGROUND: Oolonghomobisflavans are unique polyphenols found in oolong teas. Oolonghomobisflavan B (OHBFB), a dimer of (-)-epigallocatechin-3-O-gallate (EGCG), is an active compound found in green tea. PURPOSE: OHBFB has been reported to exert an inhibitory effect on lipase enzyme activity. However, little is known regarding its intercellular signaling induction effect. Further, there are no reports describing the anti-cancer effects of OHBFB. METHODS: The effect of OFBFB on B16 melanoma cells was evaluated by cell counting, and its mechanisms were determined by western blot analysis with or without protein phosphatase 2A (PP2A) inhibitor treatment. Intracellular cyclic adenosine monophosphate (cAMP) levels were evaluated by time-resolved fluorescence resonance energy transfer analysis. Quartz crystal microbalance (QCM) analysis was performed to assess the binding of OHBFB to 67LR. RESULTS: Cell growth assay and western blot analyses showed that OHBFB inhibited melanoma cell growth, followed by myosin phosphatase target subunit 1 (MYPT1) and myosin regulatory light chain (MRLC) dephosphorylation via protein phosphatase 2A (PP2A)-dependent mechanisms. These effects are mediated by intracellular cAMP- and protein kinase A (PKA) A-dependent mechanisms. QCM analysis identified the 67-kDa laminin receptor (67LR) as an OHBFB receptor with a Kd of 3.7 µM. We also demonstrated for the first time that OHBFB intake suppresses tumor growth in vivo. CONCLUSIONS: Taken together, these results indicate that the cAMP/PKA/PP2A/MYPT1/MRLC pathway is a key mediator of melanoma cell growth inhibition following OHBFB binding to 67LR and that OHBFB suppresses tumor growth in vivo.

Piper nigrum extract suppresses tumor growth and enhances the antitumor immune response in murine models of breast cancer and melanoma.

Although the antitumor effect of P. nigrum has been widely studied, research related to its possible immunomodulatory effects is relatively scarce. Here, the antitumor and immunomodulatory activity of an ethanolic extract of P. nigrum were evaluated in the murine models of 4T1 breast cancer and B16-F10 melanoma. In vitro evaluations showed that the P. nigrum extract has cytotoxic activity, induces apoptotic cell death, and has a pro-oxidant effect in both cell lines, but it regulates glucose uptake differently in both lines, decreasing it in 4T1 but not in B16-F10. P. nigrum extract significantly reduced tumor size in both models and decreased the occurrence of macrometastases in 4T1 model. Evaluation of immune subpopulations by flow cytometry revealed that the P. nigrum extract significantly increases the frequency of dendritic cells and activated CD8+ T cells and decreases the frequency of myeloid-derived suppressor like cells and Tregs in the tumor microenvironment of both models but with different dynamics. Our findings strongly suggest that the P. nigrum extract exerts immunomodulatory functions, slightly related to the modulation of cellular energy metabolism, which could ultimately contribute to the promising antitumor effect of P. nigrum.

Combinational photodynamic and photothermal - based therapies for melanoma in mouse models.

BACKGROUND: Melanoma is a highly metastatic skin cancer with limited response to current therapies in advanced patients. To overcome resistance, novel treatments based on photodynamic and photothermal therapies (PDT and PTT, respectively) have been developed to treat melanoma in preclinical murine models. Despite success inhibiting implanted tumors' growth, there has been limited evaluation of their long-term effectiveness in preventing metastasis, recurrence, or improving survival rates. METHODS: Combined and multidrug therapies based on PDT and/or PTT to treat cutaneous malignant melanoma in the preclinical mouse model were reviewed from 2016 onwards. PubMed® was the database in which the search was performed using mesh search algorithms resulting in fifty-one studies that comply with strict inclusion rules of screening. RESULTS: B16 melanoma-bearing C57BLACK6 mice model was the most used to evaluate immunotherapies, chemotherapies, and targeted therapies in combination with PDT and/or PTT. Combined therapies demonstrated a synergistic effect, resulting in intense antitumor activity. The most extensively studied protocol for developing metastatic models involved the intravenous administration of malignant cells, with some combined therapies being tested. Furthermore, the review presents the composition of the nanostructures utilized for delivering the drugs and light-responsive agents and the treatment plans for each combined approach. CONCLUSIONS: The identified mechanisms to simulate metastatic melanoma models and the therapeutic combinations may aid in evaluating the systemic protection of combined PDT and PTT-based therapies, particularly in conducting short-term preclinical experiments. Such simulations could have relevance to clinical studies.

Hydrolyzed Conchiolin Protein (HCP) Extracted from Pearls Antagonizes both ET-1 and α-MSH for Skin Whitening.

Pearl powder is a famous traditional Chinese medicine that has a long history in treating palpitations, insomnia, convulsions, epilepsy, ulcers, and skin lightining. Recently, several studies have demonstrated the effects of pearl extracts on protection of ultraviolet A (UVA) induced irritation on human skin fibroblasts and inhibition of melanin genesis on B16F10 mouse melanoma cells. To further explore the effect we focused on the whitening efficacy of pearl hydrolyzed conchiolin protein (HCP) on human melanoma MNT-1 cells under the irritation of alpha-melanocyte-stimulating hormone (α-MSH) or endothelin 1 (ET-1) to evaluate the intracellular tyrosinase and melanin contents, as well as the expression levels of tyrosinase (TYR), tyrosinase related protein 1 (TRP-1), and dopachrome tautomerase (DCT) genes and related proteins. We found that HCP could decrease the intracellular melanin content by reducing the activity of intracellular tyrosinase and inhibiting the expression of TYR, TRP-1, DCT genes and proteins. At the same time, the effect of HCP on melanosome transfer effect was also investigated in the co-culture system of immortalized human keratinocyte HaCaT cells with MNT-1. The result indicated that HCP could promote the transfer of melanosomes in MNT-1 melanocytes to HaCaT cells, which might accelerate the skin whitening process by quickly transferring and metabolizing melanosomes during keratinocyte differentiation. Further study is needed to explore the mechanism of melanosome transfer with depigmentation.

Melanogenesis Inhibitory Activity, Chemical Components and Molecular Docking Studies of Prunus cerasoides Buch.-Ham. D. Don. Flowers.

Safe depigmenting agents are currently increasing in the cosmetic or pharmaceutical industry because various compounds have been found to have undesirable side effects. Therefore, the present study aimed to investigate the melanogenesis inhibitory effects of Prunus cerasoides Buch. -Ham. D. Don. flower extracts and their molecular mechanism in B16F10 mouse melanoma cells. Moreover, we also examined phenolic and flavonoid contents, antioxidant activity, chemical constituents of potential extracts, and molecular docking. The highest phenolic and flavonoid contents with the greatest scavenging activity were found in the butanol extract of the P. cerasoides flower compared to other extracts. From all extracts, only crude, diethyl ether, and butanol extracts showed an inhibition of mushroom tyrosinase activity, cellular tyrosinase activity, and melanin content as well as the downregulation of the gene expression of the microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2) in α-MSH-stimulated B16F10 cells. Based on the molecular docking study, n-hexadecanoic acid, heptadecanoic acid, octadecanoic acid, 9,12-octadecadienoic acid, 9,12,15-octadecanoic acid, and eicosanoic acid might show an inhibitory effect against tyrosinase and MITF. In conclusion, this finding demonstrates that both the diethyl ether and butanol extracts of the P. cerasoides flower can effectively reduce tyrosinase activity and melanin synthesis through the downregulation of the melanogenic gene expression in B16F10 cells and through the molecular docking study. Taken together, the diethyl ether and butanol extracts of the P. cerasoides flower could be an anti-melanogenic ingredient for hyperpigmentary or melasma treatment.

Pueraria protein extract inhibits melanogenesis and promotes melanoma cell apoptosis through the regulation of MITF and mitochondrial­related pathways.

Pueraria Lobata Radix (P. Lobata Radix) is an edible traditional Chinese medicine that contains various active compounds. Proteins from P. Lobata Radix have become the subject of increased interest in recent years. In evaluating the whitening effect on the skin, the present study found that the P. Lobata Radix water­soluble total protein extract (PLP) had the strongest inhibitory effect on tyrosinase activity. In the present study, the anti­melanogenic effect of PLP and the inhibitory effect on B16 melanoma cells were investigated. PLP significantly reduced the tyrosinase activity and melanin content in B16 melanoma cells. Mechanistically, PLP inhibited melanogenesis by decreasing the expression of tyrosinase, tyrosinase­related protein (TRP)­1 and TRP­2 through downregulation of the microphthalmia­associated transcription factor (MITF) gene, which was mediated by inhibition of p38 mitogen­activated protein kinase signaling. In addition, PLP inhibited cell viability and triggered apoptosis of B16 cells in a dose­dependent manner. Exposure to PLP reduced the mitochondrial membrane potential (MMP) and decreased ATP generation, leading to mitochondria­related apoptosis of B16 melanoma cells. The expression levels of succinate dehydrogenase (SDH) and its two related subunits (SDHA and SDHB) were downregulated significantly by PLP, which may be associated with the regulation of mitochondrial energy metabolism by PLP. These results may explain why MMP collapse and reduced ATP generation were observed in B16 melanoma cells treated with PLP. Finally, the present study demonstrated that the inhibition of melanin synthesis by PLP was correlated with the regulation of antioxidant enzymes to reduce reactive oxygen species levels. These results suggested that PLP inhibits melanogenesis by downregulating the expression of MITF­related melanogenic enzymes and triggering apoptosis through mitochondria­related pathways.

Mechanism and inhibitory effects of cactus (Opuntia dillenii) extract on melanocytes and its potential application for whitening cosmetics.

Penghu cactus (Opuntia dillenii [Ker.] Haw) is a cactus plant that commonly grows in Penghu Island, Taiwan, Republic of China (ROC). However, still lack of scientific study on the Opuntia dillenii [Ker.] Haw extract on skin-whitening-associated tyrosinase activity and melanin production. The activities of its extract in melanogenesis were investigated in this article. In this experiment, we used an extract from the Penghu cactus (Opuntia dillenii [Ker.] Haw) to study its tyrosinase inhibition, anti-melanin generation, UV-protection effects and wound healing capacity in B16-F10 melanocytes. Without reducing cell growth greatly or causing cell death, 20 g/L cactus extract effectively inhibited the melanin production of B16-F10 cells, and melanogenesis was induced by 3-isobutyl-1-methylxanthine. The cactus extract could also promote cell proliferation. Cactus extract treatment decreased the mRNA expression of insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) and increased that of transforming growth factor ß (TGF-ß). Thus, it could reduce cell melanin production and promote cell growth but by also reducing IGF-1 and VEGF mRNA expression, may reduce wound scarring and prevent tumor proliferation and swelling. Increasing TGF-ß mRNA expression can help increase collagen to remove wrinkles and help in wound healing. Skin patch test results agreed with in vitro results with B16-F10 melanoma cells. The cactus extract significantly inhibited tyrosinase activity and reduced melanin production, showing a whitening effect on skin tests. Cactus may be a good natural candidate for inhibiting melanin production and promoting cell proliferation.

Morus alba L. root decreases melanin synthesis via sphingosine-1-phosphate signaling in B16F10 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Morus alba L. has long been used for beauty in many Asian countries and regions, including anti-aging and hyperpigmentation. AIM OF THE STUDY: This study aimed at the inhibitory effect of Morus alba L. root on melanogenesis in B16F10 melanoma cells and the mechanism involved. MATERIALS AND METHODS: This study evaluated the anti-melanogenic effect of Morus alba L. root extract (MAR) on B16F10 melanoma cells by assessing cell viability, melanin accumulation, cellular tyrosinase activity, intra/inter-cellular S1P levels, cellular S1P-related metabolic enzyme activity, and western blot analysis. In addition, the potential S1P lyase (S1PL) inhibitory constituents in MAR were identified by LC-MS/MS. RESULTS: Without affecting the viability of B16F10 melanoma cells, MAR inhibited intracellular tyrosinase activity in a dose-dependent manner, thereby reducing the accumulation of melanin. MAR also downregulated the expression level of MITF via activating the ERK signaling pathway. Furthermore, MAR increased the intra/inter-cellular S1P by inhibiting S1PL. Several compounds with inhibitory S1PL activity have been identified in MAR, such as mulberroside A and oxyresveratrol. CONCLUSIONS: The anti-melanogenic effects of MAR mainly involve promoting MITF degradation mediated via S1P-S1PR3-ERK signaling through increasing cellular S1P levels by inhibiting S1PL activity.

Combination of magnetic hyperthermia and immunomodulators to drive complete tumor regression of poorly immunogenic melanoma.

Hyperthermia using magnetic nanoparticles enables tumor-specific heating and can destroy tumor tissues. This approach works as in situ vaccination with tumor antigens released from dying tumor cells. However, in situ vaccination caused by magnetic hyperthermia is often insufficient to induce complete regression of poorly immunogenic tumors surrounded by an immunosuppressive microenvironment. In this study, we explored a novel strategy for immunotherapy using magnetic hyperthermia to regress poorly immunogenic melanoma. Magnetic hyperthermia induced tumor cell death in a B16-F10 melanoma mouse model. After hyperthermia treatment, we found elevated levels of HMGB1, which is known to be released from dying cells to promote inflammation, and the proinflammatory cytokine TNF-α was increased in serum of the mice. Systemic administration of glycyrrhizin, an HMGB1 inhibitor, reduced the levels of TNF-α in serum and successfully delayed the regrowth of tumors after magnetic hyperthermia. To achieve complete tumor regression, TLR9 activation by intratumor injection of CpG was combined with systemic administration of anti-PD-1 antibody and anti-CTLA-4 antibody. The combination therapy of magnetic hyperthermia at 46°C with the immunomodulators (glycyrrhizin+CpG+anti-PD-1+anti-CTLA-4) achieved complete tumor regression in 80% of growing 5-mm B16-F10 tumors. These findings have important implications for the development of novel cancer immunotherapy using magnetic hyperthermia for poorly immunogenic tumors.

Alantolactone Inhibits Melanoma Cell Culture Viability and Migration and Promotes Apoptosis by Inhibiting Wnt/ß-Catenin Signaling.

BACKGROUND: Melanoma is a highly invasive and metastatic malignant tumor originating from melanocytes and is associated with a poor prognosis. Surgical resection and chemotherapy are currently the main therapeutic options for malignant melanoma; however, their efficacy is poor, highlighting the need for the development of new, safe, and effective drugs for the treatment of this cancer. OBJECTIVE: To investigate the effects of alantolactone (ALT) on the proliferative, migratory, invasive, and apoptotic ability of malignant melanoma cells and explore its potential anticancer mechanism. METHODS: Melanoma cells (A375 and B16) were treated with different concentrations (4, 6, 8, and 10 µmol/L) of ALT, with DMSO and no treatment serving as controls. The effects of the different concentrations of the drug on cell proliferation were assessed by crystal violet staining and CCK-8 assay. The effects on cell migration and invasion were detected by wound healing and Transwell assays, respectively. Flow cytometry was used to evaluate the effects of the drug on apoptosis and the cell cycle. ALT target genes in melanoma were screened using network pharmacology. Western blotting was used to measure the expression levels of the proliferation-related protein PCNA; the apoptosisrelated proteins Bax, Bcl-2, and caspase-3; the invasion and metastasis-related proteins MMP-2, MMP-7, MMP-9, vimentin, E-cadherin, and N-cadherin; and the canonical Wnt signaling pathway-related proteins ß-catenin, c-Myc, and p-GSK3ß. In addition, an l model of melanoma was established by the subcutaneous injection of A375 melanoma cells into nude mice, following which the effects of ALT treatment on malignant melanoma were determined in vivo. RESULTS: Compared with the controls, the proliferative, migratory, and invasive capacity of ALT-treated melanoma cells was significantly inhibited, whereas apoptosis was enhanced (P<0.01), showing effects that were exerted in a dose-dependent manner. The expression levels of the pro-apoptotic proteins Bax and caspase-3, as well as those of the interstitial marker E-cadherin, were upregulated in melanoma cells irrespective of the ALT concentration (P<0.05). In contrast, the expression levels of the anti-apoptotic protein Bcl-2, the proliferation-related protein PCNA, and the invasion and metastasis-related proteins MMP-2, MMP-7, MMP-9, N-cadherin, and vimentin were downregulated (P<0.05). The network pharmacology results indicated that GSK3ß may be a key ALT target in melanoma. Meanwhile, western blotting assays showed that ALT treatment markedly suppressed the expression of ß-catenin as well as that of its downstream effector c-Myc, and could also inhibit GSK3ß phosphorylation. CONCLUSION: ALT can effectively inhibit the culture viability, migration, and invasion of A375 and B16 melanoma cells while also promoting their apoptosis. ALT may exert its anti-melanoma effects by inhibiting the Wnt/ß-catenin signaling pathway. Combined, our data indicate that ALT has the potential as an effective and safe therapeutic drug for the treatment of melanoma.

Chemical Composition and Tyrosinase Inhibitory Activities of Fatty Acids Obtained from Heterotrophic Microalgae, S. limacinum and C. cohnii.

Tyrosinase is the rate-limiting enzyme for melanin production in plant and mammalian cells. Upregulation of this enzyme results in hyperpigmentation disorders. In order to treat pigmentation problems, novel skin whitening compounds are extremely screened. It is found that fatty acids based on their saturation levels either increase or decrease tyrosinase enzyme activity. Thus, fatty acids and their compositions are promising candidates for the treatment of hyperpigmentation or hypopigmentation disorders. Microalgae are rich in both saturated and unsaturated fatty acids, as well. In this study, C. cohnii and S. limacinum fatty acids were evaluated as tyrosinase inhibitor candidates. Mushroom tyrosinase activity studies displayed that both extracts increase tyrosinase enzyme activity dose-dependently. On the other hand, S. limacinum at 200 µg ml-1 concentration almost decreased half of tyrosinase enzyme activity in B16-F10 cells. Besides, it was 3 times more efficient for tyrosinase enzyme activity inhibition and 2 times more effective to decrease melanin synthesis compared to C. cohnii. Considering low toxicity to B16-F10 melanoma and healthy keratinocyte cells (HaCaT), S. limacinum fatty acids could be a suitable source for lipid-based tyrosinase inhibitory functional cosmetics products.

Guttiferone E Displays Antineoplastic Activity Against Melanoma Cells.

Guttiferone E (GE) is a benzophenone found in Brazilian red propolis. In the present study, the effect of GE on human (A-375) and murine (B16-F10) melanoma cells was investigated. GE significantly reduced the cellular viability of melanoma cells in a time-dependent manner. In addition, GE demonstrated antiproliferative effect, with IC50 values equivalent to 9.0 and 6.6 µM for A-375 and B16-F10 cells, respectively. The treatment of A-375 cells with GE significantly increased cell populations in G0/G1 phase and decreased those in G2/M phase. Conversely, on B16-F10 cells, GE led to a significant decrease in the populations of cells in G0/G1 phase and concomitantly an increase in the population of cells in phase S. A significantly higher percentage of apoptotic cells was observed in A-375 (43.5%) and B16-F10 (49.9%) cultures after treatment with GE. Treatments with GE caused morphological changes and significant decrease to the melanoma cells' density. GE (10 µM) inhibited the migration of melanoma cells, with a higher rate of inhibition in B16-F10 cells (73.4%) observed. In addition, GE significantly reduced the adhesion of A375 cells, but showed no effect on B16-F10. Treatment with GE did not induce changes in P53 levels in A375 cultures. Molecular docking calculations showed that GE is stable in the active sites of the tubulin dimer with a similar energy to taxol chemotherapy. Taken together, the data suggest that GE has promising antineoplastic potential against melanoma.

SNA077, an Extract of Marine Streptomyces sp., Inhibits Melanogenesis by Downregulating Melanogenic Proteins via Inactivation of cAMP/PKA/CREB Signaling.

Excess melanin in skin is known to be the main cause of hyper-pigmentary skin diseases such as freckles and lentigo. This study aimed to evaluate the depigmenting efficacy of an extract from the marine microorganism strain, Streptomyces sp. SNA077. To determine the anti-melanogenic efficacy of SNA077, we assessed the melanin contents of SNA077-treated B16, Melan-a, and MNT-1 cells. We observed the expression of key enzymes in melanogenesis via qRT-PCR and Western blot analyses. We further estimated the skin-whitening effect of SNA077 using a skin-equivalent model. SNA077 dramatically decreased the melanin production of B16 cells, Melan-a, and MNT-1 cells. In B16 cells treated with SNA077, the activity of cellular tyrosinase was clearly inhibited. In addition, the mRNA and protein expression levels of melanogenic genes were suppressed by SNA077 treatment in B16 and MNT-1 cells. Upstream of tyrosinase, the expression levels of phospho-CREB, phospho-p38, PKA activity, cyclic AMP production, and MC1R gene expression were inhibited by SNA077. Finally, SNA077 clearly showed a skin-brightening effect with a reduced melanin content in the skin tissue model. Collectively, our results suggest for the first time that an extract of marine Streptomyces sp. SNA077 could be a novel anti-melanogenic material for skin whitening.