B16F10 Melanoma Cells Research Articles

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-3β (GSK-3β) 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 GSK3β/β-catenin, AKT, PKA/CREB, and MAPK. These findings suggest that 7H-4M has the potential to prevent the development of pigmentation diseases.

Norcantharidin Nanoemulsion Development, Characterization, and In Vitro Antiproliferation Effect on B16F1 Melanoma Cells.

Melanoma is a highly lethal type of cancer that has had an increase in incidence in the last decades. Nevertheless, current therapies lack effectiveness and have highly disabling side effects, which calls for new therapeutic strategies. Norcantharidin (NCTD) is an acid derivative with potential antitumor activity isolated from natural blister beetles. However, its solubility limitations restrict its use. To address this issue, we developed an oil-in-water nanoemulsion using commonly available cosmetic ingredients, which increased NCTD solubility 10-fold compared to water. The developed nanoemulsion showed a good droplet size and homogeneity, with adequate pH and viscosity for skin application. In vitro drug release studies showed a sustained release profile, ideal for prolonged therapeutic effects. Accelerated stability studies proved that the formulation was reasonably stable under stress conditions, with particle separation fingerprints, instability index, particle size, and sedimentation velocity analyses being conducted. To assess the therapeutic potential of the developed formulation, in vitro studies were conducted on melanoma B16F1 cells; results showed an IC50 of 1.026 +/- 0.370 mg/kg, and the cells' metabolic activity decreased after exposure to the NCTD nanoemulsion. Hence, a new "easy-to-make" nanoformulation with therapeutic potential on melanoma cells was developed, as a possible adjuvant for future melanoma treatment.

Fermented Aronia melanocarpa Inhibits Melanogenesis through Dual Mechanisms of the PI3K/AKT/GSK-3β and PKA/CREB Pathways.

UV light causes excessive oxidative stress and abnormal melanin synthesis, which results in skin hyperpigmentation disorders such as freckles, sunspots, and age spots. Much research has been carried out to discover natural plants for ameliorating these disorders. Aronia melanocarpa contains various polyphenolic compounds with antioxidative activities, but its effects on melanogenesis have not been fully elucidated. In this study, we investigated the inhibitory effect of fermented Aronia melanocarpa (FA) fermented with Monascus purpureus on melanogenesis and its underlying mechanism in the B16F10 melanoma cell line. Our results indicate that FA inhibited tyrosinase activity and melanogenesis in alpha-melanocyte-stimulating hormone (α-MSH)-induced B16F10 cells. FA significantly downregulated the PKA/CREB pathway, resulting in decreased protein levels of tyrosinase, TRP-1, and MITF. FA also inhibited the transcription of MITF by increasing the phosphorylation levels of both GSK3β and AKT. Interestingly, we demonstrated that these results were owing to the significant increase in gallic acid, a phenolic compound of Aronia melanocarpa produced after the fermentation of Monascus purpureus. Taken together, our research suggests that Aronia melanocarpa fermented with Monascus purpureus acts as a melanin inhibitor and can be used as a potential cosmetic or therapeutic for improving hyperpigmentation disorders.

Therapeutic Effects of WT1 Silencing via Respiratory Administration of Neutral DOPC Liposomal-siRNA in a Lung Metastasis Melanoma Murine Model

The lungs represent a frequent target for metastatic melanoma as they offer a high-oxygen environment for tumor development. The overexpression of the WT1 protein has been associated with the occurrence of melanoma. In this study, we evaluated the effects of silencing the WT1 protein by siRNA in both in vitro in the B16F10 melanoma cell line and in vivo in a murine model of lung metastatic melanoma. We did this by implementing a novel respiratory delivery strategy of a neutral DOPC liposomal-siRNA system (L-siRNA). In vitro studies showed an effective silencing of the WT1 protein in the siRNAs' WT1-treated cells when compared with controls, resulting in a loss of the cell's viability and proliferation by inducing G1 arrest, the inhibition of the migration and invasion capacities of the cells, as well as the induction of apoptosis. In vivo, the respiratory administration of L-WT1 siRNA showed an efficient biodistribution on the lungs. After two weeks of treatment, the silencing of the WT1 protein resulted in an important antitumor activity that reduced the tumor weight. In the survival study, L-WT1 treatment could significantly delay the death of the animals. This work demonstrates the efficacy of the L-siRNA respiratory administration as a novel therapy to reduce pulmonary tumors and to increase survivability by silencing specific cancer oncogenes as WT1.

Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation

Filopodia are dynamic, finger-like actin-filament bundles that overcome membrane tension by forces generated through actin polymerization at their tips to allow extension of these structures a few microns beyond the cell periphery. Actin assembly of these protrusions is regulated by accessory proteins including heterodimeric capping protein (CP) or Ena/VASP actin polymerases to either terminate or promote filament growth. Accordingly, the depletion of CP in B16-F1 melanoma cells was previously shown to cause an explosive formation of filopodia. In Ena/VASP-deficient cells, CP depletion appeared to result in ruffling instead of inducing filopodia, implying that Ena/VASP proteins are absolutely essential for filopodia formation. However, this hypothesis was not yet experimentally confirmed. Here, we used B16-F1 cells and CRISPR/Cas9 technology to eliminate CP either alone or in combination with Ena/VASP or other factors residing at filopodia tips, followed by quantifications of filopodia length and number. Unexpectedly, we find massive formations of filopodia even in the absence of CP and Ena/VASP proteins. Notably, combined inactivation of Ena/VASP, unconventional myosin-X and the formin FMNL3 was required to markedly impair filopodia formation in CP-deficient cells. Taken together, our results reveal that, besides Ena/VASP proteins, numerous other factors contribute to filopodia formation.

A Sulfur Containing Melanogenesis Substrate, N-Pr-4-S-CAP as a Potential Source for Selective Chemoimmunotherapy of Malignant Melanoma

N-propionyl-4-S-cysteaminylphenol (N-Pr-4-S-CAP) is a substrate for tyrosinase, which is a melanin biosynthesis enzyme and has been shown to be selectively incorporated into melanoma cells. It was found to cause selective cytotoxicity against melanocytes and melanoma cells after selective incorporation, resulting in the induction of anti-melanoma immunity. However, the underlying mechanisms for the induction of anti-melanoma immunity remain unclear. This study aimed to elucidate the cellular mechanism for the induction of anti-melanoma immunity and clarify whether N-Pr-4-S-CAP administration could be a new immunotherapeutic approach against melanoma, including local recurrence and distant metastasis. A T cell depletion assay was used for the identification of the effector cells responsible for N-Pr-4-S-CAP-mediated anti-melanoma immunity. A cross-presentation assay was carried out by using N-Pr-4-S-CAP-treated B16-OVA melanoma-loaded bone marrow-derived dendritic cells (BMDCs) and OVA-specific T cells. Administration of N-Pr-4-S-CAP induced CD8+ T cell-dependent anti-melanoma immunity and inhibited the growth of challenged B16F1 melanoma cells, indicating that the administration of N-Pr-4-S-CAP can be a prophylactic therapy against recurrence and metastasis of melanoma. Moreover, intratumoral injection of N-Pr-4-S-CAP in combination with BMDCs augmented the tumor growth inhibition when compared with administration of N-Pr-4-S-CAP alone. BMDCs cross-presented a melanoma-specific antigen to CD8+ T cells through N-Pr-4-S-CAP-mediated melanoma cell death. Combination therapy using N-Pr-4-S-CAP and BMDCs elicited a superior anti-melanoma effect. These results suggest that the administration of N-Pr-4-S-CAP could be a new strategy for the prevention of local recurrence and distant metastasis of melanoma.

Extract of Bletilla formosana callus elevates cellular antioxidative activity via Nrf2/HO-1 signaling pathway and inhibits melanogenesis in zebrafish

BackgroundBletilla species are endangered terrestrial orchids used in natural skin care formulas in Asia for a long history. In order to explore the bioactivity potential of Bletilla species as a cosmetic ingredient in a sustainable resource manner, the callus of Bletilla formosana (Hayata) Schltr. was established and extracted by an eco-friendly supercritical fluid CO2 extraction (SFE-CO2) method. The intracellular reactive oxygen species (ROS) scavenging activity and antioxidation-related gene expression of the callus extract were evaluated in both Hs68 fibroblast cells and HaCaT keratinocytes. The melanogenesis-inhibitory effect was investigated in B16F10 melanoma cells and in an in vivo zebrafish model. ResultsThe calli of B. formosana were propagated for 10–15 generations with a consistent yellow friable appearance and then subjected to SFE-CO2 extraction to obtain a yellow pasty extract. Obvious intracellular ROS scavenging activity of the extract was detected in both Hs68 and HaCaT cells with 64.30 ± 8.27% and 32.50 ± 4.05% reduction at the concentration of 250 μg/mL. Moreover, marked expression levels of heme oxygenase-1 (HO-1) and (NAD(P)H) quinone oxidoreductase-1 (NQO1) genes were detected after 6-h and 24-h treatments. These results indicate the cellular antioxidative activity of B. formosana callus extract was probably activated via the nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 signaling pathway. Melanogenesis-inhibitory effect of the extract was observed in α-MSH stimuli-inducing B16F10 cells with 28.46% inhibition of intracellular melanin content at the concentration of 50 μg/ml. The effect was confirmed with in vivo zebrafish embryos that showed a relative pigmentation density of 80.27 ± 7.98% at the concentration of 100 μg/mL without toxicity. ConclusionOur results shed light on a sustainable utilization of Bletilla species as a potential ingredient for skin.

Artocarpus heterophyllus LAM. Stem Bark Inhibits Melanogenesis Through Regulation of ROS, cAMP and MAPK Pathways

Natural-based skin-lightening cosmeceutical products are attracting high popularity nowadays due to their relatively high bioavailability upon application. Artocarpus species have been highlighted with such potential, and our previous studies have reported that Artocarpus heterophyllusLam. stem bark extract exhibited a potent anti-melanogenic activity by reducing melanin content and inhibiting cellular tyrosinase activity in B16F10 melanoma cells. Hence, this study aimed to identify the bioactive fraction from A. heterophyllus Lam. stem bark and determine its anti-melanogenic mechanisms in B16F10 melanoma cells. Our results showed that a fraction (H-3) demonstrated the most pronounced anti-melanogenic effect at 12.00 µg/mL by reducing melanin content to 22.86 ± 2.90% and inhibiting cellular tyrosinase activity at treatment concentration 33-fold lower than kojic acid, without being cytotoxic against B16F10 melanoma cells. Moreover, treatment with H-3 for 24 and 48 h substantially scavenged intracellular reactive oxygen species (ROS) of hydrogen peroxide-challenged B16F10 melanoma cells by 1.8 and 4.4%, respectively. Based on the microarray profiling and qPCR analysis, H-3 downregulated Creb3l1, Creb3l2, Creb3l3, Mitf, Tyr, Tyrp1, and Dct genes in B16F10 melanoma cells, whereas the expression of Map3k20, Mapk14 (p38), and Foxo3 genes was markedly increased. Altogether, these results demonstrated that H-3 exhibited its anti-melanogenic activity in B16F10 melanoma cells through scavenging ROS and concurrent inhibition of the cAMP and activation of the p38/MAPK signaling pathways. These findings indicate that H-3 has the potential to be used as a skin lightening cosmeceutical agent in the treatment of skin hyperpigmentation.

Studies of Erk1/2 with Respect to Tumor Aggressiveness in B16f10 Murine Melanoma Cells

Melanoma is a very aggressive form of skin cancer and have the potential to spread from a small sized primary tumor and metastasizes to different locations like lungs, bones, lymph nodes, liver and even brain. Extracellular signal regulated kinase (ERK) molecule present in MAPK pathway plays a major role in different carcinogenic processes like cell migration, cell invasiveness by altering the extracellular matrix (ECM). Cell invasiveness is facilitated by matrix metalloproteins (MMPs) by means of degradation of gelatinase enzyme regulated by a family of receptor proteins called integrins. ECM ligand fibronectin causes increased secretion of MMPs, specially MMP2 and MMP9 facilitating invasiveness and metastasis in cancer cells. This study aims to find the modulatory role of ERK 1/ 2 with respect to tumor aggressiveness on both MMP2 and MMP9 on melanoma metastasis and invasion. ERK pathway was downregulated through administration of inhibitors and ERK 1/ 2 siRNA gene silencing the effect on MMP2 and MMP9 was studied. The studies were done in B16F10 melanoma cell line and mice model. Significant changes were found in levels of MMP2 and MMP9 after downregulating the ERK pathway and can therefore a correlation between ERK and MMP activities was concluded. Elucidating these molecular phenomena in melanoma, this result can help in developing and improving targeted cancer therapies with multiple options in drug discovery.

Comparison of direct and indirect low‐temperature plasma triggering immunogenic cell death in B16F10 melanoma

AbstractThe potential antitumor application of low‐temperature plasma (LTP) in immunotherapy has attracted wide attention from researchers. The induction of immunogenic cell death (ICD) is one of the possible mechanisms underlying this event. In this work, the immunogenicity of B16F10 melanoma cells treated with direct and indirect LTP protocols was investigated. The results revealed the concentrations of H2O2, NO, and NO2− increased as LTP treatment time was longer, while the cell viability decreased. By comparison, the direct treatment induced a stronger expression of ICD markers. In vivo, the LTP‐treated B16F10 reduced tumor burden in C57BL/6 mice. This study demonstrated both direct and indirect LTP treatments triggered immunogenic cell death and were expected to provide an effective immunotherapy strategy.

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

Oleaginous Yeast Extracts and Their Possible Effects on Human Health

Four non-conventional oleaginous and pigmented yeast strains of Metschnikowia pulcherrima, Cystofilobasidium infirmominiatum, Phaffia rhodozyma, and Rhodotorula kratochvilovae were used in this study. Complex yeast extracts were prepared and tested for biological activity, safety, and effect on human health. In this paper, we measured the antioxidant activity and antimicrobial effect of yeast biomass as a whole and their extracts to compare the influence of carotenoids and other bioactive substances in the studied biomass. All yeast extracts exhibited a significant dose-dependent antimicrobial effect against both G+ and G- bacteria and had a strong antioxidant effect. No cytotoxicity in the mouse melanoma B16F1 cell line was found in concentrations up to 20% of rehydrated biomass in cell medium. All of the extracts were cytotoxic at a concentration of 5 mg of extract/g of dry biomass. All the pigmented yeast extracts showed some positive results for apoptosis of murine melanoma cell lines and are therefore strong candidates positively effect human health. Red yeast cell biomass is a prospective material with many attractive biological functions and can be used in the food industry, as a pharmaceutical material, or in the feed industry.

The pulse light mode enhances the effect of photobiomodulation on B16F10 melanoma cells through autophagy pathway.

Photobiomodulation (PBM) is the use of low irradiance light of specific wavelengths to generate physiological changes and therapeutic effects. However, there are few studies on the effects of PBM of different LED light modes on cells. Here, we investigated the difference of influence between continuous wave (CW) and pulse-PBM on B16F10 melanoma cells. Our results suggested that the pulse mode had a more significant PBM than the CW mode on B16F10 melanoma cells. Our study confirmed that ROS and Ca2+ levels in B16F10 melanoma cells treated with pulse-PBM were significantly higher than those in the control and CW-PBM groups. One mechanism that causes the difference in CW and pulse-PBM action is that pulse-PBM activates autophagy of melanoma cells through the ROS/OPN3/Ca2+ signaling pathway, and excessive autophagy activation inhibits proliferation and apoptosis of melanoma cells. Autophagy may be one of the reasons for the difference between pulse- and CW-PBM on melanoma cells. More importantly, melanoma cells responded to brief PBM pulses by increasing intracellular Ca2+ levels.

Efficient Synthesis of Chlorin e6 and Its Potential Photodynamic Immunotherapy in Mouse Melanoma by the Abscopal Effect.

Photodynamic therapy (PDT) can eradicate not only cancer cells but also stimulate an antitumor immune response. Herein, we describe two efficient synthetic methodologies for the preparation of Chlorin e6 (Ce6) from Spirulina platensis and address the phototoxic effect of Ce6 in vitro along with antitumor activity in vivo. Melanoma B16F10 cells were seeded and phototoxicity was monitored by the MTT assay. The C57BL/6 mice were subcutaneously inoculated on the left and right flank with B16F10 cells. The mice were intravenously injected with Ce6 of 2.5 mg/kg and then exposed to red light (660 nm) on the left flank tumors 3 h after the injection. The immune response was studied by analyzing Interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and Interleukin-2 (IL-2) of the right flank tumors through qPCR. Our results revealed that the tumor was suppressed not only in the left flank but also in the right flank, where no PDT was given. The upregulated gene and protein expression of IFN-γ, TNF-α, and IL-2 revealed antitumor immunity due to Ce6-PDT. The findings of this study suggest an efficient methodology of Ce6 preparation and the efficacy of Ce6-PDT as a promising antitumor immune response.

Integrin Targeting Enhances the Antimelanoma Effect of Annexin V in Mice.

Malignant melanoma, an increasingly common form of skin cancer, is a major threat to public health, especially when the disease progresses past skin lesions to the stage of advanced metastasis. Targeted drug development is an effective strategy for the treatment of malignant melanoma. In this work, a new antimelanoma tumor peptide, the lebestatin-annexin V (designated LbtA5) fusion protein, was developed and synthesized by recombinant DNA techniques. As a control, annexin V (designated ANV) was also synthesized by the same method. The fusion protein combines annexin V, which specifically recognizes and binds phosphatidylserine, with the disintegrin lebestatin (lbt), a polypeptide that specifically recognizes and binds integrin α1β1. LbtA5 was successfully prepared with good stability and high purity while retaining the dual biological activity of ANV and lbt. MTT assays demonstrated that both ANV and LbtA5 could reduce the viability of melanoma B16F10 cells, but the activity of the fusion protein LbtA5 was superior to that of ANV. The tumor volume growth was slowed in a mouse xenograft model treated with ANV and LbtA5, and the inhibitory effect of high concentrations of LbtA5 was significantly better than that of the same dose of ANV and was comparable to that of DTIC, a drug used clinically for melanoma treatment. The hematoxylin and eosin (H&E) staining test showed that ANV and LbtA5 had antitumor effects, but LbtA5 showed a stronger ability to induce melanoma necrosis in mice. Immunohistochemical experiments further showed that ANV and LbtA5 may inhibit tumor growth by inhibiting angiogenesis in tumor tissue. Fluorescence labeling experiments showed that the fusion of ANV with lbt enhanced the targeting of LbtA5 to mouse melanoma tumor tissue, and the amount of target protein in tumor tissue was significantly increased. In conclusion, effective coupling of the integrin α1β1-specific recognition molecule lbt confers stronger biological antimelanoma effects of ANV, which may be achieved by the dual effects of effective inhibition of B16F10 melanoma cell viability and inhibition of tumor tissue angiogenesis. The present study describes a new potential strategy for the application of the promising recombinant fusion protein LbtA5 in the treatment of various cancers, including malignant melanoma.

Cytotoxic, Antibacterial, and Antioxidant Activities of the Leaf Extract of Sinningia bullata.

Sinningia bullata is a tuberous member of the flowering plant family Gesneriaceae. Prior to this work, the antibacterial, antioxidant, and cytotoxic properties of S. bullata were undetermined. Here, we prepared different extracts from the leaf, stem, and tuber of S. bullata and investigated their pharmacological activities. The leaf extract of S. bullata, obtained by 100% acetone (Sb-L-A), had the highest total flavonoid content, antioxidation capacity, and cytotoxic and antibacterial activities. Sb-L-A displayed a broad range of antibacterial activities against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. The inhibition zones of Sb-L-A ranged from 8 to 30 mm and were in the following order: S. aureus > E. coli > P. aeruginosa. Incubation of B16F10 melanoma cells with Sb-L-A at a concentration of 80 μg/mL caused deaths at the rate of 96%, reduced migration by 100%, suppressed proliferation and colony formation by 99%, and induced apoptosis, which was observed in 96% of the B16F10 cells. In addition, the cytotoxic activities of Sb-L-A were synergistically enhanced when coacting with the antitumor drug epothilone B. Sb-L-A was also used to determine the cytotoxic effects against 4T1 mammary carcinoma cells. Sb-L-A of 60 μg/mL boosted the distribution of the G2 phase from 1.4% to 24.4% in the B16F10 cells. Accordingly, Sb-L-A might suppress melanoma cell proliferation by inducing G2 cell-cycle arrest. The most abundant compounds in Sb-L-A were identified using gas chromatography-mass spectrometry. The top two contents in this extract were adlupulone and villosin. Overall, the collective data in this study may indicate the pharmacological potentials of Sb-L-A for possible medical applications.

Synthesis and discovery of potential tyrosinase inhibitor of new coumarin-based thiophenyl-pyrazolylthiazole nuclei: In vitro evaluation, cytotoxicity, kinetic, and computational studies.

A well-known key enzyme in melanogenesis and hyperpigmentation is tyrosinase. The present study introduces a novel series of thiophenyl-pyrazolylthiazole-coumarin hybrids (6a-6h) as tyrosinase inhibitors. The in-vitro tyrosinase inhibition results indicated that all compounds have strong tyrosinase inhibitory activity, particularly compound 6g (IC50 = 0.043 ± 0.006 μM), was identified as the most active compound compared to the positive control (kojic acid, IC50 = 18.521 ± 1.162 μM). Lineweaver-Burk plots were employed to analyze the kinetic mechanism, and compound 6g formed an enzyme-inhibitor complex by inhibiting tyrosinase non-competitively. Furthermore, all compounds demonstrated excellent antioxidant activity against DPPH. MTT assay was used to screen the cytotoxicity of all compounds on B16F10 melanoma cells, and they had no toxic effect on the cells. The binding affinity of compounds with tyrosinase was also investigated using molecular docking, and the ligands displayed good binding energy values. These molecules could be a promising lead for skin pigmentation and associated diseases as nontoxic pharmacological scaffolds.

Gold-containing liposomes and glucose-coated gold nanoparticles enhances the radiosensitivity of B16F0 melanoma cells via increasing apoptosis and ROS production

AimTo increase the effectiveness of radiation therapy, metals with high Z number are used as radiosensitizers. In this regard, the effectiveness of various gold nanoparticles as radiosensitizer has been proven. Therefore, this study aimed to evaluate the effects of liposomes containing gold ions (Gold-Lips) and glucose-coated gold nanoparticles (Glu-GNPs) on radiation sensitivity of B16F0 melanoma cells. Main methodsNaked GNPs, Glu-GNPs and Gold-Lips were synthesized and their physicochemical properties were evaluated using DLS. The cytotoxicity and sensitivity of the nanoparticles to radiation were evaluated using MTT and colony formation assay, respectively. Flow cytometry was performed to evaluate the apoptotic effect of nanoparticles on B16F0 cells. The intracellular ROS levels and mRNA expression of Bax, Bcl-2, p53, Caspase-3, and Caspase-7 genes were also evaluated. Finally, caspase 3/7 activity was determined using a luminescence assay kit. Key findingsThe results revealed that GNPs, Glu-GNPs, and Gold-Lips had a desired size and zeta potential. Results from the colony assay showed that the all non-toxic concentrations of Gold-Lips significantly increased cell death in B16F0 cells compared with the Glu-GNPs (p > 0.05). Flow cytometry and Caspase-3/-7 activity confirmed the results of the colony assay and showed that increasing the sensitivity of cells to radiation increases apoptosis. Moreover, we found that Gold-Lips increased the mRNA expression of p53, Bax, and Caspase-3/-7, and decreased the Bcl-2 mRNA expression. SignificanceOverall, both Gold-Lips and Glu-GNPs enhanced the radiosensitivity of B16F0 cells, however, Gold-Lips had better effects, which could make them a promising tools in cancer radiotherapy.

Anti-Melanogenic and Anti-Oxidative Effects of Nostoc verrucosum (ashitsuki) Extracts

Nostoc verrucosum, an edible microalgal species, forms colonies in streams. Here, we investigated the antimelanogenic and anti-oxidative effects of N. verrucosum extracts. We collected N. verrucosum from Toyama Prefecture, Japan, and successfully cultured it in indoor cultivation systems. Aqueous, methanol, and hexane extracts of N. verrucosum were prepared for various experiments. To elucidate the antimelanogenic effects of N. verrucosum, we performed tyrosinase assay, melanin content assay, western blotting, and real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Anti-oxidative effects were evaluated using the 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) assay. The methanol and hexane extracts significantly inhibited melanin synthesis in B16F1 melanoma cells. Western blotting showed that 12.5 and 25.0 µg/mL N. verrucosum hexane extract suppressed tyrosinase activity. The qRT-PCR analysis revealed that N. verrucosum hexane extract inhibited α-melanocyte stimulating hormone-enhanced tyrosinase, tyrosinase-related protein (TRP)-1, TRP-2, and melanocortin 1 receptor (MC1R) mRNA expression. On the contrary, N. verrucosum hexane extract did not alter microphthalmia-associated transcription factor (Mitf) transcription. The ABTS assay showed that all extracts had radical scavenging activities, and the IC50 values of the aqueous and methanol extracts were 294.6 and 172.8 µg/mL, respectively. Our findings demonstrate that N. verrucosum is a suitable candidate for the development of antimelanogenic agents, cosmetics, or functional food ingredients.