Induction of immunogenic cell death and enhancement of the radiation-induced immunogenicity by chrysin in melanoma cancer cells.

Chrysin is a natural flavonoid with anti-cancer effects. Despite its beneficial effects, little information is available regarding its immunogenic cell death (ICD) properties. In this work, we hypothesized that chrysin can potentiate radiotherapy(RT)-induced immunogenicity in melanoma cell line (B16-F10). We examined the effects of chrysin alone and in combination with radiation on ICD induction in B16-F10 cells. Cell viability was assessed using an MTT assay. Cell apoptosis and calreticulin (CRT) exposure were determined using flow cytometry. Western blotting and ELISA assay were employed to examine changes in protein expression. Combination therapy exhibited a synergistic effect, with an optimum combination index of 0.66. The synergistic anti-cancer effect correlated with increased cell apoptosis in cancer cells. Compared to the untreated control, chrysin alone and in combination with RT induced higher levels of DAMPs, such as CRT, HSP70, HMGB1, and ATP. The protein expression of p-STAT3/STAT3 and PD-L1 was reduced in B16-F10 cells exposed to chrysin alone and in combination with RT. Conditioned media from B16-F10 cells exposed to mono-and combination treatments elicited IL-12 secretion in dendritic cells (DCs), inducing a Th1 response. Our findings revealed that chrysin could induce ICD and intensify the RT-induced immunogenicity.

MerTK+ macrophages promote melanoma progression and immunotherapy resistance through AhR-ALKAL1 activation.

Despite our increasing understanding of macrophage heterogeneity, drivers of macrophage phenotypic and functional polarization in the microenvironment are not fully elucidated. Here, our single-cell RNA sequencing data identify a subpopulation of macrophages expressing high levels of the phagocytic receptor MER proto-oncogene tyrosine kinase (MerTK+ macrophages), which is closely associated with melanoma progression and immunotherapy resistance. Adoptive transfer of the MerTK+ macrophages into recipient mice notably accelerated tumor growth regardless of macrophage depletion. Mechanistic studies further revealed that ALK And LTK Ligand 1 (ALKAL1), a target gene of aryl hydrocarbon receptor (AhR), facilitated MerTK phosphorylation, resulting in heightened phagocytic activity of MerTK+ macrophages and their subsequent polarization toward an immunosuppressive phenotype. Specifically targeted delivery of AhR antagonist to tumor-associated macrophages with mannosylated micelles could suppress MerTK expression and improved the therapeutic efficacy of anti-programmed cell death ligand 1 therapy. Our findings shed light on the regulatory mechanism of MerTK+ macrophages and provide strategies for improving the efficacy of melanoma immunotherapy.

Pericyte phenotype switching alleviates immunosuppression and sensitizes vascularized tumors to immunotherapy in preclinical models.

T cell-based immunotherapies are a promising therapeutic approach for multiple malignancies, but their efficacy is limited by tumor hypoxia arising from dysfunctional blood vessels. Here, we report that cell-intrinsic properties of a single vascular component, namely the pericyte, contribute to the control of tumor oxygenation, macrophage polarization, vessel inflammation, and T cell infiltration. Switching pericyte phenotype from a synthetic to a differentiated state reverses immune suppression and sensitizes tumors to adoptive T cell therapy, leading to regression of melanoma in mice. In melanoma patients, improved survival is correlated with enhanced pericyte maturity. Importantly, pericyte plasticity is regulated by signaling pathways converging on Rho kinase activity, with pericyte maturity being inducible by selective low-dose therapeutics that suppress pericyte MEK, AKT, or notch signaling. We also show that low-dose targeted anticancer therapy can durably change the tumor microenvironment without inducing adaptive resistance, creating a highly translatable pathway for redosing anticancer targeted therapies in combination with immunotherapy to improve outcome.

Inhibition of Autophagy by Berbamine Hydrochloride Mitigates Tumor Immune Escape by Elevating MHC-I in Melanoma Cells.

Impaired tumor cell antigen presentation contributes significantly to immune evasion. This study identifies Berbamine hydrochloride (Ber), a compound derived from traditional Chinese medicine, as an effective inhibitor of autophagy that enhances antigen presentation in tumor cells. Ber increases MHC-I-mediated antigen presentation in melanoma cells, improving recognition and elimination by CD8+ T cells. Mutation of Atg4b, which blocks autophagy, also raises MHC-I levels on the cell surface, and further treatment with Ber under these conditions does not increase MHC-I, indicating Ber's role in blocking autophagy to enhance MHC-I expression. Additionally, Ber treatment leads to the accumulation of autophagosomes, with elevated levels of LC3-II and p62, suggesting a disrupted autophagic flux. Fluorescence staining and co-localization analyses reveal that Ber likely inhibits lysosomal acidification without hindering autophagosome-lysosome fusion. Importantly, Ber treatment suppresses melanoma growth in mice and enhances CD8+ T cell infiltration, supporting its therapeutic potential. Our findings demonstrate that Ber disturbs late-stage autophagic flux through abnormal lysosomal acidification, enhancing MHC-I-mediated antigen presentation and curtailing tumor immune escape.

Scar/WAVE drives actin protrusions independently of its VCA domain using proline-rich domains.

Cell migration requires the constant modification of cellular shape by reorganization of the actin cytoskeleton. Fine-tuning of this process is critical to ensure new actin filaments are formed only at specific times and in defined regions of the cell. The Scar/WAVE complex is the main catalyst of pseudopod and lamellipodium formation during cell migration. It is a pentameric complex highly conserved through eukaryotic evolution and composed of Scar/WAVE, Abi, Nap1/NCKAP1, Pir121/CYFIP, and HSPC300/Brk1. Its function is usually attributed to activation of the Arp2/3 complex through Scar/WAVE's VCA domain, while other parts of the complex are expected to mediate spatial-temporal regulation and have no direct role in actin polymerization. Here, we show in both B16-F1 mouse melanoma and Dictyostelium discoideum cells that Scar/WAVE without its VCA domain still induces the formation of morphologically normal, actin-rich protrusions, extending at comparable speeds despite a drastic reduction of Arp2/3 recruitment. However, the proline-rich regions in Scar/WAVE and Abi subunits are essential, though either is sufficient for the generation of actin protrusions in B16-F1 cells. We further demonstrate that N-WASP can compensate for the absence of Scar/WAVE's VCA domain and induce lamellipodia formation, but it still requires an intact WAVE complex, even if without its VCA domain. We conclude that the Scar/WAVE complex does more than directly activating Arp2/3, with proline-rich domains playing a central role in promoting actin protrusions. This implies a broader function for the Scar/WAVE complex, concentrating and simultaneously activating many actin-regulating proteins as a lamellipodium-producing core.

Formulating a TMEM176B blocker in chitosan nanoparticles uncouples its paradoxical roles in innate and adaptive antitumoral immunity.

The immunoregulatory cation channel TMEM176B plays a dual role in tumor immunity. On the one hand, TMEM176B promotes antigen cross-presentation to CD8+ T cells by regulating phagosomal pH in dendritic cells (DCs). On the other hand, it inhibits NLRP3 inflammasome activation through ionic mechanisms in DCs, monocytes and macrophages. We speculated that formulating BayK8644 in PEGylated chitosan nanoparticles (NP-PEG-BayK8644) should slowly release the compound and by that mean avoid cross-presentation inhibition (which happens with a fast 30 min kinetics) while still triggering inflammasome activation. Chitosan nanocarriers were successfully obtained, exhibiting a particle size within the range of 200 nm; they had a high positive surface charge and a 99 % encapsulation efficiency. In in vitro studies, NP-PEG-BayK8644 did not inhibit antigen cross-presentation by DCs, unlike the free compound. The NP-PEG-BayK8644 activated the inflammasome in a Tmem176b-dependent manner in DCs. We administered either empty (eNP-PEG) or NP-PEG-BayK8644 to mice with established tumors. NP-PEG-BayK8644 significantly controlled tumor growth and improved mice survival compared to both eNP-PEG and free BayK8644 in melanoma and lymphoma models. This effect was associated with enhanced inflammasome activation by DCs in the tumor-draining lymph node and infiltration of the tumor by CD8+ T cells. Thus, encapsulation of BayK8644 in chitosan NPs improves the anti-tumoral properties of the compound by avoiding inhibition of antigen cross-presentation.

Carcinomembrane-Camouflaged Perfluorochemical Dual-Layer Nanopolymersomes Bearing Indocyanine Green and Camptothecin Effectuate Targeting Photochemotherapy of Cancer.

Photochemotherapy has been recognized as a promising combinational modality for cancer treatment. However, difficulties such as off-target drug delivery, systemic toxicity, and the hypoxic nature of the tumor microenvironment remain hindrances to its application. To overcome these challenges, cancer cell membrane camouflaged perfluorooctyl bromide (PFOB) dual-layer nanopolymersomes bearing indocyanine green (ICG) and camptothecin (CPT), named MICFNS, were developed in this study, and melanoma was exploited as the model for MICFNS manufacture and therapeutic application. Our data showed that MICFNS were able to stabilize both ICG and CPT in the nanocarriers and can be quickly internalized by B16F10 cells due to melanoma membrane-mediated homology. Upon NIR irradiation, MICFNS can trigger hyperthermia and offer enhanced singlet oxygen production due to the incorporation of PFOB. With ≥10/2.5 µM ICG/CPT, MICFNS + NIR can provide comparable in vitro cancericidal effects to those caused by using an 8-fold higher dose of encapsulated CPT alone. Through the animal study, we further demonstrated that MICFNS can be quickly brought to tumors and have a longer retention time than those of free agents in vivo. Moreover, the MICFNS with 40/10 µM ICG/CPT in combination with 30 s NIR irradiation can successfully inhibit tumor growth without systemic toxicity in mice within the 14 day treatment. We speculate that such an antitumoral effect was achieved by phototherapy followed by chemotherapy, a two-stage tumoricidal process performed by MICFNS. Taken together, we anticipate that MICFNS, a photochemotherapeutic nanoplatform, has high potential for use in clinical anticancer treatment.

Evaluation of Efficiency of Liposome-Entrapped Iridium(III) Complexes Inhibiting Tumor Growth In Vitro and In Vivo.

In this paper, three new iridium(III) complexes: [Ir(piq)2(DFIPP)]PF6 (piq = deprotonated 1-phenylisoquinoline, DFIPP = 3,4-difluoro-2-(1H-imidazo[4,5-f][1,10]phenenthrolin-2-yl)phenol, 3a), [Ir(bzq)2(DFIPP)]PF6 (bzq = deprotonated benzo[h]quinoline, 3b), and [Ir(ppy)2(DFIPP)]PF6 (ppy = deprotonated 1-phenylpyridine, 3c), were synthesized and characterized. The complexes were found to be nontoxic to tumor cells via 3-(4,5-dimethylthiazole-2-yl)-diphenyltetrazolium bromide (MTT) assay. Surprisingly, its liposome-entrapped complexes 3alip, 3blip, and 3clip on B16 cells showed strong cytotoxicity (IC50 = 13.6 ± 2.8, 9.6 ± 1.1, and 18.9 ± 2.1 µM). Entry of 3alip, 3blip, and 3clip into B16 cells decreases mitochondrial membrane potential, regulates Bcl-2 family proteins, releases cytochrome c, triggers caspase family cascade reaction, and induces apoptosis. In addition, we also found that 3alip, 3blip, and 3clip triggered ferroptosis and autophagy. In vivo studies demonstrated that 3blip inhibited melanoma growth in C57 mice with a high inhibitory rate of 83.95%, and no organic damage was found in C57 mice.

Pharmacologic LDH inhibition redirects intratumoral glucose uptake and improves antitumor immunity in solid tumor models.

Tumor reliance on glycolysis is a hallmark of cancer. Immunotherapy is more effective in controlling glycolysis-low tumors lacking lactate dehydrogenase (LDH) due to reduced tumor lactate efflux and enhanced glucose availability within the tumor microenvironment (TME). LDH inhibitors (LDHi) reduce glucose uptake and tumor growth in preclinical models, but their impact on tumor-infiltrating T cells is not fully elucidated. Tumor cells have higher basal LDH expression and glycolysis levels compared with infiltrating T cells, creating a therapeutic opportunity for tumor-specific targeting of glycolysis. We demonstrate that LDHi treatment (a) decreases tumor cell glucose uptake, expression of the glucose transporter GLUT1, and tumor cell proliferation while (b) increasing glucose uptake, GLUT1 expression, and proliferation of tumor-infiltrating T cells. Accordingly, increasing glucose availability in the microenvironment via LDH inhibition leads to improved tumor-killing T cell function and impaired Treg immunosuppressive activity in vitro. Moreover, combining LDH inhibition with immune checkpoint blockade therapy effectively controls murine melanoma and colon cancer progression by promoting effector T cell infiltration and activation while destabilizing Tregs. Our results establish LDH inhibition as an effective strategy for rebalancing glucose availability for T cells within the TME, which can enhance T cell function and antitumor immunity.

Crosslinking of Ly6a metabolically reprograms CD8 T cells for cancer immunotherapy.

T cell inhibitory mechanisms prevent autoimmune reactions, while cancer immunotherapy aims to remove these inhibitory signals. Chronic ultraviolet (UV) exposure attenuates autoimmunity through promotion of poorly understood immune-suppressive mechanisms. Here we show that mice with subcutaneous melanoma are not responsive to anti-PD1 immunotherapy following chronic UV irradiation, given prior to tumor injection, due to the suppression of T cell killing ability in skin-draining lymph nodes. Using mass cytometry and single-cell RNA-sequencing analyzes, we discover that skin-specific, UV-induced suppression of T-cells killing activity is mediated by upregulation of a Ly6ahigh T-cell subpopulation. Independently of the UV effect, Ly6ahigh T cells are induced by chronic type-1 interferon in the tumor microenvironment. Treatment with an anti-Ly6a antibody enhances the anti-tumoral cytotoxic activity of T cells and reprograms their mitochondrial metabolism via the Erk/cMyc axis. Treatment with an anti-Ly6a antibody inhibits tumor growth in mice resistant to anti-PD1 therapy. Applying our findings in humans could lead to an immunotherapy treatment for patients with resistance to existing treatments.

Anti-tumor and anti-metastasis of water-soluble sulfated ß-glucan derivatives from Saccharomyces cerevisiae.

Traditional fungi ß-glucan commonly possesses high molecular weight with poor water solubility, which remains significant challenge in the drug development and medical application. Water-soluble ß-glucan with high molecular weight (dHSCG) of 560 kDa, low molecular weight (dLSCG) of 60 kDa, and sulfated derivative (SCGS) with a molecular weight of 146 kDa and sulfate degree at 2.04 were obtained through well-controlled degradation and sulfated modification from Saccharomyces cerevisiae in this study. The structural characteristics were confirmed as ß-1,3/6-glucan by FT-IR and NMR spectroscopy. Carbohydrate microarrays and surface plasmon resonance revealed distinct and contrasting binding affinities between the natural ß-glucans and sulfated derivatives. SCGS exhibited strong binding to FGF and VEGF, while natural ß-glucan showed no response, suggesting its potential as a novel antitumor agent. Moreover, SCGS significantly inhibited the migration rate of the highly metastatic melanoma (B16F10) cells. The lung metastasis mouse model also demonstrated that SCGS significantly reduced and eliminated the nodules, achieving an inhibition rate of 86.7% in vivo, with a dramatic improvement in IFN-α, TNF-α, and IL-1ß levels. Through analysis of protein content and distribution in lung tissues, the anti-tumor and anti-metastasis mechanism of SCGS involves the regulation of degrading enzymes to protect extracellular matrix (ECM), as well as the reduction of angiogenic factor release. These findings provide a foundation for exploring the potential of SCGS in the development of new anti-tumor and anti-metastasis drugs and open up a new field in cancer research.

Stat3-mediated Atg7 expression regulates anti-tumor immunity in mouse melanoma.

Epigenetic modifications to DNA and chromatin control oncogenic and tumor-suppressive mechanisms in melanoma. Ezh2, the catalytic component of the Polycomb Repressive Complex 2 (PRC2), which mediates methylation of lysine 27 on histone 3 (H3K27me3), can regulate both melanoma initiation and progression. We previously found that mutant Ezh2Y641F interacts with the immune regulator Stat3 and together they affect anti-tumor immunity. However, given the numerous downstream targets and pathways affected by Ezh2, many mechanisms that determine its oncogenic activity remain largely unexplored. Using genetically engineered mouse models, we further investigated the role of pathways downstream of Ezh2 in melanoma carcinogenesis and identified significant enrichment in several autophagy signatures, along with increased expression of autophagy regulators, such as Atg7. In this study, we investigated the effect of Atg7 on melanoma growth and tumor immunity within the context of a wild-type or Ezh2Y641F epigenetic state. We found that the Atg7 locus is controlled by multiple Ezh2 and Stat3 binding sites, Atg7 expression is dependent on Stat3 expression, and that deletion of Atg7 slows down melanoma cell growth in vivo, but not in vitro. Atg7 deletion also results in increased CD8 + T cells in Ezh2Y641F melanomas and reduced myelosuppressive cell infiltration in the tumor microenvironment, particularly in Ezh2WT melanomas, suggesting a strong immune system contribution in the role of Atg7 in melanoma progression. These findings highlight the complex interplay between genetic mutations, epigenetic regulators, and autophagy in shaping tumor immunity in melanoma.

A novel amphiphilic squalene-based compound with open-chain polyethers reduces malignant melanoma metastasis in-vitro and in-vivo.

Squalene (SQ) is a well-known antioxidant and anti-inflammatory agent that provides promising anti-aging and UV-protective roles on human skin. However, its strong hydrophobic nature, accompanied by issues such as poor solubility and limited tissue permeation, has created challenges for scientists to investigate its untapped potential in more complex conditions, including cancer progression. The present study assessed the potent anti-metastatic properties of a newly synthesized amphiphilic ethylene glycol SQ derivative (SQ-diEG) in melanoma, the most fatal skin cancer. In vitro and in vivo experiments have discovered that SQ-diEG may exert its potential on melanoma malignancy through the mitochondria-mediated caspase activation apoptotic signaling pathway. The potent anti-metastatic effect of SQ-diEG was observed in vitro using highly proliferative and aggressive melanoma cells. Administration of SQ-diEG (25 mg/kg) significantly decreased the tumor burden on the lung and inhibited the metastasis-associated proteins and gene markers in B16F10 lung colonization mice model. Furthermore, global gene profiling also revealed a promising role of SQ-diEG in tumor microenvironment. We anticipated that the amphiphilic nature of the SQ compound bearing ethylene glycol oligomers could potentially augment its ability to reach the pathology site, thus enhancing its therapeutic potential in melanoma.

Harnessing Spatiotemporal-Specific Tumorous Exosome Dynamics: Ultra-Sensitive Lanthanide Luminescence Detection Strategy Enabled by Exosomal Membrane Engineering for Melanoma Immunotherapy Monitoring.

Focused on the newly secreted tumorous exosomes during melanoma immunotherapy, this work has pioneered an ultra-sensitive spatiotemporal-specific exosome detection strategy, leveraging advanced exosomal membrane engineering techniques. The proposed strategy harnesses the power of amplified lanthanide luminescence signals on these exosomes, enabling precise and real-time monitoring of the efficacy of melanoma immunotherapy. The methodology comprises two pivotal steps. Initially, Ac4ManNAz-associated metabolic labeling is employed to evolve azide groups onto the membranes of newly secreted exosomes with remarkable selectivity. These azide groups serve as versatile clickable artificial tags, enabling the precise identification of melanoma exosomes emerging during immunotherapy. Subsequently, lanthanide-nanoparticle-functionalized polymer chains are controllably grafted onto the exosome surfaces through click chemistry and in situ Fenton-RAFT polymerization, serving as robust signal amplifiers. When integrated with time-resolved fluorescence detection, this strategy yields detection signals with an exceptionally high signal-to-noise ratio, enabling ultra-sensitive detection of PD-L1 antigen expression levels on the spatiotemporal-specific exosomes. The detection strategy boasts a wide linear concentration range spanning from 1.7 × 104 to 1.7 × 109 particles/mL, with a remarkable theoretical detection limit of 1.28 × 103 particles/mL. The remarkable enhancements in detection sensitivity and accuracy facilitate the evaluation of the efficacy of immunotherapeutic interventions in the mouse B16 melanoma model, notably revealing a substantial disparity in PD-L1 levels between immunotherapy-treated and untreated groups (P < 0.01) and further emphasizing the cumulative therapeutic effect that intensifies with repeated treatments (P < 0.001).

Modulation of PD-L1 by Astragalus polysaccharide attenuates the induction of melanoma stem cell properties and overcomes immune evasion.

BACKGROUND: Melanoma is a highly aggressive form of skin cancer. The existence of cancer stem cells (CSCs) and tumor immune evasion are two major causes of melanoma progression, but no effective treatment has been found at present. Astragalus polysaccharide (APS) is a principal active component derived from Astragalus membranaceus, showing anti-tumor effects in various tumors including melanoma. However, the underlying mechanism is still unclear. METHODS: The regulation of APS on self-renewal ability and CSC markers expression in melanoma stem cells (MSCs) was measured by tumor sphere formation and tumorigenicity assays, RT-qPCR, and western blot. Flow cytometry was conducted to evaluate the activation of immune system by APS in melanoma mice. Further, the mechanism was explored based on PD-L1 overexpression and knock-down B16 cells. RESULTS: APS attenuated the tumor sphere formation of MSCs in vitro as well as the tumorigenicity in vivo. It also decreased the expression of CD133, BMI1 and CD47. Based on the PD-L1 overexpression and knock-down B16 cells, it was confirmed that APS inhibited the induction of MSCs by down-regulating PD-L1 expression. Meanwhile, APS increased the infiltration of CD4+ and CD8+T cells in tumor tissues because of its inhibitory effect on PD-L1. CONCLUSIONS: APS inhibited MSC induction and overcame tumor immune evasion through reducing PD-L1 expression. This study provided compelling evidence that APS could be a prospective therapeutic agent for treating melanoma.

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

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

Inhibitive Mechanism of Loquat Flower Isolate on Tyrosinase Activity and Melanin Synthesis in Mouse Melanoma B16 Cells.

Melanin naturally exists in organisms and is synthetized by tyrosinase (TYR); however, its over-production may lead to aberrant pigmentation and skin conditions. Loquat (Eriobotrya japonica (Thunb.) Lindl.) flowers contain a variety of bioactive compounds, while studies on their suppressive capabilities against melanin synthesis are limited. Loquat flower isolate product (LFP) was obtained by ethanol extraction and resin purification, and its inhibitory efficiency against TYR activity was investigated by enzyme kinetics and multiple spectroscopy analyses. In addition, the impact of LFP on melanin synthesis-related proteins' expression in mouse melanoma B16 cells was analyzed using Western blotting. HPLC-MS/MS analysis indicated that LFP was composed of 137 compounds, of which 12 compounds, including flavonoids (quercetin, isorhamnoin, p-coumaric acid, etc.) and cinnamic acid and its derivatives, as well as benzene and its derivatives, might have TYR inhibitory activities. LFP inhibited TYR activity in a concentration-dependent manner with its IC50 value being 2.8 mg/mL. The inhibition was an anti-competitive one through altering the enzyme's conformation rather than chelating copper ions at the active center. LFP reduced the expression of TYR, tyrosinase-related protein (TRP) 1, and TRP2 in melanoma B16 cells, hence inhibiting the synthesis of melanin. The research suggested that LFP had the potential to reduce the risks of hyperpigmentation caused by tyrosinase and provided a foundation for the utilization of loquat flower as a natural resource in the development of beauty and aging-related functional products.

Tumour-intrinsic endomembrane trafficking by ARF6 shapes an immunosuppressive microenvironment that drives melanomagenesis and response to checkpoint blockade therapy.

Tumour-host immune interactions lead to complex changes in the tumour microenvironment (TME), impacting progression, metastasis and response to therapy. While it is clear that cancer cells can have the capacity to alter immune landscapes, our understanding of this process is incomplete. Herein we show that endocytic trafficking at the plasma membrane, mediated by the small GTPase ARF6, enables melanoma cells to impose an immunosuppressive TME that accelerates tumour development. This ARF6-dependent TME is vulnerable to immune checkpoint blockade therapy (ICB) but in murine melanoma, loss of Arf6 causes resistance to ICB. Likewise, downregulation of ARF6 in patient tumours correlates with inferior overall survival after ICB. Mechanistically, these phenotypes are at least partially explained by ARF6-dependent recycling, which controls plasma membrane density of the interferon-gamma receptor. Collectively, our findings reveal the importance of endomembrane trafficking in outfitting tumour cells with the ability to shape their immune microenvironment and respond to immunotherapy.

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

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

Ceramide Ehux-C22 Targets the miR-199a-3p/mTOR Signaling Pathway to Regulate Melanosomal Autophagy in Mouse B16 Cells.

Melanosomes are specialized membrane-bound organelles where melanin is synthesized and stored. The levels of melanin can be effectively reduced by inhibiting melanin synthesis or promoting melanosome degradation via autophagy. Ceramide, a key component in the metabolism of sphingolipids, is crucial for preserving the skin barrier, keeping it hydrated, and warding off the signs of aging. Our preliminary study indicated that a long-chain C22-ceramide compound (Ehux-C22) isolated from the marine microalga Emiliania huxleyi, reduced melanin levels via melanosomal autophagy in B16 cells. Recently, microRNAs (miRNAs) were shown to act as melanogenesis-regulating molecules in melanocytes. However, whether the ceramide Ehux-C22 can induce melanosome autophagy at the post-transcriptional level, and which potential autophagy-dependent mechanisms are involved, remains unknown. Here, miR-199a-3p was screened and identified as a novel upregulated miRNA in Ehux-C22-treated B16 cells. An in vitro high melanin expression model in cultured mouse melanoma cells (B16 cells) was established by using 0.2 µM alpha-melanocyte-stimulating hormone(α-MSH) and used for subsequent analyses. miR-199a-3p overexpression significantly enhanced melanin degradation, as indicated by a reduction in the melanin level and an increase in melanosome autophagy. Further investigation demonstrated that in B16 cells, Ehux-C22 activated miR-199a-3p and inhibited mammalian target of rapamycin(mTOR) level, thus activating the mTOR-ULK1 signaling pathway by promoting the expression of unc-51-like autophagy activating kinase 1 (ULK1), B-cell lymphoma-2 (Bcl-2), Beclin-1, autophagy-related gene 5 (ATG5), and microtubule-associated protein light chain 3 (LC3-II) and degrading p62. Therefore, the roles of Ehux-C22-regulated miR-199a-3p and the mTOR pathway in melanosomal autophagy were elucidated. This research may provide novel perspectives on the post-translational regulation of melanin metabolism, which involves the coordinated control of melanosomes.