The Hippo Pathway Kinases LATS1/2 Suppress Cancer Immunity.

Poorly immunogenic tumor cells evade host immunity and grow even in the presence of an intact immune system, but the complex mechanisms regulating tumor immunogenicity have not been elucidated. Here, we discovered an unexpected role of the Hippo pathway in suppressing anti-tumor immunity. We demonstrate that, in three different murine syngeneic tumor models (B16, SCC7, and 4T1), loss of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in tumor cells inhibits tumor growth. Tumor regression by LATS1/2 deletion requires adaptive immune responses, and LATS1/2 deficiency enhances tumor vaccine efficacy. Mechanistically, LATS1/2-null tumor cells secrete nucleic-acid-rich extracellular vesicles, which induce a type I interferon response via the Toll-like receptors-MYD88/TRIF pathway. LATS1/2 deletion in tumors thus improves tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. Our observations uncover a key role of the Hippo pathway in modulating tumor immunogenicity and demonstrate a proof of concept for targeting LATS1/2 in cancer immunotherapy.

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

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

CD24 flags anastasis in melanoma cells.

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

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

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

Enhanced Therapeutic Efficacy Against Melanoma through Exosomal Delivery of Hesperidin.

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

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

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

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

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

Electrospun fibrillary scaffold for electrochemical cell biomarkers detection.

A novel scaffold for in situ electrochemical detection of cell biomarkers was developed using electrospun nanofibers and commercial adhesive polymeric membranes. The electrochemical sensing of cell biomarkers requires the cultivation of the cells on/near the (bio)sensor surface in a manner to preserve an appropriate electroactive available surface and to avoid the surface passivation and sensor damage. This can be achieved by employing biocompatible nanofiber meshes that allow the cells to have a normal behavior and do not alter the electrochemical detection. For a better mechanical stability and ease of handling, nylon 6/6 nanofibers were collected on commercial polymeric membranes, at an optimal fiber density, obtaining a double-layered platform. To demonstrate the functionality of the fabricated scaffold, the screening of cellular stress has been achieved integrating melanoma B16-F10 cells and the (bio)sensor components on the transducer whereas the melanin exocytosis was successfully quantified using a commercial electrode. Either directly on the surface of the (bio)sensor or spatially detached from it, the integration of cell cultures in biosensing platforms based on electrospun nanofibers represents a powerful bioanalytical tool able to provide real-time information about the biomarker release, enzyme activity or inhibition, and monitoring of various cellular events.

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.

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

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

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

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

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

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

Putrescine Upregulates Melanogenesis Through Modulation of MITF Transcription Factor in B16F1 Mouse Melanoma Cells.

Research background: Ageing is a biochemical, metabolic and genetic physiological phenomenon. The suppression of melanin biosynthesis, evident in the greying of the hair, is a hallmark of ageing resulting from translation failure, reduced enzyme activity and cellular senescence. Putrescine, the smallest member of the polyamine family and an organic chemical, is present in living mammalian cells and plays a crucial role in regulating skin melanogenesis. Therefore, the purpose of this study is to explore the effect of putrescine on the signalling pathways of melanogenesis in melanoma cells. Experimental approach: Melanin production capacity of putrescine was analysed using a tyrosinase activity assay. To assess the cell viability of B16F1 cells exposed to putrescine, a tetrazolium dye MTT assay was performed. The effect of putrescine on melanin synthesis in the presence of H2O2 was evaluated using various in vitro assays in B16F1 cells. The effect of putrescine on melanin production in B16F1 cells was determined using a specific melanin production assay. Gene expression was analysed using real-time polymerase chain reaction (RT-PCR). Furthermore, the effect of putrescine on the expression of proteins related to melanin production in the cells treated with H2O2 was analysed by immunofluorescence and Western blot analysis. Results and conclusions: Putrescine increased tyrosinase activity and showed no cytotoxicity in B16F1 cells. In addition, putrescine effectively scavenged H2O2, as shown by the reduction of intracellular H2O2 amounts in 2',7'-dichlorofluorescin diacetate analysis, and promoted melanin production in living cells. The stimulation of melanogenesis by putrescine was attributed to the increased expression of Mitf, Tyr, Trp-1 and Trp-2 genes. Immunofluorescence assays revealed that putrescine enhanced the expression of proteins associated with melanogenesis and upregulated TYR, TRP-1 and TRP-2 via the microphthalmia-associated transcription factor (MITF) and increased the expression of methionine sulfoxide reductases A (MSRA) and B (MSRB) in the cells treated with H2O2, effectively promoting melanogenesis. These results suggest that putrescine can be used to stimulate melanin synthesis. Novelty and scientific contribution: This is the first study to investigate the effect of putrescine on the signalling pathways of melanogenesis in B16F1 melanoma cells. The results confirm that putrescine can promote melanogenesis through the expression of TYR, TRP-1 and TRP-2 via the MITF in cells treated with H2O2. Putrescine can be used exclusively as a cosmetic product to prevent premature greying of hair.

Elsinochrome A induces cell apoptosis and autophagy in photodynamic therapy.

Elsinochrome A (EA) is a perylene quinone natural photosensitizer, photosensitizer under light excitation generates reactive oxygen species (ROS) to induce apoptosis, so can be used for treating tumors, that is so-called photodynamic therapy (PDT). However, the molecular mechanism, especially related to apoptosis and autophagy, is still unclear. In this study, we aimed to explore the mechanism of EA-PDT-induced B16 cells apoptosis and autophagy. The action of EA-PDT on mitochondrial permeability transition pore (MPTP), mitochondrial membrane potential (MMP) and the mitochondrial function were researched by fluorescence technique and Extracellular Flux Analyzer. Illumina sequencing, tandem mass tags Quantitative Proteomics and Western Blot studied the mechanism at the gene and protein levels. The results indicated that EA-PDT had excellent phototoxicity in vitro. EA could bind to the mitochondria. EA-PDT for 5 min caused MPTP opening, MMP decreasing and abnormal mitochondrial function with a concentration-dependent characteristic. EA-PDT resulted in an increase intracellular ROS and the number of autophagosomes. Caspase2, caspase9 and tnf were upregulated, and bcl2, prkn, atg2, atg9 and atg10 were downregulated. Our results indicated that EA-PDT induced cell apoptosis and autophagy through the mediation of ROS/Atg/Parkin. This study can provide enlightenment for exploring potential targets of drug development for the PDT of melanoma.

The Skin-Whitening and Antioxidant Effects of Protocatechuic Acid (PCA) Derivatives in Melanoma and Fibroblast Cell Lines.

The skin is the most voluminous organ of the human body and is exposed to the outer environment. Such exposed skin suffers from the effects of various intrinsic and extrinsic aging factors. Skin aging is characterized by features such as wrinkling, loss of elasticity, and skin pigmentation. Skin pigmentation occurs in skin aging and is caused by hyper-melanogenesis and oxidative stress. Protocatechuic acid (PCA) is a natural secondary metabolite from a plant-based source widely used as a cosmetic ingredient. We chemically designed and synthesized PCA derivatives conjugated with alkyl esters to develop effective chemicals that have skin-whitening and antioxidant effects and enhance the pharmacological activities of PCA. We identified that melanin biosynthesis in B16 melanoma cells treated with alpha-melanocyte-stimulating hormone (α-MSH) is decreased by PCA derivatives. We also found that PCA derivatives effectively have antioxidant effects in HS68 fibroblast cells. In this study, we suggest that our PCA derivatives are potent ingredients for developing cosmetics with skin-whitening and antioxidant effects.

Sub-lineage B.1.6 of hMPXV in a global context: Phylogeny and epidemiology.

During the 2022 COVID-19 pandemic, monkeypox emerged as a significant threat to global health. The virus responsible for the disease, the human monkeypox virus (hMPXV), underwent various genetic changes, resulting in the emergence of over a dozen distinct lineages, which could be identified by only a small number of unique mutations. As of January 25, 2023, genomic information of hMPXV generated had reached 4632 accessions in the GISAID database. In this study, we aimed to investigate the epidemiological and phylogenetic characteristics of the B.1.6 sub-lineage of hMPXV in Peru, compared with other circulating sub-lineages during the global outbreak. The B.1.6 sub-lineage, characterized by the 111029G>A mutation, was estimated to have emerged in June 2022 and was found mainly in Peru. Most cases (95.8%) were men with an average age of 33 years, and nearly half of the patients had HIV, of whom only 77.35% received antiretroviral therapy. Our findings revealed that the B.1.6, B.1.4, and B.1.2 sub-lineages were well represented and had a higher number of mutations despite having the lowest media substitution rates per site per year. Moreover, it was estimated that B.1.2 and B.1.4 appeared in February 2022 and were the first two sub-lineages to emerge. A mutation profile was also obtained for each sub-lineage, reflecting that several mutations had a pattern similar to the characteristic mutation. This study provides the first estimation of the substitution rate and ancestry of each monkeypox sub-lineage belonging to the 2022 outbreak. Based on our findings, continued genomic surveillance of monkeypox is necessary to understand better and track the evolution of the virus.

Loss of two-pore channel 2 function in melanoma-derived tumours reduces tumour growth in vivo but greatly increases tumour-related toxicity in the organism.

BACKGROUND: Melanoma, a severe form of skin cancer, poses significant health risks due to its aggressive nature and potential for metastasis. The role of two-pore channel 2 (TPC2) in the development and progression of melanoma remains poorly understood. This study aims to investigate the impact of TPC2 knockout (KO) on melanoma-derived tumors, focusing on tumour growth and related toxicity in the organism. METHODS: The study utilized CHL-1 and B16 melanoma cell lines with TPC2 KO to assess the changes in proliferation dynamics. Methods included real-time monitoring of cell proliferation using the xCELLigence system, in vivo tumour growth assays in mice, histopathological analyses, inflammation marker assessment, and quantitative PCR (qPCR) for gene expression analysis RESULTS: TPC2 KO was found to significantly alter the proliferation dynamics of CHL-1 and B16 melanoma cells. The in vivo studies demonstrated reduced tumor growth in TPC2 KO cell-derived tumors. However, a notable increase in tumor-related toxicity in affected organs, such as the liver and spleen, was observed, indicating a complex role of TPC2 in melanoma pathology. CONCLUSIONS: The loss of TPC2 function in melanoma cells leads to reduced tumour growth but exacerbates tumour-related toxicity in the organism. These findings highlight the dual role of TPC2 in melanoma progression and its potential as a therapeutic target. Further research is needed to fully understand the mechanisms underlying these effects and to explore TPC2 as a treatment target in melanoma.

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

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

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

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

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

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