B16 Cells Research Articles

Antioxidant activity and whitening effect of vitamin C and superoxide dismutase from Rosa roxburghii Tratt

Abstract With growing concerns about the negative impact of environmental pollution on skin health, there is an increased demand for natural active ingredients to combat free radicals and excessive melanin production. We focus on the Rosa roxburghii Tratt (RR) as a potential source of skin-lightening compounds. Vitamin C (Vc) and superoxide dismutase (SOD)-rich extracts (RRVC and RRSOD) were extracted from RR using macroporous resin column chromatography and graded ammonium sulfate precipitation. The antioxidant and skin-lightening effects of these extracts were evaluated. RRVC and RRSOD showed strong inhibitory activity against various radicals and tyrosinase. When formulated together, their antioxidant and skin-lightening abilities were significantly enhanced, with an optimal ratio of 5:32 (RRVS). In vivo studies on C57BL/6J mice revealed that these two extracts and their formulations effectively lowered oxidative stress levels in a model of D-galactose-induced aging in mice. Furthermore, they inhibited melanogenesis, and tyrosinase activity in B16 cells and reduced UV (ultraviolet radiation)-induced melanin deposition in mouse ears. The combination of RRVC and RRSOD had a more potent melanin-scavenging effect compared with the individual extracts. These findings suggest that RRVC and RRSOD extracts, especially when combined in the optimal ratio, can whiten skin, both enhancing the value of RR products and offering natural alternatives for skin lightening.

Recombinant Methioninase Synergistically Reverses High-docetaxel Resistance Developed in Osteosarcoma Cells.

Docetaxel combined with gemcitabine is a second-line therapy for osteosarcoma, but its efficacy is limited by the development of docetaxel resistance. The aim of the present study was to determine whether recombinant methioninase (rMETase) could reverse docetaxel resistance developed in osteosarcoma cells. Docetaxel-resistant 143B (DTR-143B) osteosarcoma cells were established by treating the parental 143B cells to increasing docetaxel concentrations (0.14-24 nM) over 5 months. The 50% inhibitory concentration (IC50) of docetaxel and rMETase as well as their combination on human osteosarcoma cells 143B and DTR-143B were determined. Four groups were analysed in vitro: untreated control; docetaxel; rMETase; docetaxel plus rMETase. The IC50 value of docetaxel for DTR-143B cells was 31.1 nM, compared to 4.38 nM for the parental 143B cells, a 7-fold increase. The combination of rMETase (0.53 U/ml) and docetaxel (4.38 nM) sensitized DTR-143B cells to docetaxel resulting in an inhibition of 73.7% compared to docetaxel alone (7.3%) or rMETase alone (54.6%) (p<0.05). rMETase thus increased the efficacy of docetaxel 10-fold on docetaxel-resistant osteosarcoma cells. rMETase reversed docetaxel resistance of DTR-143B in vitro. The present results indicate the clinical potential of rMETase to overcome docetaxel resistance in osteosarcoma patients.

Physicochemical and biological characterization of the lipid particles with bovine serum albumin corona

Diacylglycerol–based nanoparticles are promising bioactive delivery systems. However, limited understanding of their interaction with biological entities restricts their clinical use. This study investigated the protein corona formed on medium and long chain diacylglycerol (MLCD)-based solid lipid nanoparticles (NPs) modified by Polyoxethylene stearate (PEG) and compared to glyceryl tristearate (TG) and cetyl palmitate (CP) nanoparticles. Bovine serum albumin (BSA) formed corona with MLCD NPs through hydrophobic interactions and hydrogen bonding, contributing to a decrease in α–helix, an increase in β–sheet and a change in the microenvironment of Tyr residues. Owing to higher lipid hydrophilicity, MLCD NPs showed a much lower affinity for BSA than TG and CP NPs, and the binding constant with BSA was increased for larger NPs. PEG modification and the protein corona reduced the uptake of NPs by macrophages but exerted little influence on B16 cell. Among the NPs with different lipid core, the MLCD NPs showed a lower macrophages cell uptake but higher B16 cell uptake, suggesting a longer circulation time in blood but higher cancer cell internalization. This work shed light on the interactions between MLCD NPs and proteins, which is significant for application as nanocarriers with improved biological efficacy.

Rare earth oxides nanoparticles formed on the surface of WE43 magnesium alloy subjected to anodic oxidation plus heat treatment for anti-bone tumor

It was well known that rare earth elements (REE) oxides nanoparticles could inhibit tumor growth and promote osteogenesis. In this works, four WE43 magnesium alloys (AO-10, AO-30, AO-1h and AO-2h) containing different REE oxides nanoparticles were prepared by anodic oxidation plus heat treatment. The REE oxides nanoparticles on AO-10, AO-30, AO-1h and AO-2h surface were Gd2O3 and Nd2O3, (Gd0.745Y1.255)O3 and Nd2O3, (Gd0.18Y1.82)O3 and Gd2O3 as well as Y2O3 and Gd2O3, respectively. Subsequently, the bioactivity, antitumor property and osteogenic property of all materials in vitro and in vivo were studied, and their antitumor property, bioactivity and osteogenic property were all AO-2h > AO-1h > AO-30 > AO-10. The degradation behaviors of materials in vitro and vivo were further investigated, and the corrosion resistance of materials was AO-10 > AO-30 > AO-1h > AO-2h. AO-2h released the most H2 and produced the highest pH value in all materials during degradation. The results showed that the effects of different REE oxides nanoparticles on corrosion resistance, biactivity, osteogenic property and antitumor property of materials were different completely. In all materials, AO-30 had moderate inhibition of MG63 and 143B cells in vitro, and it could inhibit the growth of bone tumor in mice in vivo. Then, the osteogenic property of AO-30 was moderate. It could promote the adhesion and proliferation of MC3T3-E1 cells. Again, the corrosion resistance of AO-30 was relatively excellent. It release few H2, Mg2+ and REE. The REE levels in normal tissues were relatively low, whose effects was negligible. Hence, AO-30 was the most suitable material for anti-bone tumor in all materials.

A new isocoumarin from sponge endophytic fungus Aspergillus ochraceopetaliformis with cytotoxic activity

A new isocoumarin, 8-hydroxy-3-methyliscoumarin-6-yl acetate (1), was isolated together with five known compounds from sponge endophytic fungus Aspergillus ochraceopetaliformis. The structure of the new compound was established based on 1D and 2D NMR spectral analysis. Compounds 1–6 were evaluated for their cytotoxic activities and antimicrobial activities. Compounds 1 and 6 display cytotoxic activities against mouse melanoma cells B16 with IC50 values 72.5 ± 2.6 and 1.0 ± 0.5 µM, respectively. Compounds 4 and 5 displayed faint antimicrobial activities of Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus.

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.

Comparative Analysis of Chemical Profiles and Biological Activities of Essential Oils Derived from Torreya grandis Arils and Leaves: In Vitro and In Silico Studies.

Torreya grandis (T. grandis, Taxaceae) is a well-known nut tree species. Its fruit aril and leaves possess a unique aroma, making it an ideal natural raw material for extracting essential oils (EOs). This study aims to comprehensively compare the composition, biological activities, and pharmacological mechanism of EOs extracted from the arils (AEO) and leaves (LEO) of T. grandis. The results revealed that the chemical composition of the two EOs was highly consistent, with α-pinene and D-limonene as the main components. Both EOs significantly reduced cellular melanin production and inhibited tyrosinase activity in α-MSH-stimulated B16 cells (p < 0.05). AEO and LEO suppressed inflammatory responses in LPS-stimulated RAW 264.7 macrophages, significantly inhibiting cellular NO production and proinflammatory cytokines such as TNF-α and IL-6 (p < 0.05). A network pharmacology analysis reveals that AEO and LEO share similar molecular mechanisms and pharmacological pathways for treating skin pigmentation and inflammation. Regulating inflammatory cytokines may be a critical pathway for AEO and LEO in treating skin pigmentation. These findings suggest that AEO and LEO have potential for cosmetic applications. The leaves of T. grandis could be a valuable source of supplementary materials for producing T. grandis aril EO.

Quercetin and kaempferol from saffron petals alleviated hydrogen peroxide-induced oxidative damage in B16 cells.

Saffron petals are usually considered as waste after saffron harvest. However, saffron petals contain many important phytochemical components (e.g. quercetin and kaempferol), which may alleviate oxidative damage in human cells. The contents of flavonoids and crocin in different parts of saffron were analyzed. The protective effects of flavonoids from saffron on oxidative damage of B16 cells were investigated. Saffron stigma contained high contents of crocin and picrocrocin, whereas flavonoid content (quercetin, 4.03 ± 0.33 mg g-1 DW; kaempferol, 47.80 ± 0.60 mg g-1 DW) was higher in saffron petals than in other parts. Incubation of B16 cells with quercetin (10-30 μmol L-1) and kaempferol (20-30 μmol L-1) obtained from saffron extracts could significantly increase the total antioxidant capacity (T-AOC) and the activity of NADPH:dehydrogenase quinone-1 (NQO1) to alleviate H2O2-induced oxidative damage. Quercetin was better than kaempferol in increasing NQO1 activity and T-AOC. Quercetin extracted from saffron petals could induce NQO1 expression through regulating kelch-like ECH-associated protein-1/nuclear factor erythroid 2-related factor-2 signaling pathway to protect B16 cells from oxidative damage. The content of kaempferol-3-O-sophoroside and quercetin-3-O-sophoroside was higher in saffron petals than in other parts of saffron. The kaempferol and quercetin obtained from saffron petals could enhance the activity of antioxidant enzyme NQO1 and T-AOC in B16 cells. This indicated that saffron petals, as a potential functional food, may prevent diseases caused by oxidative stress. © 2024 Society of Chemical Industry.

A dual-pathway pyroptosis inducer based on Au–Cu2-xSe@ZIF-8 enhances tumor immunotherapy by disrupting the zinc ion homeostasis

The regulation of intracellular ionic homeostasis to trigger antigen-specific immune responses has attracted extensive interest in tumor therapy. In this study, we developed a dual-pathway nanoreactor, Au-Cu2-xSe@ZIF-8@P18 NPs (ACS-Z-P NPs), which targets danger-associated molecular patterns (DAMPs) and releases Zn2+ and reactive oxygen species (ROS) within the tumor microenvironment (TME). Zn2+ released from the metal–organic frameworks (MOFs) was deposited in the cytoplasm, leading to aberrant transcription levels of intracellular zinc-regulated proteins and DNA damage, thereby inducing pyroptosis and immunogenic cell death (ICD) dependent on caspase1/gasdermin D (GSDMD) pathway. Furthermore, upon laser irradiation, ACS-Z-P NPs could break through the limitations of inherent defects of immunosuppression in TME, enhance ROS generation through a Fenton-like reaction cascade, which subsequently triggered the activation of inflammatory vesicles and the release of damage-associated molecular patterns (DAMPs). This cascade effect led to the amplification of pyroptosis and immunogenic cell death (ICD), thereby remodeling the immunosuppressed TME. Consequently, this process improved dendritic cell (DC) antigen presentation and augmented anti-tumor T-cell responses, effectively initiating antigen-specific immune responses and further enhancing pyroptosis and ICD. This study explores the therapeutic properties of these mechanisms in detail. Statement of significanceThe synthesized Au-Cu2-xSe@ZIF-8@P18 nanoparticles (ACS-Z-Ps) can effectively enhance the bodyʼs immune response by regulating zinc ion levels within cells. This regulation leads to abnormal levels of zinc-regulated protein transcription and DNA damage, which induces cellular pyroptosis. As a result, antigen presentation to dendritic cells (DCs) is improved, and anti-tumor T-cell responses are enhanced.The ACS-Z-P NPs overcome the limitations of ROS deficiency and immunosuppression in the tumor microenvironment by using H2O2 in the tumor microenvironment through a Fenton-like reaction. This leads to an increased production of ROS and O2, remodeling of the immunosuppressed tumor microenvironment, and enhanced induction of cell pyroptosis and immunogenic cell death.ACS-Z-P NPs targeted B16 cells using the photosensitizer P18 in combination with PDT treatment. This approach significantly inhibited the proliferation of B16 cells and effectively inhibited tumor growth.

Efficient Elimination of mtDNA from Mammalian Cells with 2',3'-Dideoxycytidine.

Mammalian cell lines devoid of mitochondrial DNA (mtDNA) are indispensable in studies aimed at elucidating the contribution of mtDNA to various cellular processes or interactions between nuclear and mitochondrial genomes. However, the repertoire of tools for generating such cells (also known as rho-0 or ρ0 cells) remains limited, and approaches remain time- and labor-intensive, ultimately limiting their availability. Ethidium bromide (EtBr), which is most commonly used to induce mtDNA loss in mammalian cells, is cytostatic and mutagenic as it affects both nuclear and mitochondrial genomes. Therefore, there is growing interest in new tools for generating ρ0 cell lines. Here, we examined the utility of 2',3'-dideoxycytidine (ddC, zalcitabine) alone or in combination with EtBr for generating ρ0 cell lines of mouse and human origin as well as inducing the ρ0 state in mouse/human somatic cell hybrids. We report that ddC is superior to EtBr in both immortalized mouse fibroblasts and human 143B cells. Also, unlike EtBr, ddC exhibits no cytostatic effects at the highest concentration tested (200 μM), making it more suitable for general use. We conclude that ddC is a promising new tool for generating mammalian ρ0 cell lines.

Fast Screening of Tyrosinase Inhibitors in Coreopsis tinctoria Nutt. by Ligand Fishing Based on Paper-Immobilized Tyrosinase.

Coreopsis tinctoria Nutt. is an important medicinal plant in traditional Uyghur medicine. The skin-lightening potential of the flower has been recognized recently; however, the active compounds responsible for that are not clear. In this work, tyrosinase, a target protein for regulating melanin synthesis, was immobilized on the Whatman paper for the first time to screen skin-lightening compounds present in the flower. Quercetagetin-7-O-glucoside (1), marein (2), and okanin (3) were found to be the enzyme inhibitors. The IC50 values of quercetagetin-7-O-glucoside (1) and okanin (3) were 79.06 ± 1.08 μM and 30.25 ± 1.11 μM, respectively, which is smaller than 100.21 ± 0.11 μM of the positive control kojic acid. Enzyme kinetic analysis and molecular docking were carried out to investigate their inhibition mechanism. Although marein (2) showed a weak inhibition effect in vitro, it inhibited the intracellular tyrosinase activity and diminished melanin production in melanoma B16 cells as did the other two inhibitors. The paper-based ligand fishing method developed in this work makes it effective to quickly screen tyrosinase inhibitors from natural products. This is the first report on the tyrosinase inhibitory effect of those three compounds, showing the promising potential of Coreopsis tinctoria for the development of herbal skin-lightening products.

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

BackgroundMelanoma 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.MethodsThe 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.ResultsAPS 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.ConclusionsAPS 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.

Therapeutic impacts of GNE‑477‑loaded H2O2 stimulus‑responsive dodecanoic acid‑phenylborate ester‑dextran polymeric micelles on osteosarcoma.

Osteosarcoma (OS) is a highly malignant primary bone neoplasm that is the leading cause of cancer‑associated death in young people. GNE‑477 belongs to the second generation of mTOR inhibitors and possesses promising potential in the treatment of OS but dose tolerance and drug toxicity limit its development and utilization. The present study aimed to prepare a novel H2O2 stimulus‑responsive dodecanoic acid (DA)‑phenylborate ester‑dextran (DA‑B‑DEX) polymeric micelle delivery system for GNE‑477 and evaluate its efficacy. The polymer micelles were characterized by morphology, size and critical micelle concentration. The GNE‑477 loaded DA‑B‑DEX (GNE‑477@DBD) tumor‑targeting drug delivery system was established and the release of GNE‑477 was measured. The cellular uptake of GNE‑477@DBD by three OS cell lines (MG‑63, U2OS and 143B cells) was analyzed utilizing a fluorescent tracer technique. The hydroxylated DA‑B was successfully grafted onto dextran at a grafting rate of 3%, suitable for forming amphiphilic micelles. Following exposure to H2O2, the DA‑B‑DEX micelles ruptured and released the drug rapidly, leading to increased uptake of GNE‑477@DBD by cells with sustained release of GNE‑477. The in vitro experiments, including MTT assay, flow cytometry, western blotting and RT‑qPCR, demonstrated that GNE‑477@DBD inhibited tumor cell viability, arrested cell cycle in G1 phase, induced apoptosis and blocked the PI3K/Akt/mTOR cascade response. In vivo, through the observation of mice tumor growth and the results of H&E staining, the GNE‑477@DBD group exhibited more positive therapeutic outcomes than the free drug group with almost no adverse effects on other organs. In conclusion, H2O2‑responsive DA‑B‑DEX presents a promising delivery system for hydrophobic anti‑tumor drugs for OS therapy.

Rolling Circle Amplification-Based Self-Assembly to Form a "GPS-Nanoconveyor" for In Vitro Targeted Imaging and Enhanced Gene/Chemo (CRISPR/DOX) Synergistic Therapy.

The GPS-Nanoconveyor (MA-NV@DOX-Cas13a) is a targeted nanoplatform designed for the imaging and gene/chemotherapy synergistic treatment of melanoma. It utilizes rolling circle amplification (RCA) products as a scaffold to construct a DNA "Nanoconveyor" (NV), which incorporates a multivalent aptamer (MA) as a "GPS", encapsulates doxorubicin (DOX) in the transporter, and equips it with CRISPR/Cas13a ribonucleoproteins (Cas13a RNP). Carrying MA enhances the ability to recognize the overexpressed receptor nucleolin on B16 cells, enabling targeted imaging and precise delivery of MA-NV@DOX-Cas13a through receptor-mediated endocytosis. The activation of signal transducer and activator of transcription 3 (STAT3) in cancer cells triggers cis-cleavage of CRISPR/Cas13a, initiating its trans-cleavage function. Additionally, deoxyribonuclease I (DNase I) degrades MA-NV, releasing DOX for intracellular imaging and as a chemotherapeutic agent. Experiments demonstrate the superior capabilities of this versatile nanoplatform for cellular imaging and co-treatment while highlighting the advantages of these nanodrug delivery systems in mitigating DOX side effects.

Sodium copper chlorophyllin-loaded chitosan nanoparticle-based photodynamic therapy for B16 melanoma cancer cells.

Melanoma is one of the most aggressive and fatal skin cancers owing to its ability to metastasize and develop resistance to chemotherapy. Photodynamic therapy (PDT) is a minimally noninvasive treatment modality comprising photosensitizers (PSs), light sources, and endogenous molecular oxygen that exert a localized cytotoxic effect on cancer cells. The current study explores the therapeutic potential of sodium copper chlorophyllin-loaded chitosan nanoparticles (CH-SCC NPs) along with handheld laser-based PDT on B16 cancer cells. A modified chlorophyll derivative identified as sodium copper chlorophyllin (SCC) is a dietary supplement that has anticancer properties. Herein, we have synthesized CH-SCC NPs using the ionic gelation method to enhance the PS's bioavailability and efficiency. Chitosan nanoparticles exhibited high biocompatibility in a normal cell line L929, zebrafish, and chick embryos, and were successfully employed to deliver the SCC to cancer cells. CH-SCC NPs showed an enhanced PDT effect that killed approximately 80%-85% of B16 cells. CH-SCC NPs in combination with a handheld portable laser source showed significant therapeutic potential against the B16 skin cancer cell line. The experimental findings further strengthen our device-repurposing strategy, which suggests that SCC nanoformulations along with handheld laser can be a suitable treatment for skin cancer even in remote areas where power source and treatment cost can be a limitation.

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.

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.

Release of Exosomal PD-L1 in Bone and Soft Tissue Sarcomas and Its Relationship to Radiotherapy.

(1) Background: Exosomal PD-L1 has garnered attention owing to its role in instigating systemic immune suppression. The objective of this study is to elucidate whether bone and soft tissue sarcoma cells possess the capacity to secrete functionally active exosomal PD-L1 and whether radiotherapy (RT) induces the exosomal PD-L1 release. (2) Methods: Human osteosarcoma cell line 143B and human fibrosarcoma cell line HT1080 were utilized. Exosomes were isolated from the culture medium and blood via ultracentrifugation. The expression of PD-L1 on both tumor cells and exosomes was evaluated. The inhibitory effect on PBMC was employed to assess the activity of exosomal PD-L1. Post radiotherapy, changes in PD-L1 expression were compared. (3) Results: Exosomal PD-L1 was detected in the culture medium of tumor cells but was absent in the culture medium of PD-L1 knockout cells. Exosomal PD-L1 exhibited an inhibitory effect on PBMC activation. In tumor-bearing mice, human-derived exosomal PD-L1 was detected in the bloodstream. Following radiotherapy, tumor cells upregulated PD-L1, and human-derived exosomal PD-L1 were detected in the bloodstream. (4) Conclusions: Exosomal PD-L1 emanates from bone and soft tissue sarcoma cells and is disseminated into the circulatory system. The levels of PD-L1 in tumor cells and the release of exosomal PD-L1 were augmented after irradiation with RT.

Alterations in coenzyme Q10 status in a cybrid line harboring the 3243A>G mutation of mitochondrial DNA is associated with abnormal mitochondrial bioenergetics and dysregulated mitochondrial biogenesis

Mitochondrial DNA (mtDNA) mutations, including the m.3243A>G mutation that causes mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with secondary coenzyme Q10 (CoQ10) deficiency. We previously demonstrated that PPARGC1A knockdown repressed the expression of PDSS2 and several COQ genes. In the present study, we compared the mitochondrial function, CoQ10 status, and levels of PDSS and COQ proteins and genes between mutant cybrids harboring the m.3243A>G mutation and wild-type cybrids. Decreased mitochondrial energy production, defective respiratory function, and reduced CoQ10 levels were observed in the mutant cybrids. The ubiquinol-10:ubiquinone-10 ratio was lower in the mutant cybrids, indicating blockage of the electron transfer upstream of CoQ, as evident from the reduced ratio upon rotenone treatment and increased ratio upon antimycin A treatment in 143B cells. The mutant cybrids exhibited downregulation of PDSS2 and several COQ genes and upregulation of COQ8A. In these cybrids, the levels of PDSS2, COQ3-a isoform, COQ4, and COQ9 were reduced, whereas those of COQ3-b and COQ8A were elevated. The mutant cybrids had repressed PPARGC1A expression, elevated ATP5A levels, and reduced levels of mtDNA-encoded proteins, nuclear DNA-encoded subunits of respiratory enzyme complexes, MNRR1, cytochrome c, and DHODH, but no change in TFAM, TOM20, and VDAC1 levels. Alterations in the CoQ10 level in MELAS may be associated with mitochondrial energy deficiency and abnormal gene regulation. The finding of a reduction in the ubiquinol-10:ubiquinone-10 ratio in the MELAS mutant cybrids differs from our previous discovery that cybrids harboring the m.8344A>G mutation exhibit a high ubiquinol-10:ubiquinone-10 ratio.

Oligoribonucleotides in complexes with D-mannitol alter cell cycle and cause apoptosis in murine melanoma B16 cells

Oligoribonucleotides in complexes with D-mannitol alter cell cycle and cause apoptosis in murine melanoma B16 cells