B16 Mouse Melanoma Cells Research Articles

Preparation of Micelles Encapsulating Doxorubicin and Their Anticancer Effect in Combination With Tranilast in 3D Spheroids and In Vivo.

The objective of this study was to prepare doxorubicin encapsulated in micelles (DOX-micelles) using poly(hexadecanyloxyethylene glycol-lactate phosphate), which we recently synthesized, and to evaluate the anticancer effect of DOX-micelles in vitro and in vivo. To evaluate the anticancer effect of DOX-micelles in vitro, three-dimensional spheroids composed of B16 mouse melanoma cells and fibroblasts were prepared by changing the ratio of cancer cells to fibroblasts. In addition, for efficient doxorubicin treatment of the cells present in the center of the spheroids, tranilast, an anti-fibrotic drug was added to the spheroids before treatment with DOX-micelles, then the amount of doxorubicin and cell viability of spheroids were evaluated. Moreover, to assess the effects of the combination of DOX-micelles with tranilast in vivo, relative tumor volume was investigated in a mouse model of melanoma. The mean diameter and doxorubicin content of DOX-micelles were 93.3 nm and 3.5%, respectively. When the ratio of cancer cells to fibroblasts was 20:80, spheroids with spherical and rigid shapes were obtained. In addition, the amount of doxorubicin in the spheroids was increased by tranilast treatment, and an efficient anticancer effect was also observed. The anticancer effect of the combination of tranilast and DOX-micelles was confirmed in vivo. Micelles encapsulating doxorubicin are promising for cancer therapy, and their anticancer effect is improved by tranilast pretreatment in 3D spheroids in vivo.

Role and mechanism of Vγ4 T cells in impaired wound healing of rapamycin-induced full-thickness skin defects in mice

Role and mechanism of Vγ4 T cells in impaired wound healing of rapamycin-induced full-thickness skin defects in mice

Synthesis and anticancer activity in vitro and in vivo evaluation of iridium(III) complexes on mouse melanoma B16 cells

Combining the ligand NPIP (2-(2-nitrophenyl)-1H-imidazo[4,5-f][1,10]phenanthroline) with piq (1-phenylisoquinoline) and bzq (benzo[h]quinolone) gave [Ir(piq)2(NPIP)](PF6) (Ir1), and [Ir(bzq)2(NPIP)](PF6) (Ir2). The newly synthesized complexes were characterized by high-resolution mass spectrometry (HRMS), 1H NMR and 13C NMR. The complexes showed high antiproliferative activity against B16 cells. Three-dimensional (3D) cell model in vitro was used to evaluate the inhibitory effect of iridium (III) complex on B16 cells. The cellular uptake, mitochondrial localization, and intracellular distribution of the drugs confirmed that the iridium (III) complexes targeted the mitochondria, and the complexes can lead to the loss of mitochondrial membrane potential (MMP), increases the intracellular ROS content, further induces apoptosis. We also found that Ir1 and Ir2 can trigger the release of damage-associated molecular patterns (DAMPs) (cell surface calreticulin (CRT), heat-shock protein 70 (HSP70) and high mobility group box 1 (HMGB1)). In addition, Ir1 and Ir2 inhibited glutathione (GSH) synthesis and thus induced oxidative stress, Ir1 and Ir2 promoted malondialdehyde (MDA) production which is the stable metabolite of lipid peroxidation products. Finally, mice xenograft assay was performed to demonstrate that the complex shows higher antitumor activity in vivo than cisplatin. The inhibitory rates for cisplatin and Ir1 are 38.95% and 69.67%, respectively.

EPITHELIAL-MESENCHYMAL TRANSITION IN MELANOMA PROGRESSION: THE CONTRIBUTION OF ADAPTOR PROTEIN RUK/CIN85

The purpose of this study was to test the hypothesis that Ruk/CIN85 overexpression/knockdown in melanoma cells may be involved in the regulation of EMT. Materials and methods. The mouse melanoma cell line B16-F10 and its sublines with up-/down-regulation of Ruk/CIN85 (generated early using lentiviral technology) were used as a model for research. Melanoma cells were cultured in the complete RPMI 1610 medium under standard conditions. Proliferative activity of the cells was estimated using the MTT-test, and cell migratory potential was studied by the wound-healing assay. The data obtained were analyzed with parametric Student`s t-test. Results were expressed as mean ± SEM and significance was set at P<0.05. Results and Discussion. Cutaneous melanoma genesis is a multi-step process initiated by the transformation of a normal melanocyte following an oncogenic insult. Due to the transcriptome and metabolome reprogramming in the course of EMT, transformed melanoma cells change their phenotype and acquire increased proliferative rate, cell motility, invasiveness, and metastatic potential. According to the data obtained, overexpression of Ruk/CIN85 in B16 mouse melanoma cells (subclones Up7 and Up21) led to an increase in their proliferative activity by 1,6 and 1.8 times, respectively, at 24th hour in comparison with control Mock cells . At the 48th hour, when the cells reached confluence, the cell viability of subclones did not differ from the control ones. No statistically significant changes in the proliferative activity of B16 cells with suppressed expression of the adaptor protein (subclone Down) were found. In accordance with previous data, B16 cells overexpressing Ruk/CIN85 were characterized by strongly increased motility rate (more than twofold for both Up7 and Up21 subclones compared to control Mock cells). At the same time, knockdown of Ruk/CIN85 in B16 cells resulted in a decrease in their migratory activity by about 30%. Conclusions. All findings obtained demonstrated that the malignancy traits of melanoma B16 cells are inversely modulated upon up- and down-changes in adaptor protein Ruk/CIN85 expression levels suggesting its possible role in the control of EMT.

A noncoding regulatory RNA Gm31932 induces cell cycle arrest and differentiation in melanoma via the miR-344d-3-5p/Prc1 (and Nuf2) axis

Emerging evidence has shown that long non-coding RNAs (lncRNAs) play an important role in inhibiting tumor cell proliferation and inducing differentiation. In this study, integrative analysis of whole transcriptome sequencing data demonstrated that lncRNA-Gm31932 is significantly decreased in all-trans retinoic acid (ATRA)-induced and sodium 4-phenylbutanoate (PB-4)-induced mouse melanoma B16 cells. Silencing lncRNA-Gm31932 could inhibit B16 cell proliferation, with cell cycle arrest at the G0/G1 phase and obvious differentiation characteristics, e.g., increased cell volume, melanin content and tyrosinase (Tyr) activity. Furthermore, a series of experiments (luciferase reporter assay, RNA pull-down assay, and western blotting) showed that lncRNA-Gm3932 down-regulated Prc1 and Nuf2 by competitively sponging miR-344d-3-5p, which subsequently reduced the expression of cell cycle-related proteins CDK2, CDC2, and Cyclin B1, and increased the expression of P21 and P27. Moreover, silencing lncRNA-Gm31932 could significantly inhibit tumor growth in B16 melanoma-bearing mice. Taken together, these results indicate that as a possible signaling pathway for ATRA and PB-4, lncRNA-Gm31932 can induce cell cycle arrest and differentiation via miR-344d-3-5p/Prc1 (and Nuf2) axis.

TRAF6-overexpressing dendritic cells loaded with MUC1 peptide enhance anti-tumor activity in B16-MUC1 melanoma-bearing mice

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) signaling is a critical positive mechanism for the development, homeostasis and activation of immune cells. We investigated the effect of TRAF6 overexpression on dendritic cells (DCs) maturation. TRAF6-overexpressing DCs had increased expression of costimulatory molecules, major histocompatibility complex (MHC) molecules and IL-12 expression. This indicated that TRAF6 promoted the maturation of DCs and indirectly promoted Th1 activation. The antitumor activities between TRAF6-overexpressing DCs and control DCs were compared by administering DCs pulsed with mucin 1 (MUC1) Ag peptide in a therapeutic human MUC1-overexpressing mouse B16 melanoma cells (B16-MUC1) model. Administration of TRAF6-overexpressing DCs significantly inhibited the growth of B16-MUC1 tumors, accompanied by an increase in MUC1-specific Th1 responses and Tc1 responses, as well as a decrease in Tregs levels. TRAF6 signaling has been found to be involved in DCs maturation and Th1 activation in vitro, as well as therapeutic tumor models in vivo, indicating that TRAF6-overexpressing DCs may be a promising approach for cancer immunotherapy.

Mitofusin-2 Negatively Regulates Melanogenesis by Modulating Mitochondrial ROS Generation.

Inter-organellar communication is emerging as one of the most crucial regulators of cellular physiology. One of the key regulators of inter-organellar communication is Mitofusin-2 (MFN2). MFN2 is also involved in mediating mitochondrial fusion–fission dynamics. Further, it facilitates mitochondrial crosstalk with the endoplasmic reticulum, lysosomes and melanosomes, which are lysosome-related organelles specialized in melanin synthesis within melanocytes. However, the role of MFN2 in regulating melanocyte-specific cellular function, i.e., melanogenesis, remains poorly understood. Here, using a B16 mouse melanoma cell line and primary human melanocytes, we report that MFN2 negatively regulates melanogenesis. Both the transient and stable knockdown of MFN2 leads to enhanced melanogenesis, which is associated with an increase in the number of mature (stage III and IV) melanosomes and the augmented expression of key melanogenic enzymes. Further, the ectopic expression of MFN2 in MFN2-silenced cells leads to the complete rescue of the phenotype at the cellular and molecular levels. Mechanistically, MFN2-silencing elevates mitochondrial reactive-oxygen-species (ROS) levels which in turn increases melanogenesis. ROS quenching with the antioxidant N-acetyl cysteine (NAC) reverses the MFN2-knockdown-mediated increase in melanogenesis. Moreover, MFN2 expression is significantly lower in the darkly pigmented primary human melanocytes in comparison to lightly pigmented melanocytes, highlighting a potential contribution of lower MFN2 levels to higher physiological pigmentation. Taken together, our work establishes MFN2 as a novel negative regulator of melanogenesis.

Physicochemical Properties and Biological Activities of Silver Carp Scale Peptide and Its Nanofiltration Fractions.

To explore the physicochemical properties and biological functions of silver carp scale peptide (SCSP), its molecular-weight fractions SCSP-I, II, and III obtained by nanofiltration were assessed for their solubility, emulsibility, free radical scavenging ability, effect on the proliferation of mouse B16 cells. The results showed that the solubility of each fraction of SCSP was higher than 90%, SCSP-II and III were higher than 95%. The antioxidant powers on ⦁OH, ⦁ and Fe3+ were ranked as SCSP-III > SCSP-II > SCSP-I > SCSP. All fractions of SCSP had no toxic or side effects in mouse B16 melanoma cells experiments in vitro. At a concentration of 0.01 mg/mL, the tyrosinase activity of B16 cells in the SCSP-II fraction was significantly lower than that of the α-arbutin (P < 0.05), at 65.37%. The molecular weight distribution of SCSP was 399–1404 Dalton and 13 peptide sequences were detected. Among them, SCSP-II contained many hydrophobic amino acids, and SCSP-III stood out for combining arginine with hydrophobic amino acids. This may be the reason why the low molecular-weight SCSPs show the strong antioxidant activity and strong tyrosinase inhibition. The work provides a data base for the development of SCSP and increases the possibility of its application.

Heterogeneity of tyrosine-based melanin anabolism regulates pulmonary and cerebral organotropic colonization microenvironment of melanoma cells.

Background: Dietary tyrosine regulating melanoma progression has been well-recognized. However, whether tyrosine-based melanin anabolism contributes to pulmonary and cerebral organotropic colonization of melanoma remains elusive. Furthermore, approaches based on targeting tyrosinase activity to inhibiting multi-organ metastasis of melanoma cells need to be designed and validated.Methods: Patients derived melanoma cells and mouse B16 melanoma cells with different pigmentation were employed in this investigation. Tyrosine content dynamics in tumors and multiple organs during the melanoma progression was monitored, and tyrosine-based melanin synthesis of melanoma cells derived from multi-organ was determined. Additionally, we also adopted RNA-seq, flow cytometry, real-time PCR and composite metastasis mouse model to analyze organotropic colonization and to validate designed therapeutic strategies.Results: B16 melanoma cells with high activity of tyrosinase and sensitivity of tyrosine utilization for melanin synthesis (Tyr-H cells) easily colonized in the lung, while B16 melanoma cells lacking above characteristics (Tyr-L cells) exhibited potent proliferation in the brain. Mechanistically, Tyr-H cells recruited and trained neutrophils and macrophages to establish pulmonary metastatic niche dependent on highly secreted CXCL1 and CXCL2 and an excessive melanosome accumulation-induced cell death. Tyr-L cells enhanced PD-L1 expression in tumor-infiltrated macrophages when they are progressing in the brain. Accordingly, intervention of tyrosinase activity (2-Ethoxybenzamide or hydroquinone) in combination with inhibitors of phagocytosis (GSK343) or chemotaxis (SB225002) suppressed organotropic colonization and significantly improved the survival of melanoma- bearing mice treated with immune checkpoint blockade (PD1 antibody).Conclusions: The heterogeneity of melanoma cells in utilization of tyrosine is associated with organotropic colonization, providing the basis for developing new strategies to combat melanoma.

Comparative Study of High-throughput Screening Models for Anti-hyperpigmentation Compounds

HIGHLIGHTS It is the first time to compare mushroom tyrosinase model, B16 mouse melanoma cell model and zebrafish model. A high-throughput method for the quantification of melanin in zebrafish skin based on the Phyton for the first time.

The RGD-binding integrins αvβ6 and αvβ8 are receptors for mouse adenovirus-1 and -3 infection.

Mammalian adenoviruses (AdVs) comprise more than ~350 types including over 100 human (HAdVs) and just three mouse AdVs (MAdVs). While most HAdVs initiate infection by high affinity/avidity binding of their fiber knob (FK) protein to either coxsackievirus AdV receptor (CAR), CD46 or desmoglein (DSG)-2, MAdV-1 (M1) infection requires arginine-glycine-aspartate (RGD) binding integrins. To identify the receptors mediating MAdV infection we generated five novel reporter viruses for MAdV-1/-2/-3 (M1, M2, M3) transducing permissive murine (m) CMT-93 cells, but not B16 mouse melanoma cells expressing mCAR, human (h) CD46 or hDSG-2. Recombinant M1 or M3 FKs cross-blocked M1 and M3 but not M2 infections. Profiling of murine and human cells expressing RGD-binding integrins suggested that αvβ6 and αvβ8 heterodimers are associated with M1 and M3 infections. Ectopic expression of mβ6 in B16 cells strongly enhanced M1 and M3 binding, infection, and progeny production comparable with mαvβ6-positive CMT-93 cells, whereas mβ8 expressing cells were more permissive to M1 than M3. Anti-integrin antibodies potently blocked M1 and M3 binding and infection of CMT-93 cells and hαvβ8-positive M000216 cells. Soluble integrin αvβ6, and synthetic peptides containing the RGDLXXL sequence derived from FK-M1, FK-M3 and foot and mouth disease virus coat protein strongly interfered with M1/M3 infections, in agreement with high affinity interactions of FK-M1/FK-M3 with αvβ6/αvβ8, determined by surface plasmon resonance measurements. Molecular docking simulations of ternary complexes revealed a bent conformation of RGDLXXL-containing FK-M3 peptides on the subunit interface of αvβ6/β8, where the distal leucine residue dips into a hydrophobic pocket of β6/8, the arginine residue ionically engages αv aspartate215, and the aspartate residue coordinates a divalent cation in αvβ6/β8. Together, the RGDLXXL-bearing FKs are part of an essential mechanism for M1/M3 infection engaging murine and human αvβ6/8 integrins. These integrins are highly conserved in other mammals, and may favour cross-species virus transmission.

Suppressive Effects of Anisomycin on the Proliferation of B16 Mouse Melanoma Cells In Vitro.

Anisomycin, a potential anticancer therapeutic drug, exerts an antitumor effect on melanoma cells at a lower concentration than that required for other cancer cells. However, the molecular mechanisms remain unclear. The sensitivity to and cytotoxicity of anisomycin, as well as the effects of anisomycin on glucose metabolism and relative mRNA expression of senescence- and cancer-associated genes, were studied using B16 mouse melanoma cells. The viability of anisomycin-treated cells decreased in a concentration-dependent manner, and the growth of cell spheroids was suppressed by 50 nM anisomycin. Glucose metabolism was reduced by anisomycin treatment, and the mRNA expression of genes responsible for growth inhibition, such as p21, p53 and Txnip was upregulated. The results suggest that anisomycin may be a promising future anticancer drug that is effective at low concentrations against melanoma by reducing glucose metabolism, causing cell senescence-like phenomena.

Soy isoflavone metabolite equol inhibits cancer cell proliferation in a PAP associated domain containing 5-dependent and an estrogen receptor-independent manner

Isoflavone is a species of polyphenol found mainly in soy and soy products. Many studies have demonstrated its estrogen receptor (ER)-dependent action. Equol is an intestinal metabolite of a major soy isoflavone daidzein. We aimed to elucidate the mechanism for ER-independent actions of equol. Equol has been shown to inhibit proliferation of HeLa human cervical cancer cells and mouse melanoma B16 cells in an ER-independent manner. Using functional genetic screening, PAP associated domain containing 5 (PAPD5), which is a non-canonical poly(A) polymerase, was identified as an essential molecule in the ER-independent action. While peroral administration of equol inhibited tumor growth of control B16 cells subcutaneously inoculated in mice, it had little effect on the growth of PAPD5-ablated B16 cells. Intriguingly, equol progressed tumor growth of the PAPD5-ablated human breast cancer MCF-7 cells, which have high ERα expression. Equol has been found to induce polyadenylation of snoRNAs in a PAPD5-depdendent manner. Furthermore, peroral equol administration increased microRNA miR-320a expression in tumors. Together, these results suggest that equol may have a dual effect on ER-positive cancer cells, acting with, antiproliferative activity through PAPD5 and exhibiting proliferative activity via ERα and the former could be associated with miR-320a.

Comprehensive chemical proteomics analyses reveal that the new TRi-1 and TRi-2 compounds are more specific thioredoxin reductase 1 inhibitors than auranofin

Anticancer drugs that target cellular antioxidant systems have recently attracted much attention. Auranofin (AF) is currently evaluated in several clinical trials as an anticancer agent that targets the cytosolic and mitochondrial forms of the selenoprotein thioredoxin reductase, TXNRD1 and TXNRD2. Recently, two novel TXNRD1 inhibitors (TRi-1 and TRi-2) have been developed that showed anticancer efficacy comparable to AF, but with lower mitochondrial toxicity. However, the cellular action mechanisms of these drugs have not yet been thoroughly studied. Here we used several proteomics approaches to determine the effects of AF, TRi-1 and TRi-2 when used at IC50 concentrations with the mouse B16 melanoma and LLC lung adenocarcinoma cells, as these are often used for preclinical mouse models in evaluation of anticancer drugs. The results demonstrate that TRi-1 and TRi-2 are more specific TXNRD1 inhibitors than AF and reveal additional AF-specific effects on the cellular proteome. Interestingly, AF triggered stronger Nrf2-driven antioxidant responses than the other two compounds. Furthermore, AF affected several additional proteins, including GSK3A, GSK3B, MCMBP and EEFSEC, implicating additional effects on glycogen metabolism, cellular differentiation, inflammatory pathways, DNA replication and selenoprotein synthesis processes. Our proteomics data provide a resource for researchers interested in the multidimensional analysis of proteome changes associated with oxidative stress in general, and the effects of TXNRD1 inhibitors and AF protein targets in particular.

Bone morphogenetic protein induces bone invasion of melanoma by epithelial–mesenchymal transition via the Smad1/5 signaling pathway

Oral malignant melanoma, which frequently invades the hard palate or maxillary bone, is extremely rare and has a poor prognosis. Bone morphogenetic protein (BMP) is abundantly expressed in bone matrix and is highly expressed in malignant melanoma, inducing an aggressive phenotype. We examined the role of BMP signaling in the acquisition of an aggressive phenotype in melanoma cells in vitro and in vivo. In five cases, immunohistochemistry indicated the phosphorylation of Smad1/5 (p-Smad1/5) in the nuclei of melanoma cells. In the B16 mouse and A2058 human melanoma cell lines, BMP2, BMP4, or BMP7 induces morphological changes accompanied by the downregulation of E-cadherin, and the upregulation of N-cadherin and Snail, markers of epithelial–mesenchymal transition (EMT). BMP2 also stimulates cell invasion by increasing matrix metalloproteinase activity in B16 cells. These effects were canceled by the addition of LDN193189, a specific inhibitor of Smad1/5 signaling. In vivo, the injection of B16 cells expressing constitutively activated ALK3 enhanced zygoma destruction in comparison to empty B16 cells by increasing osteoclast numbers. These results suggest that the activation of BMP signaling induces EMT, thus driving the acquisition of an aggressive phenotype in malignant melanoma.Bone morphogenetic protein (BMP) induces epithelial–mesenchymal transition (EMT) both B16 mouse and A2058 human melanoma cell lines. The injection of B16 cells expressing constitutively activated ALK3 enhanced zygoma destruction compared with empty B16 cells, suggesting that the activation of BMP signaling enhances bone invasion by inducing EMT in melanoma cells.

Bioaccessibility and intracellular antioxidant activity of phloretin embodied by gliadin/sodium carboxymethyl cellulose nanoparticles

In this work, gliadin/sodium carboxymethyl cellulose nanoparticles (G/CMC) were prepared to encapsulate bioactive phloretin (Phl) for improving the stability and bioaccessibility by simple antisolvent precipitation. Scanning electron microscopy (SEM) and dynamic light scattering showed that G/CMC nanoparticles were spherical with the diameter below 350 nm. Electrostatic interaction and hydrogen bond were dominant driving force during the formation of G/CMC nanoparticles. Phloretin-loaded G/CMC nanoparticles (G/CMC-Phl) had robust resistance to pH shift, heat treatment and UV irradiation. The bioaccessibility of encapsulated phloretin increased from 23% to 55% via the strategy, as compared with free phloretin. Meanwhile, G/CMC-Phl had a strongly protective effect on erythrocyte hemolysis induced by 2, 2′-azobis-2-methyl-propanimidamide dihydrochloride (AAPH), and inhibited the generation of reactive oxygen species (ROS). It could recover the intracellular antioxidant enzymes (superoxide dismutase, SOD and glutathione peroxidase, GSH-Px) activity to normal levels and inhibit the production of malondialdehyde (MDA). Moreover, the protection and control release behavior of G/CMC nanoparticles was investigated by B16 mouse melanoma cells. This study provides a simple way to fabricate food-grade nanoparticles for the encapsulation of water-insoluble phloretin to broaden the application in pharmaceutical, cosmetics and food industry.

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

Biomimetic Recognition-Based Bioorthogonal Host–Guest Pairs for Cell Targeting and Tissue Imaging in Living Animals

A Combination of GM-CSF and Released Factors from Gamma-Irradiated Tumor Cells Enhances the Differentiation of Macrophages from Bone Marrow Cells and Their Antigen-Presenting Function and Polarization to Type 1.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes dendritic cell differentiation from precursors, and consequently, enhances the antigen presentation process and adaptive immune responses. With such functions, GM-CSF has been used as immunotherapy in combination with radiotherapy for cancer treatment to augment the survival and activity of immune cells. However, an immune-suppressive tumor microenvironment may cause anergy of T cells. It has also been reported that GM-CSF contributes to the development of myeloid-derived suppressor cells from the precursors. In this study, to analyze the combined effect of GM-CSF and released factors from cancer cells after gamma-ray irradiation on bone marrow cell differentiation and dynamics, we established an in vitro culture system using mouse bone marrow cells, GM-CSF, and conditioned medium from gamma ray irradiated mouse melanoma B16 cells at 24 Gy. We analyzed the gene expression changes of the bone marrow-derived cells on day 6. The results showed that GM-CSF dose-dependently enhanced the differentiation of macrophages from bone marrow cells, their antigen-presenting function and polarization to type I. The results implied the induced macrophages from the bone marrow may potentially contribute to tumor immune responses in a systemic manner when GM-CSF is boosted during photon-beam radiation therapy.

Abstract 1845: Enhancing the efficacy of antitumor antibodies with immune checkpoint inhibitors and targeted therapy in melanoma

Abstract The therapeutic outcomes of current strategies in melanoma treatment have greatly improved within the last decade. However, the persistent relatively low success rate of current standard-of-care strategies underscores the urgent need for improvement of these therapeutic approaches. The use of specific antitumor antibodies recently transformed the cancer therapeutics landscape with successes such as anti-HER2 in breast cancer, and anti-EGFR in HNSCC and colorectal cancer. However, antitumor antibodies have not been developed for the treatment of melanoma. Here, using mouse preclinical models of melanoma (both solid tumor and lung metastasis), we found that the therapeutic efficacy of the specific antitumor antibody TA99 (anti-TYRP1) is significantly enhanced by immune checkpoint blockade. This was associated with a greater CD8+, CD8+/Foxp3+, NK1.1+ and dendritic cell infiltrate, suggestive of an increased anti-tumor immune response. Further, MEK inhibition (MEKi) increased the expression of melanosomal antigens in B16 and YUMM mouse melanoma cell lines in vitro. We next combined TA99 and MEKi to treat B16 melanoma in vivo and observed a synergistic reduction in tumor growth. Treatment of mice bearing YUMM melanoma (BRAF mutant) with TA99/MEKi resulted in the complete elimination of tumors in the majority of animals, and produced durable responses. Moreover, we found a synergistic effect when B16 tumor-bearing mice were treated with TA99 combined with MEKi and immune checkpoint blockade (anti-PD1 and anti-CTLA4). Our findings suggest that MEKi induced an increased expression of tumor-associated antigens, which, in combination with antitumor antibodies, generated a robust adaptive antitumor response that was sustained by immune checkpoint inhibition therapy. These improved therapeutic effects of specific antitumor antibodies by current standard-of-care therapeutics have potential near-term translation to the clinic, in light of fully humanized anti-TYRP1 antibodies currently in clinical development. Citation Format: Rolando Pérez-Lorenzo, Stephanie O. Erjavec, Raphael Clynes, Angela M. Christiano. Enhancing the efficacy of antitumor antibodies with immune checkpoint inhibitors and targeted therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1845.

Antityrosinase Mechanism and Antimelanogenic Effect of Arbutin Esters Synthesis Catalyzed by Whole-Cell Biocatalyst.

Tyrosinase is a key enzyme responsible for enzymatic browning of fruits and vegetables and skin disorders due to overproduction of melanin. Arbutin is an inhibitor of tyrosinase; however, its high polarity and weak transdermal absorption capacity limit its applications. In this paper, a green solvent system was developed to successfully synthesize arbutin esters with improved liposolubilities (Clog P values = 0.27-5.03). Among the obtained esters, arbutin undecenoate (AU) showed the strongest tyrosinase-inhibiting activity (15.6%), which was 9.0 times higher than that of arbutin. An enzyme kinetics study indicated that AU was a competitive inhibitor with reversible inhibition. The esters inhibited tyrosinase by making the secondary structure of tyrosinase looser and less stable; moreover, the interactions between tyrosinase and AU driven by metal interactions and hydrogen bonds also offered a mechanism for inhibition of AU on tyrosinase. In addition, AU (100 μM) reduced the melanin content of B16 mouse melanoma cells to 61.3% of the control group.