Mouse Melanoma Cell Line Research Articles

Melanin Quantification by Chemical Methods in Healthy and Melanoma Cell Lines Correlated with the Importance in the Development of Melanoma

In the last decade, the incidence of skin cancer has increased significantly and there is data that rank it as the most common form of malignancy in humans. Among the classified skin cancers, melanoma is the most severe with a significant mortality rate. The determining factor in the occurrence of skin cancer is generally recognised to be ultraviolet radiation. Skin pigmentation has been shown to provide a high degree of protection and this is due to melanin. In order to be able to consider certain hypotheses and to develop new approaches to melanoma, in this study it was studied the melanin content in certain cell types that have a direct connection with melanoma. The cell lines used in the study were: (i) human melanoma cell lines: A375, SK-MEL-1, SK-MEL-3, SK-MEL-5, SK-MEL-28, SH-4, and COLO 829; (ii) mouse melanoma cell lines: B164A5, B16F0, and B16F10; and (iii) healthy cell lines of human origin: HEMa (primary melanocytes), HaCaT (immortalised keratinocytes) and 1BR3 (human fibroblasts). The results obtained pointed out that A375, SK-MEL-28 and SK-MEL-5 cells do not produce melanin, SK-MEL-1, SH-4 and SK-MEL-3 cells produce melanin while COLO 829 cells � only in small passages produce melanin. Of the 3 lines of murine melanoma tested, the one that produces melanin in a higher concentration is B16F10, followed by B16F0 and B164A5.

Phytochemical analysis of two Weigela florida cultivars, “Pink Poppet” and “Jean’s Gold”

Nine different oleanane-type glycosides were extracted and isolated by various chromatographic methods from two Weigela florida cultivars, “Pink Poppet” and “Jean’s Gold”. From the roots of W. florida “Pink Poppet”, three monodesmosidic oleanolic acid saponins 1, 4, 5 were obtained, together with one hederagenin ester 6 from the leaves, and six bidesmosidic saponins 2, 3, 6-9 were isolated from the leaves of W. florida “Jean’s Gold”. Among compounds 1-9, three were previously undescribed (1-3) and six (4-9) were already published in the literature. Their structures were assigned by spectroscopic analysis mainly 2D NMR and mass spectrometry (ESI-MS).The cytotoxicity of the isolated compounds 1, 3, 4, 6, 7, 9, against mouse colon carcinoma (CT26), mouse melanoma (B16) and human liver cancer (HepG2) cell lines, was evaluated by MTS assay. The ester group at C-28 and/or the primary alcohol function at C-23 led to a negative effect, whereas monodesmosidic oleanolic acid glycosides displayed a good cytotoxicity with IC50 ranging from 2.19 to 2.83 μM. A key sequence in the structure of the molecules was proposed for the cytotoxicity.

Regulation of eIF2α by RNF4 Promotes Melanoma Tumorigenesis and Therapy Resistance

Among the hallmarks of melanoma are impaired proteostasis and rapid development of resistance to targeted therapy that represent a major clinical challenge. However, the molecular machinery that links these processes is unknown. Here we describe that by stabilizing key melanoma oncoproteins, the ubiquitin ligase RNF4 promotes tumorigenesis and confers resistance to targeted therapy in melanoma cells, xenograft mouse models, and patient samples. In patients, RNF4 protein and mRNA levels correlate with poor prognosis and with resistance to MAPK inhibitors. Remarkably, RNF4 tumorigenic properties, including therapy resistance, require the translation initiation factor initiation elongation factor alpha (eIF2α). RNF4 binds, ubiquitinates, and stabilizes the phosphorylated eIF2α (p-eIF2α) but not activating transcription factor 4 or C/EBP homologous protein that mediates the eIF2α-dependent integrated stress response. In accordance, p-eIF2α levels were significantly elevated in high-RNF4 patient-derived melanomas. Thus, RNF4 and p-eIF2α establish a positive feed-forward loop connecting oncogenic translation and ubiquitin-dependent protein stabilization in melanoma.

Investigating the Effects of Shikonin, Deoxyshikonin, and (β,β-Dimethylacryl)Shikonin on Melanoma Cell Lines

Melanoma is the most lethal form of various skin cancers and contributes to more than 79% of all skin cancer deaths. Although there are numerous therapies available for melanoma, the high rate of recurrence in melanoma post-therapy remains a challenging issue for both patients and clinicians. Apoptosis is one of the foundations for cancer treatment as deficient apoptosis is one of the most essential reasons for the formation of tumour tissues. Shikonin (SHI), an active component extracted from Lithospermum erythrorhizon, has been broadly demonstrated to possess antitumorigenic property due to its apoptosis-inducing ability in various cancer cell lines. The analogs of SHI, such as deoxyshikonin (DO-SHI) and (β,β-dimethylacryl)shikonin (β,β-SHI), have also been found to possess similar bioactivities. The apoptosis-inducing ability of SHI and its analogs enable them to be potential anticancer therapies. In this study reported herein, we investigated the effects of SHI, DO-SHI, and β,β-SHI on both human (A375) and mouse (B16-F0 and B16-F10) melanoma cell lines. Cell viability was measured using Alamar blue assay, while cell migration was detected using scratch assay. Cell apoptosis was captured using terminal deoxynucleotidyl dUTP nick end labeling and fluorescence activated cell sorting. Signaling pathway activation was detected using Western blotting. Our results revealed that SHI, DO-SHI, and β,β-SHI reduce cell viability, inhibit cell migration, and induce apoptosis in melanoma cell lines. These 3 molecules-induced apoptosis in A375 is regulated via mitogen-activated protein kinase/caspase 3 signaling pathway. In particular, DO-SHI and β,β-SHI induce higher apoptosis rate in A375 and B16-F0 compared to SHI. The data from this study demonstrate that DO-SHI and β,β-SHI offer potential new reagents for managing melanoma.

Abstract A79: Inhibition of p53-MDM2 protein interaction reduces tumor growth in a mouse melanoma model

Abstract Tumor suppressor protein p53 is mutated in close to 50% of human tumors. Under steady state conditions, the two E3 ligases MDM2 and MDM4 interact with and inhibit the transcriptional activity of p53. Inhibition of p53-MDM2/4 interaction to reactivate p53 has therefore been considered a therapeutic strategy. In this study, we tested p53-MDM2 protein-protein interaction inhibition using the small molecule AMG 232, which is currently being tested in phase 1b/2a clinical trials. In vitro, AMG 232 induced a significant, p53-dependent growth arrest in the mouse melanoma cell line B16-F10. Using mass spectrometry-based proteomics, we identified differential protein expression patterns following AMG 232 treatment of B16-F10 melanoma cells. In vivo, the growth of B16-F10 melanoma cells implanted in WT C57BL/6 mice was significantly reduced by AMG 232 treatment. Our data demonstrate that AMG 232 induces a p53-dependent tumor growth arrest in an immunocompetent mouse model, and we are currently testing whether AMG232 can synergize with checkpoint immunotherapy. Citation Format: Katrine Ingelshed, Danai Lianoudaki, Dilraj Lama, Silke Sohn, Nicolas Fritz, Long Jiang, Fredrik Wermeling, Mikael Karlsson, David P. Lane, Saikiran K. Sedimbi. Inhibition of p53-MDM2 protein interaction reduces tumor growth in a mouse melanoma model [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A79.

4-Substituted picolinohydrazonamides as a new class of potential antitubercular agents

The series of new 4-substituted picolinohydrazonamides were synthesized (6-25) and evaluated for tuberculostatic activity. Compounds having a hydrophilic cyclic amine such as morpholine and pyrrolidine at the end of the thiosemicarbazide chain, exhibited the highest antimycobacterial activity. The antimycobacterial activity of compounds 6, 11, and 15 (MIC 0.4–0.8 μg/mL) was higher than that of reference drugs. Moreover, derivative 15 exhibited lower activity against other tested microorganism such as bacteria gram-positive, gram-negative or fungi. Thus, this compound is characterized by the selectivity of antimicrobial activity. Antiproliferative study conducted against human dermal fibroblasts (HDF) and mouse melanoma cell line (B16-F10) revealed low cytotoxicity of compound 15. Conducted research allowed to identify compound 15 as leading for further research.

Anti-melanogenic effects of extracellular vesicles derived from plant leaves and stems in mouse melanoma cells and human healthy skin

ABSTRACT Consumer interest in cosmetic industry products that produce whitening effects has increased demand for agents that decrease melanin production. Many such anti-melanogenic agents are associated with side effects, such as contact dermatitis and high toxicity, and also exhibit poor skin penetration. Considerable recent research has focused on plant-derived products as alternatives to chemotherapeutic agents that possess fewer side effects. In the current study, we investigated the anti-melanogenic effects of extracellular vesicles (EVs) extracted from leaves and stems of Dendropanax morbifera. Using spectrophotometric and biochemical approaches, we found that leaf-derived extracellular vesicles (LEVs) and stem-derived extracellular vesicles (SEVs) reduced melanin content and tyrosinase (TYR) activity in the B16BL6 mouse melanoma cell line in a concentration-dependent manner. An electron microscopy analysis further confirmed that LEVs and SEVs induce a concentration-dependent decrease in melanin content in melanoma cells. Both LEVs and SEVs exerted a greater whitening effect on melanoma cells than arbutin, used as a positive control, with LEVs producing the greater effect. Notably, neither LEVs nor SEVs induced significant cytotoxicity. We also examined the effects of plant-derived EVs on the expression of tyrosinase-related proteins (TRPs) in melanoma cells. LEVs inhibited expression of melanogenesis-related genes and proteins, including microphthalmia-associated transcription factor (MITF), TYR, TRP-1 and TRP-2. In a human epidermis model, LEVs exerted a stronger inhibitory effect on melanin production than arbutin. Collectively, our data suggest that LEVs from D. morbifera may be a novel candidate natural substance for use as an anti-melanogenic agent in cosmeceutical formulations.

Alkylation enhances biocompatibility and siRNA delivery efficiency of cationic curdlan nanoparticles

Cationic curdlan derivatives are a class of promising carriers for nucleic acid delivery including short interfering RNA (siRNA). While our previous studies demonstrated the siRNA delivery efficiency of aminated curdlan derivatives, the associated cytotoxicity issue remained unsolved. To investigate the effects of alkylation on the toxicity as well as the transfection efficiency, we conjugated short alkyl chains to 6-amino-6-deoxy-curdlan (6AC-100). The cytotoxicity of alkylated 6AC-100 derivatives (denote CuVa polymers) decreased with the increase of the degree of substitution (DS). CuVa3, with the highest DS, showed a 50% decreased cytotoxicity compared to 6AC-100 to 6AC-100 at a concentration of 140 μg/mL. The CuVa polymers readily complexed with siRNA to form nanoparticles, and induced significant knockdown of a disease related gene (STAT3) in mouse melanoma cell line B16. However, B16 cells transfected with siSTAT3 complexed to CuVa3 showed the highest phenotypic changes. These findings suggest that CuVa polymers have significantly enhanced biocompatibility and may be a promising delivery system for delivery of therapeutic siRNAs.

The transcription factor PU.1 mediates enhancer-promoter looping that is required for IL-1β eRNA and mRNA transcription in mouse melanoma and macrophage cell lines

The DNA-binding protein PU.1 is a myeloid lineage-determining and pioneering transcription factor due to its ability to bind "closed" genomic sites and maintain "open" chromatin state for myeloid lineage-specific genes. The precise mechanism of PU.1 in cell type-specific programming is yet to be elucidated. The melanoma cell line B16BL6, although it is nonmyeloid lineage, expressed Toll-like receptors and activated the transcription factor NF-κB upon stimulation by the bacterial cell wall component lipopolysaccharide. However, it did not produce cytokines, such as IL-1β mRNA. Ectopic PU.1 expression induced remodeling of a novel distal enhancer (located ∼10 kbp upstream of the IL-1β transcription start site), marked by nucleosome depletion, enhancer-promoter looping, and histone H3 lysine 27 acetylation (H3K27ac). PU.1 induced enhancer-promoter looping and H3K27ac through two distinct PU.1 regions. These PU.1-dependent events were independently required for subsequent signal-dependent and co-dependent events: NF-κB recruitment and further H3K27ac, both of which were required for enhancer RNA (eRNA) transcription. In murine macrophage RAW264.7 cells, these PU.1-dependent events were constitutively established and readily expressed eRNA and subsequently IL-1β mRNA by lipopolysaccharide stimulation. In summary, this study showed a sequence of epigenetic events in programming IL-1β transcription by the distal enhancer priming and eRNA production mediated by PU.1 and the signal-dependent transcription factor NF-κB.

Cyanidin-3-o-Glucoside Pharmacologically Inhibits Tumorigenesis via Estrogen Receptor β in Melanoma Mice.

Expression patterns of estrogen receptors [ERα, ERβ, and G-protein associated ER (GPER)] in melanoma and skin may suggest their differential roles in carcinogenesis. Phytoestrogenic compound cyanidin-3-o-glucoside (C3G) has been shown to inhibit the growth and metastatic potential of melanoma, although the underlying molecular mechanism remains unclear. The aim of this study was to clarify the mechanism of action of C3G in melanoma in vitro and in vivo, as well as to characterize the functional expressions of ERs in melanoma. In normal skin or melanoma (n = 20/each), no ERα protein was detectable, whereas expression of ERβ was high in skin but weak focal or negative in melanoma; and finally high expression of GPER in all skin vs. 50% melanoma tissues (10/20) was found. These results correspond with our analysis of the melanoma survival rates (SRs) from Human Protein Atlas and The Cancer Genome Atlas GDC (362 patients), where low ERβ expression in melanoma correlate with a poor relapse-free survival, and no correlations were observed between SRs and ERα or GPER expression in melanoma. Furthermore, we demonstrated that C3G treatment arrested the cell cycle at the G2/M phase by targeting cyclin B1 (CCNB1) and promoted apoptosis via ERβ in both mouse and human melanoma cell lines, and inhibited melanoma cell growth in vivo. Our study suggested that C3G elicits an agonistic effect toward ERβ signaling enhancement, which may serve as a potential novel therapeutic and preventive approach for melanoma.

Inhibition of NLRP3 inflammasome in tumor microenvironment leads to suppression of metastatic potential of cancer cells

Tumor microenvironment favors tumor cells to promote their growth and metastasis such as migration, invasion, and angiogenesis. IL-1β, one of the inflammatory cytokines released from myeloid cells in tumor microenvironment, plays an important role in development and progress of tumor. The activation of inflammasome is a critical step to secrete mature IL-1β through stepwise reactions to activate capspase-1. Therefore, we investigated whether the inhibition of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in macrophages regulated the metastatic potential of tumor cells. NLRP3 inflammasome was activated by ATP in bone marrow-derived primary mouse macrophages. The metastatic potential of mouse melanoma cell line (B16F10) was determined by migration and invasion assays with transwell system. ATP-treated wild-type macrophages increased the migration and invasion of melanoma cells. However, NLRP3- or caspase-1-knockout macrophages exhibited greatly diminished ability to promote the migration and invasion of melanoma cells. In addition, treatment with celastrol, an inhibitor of NLRP3 inflammasome, reduced the potency of macrophages to stimulate migration and invasion of melanoma cells. The results demonstrate that inhibition of the NLRP3 inflammasome in macrophages by genetic deficiency or a pharmacological inhibitor is linked to suppression of the metastatic potential of tumor cells. The results would provide a novel anti-cancer strategy to modulate tumor microenvironment by suppressing NLRP3 inflammasome and consequently reducing IL-1β production.

HMGB1 modulates the balance between senescence and apoptosis in response to genotoxic stress

High mobility group box-1 (HMGB1) is involved in various diseases and is associated with the resistance of many types of human cancers to chemotherapy; however, its role in cancer metastasis remains unexplored. This study examined the HMGB1 status of both highly and poorly metastatic cancer cells in response to genotoxic stress. The weakly and highly metastatic mouse melanoma cell lines (B16 vs. B16-F10), human melanoma cell lines (SK-MEL-28 vs. SK-MEL-24), colon cancer cell lines (DLD-1 vs. LS174T), and wild-type (WT) vs. HMGB1 knockout (KO) mouse embryonic fibroblasts (MEFs) were treated with doxorubicin (Dox) and camptothecin (CPT), and then cellular morphology, senescence-associated β-galactosidase staining, lactate dehydrogenase release, and caspase-3 activation were used to assess cell fate. To investigate the role of HMGB1 in p21 expression, HMGB1 and p21 expressions were examined by Western blotting, and the HMGB1-mediated p21 promoter luciferase assay was performed after small interfering RNA or overexpression of HMGB1 prior to Dox treatment. Although highly metastatic mouse melanoma B16-F10 cells preferred senescence, with persistent HMGB1 expression, poorly metastatic B16 cells entered apoptosis, with decreasing HMGB1 levels via cleavage under Dox treatment. Similarly, more metastatic human melanoma SK-MEL-24 and human colon cancer LS174T cells underwent senescence, whereas fewer metastatic melanoma SK-MEL-28 and DLD-1 cells exhibited apoptosis under Dox stimulation. In senescent B16-F10, SK-MEL-24, and LS174T cells treated with Dox, p21 levels were increased by persistent HMGB1 expression. Furthermore, HMGB1 depletion caused a senescence-apoptosis shift with p21 down-regulation in B16-F10 cells, and HMGB1 overexpression switched from apoptosis to senescence concomitantly with increased p21 expression in B16 cells after Dox treatment. The same effects were observed in both cell pairs of mouse melanoma and human colon cancer cells treated with CPT, another genotoxic stressor. Indeed, although WT MEF entered senescence accompanied by p21 increase, HMGB1 KO underwent apoptosis with p21 decrease by Dox treatment. In our cell model system, we demonstrated that highly metastatic cancer cells preferentially enter senescence, whereas apoptosis predominates in weakly metastatic cancer cells under genotoxic stress, which depends on the presence or absence of HMGB1, suggesting that the HMGB1-p21 axis is required for genotoxic stress-induced senescence. These findings suggest that HMGB1 modulation of cancers with different metastatic status could be a strategy for selectively enforcing tumor suppression.-Lee, J.-J., Park, I. H., Rhee, W. J., Kim, H. S., Shin, J.-S. HMGB1 modulates the balance between senescence and apoptosis in response to genotoxic stress.

Abstract 4520: Lymphatic vessels regulate exosome trafficking from tumors

Abstract Prior to metastasis, tumor-draining lymph nodes and distant organs are altered by a number of tumor-derived factors that help to both suppress host immunity as well as form an environment that supports metastatic tumor cells, i.e., a pre-metastatic niche. Recent studies suggest that pre-metastatic niches are induced by vesicles, including exosomes, secreted by tumor cells and cells of the tumor microenvironment. Here, we investigated the contribution of lymphatic vessels, particularly the lymphatic endothelial cells (LECs), in the active regulation of tumor exosome production and trafficking. To track exosomes, we purified and fluorescently labeled exosomes from in vitro cultures of the mouse melanoma cell line B16 F10 and of the human melanoma cell line Me260LN. Upon injection into the mouse ear dermis, fluorescent exosomes were observed within minutes in draining lymphatics, but not in surrounding blood vessels. Using the B16 F10 melanoma model overexpressing the lymphangiogenic growth factor VEGF-C, we found that upon intratumoral injection of exosomes, LECs within the tumor microenvironment abundantly take up exosomes, and at higher levels than blood endothelial cells, delivering exosomes to draining lymph nodes and eventually to the blood. In transgenic mice lacking dermal lymphatics, exosomes injected intratumorally were absent in the lymph nodes and present in far lower amounts in the blood compared to those injected into wild type mice. Additionally, while the density of endogenous blood exosomes increased upon tumor growth in wild type mice, no significant change in blood exosome density was observed in transgenic mice. Using an in vitro model where LECs are seeded onto porous inserts, we found that LEC transport of tumor exosomes occurs preferentially from their basal to apical side, and that some of these exosomes were taken up and stored intracellularly. Together, these findings suggest that lymphatic vessels constitute a major route of tumor exosome distribution to the systemic circulation and that LECs actively regulate exosome transport by active uptake. Citation Format: Lea Maillat, Lambert Potin, Witold W. Kilarski, Melody A. Swartz. Lymphatic vessels regulate exosome trafficking from tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4520.

Abstract 1485: Hepatic cell cycle-related kinase shapes a metastatic-prone liver microenvironment via crosstalk between myeloid-derived suppressor cell and natural killer T cell

Abstract Metastasis is a prominent cause of cancer-related death governed by both cancer cell-intrinsic mechanisms and extrinsic microenvironment. Clinical observations demonstrated liver as a common metastatic site for various cancers, which may be due to its immune tolerant environment. Myeloid-derived suppressor cell (MDSC) is a heterogeneous cell population of immature myeloid cells that contribute to the formation of a favorable metastatic environment partially via suppression of immune effector cells. However, the underlying mechanisms in liver tropism of tumor metastasis remain poorly understood. We have previously discovered that cell cycle-related kinase (CCRK) can promote primary hepatocellular carcinoma (HCC) development via MDSCs (Gut 2018; Nat Commun accepted). Here we hypothesize that the accumulation of hepatic MDSCs induced by CCRK may contribute to the formation of a favorable metastatic liver microenvironment. By the construction of a liver-specific CCRK inducible transgenic (TG) mouse model by a Cre/loxP system, induction of CCRK expression by tamoxifen injection could increase CD11b+Gr-1+Ly6G+Ly6Clow polymorphonuclear (PMN)-MDSCs liver accumulation specifically in male mice with upregulated Cxcl1 and Gcsf expression. Intrahepatic injection of a mouse hepatoma cell line Hep1-6 in male TG mice developed larger tumors compared to control and positively associated with increased PMN-MDSCs levels in liver. Moreover, the tumorigenicity was abolished by PMN-MDSC depletion. Notably, intrasplenic injection of a mouse melanoma cell line B16F10 exhibited increased level of liver-infiltrating PMN-MDSCs and enhanced liver metastasis in male TG mice compared to control mice. Moreover, depletion of PMN-MDSCs suppressed metastasis in liver. Mechanistically, anti-tumor NKT cells, rather than NK cells and CD8+T cells, were negatively correlated with tumor weight and MDSC proportion, indicating the potential crosstalk between MDSC and NKT in liver metastasis. Our findings suggest that hepatic CCRK expression create a tumor growth- and metastasis-supportive liver microenvironment via enhancing immunosuppression. Additional colorectal cancer metastasis models will be evaluated in our established TG mice. Moreover, the roles and underlying mechanisms of CCRK-PMN-MDSC interactions in the establishment of liver metastasis await further investigation. This project is supported by Collaborative Research Fund C4017-14G and the Focused Innovations Scheme 1907309. Citation Format: Xuezhen ZENG, Jingying Zhou, Zhewen Xiong, Hanyong Sun, Weiqin Yang, Wenshu Tang, Yu Feng, Alfred Sze-Lok Cheng. Hepatic cell cycle-related kinase shapes a metastatic-prone liver microenvironment via crosstalk between myeloid-derived suppressor cell and natural killer T cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1485.

Teraphtal decreased the sensitivity tumor cells to doxorubicine in vitro but does not affect its antitumor effect in vivo .

Introduction . Anthracycline antibiotic doxorubicin (DOX) is widely used in clinical oncology. It is known that hemin, endogenious compound, has the ability to modulate DOX cytotoxicity. We found that DOX toxicity against mammalian cancer cells can be decreased in vitro in the presence of teraftal (ТF), the component anticancer binaric catalytic system (TF + ascorbic acid).Purpose . To study the influence of TF on anticancer effect of DOX.Materials and methods . The mouse melanoma cell line B16 / F10 and mouse transplanted tumor B16 were used. The TF ability to protect from DOX-induced cell death were measured by MTT-assay, flow сytometry, light microscopy, cytochemical determination of ß-galactosidase expression, radiometric assay and tumor growth inhibition assay in vivo.Results. The sensitivity of mouse melanoma cell line B16 / F10 to DOX decreased in the presence TF (10–20 mkM) in the mean by 4–6 fold. The same mechanism takes part into the decrease of DOX cytotoxicity at the presence of TF / hemin khown which connects with the cell ability to accumulate of drug. TF protect the mouse melanoma cells B16 / F10 from apoptosis, induced by DOX throwing switching on cell premature senescence programme. The antitumor effect of DOX against mouse transplanted melanoma B16 at presence of TF was the same as DOX alone.Conclusions. The TF potency to decrease the sensitivity of cancer cells to DOX in vitro does not correlate with its ability to modulate аnthracycline antibiotics anticancer effect in vivo.

Green synthesis of selenium-N-heterocyclic carbene compounds: Evaluation of antimicrobial and anticancer potential

Three benzimidazolium salts (III-V) and respective selenium adducts (VI-VIII) were designed, synthesized and characterized by various analytical techniques (FT-IR and NMR 1H, 13C). Selected salts and respective selenium N-Heterocyclic carbenes (selenium-NHC) adducts were tested in vitro against Cervical Cancer Cell line (Hela), Breast Adenocarcinoma cell line (MCF-7), Retinal Ganglion Cell line (RGC-5) and Mouse Melanoma Cell line (B16F10) using MTT assay and the results were compared with standard drug 5-Fluorouracil. Se-NHC compounds and azolium salts showed significant anticancer potential. Molecular docking studies of compounds (VI, VII and VIII) showed strong binding energies and ligand affinity toward following angiogenic factors: VEGF-A (vascular endothelial growth factor A), EGF (human epidermal growth factor), HIF (Hypoxia-inducible factor) and COX-1 (Cyclooxygenase-1) suggesting that the anticancer activity of adducts (VI, VII and VIII) may be due to their strong anti-angiogenic effect. In addition, compounds III-VIII were screened for their antibacterial and antifungal potential. Adduct VI was found to be potent anti-fungal agent against A. Niger with zone of inhibition (ZI) value 27.01 ± 0.251 mm which is better than standard drug Clotrimazole tested in parallel.

Surface programmable activation receptor (SPAR) cell platform for the rapid detection of melanoma-specific biomarkers.

e14263 Background: We have previously demonstrated that SPAR modified T cells effectively detect bacterial and viral pathogens. SPAR cells are engineered to express a modified T cell receptor (TCR) capable of antibody-directed signal transduction and respond with a Ca2+-mediated production of luminescent signal upon target detection. Here we describe the ability of SPAR cells to rapidly detect the cell surface expression of melanoma biomarkers without a requirement for extensive sample preparation. Methods: SPAR cells expressing both an engineered T cell receptor and the luminescent reporter protein aequorin were developed via the transfection of Jurkat cells with the aequorin expression vector pEF1-Aeq and an engineered TCR complex composed of mouse FcγRI fused to the CD3ζ subunit. The mFcγRI-CD3ζ receptor binds to the Fc region of full-length mouse IgG2 antibodies: SPAR cells are programmed for target detection via the addition of target-specific antibody. The ability of programmed SPAR cells to accurately detect melanoma-specific cell surface biomarkers was evaluated using whole cells from cultured mouse (B16-F10) and human (SK-Mel-28) melanoma cell lines. SPAR cells were programmed by incubation with murine antibodies directed toward the melanoma biomarkers CD133 or TRP1, then mixed with melanoma cells or K562 control cells and evaluated for signal generation. Results: SPAR cells programmed with anti-CD133 or anti-TRP1 antibody produced luminescent signal within minutes when combined with human SK-Mel-28 cells or mouse B16-F10 cells, respectively, known to abundantly express the appropriate biomarker. No signal was generated when programmed SPAR cells were incubated with K562 cells. Further studies also document cytokine production following receptor engagement. Conclusions: SPAR cells can be programmed for the rapid and specific detection of known cell surface cancer biomarkers. The Ca2+-dependent production of luminescent signal and cytokine release in response to TCR engagement suggests SPAR cell activation. Thus, in addition to biomarker detection the SPAR system may ultimately provide predictive insights into the potency of antibody-directed cell therapy.

Anti-melanin deposition activity and active constituents of Jatropha multifida stems.

Jatropha multifida is a medicinal plant that belongs to the Euphorbiaceae family. Our investigation revealed that the chloroform extract of J. multifida stems showed anti-melanin deposition activity against α-melanocyte stimulating hormone (α-MSH)- and 3-isobutyl-1-methylxanthine (IBMX)-induced melanogenesis in the mouse melanoma cell line (B16-F10). Further fractionation and purification of the major constituents led to the isolation of two coumarins (1 and 2) and seven known lignoids (3-9). All isolated compounds exhibited anti-melanin deposition activities against the mouse melanoma cell line (B16-F10) with IC50 values ranging from 37.5 to 560.1µM, without any cytotoxicity even at high concentrations, except for 8. Further mechanistic studies suggested that 9 downregulated tyrosinase mRNA expression, while the anti-melanin deposition activities of 4 and 8 appeared to be unrelated to tyrosinase inhibition and the downregulated expression of the key melanogenesis-associated mRNAs. These results suggested that J. multifida could possess potent skin whitening ingredients.

784 A new immune evasion mechanism in melanoma

We present a novel and highly effective immune evasion mechanism in melanoma through the downregulation of man1a1, which codes for mannosidase I. This enzyme modifies glycosylation of cell-surface proteins. Using mass spectrometry, the glycocalyx of four human melanoma cell lines and normal human melanocytes (NHEM) were analyzed. All cancer lines expressed abnormal surface carbohydrates, specifically, an elevated ratio of high mannose sugars. RNA arrays of the human cancer cells showed downregulation of man1a1 compared to NHEM. To determine if this resulted in an altered glycocalyx, mouse melanoma cell line (B16) was treated with Kifunensine (Kif), a mannosidase I inhibitor. Kif-treated cells showed more high mannose structures, similar to human melanoma cell lines, compared to untreated cells. NK cells express receptors against various carbohydrates, providing targeting mechanisms for cell-mediated killing of tumor cells. Cytotoxicity assays using isolated mouse NK cells led to a 5-fold decrease in cell-mediated lysis of Kif-treated B16 cells compared to untreated cells. B16 cells overexpressing man1a1(B16+man1a1) were generated by stable transfection of a mammalian vector containing man1a1. The glycocalyx of these cells appeared more normal by mass spectrometry. Cytotoxicity assay of B16+man1a1 resulted in a 2-fold increase in cell lysis, compared to B16 transfected with empty vector. To test our hypothesis in vivo, a control mouse group and an experimental mouse group were subcutaneously and intravenously injected with either B16+man1a1 or B16 cells carrying empty vector, respectively. After two weeks, mouse skin and lungs were harvested. Compared to control mice, skin tumor volume was 10-fold smaller in mice injected with B16+man1a1 cells. There were 7-fold (p < 0.005) fewer metastatic colonies in lung parenchyma of mice injected intravenously with B16+man1a1 cells compared to control mice. Our results show glycosylation is a major factor in cancer immune evasion and/or suppression and can be reversed by overexpressing a single gene.

Modeling Melanoma in 3D uGel™‐based Artificial Tumor Tissues Demonstrate Initiation, Progression and Metastatic Processes In‐vitro

In a continuing collaboration between our laboratory, the UWRF Tissue and Cellular Innovation Center, and BRTI Lifesciences, LLC we have been evaluating the application of a commercial hydrogel as a matrix for the development and study of several artificial tumor tissues. Here we report on the application of uGel™, a recently developed chitosan/hyaluronate precast matrix to generate 3D melanoma artificial tissues. In this work, we coupled uGel™ matrix with mouse melanoma cell lines B16F10 and B16F1 to generate and study 3D tumor tissues in‐vitro. As we have previously reported with another earlier and related hydrogel, Cell‐Mate™, both of these cells lines successfully produced extensive and long‐lived tumor tissues. In addition, in this series of studies with uGel™ it was possible to model not just the end stages of tumor progression and metastasis, but also the early stages of tumor establishment and local invasion of the matrix in patterns which clearly reflect early tumor development in vivo. As we had seen before, the final stages of tumor development in melanoma includes the generation of two distinct populations of cells, one pigmented and one not. Additionally these two populations also correlate tightly with differential patterns of tissue formation ranging from highly branched and pigmented to completely rounded, smooth and lacking virtually any pigment. Although the branching features are clearly associated with local invasion, our work suggests that the pale, rounded features may actually represent the metastatic stage for these tumors. Interestingly, uGel™ based melanoma developed and progressed to full‐blown tumor masses within a three week period while in our earlier work with Cell‐Mate™ constructs fully developed tumors required from 2–3 months of long‐term culture to reach the same stage. Although the two materials are biochemically identical, they do differ in mechanical properties at the time of loading which may play a role in the significant differences observed in terms of developmental timeline. This study further support our view that small‐scale and long‐term 3D artificial tissues are an effective tool to better understand the biology of cancer and to study it's progression in‐vitro.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.