Cultured B16F10 Cells Research Articles

Effects of clopidogrel bisulfate on B16-F10 cells and tumor development in a murine model of melanoma.

Metastatic melanoma is a very aggressive skin cancer. Platelets are constituents of the tumor microenvironment and, when activated, contribute to cancer progression, especially metastasis and inflammation. P2Y12 is an adenosine diphosphate receptor that triggers platelet activation. Inhibition of P2Y12 by clopidogrel bisulfate (CB) decreases platelet activation, which is also controlled by the extracellular concentration and the metabolism of purines by purinergic enzymes. We evaluated the effects of CB on the viability and proliferation of cultured B16-F10 cells. We also used a metastatic melanoma model with C57BL-6 mice to evaluate cancer development and purine metabolism modulation in platelets. B16-F10 cells were administered intraperitoneally to the mice. Two days later, the animals underwent a 12-day treatment with CB (30mg/kg by gavage). We have found that CB reduced cell viability and proliferation in B16-F10 culture in 72h at concentrations above 30µm. In vivo, CB decreased tumor nodule counts and lactate dehydrogenase levels and increased platelet purine metabolism. Our results showed that CB has significant effects on melanoma progression.

Exercise Alters Ceramide Metabolism in Melanoma

IntroductionWe previously demonstrated that exercise enhanced the efficacy of chemotherapy against B16F10 melanoma in mice. Exercise likely impacts both the tumor microenvironment and the tumor cells. Exercise has been demonstrated to modulate ceramide metabolism in healthy tissue, but the relationship between exercise and ceramide in tumors has not been evaluated. Ceramides are essential sphingolipid metabolites involved in cell death and survival pathways. Low content of ceramides in melanoma is a potential mechanism for drug resistance. Thus, increasing ceramides content in tumor cells is likely to increase sensitivity to chemotherapy. We hypothesized that exercise would increase accumulation of ceramide in tumor by regulating ceramide metabolism.MethodsC57BL/6 male mice were subcutaneously injected with B16F10 or BP melanoma cells and randomly assigned into sedentary control (No Ex) and exercise (Ex) groups. After tumor establishment, Ex mice performed treadmill running at 12 meter/min for 45 min for 5 consecutive exercise days with two days off then 5 additional exercise days. Ceramides were measured in tumor homogenates by liquid chromatography‐tandem mass spectrometry (LC/MS). Ceramide metabolizing enzymes were analyzed in tumors by immunofluorescence staining and western blotting. Cultured B16F10 or BP cells were treated with serum collected from corresponding melanoma tumor bearing mouse to examine effect of circulating factors on ceramide regulating enzymes.ResultsSeveral ceramide species were increased in Ex compared to No Ex B16F10 tumors, with the largest exercise‐induced increases in C16‐, dihydro‐C16‐, and C20‐ ceramide. Significantly increased expression of ceramide synthase 6 (CerS6), which generates C16‐ceramide using sphinganine or sphingosine, was observed in Ex B16F10 tumors by immunofluorescence staining and western blotting. Surprisingly, there was a trend toward an increase in the ceramide degrading enzyme acid ceramidase (aCDase) in Ex tumors. This may suggest a feedback loop. In contrast, Ex significantly suppressed expression of sphingosine kinase 1 (Sphk1), which converts sphingosine to the key survival sphingolipid S1P, in B16F10 tumors. Likewise, Sphk1 protein expression decreased in B16F10 cells treated in vitro with serum from Ex. Unlike the findings in B16F10 tumors and cultured B16F10 cells, no change in C16‐Ceramide, CerS6, or Sphk1 was observed in Ex BP tumors and in in vitro treatment of BP cells with Ex mice serum.DiscussionOur data suggests that exercise may contribute to B16F10 tumor cell death in response to chemotherapy by modulating ceramide metabolism toward a pro‐apoptotic ceramide/sphingosine‐1‐phosphate balance. However, these results were not consistent in BP tumors. This raises the important finding that exercise can have different effects between tumors of different patient or mouse origin, even with the same diagnosis (i.e. B16F10 vs BP melanoma models). Future work to determine the extent to which exercise regulates ceramide‐sphingolipid signaling in other melanoma tumor models, and in patients, is necessary. Future work will also determine whether exercise‐induced changes in ceramide levels are responsible for the previously reported exercise‐induced increased chemotherapy efficacy against melanoma tumors.

Kojic Acid Showed Consistent Inhibitory Activity on Tyrosinase from Mushroom and in Cultured B16F10 Cells Compared with Arbutins.

Kojic acid, β-arbutin, α-arbutin, and deoxyarbutin have been reported as tyrosinase inhibitors in many articles, but some contradictions exist in their differing results. In order to provide some explanations for these contradictions and to find the most suitable compound as a positive control for screening potential tyrosinase inhibitors, the activity and inhibition type of the aforementioned compounds on monophenolase and diphenolase of mushroom tyrosinase (MTYR) were studied. Their effects on B16F10 cells melanin content, tyrosinase (BTYR) activity, and cell viability were also exposed. Results indicated that α-arbutin competitively inhibited monophenolase activity, whereas they uncompetitively activated diphenolase activity of MTYR. β-arbutin noncompetitively and competitively inhibited monophenolase activity at high molarity (4000 µM) and moderate molarity (250–1000 µM) respectively, whereas it activated the diphenolase activity of MTYR. Deoxyarbutin competitively inhibited diphenolase activity, but could not inhibit monophenolase activity and only extended the lag time. Kojic acid competitively inhibited monophenolase activity and competitive–noncompetitive mixed-type inhibited diphenolase activity of MTYR. In a cellular experiment, deoxyarbutin effectively inhibited BTYR activity and reduced melanin content, but it also potently decreased cell viability. α-arbutin and β-arbutin dose-dependently inhibited BTYR activity, reduced melanin content, and increased cell viability. Kojic acid did not affect cell viability at 43.8–700 µM, but inhibited BTYR activity and reduced melanin content in a dose-dependent manner. Therefore, kojic acid was considered as the most suitable positive control among these four compounds, because it could inhibit both monophenolase and diphenolase activity of MTYR and reduce intercellular melanin content by inhibiting BTYR activity without cytotoxicity. Some explanations for the contradictions in the reported articles were provided.

Melanin photosensitization by green light reduces melanoma tumor size

Upon excitation by visible light (VL), melanin engages in photosensitization reactions, generating singlet oxygen (1O2) and causing oxidative damage to biomolecules via both type I and type II mechanisms. Therefore, VL irradiation may be used to perturb the survival and growth of pigmented melanoma lesions. However, this hypothesis has not been investigated previously, and there is no information regarding the effectiveness of this wavelength of light on melanomas. Therefore, this hypothesis needs to be tested in vivo, as VL has also been shown to stimulate tumor growth via photo biomodulation. This study aimed to determine the effect of melanin photosensitization following exposure to VL in cultured B16-F10 cells and melanoma lesions induced in mice. Cell viability was evaluated by the MTT assay after irradiation with blue, green, and red lights (466 nm, 532 nm, and 630 nm, respectively). Singlet oxygen (1O2) was investigated in melanin extracted from M+B16-F10 cells. Melanin-loaded M+B16-F10 cells showed a significant reduction in viability (∼30%) and 1O2 generation only after irradiation with 532 nm light. Mice were divided into groups with carcinogenesis induction (C+) or without induction (control, C-), irradiated (I+) with green light (GL 36 J.cm−2) at 7-day intervals or without irradiation (I-). The average tumor size (TAS) and pigmentation were quantified using a caliper and ImageJ software, respectively. After seven weeks, skin samples were collected from all groups for histopathological analysis. Statistical analysis was performed using Origin 2021 and GraphPad Prism 9®. The C-/I+ group showed initial pigmentation but no histological changes following irradiation. C+/I- showed tumor progression up to three times the initial volume, whereas the C+/I+ group presented a significant ∼20% decrease in TAS. These results suggest that photosensitization of melanin by GL (532 nm) reduces the viability of M+B16-F10 cells, decreases TAS, and prevents the growth of melanoma in C57BL6 mice.

Epigenetic therapy to enhance therapeutic effects of PD-1 inhibition in therapy-resistant melanoma.

Targeted therapy and immunotherapy have revolutionized the treatment of metastatic skin melanoma but around half of all patients develop resistance early or late during treatment. The situation is even worse for patients with metastatic uveal melanoma (UM). Here we hypothesized that the immunotherapy of therapy-resistant skin melanoma or UM can be enhanced by epigenetic inhibitors. Cultured B16F10 cells and human UM cells were treated with the histone deacetylase inhibitor (HDACi) entinostat or BETi JQ1. Entinostat-induced HLA expression and PD-L1, but JQ1 did not. A syngeneic mouse model carrying B16-F10 melanoma cells was treated with PD-1 and CTLA4 inhibitors, which was curative. Co-treatment with the bioavailable BETi iBET726 impaired the immunotherapy effect. Monotherapy of a B16-F10 mouse model with anti-PD-1 resulted in a moderate therapeutic effect that could be enhanced by entinostat. Mice carrying PD-L1 knockout B16-F10 cells were also sensitive to entinostat. This suggests HDAC inhibition and immunotherapy could work in concert. Indeed, co-cultures of UM with HLA-matched melanoma-specific tumor-infiltrating lymphocytes (TILs) resulted in higher TIL-mediated melanoma killing when entinostat was added. Further exploration of combined immunotherapy and epigenetic therapy in metastatic melanoma resistant to PD-1 inhibition is warranted.

The anti-UV properties of Saussurea involucrate Matsum. & Koidz. Via regulating PI3K/Akt pathway in B16F10 cells

Ethnopharmacological relevanceUltra Violet (UV) radiation is the major reason for reactive oxygen species (ROS) forming, skin cell damage, melanin production, and could horribly cause skin cancer. Saussureae Involucratae Herba (SIH) is the aerial part of Saussurea involucrata Matsum. & Koidz. This Material Medica is popular with both in Uyghur and Chinese medicines filed. SIH is one of the famous species of the Asteraceae family and which prescribed for skin protection from UV-induced damage according to China Pharmacopeia (2020). However, the detailed working mechanism involved is still not elucidated. Aim of the studyWe would like to probe the potential transduction pathway of SIH against UV-induced skin cell damages in cultured B16F10 cells. MethodsWestern blot, luciferase assay, laser confocal, RT-PCR and flow cytometer were employed here to verify the protective pharmaceutical value of SIH in cultured B16F10 cells after UV pre-treatment. ResultsOur result revealed that SIH attenuates ROS formation after UV-induced damage in B16F10 cells in a dose-dependent manner. Moreover, the transcriptional and translational anti-oxidative encoding genes were up-regulated under the presence of SIH. Further studies showed that SIH activated transcriptional activity of anti-oxidant response element (ARE). Moreover, we found that SIH dramatically stimulates PI3K/Akt phosphorylation in cultured B16F10 cells, this result was further verified by its specific inhibitors, LY294002 and Tocris. ConclusionOur findings concluded that SIH protect melanoma cells from UV damages via activating PI3K/Akt signaling and which could provide scientific evidence for anti-UV pharmaceutical values of this herbal extract.

Spatiotemporally controllable diphtheria toxin expression using a light-switchable transgene system combining multifunctional nanoparticle delivery system for targeted melanoma therapy

Gene therapy with external gene insertion (e. g. a suicide gene) and expression specifically in mutated tumor cells has shown to be a promising strategy in treatment of tumors. However, current tumor gene therapy often suffered from low efficiency in gene expression and off-target effects which may cause damage to normal tissues. To address these issues, in this study, a light-switchable transgene nanoparticle delivery system loaded with a diphtheria toxin A (DTA) segment encoded gene, a suicide gene for tumor cells, was developed. The nanoparticles contained vitamin E succinate-grafted polyethyleneimine core and arginylglycylaspartic acid (RGD)-modified pegylated hyaluronic acid shell for targeted delivery of the loaded gene to tumor cells via receptor-mediated (CD44 and αvβ3) endocytosis. Notably, the expression of target proteins in tumor cells could be conveniently regulated by adjusting the blue light intensity in the Light-On system. In in-vitro studies in cultured B16-F10 cells, the pG-DTA-loaded nano-micelles showed greatly improved inhibitory rate compared with the pG-DTA group. Moreover, in the tumor-bearing C57BL/6 mice model, the pG-DTA-loaded nanoparticle exhibited greatly improved efficacy and reduced systemic toxicity with significantly increased survival rate after 21 days. Significantly suppressed tumor angiogenesis was also identified in the nanoparticle-treated group likely due to the targeting ability of the RGD-modified nanoparticle. All the above results indicated that the combination of a light-switchable transgene system with a nanoparticle-based targeted delivery system have great potentials in gene therapy of malignant tumors with improved precision and efficacy.

Studies on the treatment of melanoma with folate acid conjugated dextran and lauryl alcohol loaded with IMD0354.

Background: IMD-0354 is a kind of hydrophobic small molecule inhibitor of IKKβ, which can effectively inhibit the NF-κB pathway. Besides, IMD-0354 can inhibit a variety of tumor cells in culture, but its poor water solubility and low utilization have limited its clinical application.Methods: In this study, IMD-0354 was synthesized through esterifying the folate acid (FA) conjugated dextran (Dex) as well as the lauryl alcohol (LA).Results:The particle (IMD/FA-Dex-LA) size was 212.13±10.62nm, the encapsulation efficiency was 89.27±6.51%, and the drug loading was 4.25±0.42%. Cell viability studies indicated that the IMD/FA-Dex-LA effectively inhibited survival of B16F10 cells in culture. Meanwhile, Western Blotting results showed that the nuclear transport of NF-κB was reduced after blocking the IKK pathway, which would thereby suppress melanoma cell division and proliferation. Moreover, subcutaneous tumor implantation experiment revealed that, the drug-loading complex had an obvious effect on suppressing melanoma cells. Findings of this study demonstrated that the IMD-0354 loaded FA-Dex-LA was more effective than IMD-0354 alone.Conclusion: In summary, FA-Dex-LA has been successfully synthesized in this study, which can serve as a carrier for hydrophobic drug. Further, it is believed the FA-Dex-LA can potentially applied in cancer treatment.

Host H67D Genotype Affects Tumor Growth in Mouse Melanoma

Background: We investigated the effects of the frequently polymorphic high iron gene (HFE), on tumor growth in a novel mouse model. We hypothesized that host mutations in the HFE protein modify tumor progression. Methods: C57BL/6 mice possessing either H67D/H67D or WT/WT HFE genotype, aged 18 months, were injected subcutaneously with 4x106 cells of the B16F10 mouse melanoma cell line. After 2 weeks, mice were sacrificed and the tumors and plasma were collected. Animal methods were approved by our IACUC, (04-166). Tumors were analyzed by RT-PCR. In parallel, bone marrow derived macrophages were cultured to determine how B16F10 cell cultures react to conditioned media from macrophages of each genotype. ELISAs were performed for ferroportin, ferritin, secreted cytokine and chemokine content of macrophage media. Results: H67D mice had significantly smaller tumors (t-test, P = 0.02) after two weeks. Exploratory qRT-PCR analysis of tumors revealed that the H67D host may suppress angiogenesis and growth. In culture, macrophages derived from H67D mice secrete higher levels of monocyte chemoattractant protein 1 (MCP1), suggesting a change in chemotactic signaling (p = 0.02). When conditioned media from the H67D macrophages were placed onto B16F10 cells in culture there was significantly less growth compared to conditioned media from WT macrophages (p < 0.01 MTT, p < 0.01 BrdU). In macrophage cell lysates, H67D is associated with lower levels of Ferroportin (p = 0.03). Conclusions: Macrophages from H67D mice do not support tumor cell proliferation as well as WT controls. Our data reveal the importance of HFE genotype on tumor growth that may be related to macrophage function.

Ardisia crenata extract stimulates melanogenesis in B16F10 melanoma cells through inhibiting ERK1/2 and Akt activation.

Melanin protects the skin against ultraviolet radiation by scattering incoming light and absorbing diverse free radicals. Agents that increase melanin synthesis in melanocytes may reduce the risk of photodamage and skin cancer. The present study investigated the effect of a methanol extract of Ardisia crenata (AC) on melanogenesis in B16F10 cells. Treatment of cultured B16F10 cells with AC extract (10, 20 and 40 µg/ml) stimulated an increase in melanin levels in a concentration-dependent manner, without cytotoxicity. Tyrosinase is key in the regulation of melanin production, thus the effect of AC extract on tyrosinase activity and protein expression was analyzed. AC extract was observed to significantly increase tyrosinase activity and protein expression in B16F10 cells. Furthermore, AC extract was found to markedly increase the protein expression of microphthalmia-associated transcription factor, which is an important transcription factor involved in tyrosinase gene expression. In addition, AC extract (40 µg/ml) was observed to suppress the activation of extracellular signal-regulated kinase (ERK) and Akt, which negatively regulate melanin synthesis in B16F10 cells. In conclusion, to the best of our knowledge, the present study is the first to show that a methanol extract of AC stimulates melanogenesis by increasing tyrosinase expression via the inhibition of ERK and Akt. Thus, methanol extract of AC may be a potential treatment for hypopigmentation diseases and may be a candidate for skin-tanning cosmetic products.

Proopiomelanocortin gene delivery induces apoptosis in melanoma through NADPH oxidase 4-mediated ROS generation

Hypoxia in the tumor microenvironment triggers differential signaling pathways for tumor survival. In this study, we characterize the involvement of hypoxia and reactive oxygen species (ROS) generation in the antineoplastic mechanism of proopiomelanocortin (POMC) gene delivery in a mouse B16-F10 melanoma model in vivo and in vitro. Histological analysis revealed increased TUNEL-positive cells and enhanced hypoxic activities in melanoma treated with adenovirus encoding POMC (Ad-POMC) but not control vector. Because the apoptotic cells were detected mainly in regions distant from blood vessels, it was hypothesized that POMC therapy might render melanoma cells vulnerable to hypoxic insult. Using a hypoxic chamber or cobalt chloride (CoCl2), we showed that POMC gene delivery elicited apoptosis and caspase-3 activation in cultured B16-F10 cells only under hypoxic conditions. The apoptosis induced by POMC gene delivery was associated with elevated ROS generation in vitro and in vivo. Blocking ROS generation using the antioxidant N-acetyl-l-cysteine abolished the apoptosis and caspase-3 activities induced by POMC gene delivery and hypoxia. We further showed that POMC-derived melanocortins, including α-MSH, β-MSH, and ACTH, but not γ-MSH, contributed to POMC-induced apoptosis and ROS generation during hypoxia. To elucidate the source of ROS generation, application of the NADPH oxidase inhibitor diphenyleneiodonium attenuated α-MSH-induced apoptosis and ROS generation, implicating the proapoptotic role of NADPH oxidase in POMC action. Of the NADPH oxidase isoforms, only Nox4 was expressed in B16-F10 cells, and Nox4 was also elevated in Ad-POMC-treated melanoma tissues. Silencing Nox4 gene expression with Nox4 siRNA suppressed the stimulatory effect of α-MSH-induced ROS generation and cell apoptosis during hypoxia. In summary, we demonstrate that POMC gene delivery suppressed melanoma growth by inducing apoptosis, which was at least partly dependent on Nox4 upregulation.

EGCG Suppresses Melanoma Tumor Angiogenesis and Growth without Affecting Angiogenesis and VEGF Expression in the Heart and Skeletal Muscles in Mice

Melanoma is a highly malignant cancer with a potent capacity to metastasize distantly and has a higher mortality. There is no effective therapy for high risk melanoma patients to prevent relapse or distant metastasis. Therefore effective chemoprevention strategies are needed. The present study mainly evaluates the effects of EGCG on melanoma angiogenesis, growth, and capillary density (CD) in the heart and skeletal muscles of mice. 5 x 10^5 B16F10 cells were inoculated into the right proximal dorsal of the back in the eight week old male mice (n=12). Then, 6 mice received EGCG at 50-100 mg/kg/d in drinking water for 4 weeks and 6 control mice received drinking water only. Tumor size was monitored using dial calipers. At the end of the experiment, blood samples, tumors, hearts, and limb muscles were collected and measured for VEGF expression using ELISA and capillary density (CD) using CD31 immunohistochemistry. Compared to the control, EGCG treatment significantly reduced tumor weight (2.9±0.5 vs. 5.9±1.1 g; P&lt;0.01; n=6), melanoma CD (117±9 vs. 167±23; P&lt;0.01), and melanoma VEGF expression (32±1.5 vs. 42±2 pg/mg; P &lt; 0.01), respectively. Also EGCG had no effects on body weight, heart weight, angiogenesis or VEGF expression in the heart and skeletal muscle of mice. EGCG (20-50 µg/ml) significantly inhibited the proliferation, migration, VEGF expression, and the activation of HIF-1α and NFαB in cultured B16F10 cells, respectively. These findings support the hypothesis that EGCG, a major green tea polyphenol, directly targets tumor cells and tumor vasculature, thereby inhibiting tumor growth, proliferation, migration, and angiogenesis of melanoma, and that the down-regulation of VEGF expression by EGCG is associated with the inhibition of HIF-1α and NFkB activation. EGCG has great potential as a chemopreventive agent because it has no effect on angiogenesis in normal tissue and has low toxicity.

Berberine in combination with doxorubicin suppresses growth of murine melanoma B16F10 cells in culture and xenograft

Melanoma is very aggressive and major cause of mortality due to skin cancer. Herein, we studied the anticancer effects of berberine, a plant alkaloid, in combination with doxorubicin on murine melanoma B16F10 cells in vitro and in vivo. This drug combination strongly inhibited cell growth and induced cell death, and caused G2/M arrest in cell cycle together with a decrease in Kip1/p27. Berberine showed stronger inhibitory effect on ERK1/2 phosphorylation as compared to Akt phosphorylation, whereas the combination of the drugs showed greater inhibitory effect on Akt phosphorylation. In murine B16F10 xenograft, cells were implanted into mice and treated with vehicle (methyl cellulose) or berberine (100mg/kg of body weight/day by oral gavage) or doxorubicin (4mg/kg of body weight/week by intraperitoneal injection) or combination of berberine and doxorubicin. Berberine alone did not show any considerable effect on tumor growth as observed with doxorubicin, however, the combination of the two drugs resulted in a significant and strong decrease in tumor volume (85%, p<0.005) and tumor weight (78%, p<0.05) as compared to control. Immunohistochemical analysis of tumor samples showed that drug combination decreased PCNA-positive cells (82%, p<0.001) and increased cleaved caspase-3 positive cells (3-fold, p<0.05) indicating inhibition of proliferation and an increase in apoptosis, respectively. Overall, our findings suggest that berberine and doxorubicin could be a novel combination to inhibit melanoma tumor growth.

PEGylated fullerene/iron oxide nanocomposites for photodynamic therapy, targeted drug delivery and MR imaging

Recently, fullerene and fullerene derivatives owning to their highly enriched physical and chemical properties have been widely explored for applications in many different fields including biomedicine. In this study, iron oxide nanoparticles (IONPs) were decorated onto the surface of fullerene (C60), and then PEGylation was performed to improve the solubility and biocompatibility of C60-IONP, obtaining a multi-functional C60-IONP-PEG nanocomposite with strong superparamagnetism and powerful photodynamic therapy capacity. Hematoporphyrin monomethyl ether (HMME), a new photodynamic anti-cancer drug, was conjugated to C60-IONP-PEG, forming a C60-IONP-PEG/HMME drug delivery system, which demonstrated an excellent magnetic targeting ability in cancer therapy. Compared with free HMME, remarkably enhanced photodynamic cancer cell killing effect using C60-IONP-PEG/HMME was realized not only in a cultured B16-F10 cells in vitro but also in an in vivo murine tumor model due to 23-fold higher HMME uptake of tumor and strong photodynamic activity of C60-IONP-PEG. Moreover, C60-IONP-PEG could be further used as a T2-contrast agent for in vivo magnetic resonance imaging. Our work showed C60-IONP-PEG/HMME had a great potential for cancer theranostic applications.

Expression of Foxp3, CD25 and IL-2 in the B16F10 cancer cell line and melanoma is correlated with tumor growth in mice

The forkhead box P3 (Foxp3) transcription factor is one of the most studied markers used to identify CD4+CD25+ regulatory T cells (Tregs), and has been identified as a key regulator in the development and function of Tregs. Foxp3 expression has been reported in a variety of solid human tumors, including melanoma. The aims of the present study were to analyze Foxp3 expression in B16F10 melanoma cells in vitro, to determine whether this expression was affected during tumor growth in a murine melanoma model and to correlate Foxp3 expression with CD25 expression, interleukin (IL)-2 production and tumor weight. Foxp3 expression was analyzed with quantitative (q)PCR, flow cytometry and confocal microscopy. CD25 expression was analyzed by flow cytometry, and cytokine production was measured by ELISA [IL-2, interferon (IFN)-γ, transforming growth factor (TGF)-β and IL-10] and flow cytometry (IL-2, IFN-γ, IL-4 and IL-5). Foxp3 and CD25 expression was detected in the B16F10 cells in culture and in the intratumoral B16F10 cells. An increase in Foxp3 and CD25 expression was observed in a time-dependent manner during tumor growth at 7, 14 and 21 days. The production of the IL-2, IL-10, IFN-γ and TGF-β cytokines was observed in the B16F10 cells and also detected in the tumoral microenvironment during tumor growth (7, 14 and 21 days). An increase in IL-2 and IL-10 production was observed, whereas IFN-γ production decreased in a time-dependent manner. The production of tumor necrosis factor (TNF)-α was not observed in culture, but was detected during tumor growth, whereas the production of IL-4 and IL-5 was not detected. These data showed a positive correlation between the expression of Foxp3, CD25 and IL-2 and tumor weight in murine melanoma. From these data, it may be suggested that Foxp3 participates in melanoma growth, the modulation of the IL-2, IFN-γ and TNF-α cytokines and CD25 expression, and that it also plays a possible role in immunosuppression.

A Novel Synthesized Tyrosinase Inhibitor: (E)-2-((2,4-Dihydroxyphenyl)diazenyl)phenyl 4-Methylbenzenesulfonate as an Azo-Resveratrol Analog

We synthesized a novel series of (E)-2-((substituted phenyl)diazenyl)phenyl 4-methylbenzenesulfonate derivatives (2 and 3) and (E)-2-((substituted phenyl)diazenyl)phenol derivatives (4 and 5), and conducted an evaluation in order to determine their inhibitory effects on mushroom tyrosinase, with the aim of discovering a tyrosinase inhibitor. Most of the compounds (3-5) exhibited higher inhibitory effects than kojic acid (IC(50) = 49.08 µM), a representative tyrosinase inhibitor. A novel synthesized compound, (E)-2-((2,4-dihydroxyphenyl)diazenyl)phenyl 4-methylbenzenesulfonate (3), showed the best results with an IC(50) of 17.85 µM, and showed competitive inhibition on Lineweaver-Burk plots, as further confirmed by the docking results. In addition, active compounds 3-5 were not cytotoxic to cultured B16F10 cells at the concentrations tested, and inhibited both tyrosinase and melanin synthesis. Therefore the active compounds (3-5) might be considered excellent candidates for use in the development of therapeutic agents for diseases associated with hyperpigmentation.

Properties of B16-F10 murine melanoma cells subjected to metabolic stress conditions.

Neoplastic cells which co-form tumors are usually subjected to various stress factors, mainly hypoxia and shortage of nutrient factors. Such cells employ different strategies that permit their survival under such conditions. Experiments in vitro are usually carried out in the presence of 21% oxygen and medium supplemented with 10% FBS. Altering these parameters can approximate the in vivo conditions found within tumor mass. The present paper reports certain properties (especially ability to metastasize) of B16-F10 cells able to grow upon exposure to altered growth conditions (medium supplemented with 0.06% FBS or presence of 1% oxygen for 24 or 72 hours). These properties were compared with those of control cells cultured in the presence of 21% oxygen and in medium supplemented with 10% FBS. Some properties of the cells exposed to medium supplemented with 0.06% FBS differ from those of cells cultured under low oxygenation conditions (ability to form metastases, to migrate, or to express various proteins). Only the partial deprivation of oxygen did increase both the number of migrating cells and the number of metastases formed. Serum deficiency enhanced only the cell ability to metastasize, but not to migrate. It appears that cultured B16-F10 cells employ different adaptation strategies under conditions of oxygen shortage and those of serum deficiency. Under oxygen deprivation, such cells most likely undergo an epithelial-mesenchymal transition, whereas serum deficiency ("starvation"), while increasing the tumorigenicity of B16-F10 cells, does not induce the epithelial-mesenchymal transition.

Effect of xanthoxylin on melanin content and melanogenic protein expression in B16F10 melanoma

Background: Reduced production of melanin and decreased or absence of melanocytes leads to various hypopigmentation disorders. Melanin synthesis is regulated by melanogenic proteins such as tyrosinase, tyrosinase-related protein 1 (TRP-1) and tyrosinase-related protein 2 (TRP -2), as well as their transcription factors. Objectives: This study elucidated the effects of xanthoxylin on melanin content, dendriticity, melanogenic protein expression and its signal transduction pathways in mouse B16F10 melanoma cells (B16F10 cells). Methods: Melanin production of B16F10 cells was measured by using a melanin content assay. The effect of xanthoxylin on the dendriticity of B16F10 cells was determined by a melanocyte dendricity assay. RT-PCR was used to investigate the effects of xanthoxylin on the melanogenic protein expression. Results: We found that xanthoxylin increased melanin production, number of dendrites, tyrosinase, and microphthalmia-associated transcription factor (MITF) expression in cultured B16F10 cells. In addition, PKA and PKC inhibitor decreased melanin production, tyrosinase, and MITF expression in xanthoxylin-treated cells. However, xanthoxylin did not inhibit TRP-1 and TRP-2 expression. Conclusion: These results indicated that xanthoxylin induces melanogenesis mainly via cAMP-mediated PKA activation. Other signaling pathways may also play a role in xanthoxylin-induced melanogenesis. Keywords: Melanocyte, melanin, xanthoxylin

Abstract 2427: The stress response controls the interorgan glutathione/interleukin-6 cycling activity: A systemic signaling mechanism promoting metastases growth.

Abstract B16 melanoma F10 (B16-F10) cells with high glutathione (GSH) content show very high metastatic activity in vivo. An intertissue flow of GSH, where the liver is the main reservoir, increases GSH content in metastatic cells and promote their growth. We studied possible tumor-derived molecular signals that could activate GSH release from hepatocytes. GSH efflux increases in hepatocytes isolated from mice bearing liver or lung metastases, thus suggesting a systemic mechanism. Fractionation of serum-free conditioned medium from cultured B16-F10 cells and monoclonal antibody-induced neutralization techniques facilitated identification of interleukin (IL)-6 as a tumor-derived molecule promoting GSH efflux in hepatocytes. IL-6 activates GSH release through a methionine-sensitive/organic anion transporter polypeptide 1- and multidrug resistance protein 1-independent channel located on the sinusoidal site of hepatocytes. Specific siRNAs were used to knock down key factors in the main signaling pathways activated by IL-6, which revealed a STAT3-dependent mechanism. Our results show that IL-6 (mainly of tumor origin) facilitates GSH release from hepatocytes and its interorgan transport to metastatic growing foci. Metastatic tumor activated the hypothalamic-pituitary-adrenal axis, thus increasing expression of hypothalamic corticotropin releasing factor and plasma levels of adrenocorticotropin hormone, corticosterone, and cathecolamines. Corticosterone and norepinephrine increased IL-6 expression and synthesis in metastatic cells, thus promoting the interorgan GSH/IL-6 cycling activity. Our results suggest possible mechanisms of cross-talk between metastases and the central nervous system of the host. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2427. doi:1538-7445.AM2012-2427

Antiangiogenic and antitumoral properties of a polysaccharide isolated from the seaweed Sargassum stenophyllum.

The potential antiangiogenic and antitumoral properties of SargA, a polysaccharide extracted from the brown marine alga Sargassum stenophyllum, were studied in assays carried out in chick embryos and mice. Gelfoam plugs containing SargA (2-1500 microg/plug) implanted in vivo into fertilized 6-day-old chicken eggs induced dose-related antiangiogenic activity in the chorioallantoic membrane (CAM). By day 8, the highest dose of SargA alone decreased the vessel number in the CAM by 64%, but coadministered with hydrocortisone (156 microg/plug, which alone caused 30% inhibition) failed to potentiate its antiangiogenic effect. Combined with basic fibroblast growth factor (50 ng/plug), SargA (1500 microg/plug) abolished angiogenesis stimulated by this factor in both chick embryo CAM and in subcutaneous (s.c.) Gelfoam plugs implanted in the dorsal skin of Swiss mice (measured as plug hemoglobin content). Repeated s.c. injections of SargA (1.5 or 150 microg per animal per day for 3 days) close to B16F10 melanoma cell tumors in the dorsal skin of mice markedly decreased tumor growth in a dose-related fashion (by 40% and 80% at 2 weeks after the first injection, respectively), without evident signs of toxicity. SargA caused graded inhibitions of migration and viability of cultured B16F10 cells and also displayed antithrombotic activity in human plasma (5 mg/ml increased thrombin time 2.5-fold relative to saline). Thus, SargA exhibits pronounced antiangiogenic as well as antitumoral properties. Although the latter action of SargA might be related to the inhibition of angiogenesis, the polysaccharide also exerts cytotoxic effects on tumor cells. Because of its chemical characteristics and polyanionic constituents, we postulate that the polysaccharide SargA might modulate the activity of heparin-binding angiogenic growth factors.