B16F10 Melanoma Cell Line Research Articles

Fibronectin-Integrin (α5β1) Modulates Migration and Invasion of Murine Melanoma Cell Line B16F10 by Involving MMP-9

Cell adhesion to extracellular matrix (ECM) initiates signaling cascade regulated by cell surface integrin receptors, which affects the proliferation and invasion of cells. Cells cultured in the presence of ECM ligand fibronectin (FN) stimulate secretion of matrix metalloproteinases (MMPs), facilitating cancer cell invasion and metastasis. Among all the members of the MMP family, MMP-9 is of crucial importance in tumor invasion and metastasis. The present study aims at studying the effects of integrin receptor alpha5beta1 and its ligand FN on expression of MMP-9 in murine melanoma cell line B16F10 and understanding the molecular mechanism(s) involved. The main experimental methods performed in the study were gelatin zymography, immunoblot, real-time RT-PCR, immunocytochemistry, enzyme linked immunosorbent assay (ELISA), transwell chamber assay, and in vivo metastasis assay in syngenic (C57BL6J) mice. The study reports that FN induces the activity, mRNA, and protein expression of MMP-9 and initiates its proteolytic activation in B16F10 cells. Blockage of the alpha5 receptor abrogated the FN-mediated stimulatory response on MMP-9 in B16F10 cells. Inhibitor studies and immunoblot analysis strongly suggest the involvement of focal adhesion kinase (FAK), extracellular regulated kinase (ERK), and phosphatidylinositol-3-kinase (PI-3K) in the FN-mediated responses. Immunocytochemical analysis showed the nuclear localization of nuclear factor-kappaB (NF-kappaB) might lead to activation of MMP-9 gene upon FN treatment. This study demonstrates that integrin receptor alpha5beta1 and FN interaction induces the invasive potential of B16F10 cells and MMP-9 induction is the downstream effectors in the process. This system serves as a novel model system to understand the molecular mechanism of melanoma growth and invasion.

The potential of Pluronic polymeric micelles encapsulated with paclitaxel for the treatment of melanoma using subcutaneous and pulmonary metastatic mice models

The increasing global incidence of malignant melanoma combined with the poor prognosis and low survival rates of patients necessitates the development of new chemotherapeutic strategies. Thus, the objective of this present study was to investigate the therapeutic efficacy of Pluronic polymeric micelles encapsulating paclitaxel (PTX) in both B16F10 melanoma subcutaneous mice model and pulmonary metastatic mice model. Herein, we developed a PTX-loaded polymeric micelles (PF-PTX) consisting of Pluronic P 123 and F127 block copolymers with small particle size (∼25 nm), high encapsulation efficiency (>90%), good stability in lyophilized form and pH-dependent in vitro release. Furthermore, influence of PF-PTX on in vitro cytotoxicity was determined by MTT assay using B16F10 melanoma cell line, while cellular distribution of PF-PTX was detected by confocal microscopy. Additionally, C57BL/6 mice bearing subcutaneous or pulmonary B16F10 melanoma tumors were treated with Taxol or PF-PTX, and antitumor effect was compared. It was found that antitumor efficacy of PF-PTX in both tumor models showed significant tumor growth delay and increased survival. In summary, the simple Pluronic-based nanocarrier could be harnessed for the delivery of anticancer drug to melanoma, with increased therapeutic index.

Selection of Brain Metastasis-Initiating Breast Cancer Cells Determined by Growth on Hard Agar

An approach that facilitates rapid isolation and characterization of tumor cells with enhanced metastatic potential is highly desirable. Here, we demonstrate that plating GI-101A human breast cancer cells on hard (0.9%) agar selects for the subpopulation of metastasis-initiating cells. The agar-selected cells, designated GI-AGR, were homogeneous for CD44(+) and CD133(+) and five times more invasive than the parental GI-101A cells. Moreover, mice injected with GI-AGR cells had significantly more experimental brain metastases and shorter overall survival than did mice injected with GI-101A cells. Comparative gene expression analysis revealed that GI-AGR cells were markedly distinct from the parental cells but shared an overlapping pattern of gene expression with the GI-101A subline GI-BRN, which was generated by repeated in vivo recycling of GI-101A cells in an experimental brain metastasis model. Data mining on 216 genes shared between GI-AGR and GI-BRN breast cancer cells suggested that the molecular phenotype of these cells is consistent with that of cancer stem cells and the aggressive basal subtype of breast cancer. Collectively, these results demonstrate that analysis of cell growth in a hard agar assay is a powerful tool for selecting metastasis-initiating cells in a heterogeneous population of breast cancer cells, and that such selected cells have properties similar to those of tumor cells that are selected based on their potential to form metastases in mice.

Abstract 380: Magnetic nanoplatforms for tumor targeting, imaging and energy delivery

Abstract We have developed magnetic nano-liposomes (MNL), incorporating superparamagnetic iron oxide nanoparticles (SPION), that are versatile theranostic nanoplatform for enhanced drug delivery and monitoring of cancer treatment. MNL are prepared with a formuation of DPPC:DOTAP:CHOL and DOPE-PEG5000. Incorporation of SPIONs results in MNL with mean diameter of 150-250 nm. MNL are easily taken up by B16-F10 melanoma, HUMVEC-D and breast cancer cell lines. They preferentially target the tumor vasculature as shown in a dorsal skin fold chamber using fluorescently labeled MNL. MNL display superparamagnetic response that is essential for magnetic targeting, MR contrast enhancement and magnetic heating. MNL was administrated to SCID mouse with metastatic (B16-F10) melanoma grown in the right flank. Pre-injection and post-injection MR images were used to assess response to magnetic targeting effects. Biodistribution studies were conducted by 111In labeled MNL and amount of radioactivity recovered was used to confirm the effect of targeting for intratumoral administrations. We have shown that tumor signal intensities in T2 weighted images decreased an average of 20±5% and T2* values decreased and average of 14±7ms in the absence of magnetic targeting. This compares to an average signal decrease of 57±12% and a decrease in T2* relaxation times of 27±8ms with the aid of external magnet showing up to 2-fold greater accumulation by magnetic targeting. 111In radio-labeled MNL have been shown to enable multi-modal imaging in vivo using MRI and SPeCT/CT. The images show that an MNL bolus injected intra-tumorally was retained in the tumor 24 hours after injection. Application of a magnetic field enables redistribution of the MNL in the tumor. These MNL are also responsive to ac magnetic fields applied using a Copper coil at 360 kHz and 170A driving current. Both hyperthermia (upto 45C) and thermo-ablative temperatures upto 90C were achieved in 10 – 30 minutes ex vivo in buffer. The results indicate high efficiency for magnetic heating using MNL (Specific Absorption Rate ∼ 104 W/kg) and demonstrate the capability to couple ac magnetic fields to MNL to achieve any set of temperatures needed for hyperthermia and thermal ablation. To date there are no truly theranostic platforms that have been approved for clinical use that combine targeting, thermal heating and MRI imaging capabilities. Existing FDA approved magnetic nanoparticle formulations like ferridex and ferumoxytol, are optimized for MR imaging, and have not been shown to be usable for thermal therapy.The MNL platform is a novel nanoplatform combining multi-modal imaging capabilities, with magnetic targeting and thermal therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 380. doi:10.1158/1538-7445.AM2011-380

Identification of PITX1 as a TERT Suppressor Gene Located on Human Chromosome 5

Telomerase, a ribonucleoprotein enzyme that maintains telomere length, is crucial for cellular immortalization and cancer progression. Telomerase activity is attributed primarily to the expression of telomerase reverse transcriptase (TERT). Using microcell-mediated chromosome transfer (MMCT) into the mouse melanoma cell line B16F10, we previously found that human chromosome 5 carries a gene, or genes, that can negatively regulate TERT expression (H. Kugoh, K. Shigenami, K. Funaki, J. Barrett, and M. Oshimura, Genes Chromosome Cancer 36:37-47, 2003). To identify the gene responsible for the regulation of TERT transcription, we performed cDNA microarray analysis using parental B16F10 cells, telomerase-negative B16F10 microcell hybrids with a human chromosome 5 (B16F10MH5), and its revertant clones (MH5R) with reactivated telomerase. Here, we report the identification of PITX1, whose expression leads to the downregulation of mouse tert (mtert) transcription, as a TERT suppressor gene. Additionally, both human TERT (hTERT) and mouse TERT (mtert) promoter activity can be suppressed by PITX1. We show that three and one binding site within the hTERT and mtert promoters, respectively, that express a unique conserved region are responsible for the transcriptional activation of TERT. Furthermore, we showed that PITX1 binds to the TERT promoter both in vitro and in vivo. Thus, PITX1 suppresses TERT transcription through direct binding to the TERT promoter, which ultimately regulates telomerase activity.

The inflammasome component NLRP3 impairs antitumor vaccine by enhancing the accumulation of tumor-associated myeloid-derived suppressor cells.

The inflammasome is a proteolysis complex that generates the active forms of the proinflammatory cytokines interleukin (IL)-1β and IL-18. Inflammasome activation is mediated by NLR proteins that respond to microbial and nonmicrobial stimuli. Among NLRs, NLRP3 senses the widest array of stimuli and enhances adaptive immunity. However, its role in antitumor immunity is unknown. Therefore, we evaluated the function of the NLRP3 inflammasome in the immune response using dendritic cell vaccination against the poorly immunogenic melanoma cell line B16-F10. Vaccination of Nlrp3(-/-) mice led to a relative 4-fold improvement in survival relative to control animals. Immunity depended on CD8(+) T cells and exhibited immune specificity and memory. Increased vaccine efficacy in Nlrp3(-/-) hosts did not reflect differences in dendritic cells but rather differences in myeloid-derived suppressor cells (MDSC). Although Nlrp3 was expressed in MDSCs, the absence of Nlrp3 did not alter either their functional capacity to inhibit T cells or their presence in peripheral lymphoid tissues. Instead, the absence of Nlrp3 caused a 5-fold reduction in the number of tumor-associated MDSCs found in host mice. Adoptive transfer experiments also showed that Nlrp3(-/-) MDSCs were less efficient in reaching the tumor site. Depleting MDSCs with an anti-Gr-1 antibody increased the survival of tumor-bearing wild-type mice but not Nlrp3(-/-) mice. We concluded that Nlrp3 was critical for accumulation of MDSCs in tumors and for inhibition of antitumor T-cell immunity after dendritic cell vaccination. Our findings establish an unexpected role for Nlrp3 in impeding antitumor immune responses, suggesting novel approaches to improve the response to antitumor vaccines by limiting Nlrp3 signaling.

Let-7b-mediated suppression of basigin expression and metastasis in mouse melanoma cells

Basigin (Bsg), also called extracellular matrix metalloproteinase inducer (EMMPRIN), is highly expressed on the surface of tumor cells and stimulates adjacent fibroblasts or tumor cells to produce matrix metalloproteinases (mmps). It has been shown that Bsg plays an important role in growth, development, cell differentiation, and tumor progression. MicroRNAs (miRNAs) are a class of short endogenous non-protein coding RNAs of 20–25 nucleotides (nt) that function as post-transcriptional regulators of gene expression by base-pairing to their target mRNAs and thereby mediate cleavage of target mRNAs or translational repression. In this study, let-7b, one of the let-7 family members, was investigated for its effect on the growth and invasiveness of the mouse melanoma cell line B16-F10. We have shown that let-7b can suppress the expression of Bsg in B16-F10 cells and also provided evidence that this suppression could result in the indirect suppression of mmp-9. The ability of B16-F10 cells transfected with let-7b to invade or migrate was significantly reduced. In addition, let-7b transfected B16-F10 cells displayed an inhibition of both cellular proliferation and colony formation. Furthermore, it was shown that the overexpression of let-7b in B16-F10 cells could reduce lung metastasis. Taken together, the present study identifies let-7b as a tumor suppressor that represses cancer cell proliferation and migration as well as tumor metastasis in mouse melanoma cells.

Anti-angiogenic activity of Ipomoea obscura extract and Ipobscurine-A

Objective: Ipomoea obscura (L.) Ker-Gawl, is a medicinal herb with indole alkaloids as an active constituent. In this study, we investigated the anti-angiogenic activity of I. obscura extract and one of its major compounds Ipobscurine-A (IPO-A).Methods: In vivo angiogenesis was induced by injecting B16F10 melanoma cells intradermally on the shaven ventral skin of C57BL/6 mice. In vitro experiments were conducted using human umbilical vein endothelial cells.Results: I. obscura and IPO-A significantly inhibited endothelial cell proliferation, migration, invasion, and tube formation in vitro. Vascular endothelial growth factor (VEGF)-induced sprouting of endothelial cells from rat aorta ex vivo was also inhibited. A marked decrease in the production of matrix metalloproteinases (MMPs) and the expressions of VEGF, cyclooxygenase-2, and nitric oxide synthase by B16F10 cells were observed after the treatment with the extract or IPO-A. Intraperitoneal administration of the extract significantly inhibited B16F10 melanoma cell line–induced neo-vessel formation in C57BL/6 mice in vivo. Analysis of serum cytokine profile clearly showed that extract significantly reduced the elevated levels of pro-inflammatory cytokines such as interleukins (IL)-1β, IL-6, tumor necrosis factor-α, and granulocyte–monocyte colony stimulating factor and the most potent angiogenic factor VEGF in animals. Serum NO level was also found to be significantly lowered by the administration of the extract. Anti-angiogenic factors such as TIMP-1 and IL-2 level were elevated in the extract-treated animals.Conclusion: These data clearly demonstrate that I. obscura extract and IPO-A inhibit the tumor-specific angiogenesis by downregulating pro-angiogenic factors such as MMP, VEGF, and pro-inflammatory mediators and upregulating anti-angiogenic factors such as IL-2 and TIMP-1.

Galectin inhibitory disaccharides promote tumour immunity in a breast cancer model

High level galectin-1 expression results in cancer cell evasion of the immune response, increased tumour survival and aggressive metastases. Using a galectin-1 polyclonal antibody, high levels of galectin-1 protein were shown to be expressed by breast cancer cells established from FVB/N MMTV- c- neu mice as well as by the B16F10 melanoma cell line. In mixed lymphocyte cultures using tumour cells as antigenic stimulators, addition of recombinant galectin-1 dose-dependently inhibited lymphocyte production. Disaccharides were identified that inhibited galectin-1 function and increased growth and activation of CD8 + CTL’s killing cancer cells. X-ray crystallographic structures of human galectin-1 in complex with inhibitory disaccharides revealed their mode of binding. Combining galectin-blocking carbohydrates as adjuvants with vaccine immunotherapy in vivo to promote immune responses significantly decreased tumour progression and improved the outcomes for tumour challenged mice. This is the first report showing that suitably selected galectin-1 blocking disaccharides will act as adjuvants promoting vaccine stimulated immune responses against tumours in vivo.

Abstract 4768: Investigation of frameshift peptides for cancer vaccines

Abstract Background: Our overall goal is to develop a general, preventative cancer vaccine. We are systematically applying bioinformatics, proteomics and immunological methods to achieve this vision. One of our foci is the identification of framshifted (FS) neo-peptides in tumor cells as vaccine components. FS peptides are expressed in alternate reading frames which are activated by various gene mutations and abnormal splicings in tumor cells. Aim/Method: Our hypothesis is that FS peptides are recognized as foreign antigens to the immune system thus making them potential cancer vaccine candidates. We have identified a list of FS peptides by bioinformatic analysis using the publically available human tumor and normal EST databases. To set up a systematical approach of prophylactic cancer vaccine development, we evaluated one potential FS peptide on the list as cancer vaccine candidate which is produced by a splice variant in SMC1A (Structural Maintenance of Chromosomes 1A). Results: The SMC1A FS peptide is a 17 amino acid peptide which is encoded by an abnormal alternative splicing event between exon 1 and exon 4. It was detected in mouse tumor cell lines including melanoma (n=2) and a breast tumor cell lines. It was also detected in human tumor cDNAs assayed thus far including breast (n=34), pancreas (n=4), and lung cancer (n=1) cell lines and primary breast (n=7) and pancreatic tumors (n=4). The expression levels of the SMC1 FS variant increases in most primary tumors with ranges from 2 to 70 fold. Specifically we see an increased expression in 6/7 breast and 2/4 pancreatic tumors screened. Single genetic immunization with plasmids expressing SMC1 FS, mouse GM-CSF, and CpG2216 significantly delayed tumor growth in C57BL/6 mice transplanted with the B16F10 melanoma cell line. Both antibody and IFN-γ released splenocytes to SMC1 FS were detected in immunized mice. Discussion: These findings suggest that the SMC1 FS peptide can be a potential cancer vaccine candidate to prevent various tumors. We are applying this same basic approach to accumulate sufficient antigens to constitute a broadly protective, prophylactic cancer vaccine. (This research is supported by an Innovation Award from the DoD and The Keck Foundation to SAJ.) Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4768.

Experimental Anticancer Therapy with Vascular-disruptive Peptide and Liposome-entrapped Chemotherapeutic Agent

Vasculature is essential for the sustained growth of solid tumors and metastases. Tumor cells surviving vascular-disruptive therapeutic intervention (especially those present at the tumor rim) can contribute to tumor regrowth. The aim was to strengthen, by carrier-mediated delivery of a chemotherapeutic, the curative effects of a bifunctional anti-vascular oligopeptide capable of inducing vascular shutdown and tumor shrinkage. For the in vitro experiments and animal therapy, ACDCRGDCFC-GG-(D)(KLAKLAK)(2) peptide (900 microM in D-PBSA, i.e. Dulbecco's PBS without Ca(2+) and Mg(2+)) and size-calibrated, passively or actively targeted liposomes based on distearoylphosphatidylcholine, cholesterol, and N-carbamoyl-methoxypolyethyleneglycol coupled to distearoylphosphatidylethanolamine (PEG-DSPE) and containing gradient-entrapped doxorubicin were used. The KB (human nasopharyngeal carcinoma) cell line overexpressing folate receptors was used in the fluorescence studies of liposomal uptake. The B16-F10 melanoma cell line was used for confirming, by flow cytometry and confocal microscopy, doxorubicin intracellular transfer as well as to induce experimental tumors in C57BL/6 mice. Animal therapy was achieved with injections of vascular-disrupting peptide, doxorubicin-loaded liposomes, or alternating combined therapy. The results (tumor growth inhibition and survival) were compared using the Mann-Whitney U test and the log-rank test. Necrosis in H&E-stained tumor sections was assessed microscopically by pathologists. Treatment of C57BL/6 mice bearing B16-F10 experimental tumors with a combination of vascular-disruptive peptide and doxorubicin-carrying pegylated liposomes (either passively targeted liposomes (PTL) or folate receptor targeted) gave better therapeutic effects when tumor development was re-challenged with a second cycle of combined therapy. Marked inhibition of tumor growth and a statistically significant extension of the lifespan of the treated mice were observed when the re-challenge involved the use of folate receptor-targeted liposomes (FTL). Anticancer therapy involving vascular-disruptive peptide and doxorubicin delivered via pegylated folate receptor-targeted liposomes is more effective than either monotherapy, especially when tumor growth is re-challenged with the therapeutic combination.

Skin Tumor Responsiveness to IL-2 Treatment Correlates to CD8 Treg Expansion in an Immunocompetent Mouse Model. (100.15)

Abstract CD8 Tregs play an important role in graft tolerance and autoimmune disease status. Interleukin-2 (IL-2) induces Foxp3 expression by activated human CD8 T cells in vitro, and expands circulating CD8 Foxp3+ T cells in melanoma patients. IL-2 is FDA approved for the treatment of advanced melanoma, yet positive outcomes are limited. The aim of this study was to determine the phenotype and distribution of CD8 Foxp3 expressing T cells in skin tumor-bearing mice receiving IL-2. In normal or skin tumor-bearing mice, Foxp3 expressing CD8 T cells were a rare but naturally occurring cell subset. Primarily located in skin-draining lymph nodes CD8 Foxp3+ T cell expressed both activated CTL (CD28+, CD44+) and Treg (CTLA4+, PD1lo, NKG2Alo) markers. Following high dose IL-2 treatment, a dramatic increase in CD8 Treg prevalence was observed in a subcutaneous-tumor model that is non-responsive to IL-2 treatment (B16-F10 melanoma cell line). Furthermore, distribution analysis in this (B16-F10) model demonstrated increased CD8 Treg expression in the circulation and tumor-draining lymph nodes. Conversely, in the IL-2 responsive MCA-205 fibrosarcoma-bearing mouse model no significant changes in CD8 Treg were measured, either in the bloodstream or within the tumor-draining lymph nodes. In conclusion, this study characterized CD8 Foxp3+ T cells (CD8 Treg) as a new immunosuppressive cell subset, the expansion of which was associated with skin tumor that failed to respond to IL-2 treatment.

Combined effects of the proteasome inhibitor bortezomib and Hsp70 inhibitors on the B16F10 melanoma cell line.

Studies have shown that the 26S proteasome is involved in cell cycle control, transcription, DNA repair, immune response and protein synthesis. In the present study, we investigated the antiproliferative effects of the proteasome inhibitor bortezomib and heat shock protein (Hsp)70 inhibitors on the B16F10 melanoma cell line. The IC50 value of bortezomib was found to be 2.46 nM, while that of the Hsp70 inhibitor quercetin was 45 µM in the B16F10 cells. This indicates that bortezomib is more effective than quercetin in inhibiting cell growth. In response to treatment with 10 nM bortezomib for 24 h, cells underwent rounding, shrinkage and detachment. Unexpectedly, such morphological changes were not observed in cells treated with 20 µM quercetin alone, nor in cells treated with bortezomib + quercetin, indicating that quercetin inhibited the cytotoxic effects of bortezomib. Quantitation of cell viability also indicated that quercetin interfered with the cytotoxic effects of bortezomib. However, the combination of quercetin with another proteasome inhibitor, MG132, caused significant cell death as compared to single-agent treatment. A DNA ladder assay also confirmed the inhibitory effect of quercetin on the apoptosis-inducing effect of bortezomib. However, quercetin did not prevent the induction of apoptosis by MG132; on the contrary, it potentiated the apoptosis-inducing effect of MG132. These results suggest that the combination of quercetin with clinically beneficial proteasome inhibitors (except bortezomib) may have increased efficacy in the treatment of cancer. We also tested the combination of two other Hsp70 inhibitors, KNK-437 and schisandrin-B, in combination with bortezomib. Neither of these combinations was more effective than single-agent treatment.

Fluvastatin increases tyrosinase synthesis induced by UVB irradiation of B16F10 melanoma cells.

Statins are widely used to lower plasma concentrations of lipids, e.g. cholesterol. One of the main effects of statin treatment is inhibition of hydroxymethyl glutaryl-coenzyme A reductase. The role of fluvastatin, a frequently used statin, was examined in potential modulation of tyrosinase (key enzyme of melanogenesis) synthesis. Levels of tyrosinase mRNA induced by UVB irradiation of B16F10 melanoma cell line were measured by real time PCR. Fluvastatin increases tyrosinase mRNA production induced by UVB irradiation in B16F10 melanoma cell line. Fluvastatin treatment may potentially influence melanin synthesis and protection against UV irradiation.

Combined effects of the proteasome inhibitor bortezomib and Hsp70 inhibitors on the B16F10 melanoma cell line

Combined effects of the proteasome inhibitor bortezomib and Hsp70 inhibitors on the B16F10 melanoma cell line

Potent adaptive immune responses induced against HIV-1 gp140 and influenza virus HA by a polyanionic carbomer

Carbopol is a polyanionic carbomer gel used in man for a variety of topical applications and drug delivery purposes. Here we show that subcutaneous administration of carbopol with glycoprotein antigens elicits unusually strong specific adaptive immune responses in mice. Recombinant soluble HIV-1 envelope glycoprotein (Env)-based antigen formulated in carbopol was at least as potent at stimulating Env-specific B and T cell responses as Freund's Complete Adjuvant, and significantly more potent than aluminium salts. The antigen-specific T cell immune response elicited both Th1 and Th2 cytokines including high titers of IFN-γ, IL-2 and IL-4, and drove a Th1 isotype-switched antibody response. Mice immunized with a low dose of purified influenza HA in carbopol generated high titers of anti-HA antibodies and were protected from lethal challenge and disease with live virus. Similarly, immunization of mice with the melanoma cell line B16F10 formulated in carbopol significantly delayed tumor growth. We propose that carbopol, or related cross-linked polyacrylic acid analogues, may have promise for use as systemic vaccine adjuvants in man.

Synthesis and characterization of thermo-responsive copolymeric nanoparticles of poly(methyl methacrylate- co- N-vinylcaprolactam)

Copolymeric nanoparticles of methyl methacrylate (MMA) and N-vinylcaprolactam (VCL) were prepared through free radical polymerization using hydrogen peroxide and l-ascorbic acid as a redox initiator in o/w microemulsion containing sodium dodecyl sulphate (SDS). The copolymers were characterized by FTIR and gel permeation chromatography (GPC) and composition of copolymer was determined by 1H NMR spectroscopy. Reactivity ratio was determined by linear least square and non-linear least square methods. The morphology and particle size distribution of copolymer latexes was determined through transmission electron microscopy (TEM) and dynamic light scattering (DLS). Copolymers were of less than 50 nm size with spherical morphology and latexes were stable for more than 6 months. Phase transition temperature measured through UV–vis spectrometry, for the synthesized copolymer indicates their potential use in biosensors and targeted drug delivery system. Cytotoxicity of nanoparticles was determined by MTT assay on B16F10 melanoma cell lines. Cell viability data shows the IC 50 values of copolymeric nanoparticles to be in the range of 0.01–0.1 mg/mL.

Fluvastatin increases tyrosinase synthesis induced by α-melanocyte-stimulating hormone in B16F10 melanoma cells

The aim of this study was to evaluate the effect of fluvastatin on the α-melanocyte-stimulating hormone-mediated increase in tyrosinase activity in the melanoma B16F10cell line and to establish whether Akt and extracellular signal-regulated kinase (Erk) inhibition is involved in tyrosinase synthesis after fluvastatin administration. Fluvastatin modulates α-melanocyte-stimulating hormone induced melanogenesis by increasing tyrosinase mRNA production, as shown by real time PCR, or tyrosinase protein synthesis, as presented by western blot technique. The stimulatory effect of fluvastatin on melanogenesis was, in part, induced by modulation of cell proliferation (decreased melanoma cell proliferation in G2/M phase) and possibly decrease of Akt. These findings indicate that fluvastatin increases tyrosinase synthesis induced by α-melanocyte-stimulating hormone in B16F10cells and reveal an unknown effect of statin use: their influence on melanin production.

Effect of combination of taurine and azelaic acid on antimelanogenesis in murine melanoma cells

BackgroundPigmentation in human skin is an important defense mechanism against sunlight or oxidative stress. Despite the protective role of melanin, abnormal hyperpigmentation such as freckles and chloasma sometimes can be serious aesthetic problems. Because of these effects of hyperpigmentation, people have considered the effect of depigmentation. Azelaic acid (AZ) is a saturated dicarboxylic acid found naturally in wheat, rye, and barley. Previously, we showed that AZ inhibited melanogenesis. In this study, we investigated the antimelanogenic activity of combination of AZ and taurine (Tau) in B16F10 mouse melanoma cells.MethodsThe mouse melanoma cell line B16F10 was used in the study. We measured melanin contents and tyrosinase activity. To gain the change of protein expression, we carried out western blotting.ResultsWe investigated that AZ combined with taurine (Tau) show more inhibitory effects in melanocytes than the treatment of AZ alone. AZ combined with Tau inhibited the melanin production and tyrosinase activity of B16F10 melanoma cells without significant cytotoxicity. Also inhibitory effects after treatment with these combined chemical are stronger than AZ alone on melanogenesis.ConclusionsThese findings indicate that AZ with Tau might play an important role in the regulation of melanin formation and be useful as effective ingredients in antimelanogesis.

Proteolipid protein 2 is associated with melanoma metastasis

The metastasis of malignant tumor cells from the primary tumor to distant sites in the body is a complex process. To identify genes that may be essential for metastasis, we established poorly metastatic mouse melanoma cells, namely Y925F-mutated FAK-transfected cells (Y925F cells), from the highly metastatic mouse melanoma cell line B16F10, and performed expression analyses. The expression of phospholipid protein 2 (PLP2) was markedly down-regulated in the Y925F cells. To elucidate the function of PLP2, we established melanoma cells overexpressing PLP2. We found that PLP2 enhanced proliferation, adhesion, invasion, and MMP-2 secretion in vitro, and tumor metastasis in vivo. These results suggest that PLP2 aids metastasis. Furthermore, we showed that PLP2 binds specifically to PI3K, thus activating Akt.