B16F10 Mouse Melanoma Cells Research Articles

Development of a peptide-based bifunctional chelator conjugated to a cytotoxic drug for the treatment of melanotic melanoma.

The cytotoxic drug gemcitabine (GEM) has been conjugated to receptor-binding peptides to target melanoma tumors. A hexapeptide having a Lys-Gly-His-Lys sequence (pep-1), an octapeptide with an Arg-Gly-Asp-Lys-Gly-His-Lys sequence (pep-2), a GEM-conjugated Lys-Gly-His-Lys peptide (GEM-pep-3) and a GEM-conjugated Asp-Gly-Arg peptide (GEM-pep-4) were synthesized and characterized. In vitro uptake of fluorescently labeled GEM-pep-3 and GEM-pep-4 on B16F10 cells was investigated. Fluorescence microscopy studies demonstrated significant uptake of GEM-pep-3 in the B16F10 mouse melanoma cell line. The peptides and GEM-coupled peptides were radiolabeled with [99mTc(CO)3(H2O)3]+ and examined for in vitro cell binding in the B16F10 melanoma cell line and in vivo biodistribution and scintigraphic studies in a B16F10 melanoma tumor-bearing mice model. In vitro cellular uptake studies and biological evaluation confirmed significant deposition of GEM-pep-3 at the melanoma tumor site. The MTT assay depicted higher cytotoxic behaviour of GEM-pep-3 than free GEM. A considerable amount of cell apoptosis was also observed in B16F10 cells. Finally, the in vivo therapeutic efficacy study revealed a significant decrease in tumor growth in the GEM-pep-3-treated animal model. These studies reveal enough potentiality of GEM-pep-3 to treat melanoma and underline the need for further evaluation.

Artificial T Cell Mimetics to Combat Melanoma Tumor Growth

Despite recent breakthroughs in melanoma treatment with anti-PD-1 immunotherapy, innovative approaches are needed to improve off-target effects. In this study, we report a T cell mimetic microparticle delivery of soluble PD1 aiming at providing a carrier substrate for future combinatorial and targeting efforts. Microparticles of sizes varying from (5 μm to-7 μm) were conjugated with soluble mouse or human PD-1 through nearly irreversible binding between streptavidin and biotin. PD-1 conjugated microparticles (PDMPs) suppressed 3-dimensional tumor growth of human A375 and mouse B16-F10 melanoma cells compared to control microparticles conjugated with the Fc portion of human IgG1 (IgG1MPs). This can be attributed to competitive inhibition by PDMPs on a melanoma cell-intrinsic PD-1/PD-L1 pathway. A single, local administration of mPDMPs in a B16-F10 mouse melanoma model inhibited tumor growth significantly compared to control IgMPs at the same dose. CD45+ immune cells were found to infiltrate tumors treated with mPDMPs as a mechanism for tumor control. These results collectively suggest that PDMPs can target the melanoma cell-intrinsic PD-1/PD-L1 pathway and that these artificial T cell mimetics can be the scaffold for further improvements in anti-tumor immunotherapy.

Chemotherapy can promote liver metastasis by enhancing metastatic niche formation in mice

BackgroundSome chemotherapeutic agents have been reported to promote lung metastasis. However, there have been no reports regarding chemotherapy-induced liver metastasis. We hypothesized that chemotherapy might also enhance liver metastasis. The present study aimed to create a chemotherapy-enhanced liver metastasis mouse model and investigate its mechanism. Materials and methodsMice were pretreated with cisplatin, vincristine, or saline by intraperitoneal injection. Next, B16F10 mouse melanoma cells and BE(2)-C human neuroblastoma cells were injected into the spleens of C57BL/6 and BALB/c nu/nu mice, respectively, to induce experimental liver metastasis, and the number of liver nodules was determined. We also analyzed the effect of chemotherapy on changes of the liver tissue regarding representative metastasis-promoting factors using real-time quantitative polymerase chain reaction and immunohistochemical and histological analysis. ResultsCisplatin increased the number of nodules by 4.7-fold in the B16F10 liver metastasis model. Vincristine increased the number of nodules by 3.8-fold in the BE(2)-C liver metastasis model. Cisplatin increased mRNA levels of matrix-metalloproteinase (MMP)-2 and periostin, while vincristine increased MMP-9 and S100A8/9 levels in liver tissues. Cisplatin induced fibrosis, whereas vincristine induced neutrophil recruitment in liver tissues according to histological and immunohistochemical analysis. ConclusionsWe concluded that cisplatin or vincristine could enhance liver metastasis of mouse melanoma cells or human neuroblastoma cells, respectively. In addition, the mRNA expression of MMP-2 and periostin, or MMP-9 and S100A8/9 is increased by cisplatin or vincristine pretreatment, possibly resulting in fibrosis or neutrophil recruitment, respectively. These niche factors might be associated with increased liver metastasis.

Genome-wide RNA-Seq identifies Fas/FasL-mediated tumoricidal activity of embryonic stem cells.

The discovery of tumor tropism of stem cells revealed the intimate relationship between stem cells and tumor cells, but the functional role of stem cells in tumorigenesis is poorly understood. To investigate embryonic stem cell (ESC) and tumor cell interactions, we co-cultured mouse ESCs with mouse melanoma B16-F10 cells or mouse pancreatic tumor Pan02 cells, and found that ESCs significantly inhibited tumor cell proliferation. Coculture of ESCs and tumor cells resulted in significant inhibition of tumorigenesis in vivo. Histological analyses indicated that ESCs encircled apoptotic tumor cells. We carried out time course RNA-Seq analyses of ESC and tumor cell co-cultures, and identified Fas/FasL signaling as a major pathway involved in ESC-mediated apoptosis of tumor cells. We further generated FADD-deficient tumor cells by CRISPR/Cas9-mediated gene editing, and demonstrated that FADD-deficient tumor cells were obviously resistant to ESC-mediated inhibition of tumor cell proliferation. Our results indicate the Fas/FasL signaling pathway plays a critical role in ESCs-mediated tumoricidal activity.

Ras GAP-related and C-terminal domain-dependent localization and tumorigenic activities of IQGAP1 in melanoma cells.

IQGAP1 interacts with a number of binding partners through a calponin homology domain (CHD), a WW motif, IQ repeats, a Ras GAP-related domain (GRD), and a conserved C-terminal (CT) domain. Among various biological and cellular functions, IQGAP1 is known to play a role in actin cytoskeleton dynamics during membrane ruffling and lamellipodium protrusion. In addition, phosphorylation near the CT domain is thought to control IQGAP1 activity through regulation of intramolecular interaction. In a previous study, we discovered that IQGAP1 preferentially localizes to retracting areas in B16F10 mouse melanoma cells, not areas of membrane ruffling and lamellipodium protrusion. Nothing is known of the domains needed for retraction localization and very little is known of IQGAP1 function in the actin cytoskeleton of melanoma cells. Thus, we examined localization of IQGAP1 mutants to retracting areas, and characterized knock down phenotypes on tissue culture plastic and physiologic-stiffness hydrogels. Localization of IQGAP1 mutants (S1441E/S1443D, S1441A/S1443A, ΔCHD, ΔGRD or ΔCT) to retracting and protruding cell edges were measured. In retracting areas there was a decrease in S1441A/S1443A, ΔGRD and ΔCT localization, a minor decrease in ΔCHD localization, and normal localization of the S1441E/S1443D mutant. In areas of cell protrusion just behind the lamellipodium leading edge, we surprisingly observed both ΔGRD and ΔCT localization, and increased number of microtubules. IQGAP1 knock down caused loss of cell polarity on laminin-coated glass, decreased proliferation on tissue culture polystyrene, and abnormal spheroid growth on laminin-coated hydrogels. We propose that the GRD and CT domains regulate IQGAP1 localization to retracting actin networks to promote a tumorigenic role in melanoma cells.

Inhibition of tumor growth by cancer vaccine combined with metronomic chemotherapy and anti-PD-1 in a pre-clinical setting

Tumor microenvironment (TME) is characterized by multiple immune suppressive mechanisms able to suppress anti-tumor effector cell immunity. Combinatorial strategies, including vaccine and immunomodulatory drugs, need to be developed for improved immunotherapy efficacy.A novel combinatorial approach was assessed in C57BL/6 mice injected with mouse melanoma B16F10 cells. A multi-peptide vaccine (PEPT) was combined with a low dose metronomic chemotherapy (MCT) and an anti-PD-1 checkpoint inhibitor (CI). Statistical analysis were performed with the unpaired two-sided Student’s t-test and ANOVA.Animals treated with the multi-peptide vaccine combined with MCT or CI showed remarkable delay in tumor growth and prolonged survival as compared to control groups. The multi-pronged combination including PEPT+MCT+CI was able to prolong survival in all mice and inhibit tumor growth in 66.6% of mice. All animals which did not show tumor growth were re-challenged with the same melanoma cells and one of them showed complete tumor growth inhibition. The anti-tumor effect was associated with strong T cell immune response to vaccine mutated peptides and significant reduction of regulatory T cells.The combination of a vaccine with MCT and CI was highly efficient in potentiating the vaccine’s anti-tumor effects. The approach is highly promising to be moved into clinical trial.

Melanogenic mechanism of ethanolic extract of Dalbergia odorifera

Vitiligo and hair graying are acquired chronic hypopigmentary disorders of the skin. This study was carried out to investigate the effect of ethanolic extract of D. odorifera (DOE) on melanogenesis and determine its mechanism of action in B16F10 mouse melanoma cells and human epidermal melanocytes. DOE increased melanin content levels. It also enhanced the expression of microphthalmia-associated transcription factor (MITF) gene and its downstream target genes tyrosinase-related protein (TRP) 1, TRP 2, and tyrosinase. DOE increased cAMP levels, as determined by a cAMP production assay. In addition, DOE increased PKA activity and induced phosphorylation of cAMP response element binding (CRE B) protein, its downstream signaling protein. However, DOE showed no effects on mitogen-activated kinases (MAPKs) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Taken together, DOE induces melanogenesis by upregulating MITF gene through the cAMPCREB signaling pathway, which suggests its potential for the treatment of hypopigmentary skin diseases.

37 - Differential regulation of ischemic limb vascularization and tumor growth by endothelial glutaredoxin-1

Mechanisms for the lack of efficacy of anti-angiogenic strategies for cancer have not been elucidated. We previously demonstrated that up-regulated glutaredoxin-1 (Glrx) inhibited endothelial cell (EC) angiogenic properties. Glrx is a small cytosolic enzyme which reverses glutathione adducts on protein thiols. Global Glrx transgenic mice had impaired ischemic limb vascularization, while Glrx knockout mice showed enhanced ischemic revascularization in association with HIF-1a activation. Here we generated EC-specific Glrx transgenic mice (EC-Glrx TG) by crossing VE-cadherin tTA mice and tet-operated Glrx mice. Similar to global Glrx TG mice, EC-Glrx TG mice showed impaired blood flow recovery after femoral artery ligation. Subcutaneous Matrigel implants contained significantly lower hemoglobin content compared to control mice, indicating less EC migration in EC-Glrx TG mice. Potential suppression of tumor angiogenesis was tested in EC-Glrx TG mice. When B16F0 mouse melanoma cells were subcutaneously implanted for 2 weeks, isolectin B4 staining in tumors indicated fewer EC markers in EC-Glrx TG tumors. Unexpectedly, B16F0 tumor weight was not inhibited, but rather dramatically promoted in EC-Glrx TG mice (667 ± 160 vs. 1229 ± 202 mg, n=12, 8, respectively, P

Pratol, an O-Methylated Flavone, Induces Melanogenesis in B16F10 Melanoma Cells via p-p38 and p-JNK Upregulation.

Tyrosinase is the rate-limiting enzyme critical for melanin synthesis. It controls pigmentation in the skin. Activation of tyrosinase is currently the most common approach in the development of tanning and haircare products. Pratol is a 7-hydroxy-4-methoxyflavone found in Trifolium pratense. In this study, we investigated the effects of pratol on melanogenesis. We also studied the mechanism of action of pratol in B16F10 mouse melanoma cells. The cells were treated with various concentrations (6.25, 12.5, 25, and 50 μM) of pratol to observe its effects. The results showed that pratol significantly increased melanin content and tyrosinase activity in the cells without being cytotoxic. In addition, pratol strongly increased the expression of tyrosinase and tyrosinase-related protein-1 and 2 by enhancing the expression of microphthalmia-associated transcription factor. Furthermore, pratol stimulated melanogenesis via the phosphorylation of p38, c-Jun N-terminal kinases (JNK), and extracellular signal–regulated kinase (ERK). The findings from an assay searching for the inhibitor revealed that SB203580 (a specific p38 inhibitor) or SP600125 (a p-JNK inhibitor) attenuated pratol-induced cellular tyrosinase activity whereas PD98059 (an ERK inhibitor) did not. Additionally, pratol interfered with the phosphorylation of p-AKT. We also found that pratol-induced melanogenesis was reversed by H89, which is a specific protein kinase A inhibitor. The results suggest that, owing to its multi-functional properties, pratol may be a potential tanning agent or a therapeutic agent for hair depigmentation in the cosmetic industry.

The \u03b1-melanocyte stimulating hormone/peroxisome proliferator activated receptor-\u03b3 pathway down-regulates proliferation in melanoma cell lines

BackgroundThe α-Melanocyte Stimulating Hormone (αMSH)/Melanocortin-1 receptor (MC1R) interaction promotes melanogenesis through the cAMP/PKA pathway. The direct induction of this pathway by Forskolin (FSK) is also known to enhance melanocyte proliferation. αMSH acts as a mitogenic agent in melanocytes and its effect on proliferation of melanoma cells is less known. We previously identified the αMSH/Peroxisome Proliferator Activated Receptor (PPARγ) pathway as a new pathway on the B16-F10 mouse melanoma cell line. αMSH induced the translocation of PPARγ into the nucleus as an active transcription factor. This effect was independent of the cAMP/PKA pathway and was mediated by the activation of the PI(4,5)P2/PLC pathway, a pathway which we have described to be triggered by the αMSH-dependent MC1R stimulation. Moreover, in the same study, preliminary experiments showed that mouse melanoma cells responded to αMSH by reducing proliferation and that PPARγ was involved in this effect. Due to its key role in the control of cell proliferation, PPARγ agonists are used in therapeutic models for different forms of cancer, including melanoma. The purpose of this study was: (a) to confirm the different proliferative behavior in response to αMSH in healthy and in melanoma condition; (b) to verify whether the cAMP/PKA pathway and the PLC/PPARγ pathway could exert an antagonistic function in the control of proliferation; (c) to deepen the knowledge of the molecular basis responsible for the down-proliferative response of melanoma cells after exposure to αMSH.MethodsWe employed B16-F10 cell line, a human melanoma cell line (Mel 13) and two primary cultures of human melanocytes (NHM 1 and NHM 2, respectively), all expressing a wild type MC1R and responding to the αMSH in terms of pigmentation. We evaluated cell proliferation through: a) cell counting, b) cell cycle analysis c) protein expression of proliferation modulators (p27, p21, cyclin D1 and cyclin E).ResultsThe αMSH acted as a mitogenic agent in primary cultures of human melanocytes, whereas it determined a slow down of proliferation in melanoma cell lines. FSK, as an inducer of the cAMP/PKA pathway, reproduced the αMSH mediated effect on proliferation in NHMs but it did not mimic the αMSH effect on proliferation in B16-F10 and Mel 13 melanoma cell lines. Meanwhile, 3 M3-FBS (3 M3), as an inducer of PI(4,5)P2/PLC pathway, reproduced the αMSH proliferative effect. Further experiments, treating melanoma cell lines with αMSH in the presence/absence of GW9662, as an inhibitor of PPARγ, confirmed the key role of this transcription factor in decreasing cell proliferation in response to the hormone exposure.ConclusionsIn both melanoma cell lines, αMSH determined the reduction of proliferation through the PI(4,5)P2/PLC pathway, employing PPARγ as an effector element. These evidence could offer perspectives for new therapeutic approaches for melanoma.

Whitening and anti-wrinkle activities of ferulic acid isolated from Tetragonia tetragonioides in B16F10 melanoma and CCD-986sk fibroblast cells

Ferulic acid isolated from Tetragonia tetragonioides was tested for its whitening effect on the B16F10 mouse melanoma cell line and its anti-wrinkle activity on the CCD-986sk human dermal fibroblast cell line. Ferulic acid, one of the primary phenolic compounds that can be isolated from T.tetragonioides, has been reported to show potential as a functional food, for its whitening effect and anti-wrinkle activity. To measure its whitening and anti-wrinkle activities, cells were treated with ferulic acid isolated from T.tetragonioides at concentrations between 5 and 20μM. Ferulic acid showed no cytotoxicity at concentrations up to 20μM. Ferulic acid inhibited melanin synthesis, tyrosinase expression, and microphthalmia transcription factor expression in B16F10 cells stimulated with α-melanocyte stimulating hormone. Ferulic acid induced procollagen synthesis, hyaluronic acid synthesis, tissue inhibitor of metalloproteinase synthesis, and inhibited matrix metalloproteinase (MMP)-1 and MMP-9 expression in CCD-986sk cells stimulated with UV-B. On the basis of these results, we conclude that ferulic acid isolated from T.tetragonioides shows potential for use as a functional food, with whitening and anti-wrinkle activities.

The Impact of Environmental Light Intensity on Experimental Tumor Growth

Cancer research requires for consistent models that minimize environmental variables. Within the typical laboratory animal housing facility, animals may be exposed to varying intensities of light as a result of cage type, cage position, light source, and other factors; however, studies evaluating the differential effect of light intensity during the light phase on tumor growth are lacking. The effect of cage face light intensity, as determined by cage rack position was evaluated with two tumor models using the C57Bl/6NHsd mouse and transplantable B16F10 melanoma cells or Lewis lung carcinoma (LLC) cells. Animals were housed in individually-ventilated cages placed at the top, middle, or bottom of the rack in a diagonal pattern so that the top cage was closest to the ceiling light source, and cage face light intensity was measured. Following a two-week acclimation period at the assigned cage position, animals were subcutaneously administered either 1.3×106 B16F10 melanoma cells or 2.5×105 Lewis lung carcinoma cells. Weights of excised tumors were measured following euthanasia 18 days (melanoma) or 21 days (LCC) after tumor cell administration. Cage face light intensity was significantly different depending on the location of the cage, with cages closest to the light source have the greatest intensity. Mean tumor weights were significantly less (p<0.001 for melanoma; p≤0.01 for LCC) in middle light intensity mice compared to high and low light intensity mice. The environmental light intensity to which experimental animals are exposed may vary markedly with cage location and can significantly influence experimental tumor growth, thus supporting the idea that light intensity should be controlled as an experimental variable for animals used in cancer research.

Anti-Melanogenic Effects of Flavonoid Glycosides from Limonium tetragonum (Thunb.) Bullock via Inhibition of Tyrosinase and Tyrosinase-Related Proteins.

Overproduction and stimulation of tyrosinase result in increased melanogenesis of which several skin disorders such as freckles, spots, and hyperpigmentation appear as complications. Limonium tetragonum is a halophyte well-known for its antioxidative properties. This study investigated the anti-melanogenic effects of solvent-partitioned L. tetragonum extracts (LTEs) and its bioactive constituents, two isolated flavonoid glycosides. Current study followed a set of experiments on B16-F10 mouse melanoma cell model with a focus on tyrosinase activity and production. The anti-melanogenic capacity of LTEs was confirmed by their tyrosinase inhibitory effects, prevention of DOPA oxidation, and suppression of melanin production. The inhibition of tyrosinase and DOPA oxidation by LTEs was suggested to be related with the downregulation of microphthalmia-associated transcription factor, tyrosinase, tyrosinase-related protein-1, and tyrosinase-related protein-2, verified with mRNA and protein expression levels. Among all tested LTEs, 85% aq. MeOH and n-BuOH were found to be the most active fractions which later yielded the two known compounds, myricetin 3-galactoside and quercetin 3-O-β-galactopyronaside. The anti-melanogenic potential of the compounds were confirmed by their tyrosinase inhibitory effects. These results suggested that L. tetragonum may serve as a potential source of bioactive substances with effective anti-melanogenesis properties.

Benzyl isothiocyanate and phenethyl isothiocyanate inhibit murine melanoma B16F10 cell migration and invasion in vitro.

Benzyl isothiocyanate (BITC), and phenethyl isothiocyanate (PEITC) have been demonstrated to induce anticancer function in many human cancer cells and also inhibit cancer cell migration and invasion. However, there are no studies that show BITC and PEITC to inhibit cell migration and invasion in mouse melanoma B16F10 cells. In this study, we investigated anti-metastasis effects of BITC and PEITC in melanoma cancer cells invitro. Under sub-lethal concentrations (from 1, 2.5 up to 5µM), BITC and PEITC significantly inhibited cell mobility, migration and invasion nature of B16F10 cells. Gelatin zymography assay also showed that BITC and PEITC inhibited matrix metalloproteinase-2 (MMP-2) activity in B16F10 cells. PEITC reduced MAPK signaling associated proteins such as p-ERK1/2, p-p38 and p-JNK1/2 but BITC increased those MAPK signaling associated proteins. BITC and PEITC both suppressed the expression of RhoA, Ras, and SOS-1, however, PEITC increased FAK and GRB2 but BITC increased FAK at 48h. Furthermore, PEITC decreased the expression of MMP-2 and tissue inhibitors of matrix metalloproteinases (TIMP) but BITC increased them. PEITC inhibited NF-κB protein levels and DNA binding which was confirmed by electrophoretic mobility shift (EMSA) assay. Based on these observations, we suggest that BITC and PEITC can be used in anti-metastasis of melanoma cells in the future.

PD-1 blockade at the time of tumor escape potentiates the immune-mediated antitumor effects of a melanoma-targeting monoclonal antibody

ABSTRACTTumor antigen-targeting monoclonal antibodies (TA-targeting mAbs) are used as therapeutics in many malignancies and their capacity to mobilize the host immunity puts them at the forefront of anti-cancer immunotherapies. Both innate and adaptive immune cells have been associated with the therapeutic activity of such antibodies, but tumor escape from mAb-induced tumor immune surveillance remains one of the main clinical issues. In this preclinical study, we grafted immunocompetent and immunocompromised mice with the B16F10 mouse melanoma cell line and treated them with the TA99 TA-targeting mAb to analyze the immune mechanisms associated with the tumor response and resistance to TA99 monotherapy. In immunocompetent mice TA99 treatment strongly increased the fraction of CD8 and CD4 effector T cells in the tumor compared with isotype control, highlighting the specific immune modulation of the tumor microenvironment by TA99. However, in most mice, TA99 immunotherapy could not prevent immune effector exhaustion and the recruitment of regulatory CD4 T cells and consequently tumor escape from immune surveillance. Remarkably, anti-PD-1 treatment at the time of tumor emergence restored the Th1 effector functions of CD4 and CD8 T cells as well as of natural killer and γδT cells, which translated into a significant slow-down of tumor progression and extended survival. Our findings provide the first evidence that PD-1 blockade at the time of tumor emergence can efficiently boost the host anti-tumor immune response initiated several weeks before by the TA-targeting mAb. These results are promising for the design of combined therapies to sensitize non-responder or resistant patients.

SoyasaponinAg inhibits α‑MSH‑induced melanogenesis in B16F10 melanoma cells via the downregulation of TRP‑2.

Saponins, which are glycosylated, represent a diverse group of biologically functional products in plants. In the present study, we investigated the effects of soyasaponin Ag, a secondary metabolite extracted from soybean, on α-melanocyte-stimulating hormone (α-MSH)-induced melanin synthesis in B16F10 mouse melanoma cells and the underlying molecular mechanisms. To elucidate the mechanisms through which soyasaponin Ag inhibits melanin synthesis, we performed cellular tyrosinase activity assays and analyzed the expression of the melanogenesis-related genes, tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-2. We demonstrated that soyasaponin Ag inhibited α-MSH-induced melanin synthesis in melanoma cells. Of note, soyasaponin Ag had no inhibitory effect on intracellular tyrosinase activity. However, soyasaponin Ag inhibited TRP-2 expression in a dose-dependent manner. Therefore, the depigmenting effect of soyasaponin Ag may be due to the inhibition of tyrosinase expression or the enhancement of tyrosinase degradation. Moreover, soyasaponin Ag did not exert any toxic on B16F10 mouse melanoma cells, suggesting that soyasaponin is a safe component for use in skin care cosmetic formulations that are used for skin whitening.

Abstract 915: Thrombomodulin expression regulates the tumor stroma differentiation of mesenchymal stem cells for controlling tumor growth

Abstract Introduction: Thrombomodulin (TM), a thrombin receptor on endothelial cell surface, acts as an anti-coagulant factor in thrombin-catalyzed protein C activation and inhibits the pro-coagulant functions of thrombin. Owing to TM’s widely expression patterns on various cell types, our studies have demonstrated that beside anti-coagulation, TM also participates in controlling cell-cell adhesion, tumor growth, inflammation, angiogenesis, and atherosclerosis. However, the significance of TM expression in mesenchymal stem cells (MSCs) has never been investigated. The non-hematopoietic MSCs could specifically home into the developing tumors and becomes the active components of tumor stroma, which affects tumor’s growth, immunity, progression, and therapeutic resistance. Recently, we investigated the role of TM on the tumor microenvironment differentiation of MSCs for establishing the active stroma that assists tumor growth. Materials &amp; methods: Normal and TM gene-targeting transgenic mice-isolated bone marrow-derived MSCs were used as the research models to investigate the expressing significance of TM on MSC’s biological activities and differentiations during mouse melanoma B16F10 cell’s conditioning in vitro and in vivo. Results: Our data showed that MSC's TM expression was low in the quiescent state but was up-regulated while treating with B16F10 cell’s conditioned medium in the mechanism of plate-derived growth factor signaling dependence. Accordingly, using conditional gene knockout strategy, under the condition of tumor cell other than normal culture resulted in that TM gene knockout significantly affected MSC’s proliferation, migration, interleukin-6 secretion, angiogenic activity, and tumor-associated fibroblast differentiation in vitro. Furthermore, TM knockout notably reduced the capacity of MSCs on regulating the growth, vasculogenesis, and active stroma establishment of B16F10 melanoma xenograft in vivo. Conclusion: These results indicate that the secretion of tumor-associated plate-derived growth factor is a critical event for MSC’s TM expression that controls MSC’s stroma differentiation for developing active tumor microenvironment, which promotes tumor progression. This study further suggests that the modulation of TM expression on MSCs has the therapeutic potential via diminishing the establishment of functional tumor stroma that sensitizes tumor for the therapies. Citation Format: Tsung-Hsien Shih, Jhy-Ming Li, Guey-Yueh Shi, Hua-Lin Wu. Thrombomodulin expression regulates the tumor stroma differentiation of mesenchymal stem cells for controlling tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 915. doi:10.1158/1538-7445.AM2017-915

Abstract 2671: Antitumor activity of NKTR-214 in combination with pmel-1 ACT in an aggressive murine melanoma model

Abstract The adoptive cell transfer (ACT) of genetically engineered T cells expressing cancer-specific T-cell receptors (TCR) has been shown to induce effective anti-tumor response. However, tumors frequently relapse after an initial response. Another strategy towards stimulating the immune system is the use of high-dose interleukin-2 (IL-2) to target the IL-2 receptor (IL2R), leading to immune cell expansion. However, clinically approved IL-2 expands both tumor-killing CD8+ effector T cells (CD8T) as well as regulatory T cells (Tregs) through binding the IL-2Rβγ and IL-2Rαβγ complexes, respectively. Tregs in the tumor lead to immune suppression, which hampers the antitumoral response. NKTR-214 is a CD122-biased cytokine agonist conjugated with multiple releasable chains of polyethylene glycol and designed to provide sustained signaling through the heterodimeric IL-2 receptor pathway (IL-2Rβγ) to preferentially activate and expand effector CD8+ T and NK cells over Tregs. We used the pmel-1 ACT/B16 melanoma tumor model to test the anti-tumor activity of NKTR-214 and evaluate its effects on tumor-specific TCR transgenic T cells. On Day 0 (D0) C57BL/6 mice were implanted with B16-F10 mouse melanoma cells and lymphodepleted with 500 cGy on D6. On D7, mice were treated with either the combination of ACT (T lymphocytes activated in vitro with 1 μg/ml gp100) plus NKTR-214 (0.8 mg/kg, q9dx3, i.v.) or with C57/B6 T cells plus PBS (vehicle control). The tumors of the vehicle control mice (n=12) rapidly grew to the 1500mm3 endpoint in 12 days post-treatment, versus 35 days for the NKTR-214 group (n=12) with only 1 out of 12 mice reaching endpoint. Bioluminescence imaging was used to visualize the in vivo distribution and tumor-homing of antigen-specific T cells. Interestingly, the reporter T cells were retained in the spleen until D7 and could be seen migrating to the tumor at D9 reaching peak of bioluminescence at day 12, a delayed time point compared to the 5 days usually observed in mice treated with standard IL-2. The signal persisted in the NKTR-214 + ACT group until D20 versus D7 in the vehicle-control animals. These data suggest that NKTR-214 + ACT is well tolerated and provides a robust anti-tumor response in the aggressive B16F10 model. Treatment with NKTR-214 + ACT robustly mobilizes T cells into the tumor where they durably persist. The robust and long-lasting effect of NKTR214 supports its potential use in combination with cell-based therapeutics. Citation Format: Giulia Parisi, Justin Saco, Siwen Hu-Lieskovan, Ruixue Zhang, Paige Krystofinski, Cristina Puig Saus, Deborah H. Charych, Antoni Ribas. Antitumor activity of NKTR-214 in combination with pmel-1 ACT in an aggressive murine melanoma model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2671. doi:10.1158/1538-7445.AM2017-2671

Anti-melanogenic effect of gomisin N from Schisandra chinensis (Turcz.) Baillon (Schisandraceae) in melanoma cells.

Overproduction and accumulation of melanin in the skin can lead to pigmentation disorders, such as freckles and melasma. Many researchers are studying the regulation of melanogenesis in the skin with the aim of developing whitening cosmetics. In this study, bioactivity-guided separation of the hexane fraction of Schisandra chinensis (Turcz.) Baillon extract yielded five major compounds, β-chamigrenal, α-ylangenol, gomisin N, gomisin A, and schisandrin. The structures were identified by using 1H-NMR and 13C-NMR spectroscopy and comparing the spectral data with published data. Melanogenesis inhibitory activity assay in alpha-melanocyte-stimulating hormone (α-MSH)-induced B16F10 mouse melanoma cells revealed that gomisin N was the active component and significantly inhibited melanin synthesis in a concentration-dependent manner. Thus, we evaluated the mechanism underlying its anti-melanogenic effect. Gomisin N inhibited the expression of tyrosinase and microphthalmia-associated transcription factor (MITF) in B16F10 cells, while it did not affect cAMP response element binding protein (CREB) phosphorylation. Additionally, gomisin N activated AKT phosphorylation, which inhibits MITF expression. Thus, our results strongly suggest that the active compound, gomisin N, has potential for use in cosmetics to reduce hyperpigmentation.

Effective Skin Cancer Treatment by Topical Co-delivery of Curcumin and STAT3 siRNA Using Cationic Liposomes.

The aim of the present study was to evaluate the effectiveness of iontophoretic co-delivery of curcumin and anti-STAT3 siRNA using cationic liposomes against skin cancer. Curcumin was encapsulated in DOTAP-based cationic liposomes and then complexed with STAT3 siRNA. This nanocomplex was characterized for the average particle size, zeta-potential, and encapsulation efficiency. The cell viability studies in B16F10 mouse melanoma cells have shown that the co-delivery of curcumin and STAT3 siRNA significantly (p<0.05) inhibited the cancer cell growth compared with either liposomal curcumin or STAT3 siRNA alone. The curcumin-loaded liposomes were able to penetrate up to a depth of 160μm inside the skin after iontophoretic (0.47mA/cm2) application. The in vivo efficacy studies were performed in the mouse model of melanoma skin cancer. Co-administration of the curcumin and STAT3 siRNA using liposomes significantly (p<0.05) inhibited the tumor progression as measured by tumor volume and tumor weight compared with either liposomal curcumin or STAT3 siRNA alone. Furthermore, the iontophoretic administration of curcumin-loaded liposome-siRNA complex showed similar effectiveness in inhibiting tumor progression and STAT3 protein suppression compared with intratumoral administration. Taken together, cationic liposomes can be utilized for topical iontophoretic co-delivery of small molecule and siRNA for effective treatment of skin diseases.