B16F10 Melanoma Cell Line Research Articles

Antioxidant and anticancer effects of extracts from fermented Haliotis discus hannai with Cordyceps militaris mycelia.

In this study, Haliotis discus hannai (H. discus hannai) was fermented using Cordyceps militaris (C. militaris) and was investigated for the improvement of its antioxidant and anticancer potential after fermentation. Different radical scavenging activities of the extracts from fermented H. discus hannai with C. militaris mycelia (FHCM) were assessed by electron spin resonance. The antioxidant potential of FHCM was also determined on the basis of a ferric reducing antioxidant power assay and 2,2'-azinobis-(3-ethybenzothiazoline-6-sulfonic acid) radical scavenging activity. Higher antioxidant and radical scavenging activities were observed in FHCM than in the unfermented C. militaris mycelia or H. discus hannai alone. FHCM demonstrated an anticancer activity against melanoma B16F10 cell line. In addition, FHCM co-treatment with doxorubicin showed an increased anticancer effect in both in vitro and in vivo. Therefore, the present study suggests that the mycelial fermentation on H. discus hannai is highly suitable for pharmaceutical applications.

Systemic delivery and activation of the TRAIL gene in lungs, with magnetic nanoparticles of chitosan controlled by an external magnetic field.

Recently, functional therapies targeting a specific organ without affecting normal tissues have been designed. The use of magnetic force to reach this goal is studied in this work. Previously, we demonstrated that nanocarriers based on magnetic nanoparticles could be directed and retained in the lungs, with their gene expression under the control of a promoter activated by a magnetic field. Magnetic nanoparticles containing the TRAIL gene and chitosan were constructed using the ionic gelation method as a nanosystem for magnetofection and were characterized by microscopy, ζ-potential, and retention analysis. Magnetofection in the mouse melanoma cell line B16F10 in vitro induced TRAIL-protein expression and was associated with morphological changes indicative of apoptosis. Systemic administration of the nanosystem in the tail vein of mice with melanoma B16F10 at the lungs produced a very significant increase in apoptosis in tumoral cells that correlated with the number of melanoma tumor foci observed in the lungs. The high levels of apoptosis detected in the lungs were partially related to mouse survival. The data presented demonstrate that the magnetofection nanosystem described here efficiently induces apoptosis and growth inhibition of melanoma B16F10 in the lungs. This new approach for systemic delivery and activation of a gene based in a nanocomplex offers a potential application in magnetic gene delivery for cancer.

Expression of kindlin-3 in melanoma cells impedes cell migration and metastasis

ABSTRACTKindlins are a small family of 4.1-ezrin-radixin-moesin (FERM)-containing cytoplasmic proteins. Kindlin-3 is expressed in platelets, hematopoietic cells, and endothelial cells. Kindlin-3 promotes integrin activation, clustering and outside-in signaling. Aberrant expression of kindlin-3 was reported in melanoma and breast cancer. Intriguingly, kindlin-3 has been reported to either positively or negatively regulate cancer cell metastasis. In this study, we sought to clarify the expression of kindlin-3 in melanoma cells and its role in melanoma metastasis. Two widely used metastatic mouse and human melanoma cell lines B16-F10 and M10, respectively, were examined and found to lack kindlin-3 mRNA and protein expression. When kindlin-3 was ectopically expressed in these cells, cell migration was markedly reduced. These are attributed to aberrant Rac1 and RhoA activation and overt membrane ruffling. Our data demonstrate for the first time that despite its well established role as a positive regulator of integrin-mediated cell adhesion, aberrant expression of kindlin-3 could lead to imbalanced RhoGTPases signaling that impedes rather than promotes cell migration.

Sesquiterpenes from the whole plants of Parasenecio roborowskii

Six eremophilane-type (parasenolide A–F) and an eudesmane-type (parasenin) sesquiterpenoids, along with eight known sesquiterpenes, were isolated from the whole plants of Parasenecio roborowskii. The structures and absolute configurations of new compounds were elucidated using extensive spectroscopic analysis, including HRESIMS, 1D and 2D NMR experiments, the CD exciton chirality methods, and single-crystal X-ray crystallography. All isolated compounds were evaluated for cytotoxicity against five human cancer (HeLa, HepG2, K562, MDA231, and NCI-H460) cell lines and a murine melanoma B16 F10 cell line by MTT assay. Compounds 1–15 showed cytotoxic activities, especially compounds 3, 4, 8, 10, and 12. These five compounds showed broad spectrum activities against all the tested cancer cell lines with IC50 ranging from 9.2 to 35.5μM. The study supports that eremophilenolides and eudesmane-type sesquiterpenes occur mainly in the genus Parasenecio and can be used as a chemosystematic marker of the genus.

In vitro evaluation of anti-proliferative, anti-inflammatory and pro-apoptotic activities of the methanolic extracts of Andrographis nallamalayana Ellis on A375 and B16F10 melanoma cell lines.

Natural plant products have been widely used in controlling cancer with fewer or no side effects and the use of plant extracts as complementary to synthetic medicine is gaining increased popularity. Members of Andrographis plants possess important medicinal properties. In the present study, anti-cancerous properties of Andrographis nallamalayana (AN) were tested on A375 and B16F10 skin melanoma cancer cell lines. The leaf extracts of AN significantly reduced the cell viability and cell survival of skin cancer cell lines, achieved by MTT assay and clonogenic assays, respectively. Further, TUNEL assays revealed that AN extracts induces the apoptosis. Western blot analysis revealed that AN leaf extracts reduced the expression of Bcl-2, an anti-apoptotic protein and induced the expression of proapoptotic molecules such as Bcl-2 associated death promoter protein (BAD), Bcl-2 associated X protein (BAX) and cleaved caspase-3. Moreover, the qRT-PCR and western blot analysis demonstrated the reduced expression of G2/M phase proteins cdk1, cyclin B1 and increased expression of p53, cyclin-dependent kinase 1 inhibitor, p21. Further, immunofluorescence analysis revealed that AN reduced the NF-κB nuclear translocation, luciferase reporter assays demonstrated reporter gene activation. qRT-PCR assays showed that AN significantly reduced the expression of NF-κB target genes. The results concluded that the extracts of AN exhibited significant anti-proliferative, anti-inflammatory and pro-apoptotic activities on melanoma skin cancer cell lines.

Ursolic Acid Loaded PLGA Nanoparticles: in vitro and in vivo Evaluation to Explore Tumor Targeting Ability on B16F10 Melanoma Cell Lines

Ursolic acid (UA), a pentacyclic triterpenoid extracted from plants, shows promising inhibitory effect in different tumor bearing cell lines. In the present study we fabricated UA loaded PLGA nanoparticles (UA-NPs) as the drug carrier and thoroughly evaluated in vitro and in vivo the differential tumor targeting effects of UA and UA-NPs in B16F10 melanoma cells. Ursolic acid loaded PLGA nanoparticles were prepared by emulsion solvent evaporation technique and evaluated for particle size, polydispersity, zeta potential and drug release potency. MTT assay as well as flow cytometric and confocal microscopic analyses were done in B16F10 mouse melanoma cell lines. Formulations were labeled with technetium-99m to evaluate the biodistribution and perform scintigraphic imaging studies following intravenous administration in tumor bearing mice model. Single emulsification technique produced smooth spherical nanoparticles of small size with relatively narrow size distribution (154 ± 4.56nm). On B16F10 cell line, the formulation showed higher cytotoxicity compared to the free drug due to increased in vitro cellular uptake. The formulation was successfully radiolabeled and remained substantially (>90%) stable when incubated (37°C, 6h) separately in normal saline or freshly collected rat serum or histidine solution. The radiolabeled UA-NPs exhibited slower blood clearance and comparatively high uptake in tumor region as evidenced by biodistribution and scintigraphic studies. The in vitro and in vivo studies have proved the tumor targeting potential of UA-NPs in B16F10 melanoma cell lines.

Abstract 2739: Carnosic acid induces cell cycle arrest of B16F10 cells and synergizes with carmustine and lomustine in vitro and in vivo

Abstract Carnosic acid (CA), one of the purified components of rosemary, has shown anti-inflammatory, chemo-preventive, and anticancer activities. In this study, we treated melanoma cell line B16F10 with CA and detected the anticancer effects. After 24-hour treatment, CA decreased cell proliferation rate, wound healing activity, and inhibited colony formation of B16F10. Additionally, CA induced G0/G1 cell cycle arrest, up-regulated p21 expression, and down-regulate cyclin E, cyclin D and CDK4 expression. Carmustine (BCNU) and Lomustine (CCNU) are used in clinical treatment of melanoma. To assay the combinational effects of CA and BCNU or CCNU, B16F10 cells were treated with CA, BCNU, CCNU alone, or CA combined with BCNU or CCNU. Our results showed, BCNU and CCNU arrested cell cycle at G2/M phase after 24-hour treatment. Dramatically, CA enhenced BCNU- and CCNU-induced cell cycle arrest at G2/M phase, and decreased cyclin B1 and p-CDK1 expresion. In the in vivo xenograft model, B16F10 cells (5×105) were inoculated subcutaneously in C57BL/6 mice, and the tumor grew for one week. The mice were divided into six groups and provided different treated strategies: control, CA (50 mg/kg) alone, BCNU (50 mg/kg) alone, CCNU (25 mg/kg) alone, CA combined with BCNU, and CA combined with CCNU. After two-week treatment, compared with control group, CA had better treated effects than BCNU or CCNU. Compared with CA, BCNU or CCNU treatment alone, the combined strategies inhibited the tumor growth in melanoma mice model more effectively. Furthermore, BCNU and CCNU treatment in melanoma mice caused high values of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). However, CA significantly decreased AST and ALT values caused by BCNU and CCNU in combined trestment group. Therefore, CA is more safe and effective than BCNU and CCNU, and the combined treatment strategies of CA and BCNU or CCNU result in an increased antitumor efficacy. CA may be suitable for translation to future clinic. Citation Format: Kun-I Lin, Chih-Chien Lin, Ming-Feng Chen, Chang-Han Chen, Li-Yen Shiu. Carnosic acid induces cell cycle arrest of B16F10 cells and synergizes with carmustine and lomustine in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2739.

Abstract 2351: Development of a B16F10 cell line expressing mNectin1 to study the activity of OncoVEXmGM-CSF in murine syngeneic melanoma models

Abstract Background: Talimogene laherparepvec is an oncolytic immunotherapy based on a modified herpes simplex virus type 1 (HSV-1), designed to kill neoplastic cells through two mechanisms: a) direct lysis and b) stimulation of a tumor antigen-specific adaptive immune response by way of GM-CSF expression. In order to support mechanism of action studies in a mouse melanoma model relevant to talimogene laherparepvec's approved U.S. indication, we expressed the HSV-1 entry receptor Nectin1 on B16F10 melanoma cells. B16F10 cells have previously been shown to be resistant to HSV-1 due to lack of entry receptor expression (Miller et al., Mol Ther, 2001). The goal of our work was to determine if mNectin1 expression in B16F10 melanoma cells renders them sensitive to talimogene laherparepvec treatment. For preclinical studies we used OncoVEXmGM-CSF, an HSV-1 modified similarly to talimogene laherparepvec except that murine GM-CSF is expressed instead of human GM-CSF. Herein we describe studies to assess the efficacy of OncoVEXmGM-CSF in a novel B16F10-mNectin1 syngeneic melanoma model. Methods: Parental B16F10 cells were transduced with lentivirus expressing either mNectin1, or eGFP as a control. In vitro sensitivity was determined by plating both cell types in 96-well plates, infecting with serially diluted OncoVEXmGM-CSF and then measuring ATP 72 hours later. For efficacy studies, B16F10 or B16F10-mNectin1 cells were injected subcutaneously on the right flank of female C57BL/6 mice. On day 10, mice were randomized into treatment groups based on tumor volume (n = 10/group). Talimogene laherparepvec was delivered intratumorally (5×106 PFU/dose,) every 3 days during the first week. Tumor growth and body weight were measured twice per week throughout the experiment. Survival analysis was performed using a Kaplan-Meier estimator. Results: B16F10 cells were insensitive to OncoVEXmGM-CSF up to 100 MOI (multiplicity of infection). In contrast, two other syngeneic cell lines, A20 and CT-26, showed MOI IC50 of 1 and 20. B16F10 cells expressing mNectin1 showed high sensitivity to OncoVEXmGM-CSF with an MOI IC50 of 0.01. In contrast, B16F10 cells expressing eGFP were insensitive up to an MOI of 100. In vivo, B16F10 mNectin1 cells showed a similar growth pattern as the parental B16F10 when injected subcutaneously. However, OncoVEXmGM-CSF treatment of B16F10-mNectin1 tumors caused a highly significant (p<0.0001) inhibition of tumor growth and increase in median overall survival compared to the control group. B16F10 mNectin1 tumors that grew during or following OncoVEXmGM-CSF treatment were shown to maintain mNectin1 expression suggesting other mechanisms of OncoVEXmGM-CSF resistance were involved. Conclusion: Expression of mNectin 1 in the B16F10 melanoma cell line confers sensitivity to OncoVEXmGM-CSF in vitro and extends median overall survival of mice bearing B16F10-muNectin1 tumors. Citation Format: Keegan Cooke, Juan Estrada, Jinghui Zhan, Petia Mitchell, Yannick Bulliard, Pedro J. Beltran. Development of a B16F10 cell line expressing mNectin1 to study the activity of OncoVEXmGM-CSF in murine syngeneic melanoma models. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2351.

In vitro and in vivo studies of the antineoplastic activity of copper (II) compounds against human leukemia THP-1 and murine melanoma B16-F10 cell lines

We investigated the antineoplastic activities of a previously reported copper (II) coordination compound, [Cu(BMPA)Cl2]CH3OH (1), and a new compound, [Cu(HBPA)Cl2]H2O (2), where BMPA is bis(pyridin-2-ylmethyl)amine and HBPA is (2-hydroxybenzyl)(2-pyridylmethyl)amine, using various cellular models of human leukemia (THP-1, U937, HL60, Molt-4, JURKAT) and human colon cancer (COLO 205), as well as a murine highly metastatic melanoma (B16-F10) cell line. Compound (2) was characterized using several physical and chemical techniques, including X-ray diffraction studies. The IC50 values of the copper coordination complexes in the human leukemia cell lines ranged from 87.63 ± 1.02 to ≥400 μM at high cell concentrations and from 19.17 ± 1.06 to 97.67 ± 1.23 μM at low cell concentrations. Both compounds induced cell death, which was determined by cell cycle analyses and phosphatidylserine exposure studies. THP-1 cells released cytochrome c to the cytoplasm 12 h after treatment with 400 μM of compound (2). To evaluate the apoptosis pathway induced by compound (2), we measured the activities of initiator caspases 8 and 9 and executioner caspases 3 and 6. The results were suggestive of the activation of both intrinsic and extrinsic apoptosis pathways. To investigate the activities of the compounds in vivo, we selected two sensitive cell lines from leukemia (THP-1) and solid tumor (B16-F10) lineages. BALB/c nude bearing THP-1 tumors treated with 12 mg·kg−1 of compound (2) showed a 92.4% inhibition of tumor growth compared with the control group.

Posttranslational proteolytic processing of Leda-1/Pianp involves cleavage by MMPs, ADAM10/17 and gamma-secretase

Leda-1/Pianp is a type I transmembrane protein expressed by CNS cells, murine melanoma cell line B16F10 and rat liver sinusoidal endothelial cells. The early steps of posttranslational modifications of Leda-1/Pianp have been described to include glycosylation and processing by proprotein convertases. Here, we comprehensively characterized the subsequent steps of proteolytic processing of Leda-1/Pianp. For this purpose specific protease inhibitors and cell lines deficient in PS1, PS2, PS1/PS2 and ADAM10/17 were deployed. Leda-1/Pianp was cleaved at numerous cleavage sites within the N-terminal extracellular domain. The sheddases involved included MMPs and ADAM10/17. Ectodomain shedding yielded C-terminal fragments (CTF) of ∼15 kDa. The CTF was further processed by the γ (gamma)-secretase complex to generate the intracellular domain (ICD) of ∼10 kDa. Although PS1 was the dominant intramembrane protease, PS2 was also able to cleave Leda-1/Pianp in the absence of PS1. Thus, Leda-1/Pianp is constitutively processed by proprotein convertases, sheddases including MMPs and ADAM10/17 and intramembrane protease γ-secretase.

Footprintless disruption of prosurvival genes in aneuploid cancer cells using CRISPR/Cas9 technology.

CRISPR/Cas9 has emerged as a powerful methodology for the targeted editing of genomic DNA sequences. Nevertheless, the intrinsic inefficiency of transfection methods required to use this technique with cultured cells requires the selection and isolation of successfully modified cells, which invariably subjects the cells to stress. Here we report a workflow that allows the isolation of genomically modified cells, even where loss of functional alleles constitutes a selective disadvantage owing to impaired ability to survive stress. Using targeted disruption of the Id1 and Id3 genes in murine B16-F10 and Ret melanoma cell lines as an example, we show that the method allows for the footprintless isolation of CRISPR/Cas9-modified aneuploid cancer cells. We also provide evidence that serial CRISPR/Cas9 modifications can occur, for example when initial homologous recombination events introduce cryptic PAM sequences, and demonstrate that multiple alleles can be successfully targeted in aneuploid cancer cells. By sequencing individual alleles we also found evidence for CRISPR/Cas9-induced transposable element insertion, albeit at a low frequency. This workflow should have broad application in the functional analysis of prosurvival gene function in cultured cells.

Phosphate induces formation of matrix vesicles during odontoblast-initiated mineralization in vitro

Mineralization is a process of deposition of calcium phosphate crystals within a fibrous extracellular matrix (ECM). In mineralizing tissues, such as dentin, bone and hypertrophic cartilage, this process is initiated by a specific population of extracellular vesicles (EV), called matrix vesicles (MV). Although it has been proposed that MV are formed by shedding of the plasma membrane, the cellular and molecular mechanisms regulating formation of mineralization-competent MV are not fully elucidated. In these studies, 17IIA11, ST2, and MC3T3-E1 osteogenic cell lines were used to determine how formation of MV is regulated during initiation of the mineralization process. In addition, the molecular composition of MV secreted by 17IIA11 cells and exosomes from blood and B16-F10 melanoma cell line was compared to identify the molecular characteristics distinguishing MV from other EV. Western blot analyses demonstrated that MV released from 17IIA11 cells are characterized by high levels of proteins engaged in calcium and phosphate regulation, but do not express the exosomal markers CD81 and HSP70. Furthermore, we uncovered that the molecular composition of MV released by 17IIA11 cells changes upon exposure to the classical inducers of osteogenic differentiation, namely ascorbic acid and phosphate. Specifically, lysosomal proteins Lamp1 and Lamp2a were only detected in MV secreted by cells stimulated with osteogenic factors. Quantitative nanoparticle tracking analyses of MV secreted by osteogenic cells determined that standard osteogenic factors stimulate MV secretion and that phosphate is the main driver of their secretion. On the molecular level, phosphate-induced MV secretion is mediated through activation of extracellular signal-regulated kinases Erk1/2 and is accompanied by re-organization of filamentous actin. In summary, we determined that mineralization-competent MV are distinct from exosomes, and we identified a new role of phosphate in the process of ECM mineralization. These data provide novel insights into the mechanisms of MV formation during initiation of the mineralization process.

Effect of Adipocyte Secretome in Melanoma Progression and Vasculogenic Mimicry.

Obesity, favored by the modern lifestyle, acquired epidemic proportions nowadays. Obesity has been associated with various major causes of death and morbidity including malignant neoplasms. This increased prevalence has been accompanied by a worldwide increase in cutaneous melanoma incidence rates during the last decades. Obesity involvement in melanoma aetiology has been recognized, but the implicated mechanisms remain unclear. In the present study, we address this relationship and investigate the influence of adipocytes secretome on B16-F10 and MeWo melanoma cell lines. Using the 3T3-L1 adipocyte cell line, as well as ex vivo subcutaneous (SAT) and visceral (VAT) adipose tissue conditioned medium, we were able to show that adipocyte-released factors play a dual role in increasing melanoma cell overall survival, both by enhancing proliferation and decreasing apoptosis. B16-F10 cell migration and cell-cell and cell-matrix adhesion capacity were predominantly enhanced in the presence of SAT and VAT released factors. Melanocytes morphology and melanin content were also altered by exposure to adipocyte conditioned medium disclosing a more dedifferentiated phenotype of melanocytes. In addition, exposure to adipocyte-secreted molecules induced melanocytes to rearrange, on 3D cultures, into vessel-like structures, and generate characteristic vasculogenic mimicry patterns. These findings are corroborated by the released factors profile of 3T3-L1, SAT, and VAT assessed by microarrays, and led us to highlight the mechanisms by which adipose secretome from sub-cutaneous or visceral depots promote melanoma progression. J. Cell. Biochem. 117: 1697-1706, 2016. © 2015 Wiley Periodicals, Inc.

Erdr1 Suppresses Murine Melanoma Growth via Regulation of Apoptosis.

Melanoma, one of the aggressive cancers, is known to be resistant to chemotherapy. Because of its aggressive nature, effectively inducing apoptosis is necessary to treat melanoma. Erythroid differentiation regulator 1 (Erdr1) is known to be a stress-related survival factor exhibiting anti-cancer effects in several cancers. However, little is known about the functions and underlying mechanisms of Erdr1 so far. To demonstrate the effect of Erdr1 in melanoma apoptosis, recombinant murine Erdr1 was injected into mice implanted with B16F10 melanoma cells. In vivo tumor growth was significantly inhibited in mice injected with Erdr1 compared to the control. In addition, the tumor from Erdr1-injected mice showed an increased level of apoptosis. Accordingly, apoptosis-regulating factors including anti-apoptotic marker Bcl-2 and pro-apoptotic marker Bax in the tumor tissues were examined. As expected, the decreased level of Bcl-2 and increased level of Bax were detected in tumors within the mice injected with Erdr1. Based on the in vivo study, the role of Erdr1 in tumor apoptosis was further tested by incubating it with cells of the murine melanoma cell line B16F10. Erdr1-induced apoptosis in B16F10 cells was observed. Additionally, Erdr1 downregulated STAT3 activity, inhibiting apoptosis via regulation of the Bcl-2 family. Overall, data demonstrate that Erdr1 induced murine melanoma apoptosis through the regulation of Bcl-2 and Bax. These findings suggest that Erdr1 is a novel regulator of apoptosis in melanoma.

Biodegradable Nanocapsules Containing A Nanobiotechnological Complex for the In-vitro Suppression of A Melanoma Cell Line B16F10

Melanoma is a fatal skin cancer. In this paper, we design a nanobiotechnological approach for the specific depletion of tyrosine that is essential for melanoma growth. We combine two nanobiotechnological methods into a single therapeutic agent in the form of Polylactide (PLA) nanocapsules containing a nanobiotechnological complex of polyhemoglobintyrosinase. Our results show that nanocapsules can enter into the melanoma cells to deplete tyrosine. This leads to the inhibition of tumor growth, migration and colonization in a highly malignant melanoma cell line B16F10. We also analyzed possible mechanisms of action including ROS generation, apoptosis induction and effect on cell cycle

Encapsulation of palladium porphyrin photosensitizer in layered metal oxide nanoparticles for photodynamic therapy against skin melanoma

We designed a biodegradable nanocarrier of layered double hydroxide (LDH) for photodynamic therapy (PDT) based on the intercalation of a palladium porphyrin photosensitizer (PdTCPP) in the gallery of LDH for melanoma theragnosis. Physical and chemical characterizations have demonstrated the photosensitizer was stable in the layered structures. In addition, the synthesized nanocomposites rendered extremely efficacious therapy in the B16F10 melanoma cell line by improving the solubility of the hydrophobic PdTCPP photosensitizer. The detection of singlet oxygen generation under irradiation at the excitation wavelength of a 532 nm laser was indeed impressive. Furthermore, the in vivo results using a tumour xenograft model in mice indicated the apparent absence of body weight loss and relative organ weight variation to the liver and kidney demonstrated that the nanocomposites were biosafe with a significant reduction in tumour volume for the anti-cancer efficacy of PDT. This drug delivery system using the nanoparticle–photosensitizer hybrid has great potential in melanoma theragnosis.

Syntenin controls migration, growth, proliferation, and cell cycle progression in cancer cells.

The scaffold protein syntenin abounds during fetal life where it is important for developmental movements. In human adulthood, syntenin gain-of-function is increasingly associated with various cancers and poor prognosis. Depending on the cancer model analyzed, syntenin affects various signaling pathways. We previously have shown that syntenin allows syndecan heparan sulfate proteoglycans to escape degradation. This indicates that syntenin has the potential to support sustained signaling of a plethora of growth factors and adhesion molecules. Here, we aim to clarify the impact of syntenin loss-of-function on cancer cell migration, growth, and proliferation, using cells from various cancer types and syntenin shRNA and siRNA silencing approaches. We observed decreased migration, growth, and proliferation of the mouse melanoma cell line B16F10, the human colon cancer cell line HT29 and the human breast cancer cell line MCF7. We further documented that syntenin controls the presence of active β1 integrin at the cell membrane and G1/S cell cycle transition as well as the expression levels of CDK4, Cyclin D2, and Retinoblastoma proteins. These data confirm that syntenin supports the migration and growth of tumor cells, independently of their origin, and further highlight the attractiveness of syntenin as potential therapeutic target.

Antimicrobial, antioxidant and anti-tyrosinase properties of extracts of the Mediterranean parasitic plant Cytinus hypocistis.

BackgroundCytinus is an endophytic parasitic plant occurring in South Africa, Madagascar, and in the Mediterranean region. We have extracted the inflorescences (the only visible part of the plant, emerging from the host roots at the time of blossom) of Cytinus hypocistis collected in Sardinia, Italy, and explored the antimicrobial, antioxidant, anti-tyrosinase, and cytotoxic activities of the extracts.MethodsExtracts from C. hypocistis were prepared using increasing polarity solvents: cyclohexane, ethanol, and water. Phenolic composition were determined through spectrophotometric assays, and antioxidant activity with both electron-transfer and hydrogen-atom assays. Nine different bacterial strains, including clinical isolate methicillin-resistant Staphylococcus aureus, were used in agar diffusion method. Cytotoxicity was tested using against the B16F10 melanoma cell line.ResultsWhile cyclohexane extracts where biologically inactive, ethanolic and aqueous extracts displayed an intriguing activity against several Gram-positive bacterial strains, including methicillin-resistant S. aureus, and against the Gram-negative Acinetobacter baumanii. Compared to the conventional antibiotics like cloxacillin, ampicillin, and oxytetracycline, C.hypocistis extracts were less active in absolute terms, but displayed a wider spectrum (notably, cloxacillin and ampicillin were inactive against methicillin-resistant S. aureus). The ethanolic extract of C. hypocistis was found to be particularly rich in polyphenols, in most part hydrolysable tannins. The antioxidant activity of extracts, tested with several methodologies, resulted to be particularly high in the case of ethanolic extracts, in accordance with the composition in phenolics. In detail, ethanol extracts presented about a twofold higher activity than the water sample when tested through the oxygen radical absorbance capacity-pyrogallol red (ORAC-PYR) assay. Cytotoxicity analysis against the B16F10 melanoma cell line showed that both extracts have not significant cytotoxic effect, even at the highest dose (1000 µg/mL). Tests showed that ethanolic extracts also had the greatest tyrosinase inhibition activity, indicating that C. hypocistis-derived substances could find application in food formulations as anti-browning agents.ConclusionsOverall, these results point to the need of further studies on C. hypocistis extracts, aimed at isolating and fully characterizing its biologically active compounds.

Cytotoxic Activity and Antiproliferative Effects of Crude Skin Secretion from Physalaemus nattereri (Anura: Leptodactylidae) on in vitro Melanoma Cells.

Anuran secretions are rich sources of bioactive molecules, including antimicrobial and antitumoral compounds. The aims of this study were to investigate the therapeutic potential of Physalaemus nattereri skin secretion against skin cancer cells, and to assess its cytotoxic action mechanisms on the murine melanoma cell line B16F10. Our results demonstrated that the crude secretion reduced the viability of B16F10 cells, causing changes in cell morphology (e.g., round shape and structure shrinkage), reduction in mitochondrial membrane potential, increase in phosphatidylserine exposure, and cell cycle arrest in S-phase. Together, these changes suggest that tumor cells die by apoptosis. This skin secretion was also subjected to chromatographic fractioning using RP-HPLC, and eluted fractions were assayed for antiproliferative and antibacterial activities. Three active fractions showed molecular mass components in a range compatible with peptides. Although the specific mechanisms causing the reduced cell viability and cytotoxicity after the treatment with crude secretion are still unknown, it may be considered that molecules, such as the peptides found in the secretion, are effective against B16F10 tumor cells. Considering the growing need for new anticancer drugs, data presented in this study strongly reinforce the validity of P. nattereri crude secretion as a rich source of new anticancer molecules.

Abstract A52: Radiation and dual PD-L1 and CTLA4 checkpoint blockade non-redundantly improves tumor resistance, response, and immunity

Abstract Background: Immune checkpoint inhibitors such as CTLA4 and PD-1 result in impressive clinical responses, but optimal results will require combination therapy. This raises fundamental questions about non-redundancy and mechanisms of resistance. Emerging data indicate that combining immune checkpoint inhibitors with radiation (RT) may hold promise. We therefore evaluated this combination for metastatic melanoma using parallel studies in mice and humans. Methods: In a phase I clinical trial with 19 patients with multiple melanoma metastases, a single index lesion was irradiated with hypofractionated RT, delivered over two or three fractions, followed by four cycles of the anti-CTLA4 antibody ipilimumab. We reproduced this therapy in mice using the melanoma cell line B16-F10. For this, each flank of C57BL/6 mice was implanted with tumors to model multiple metastases. Mice received anti-CTLA4 (on days 5, 8, and 11), irradiation of one tumor using an image-guided micro-irradiator (20 Gy x 1 on day 8), or both treatments. Mechanistic studies were performed on material obtained from patients and mice at baseline and thereafter. Results: Overall, treatment in the phase I study was well tolerated and toxicity was similar to that reported for anti-CTLA4. Major tumor regressions were observed in a subset of patients with metastatic melanoma treated with anti-CTLA4 + RT. In mice, although combined treatment enhanced the CD8 T cell to Treg ratios and improved responses in irradiated and unirradiated tumors, resistance was common. Genome-wide and unbiased analyses revealed that resistant tumors have increased PD-L1, interferon-stimulated genes, and exhausted T cells that depress the CD8/Treg ratio. Patients and mice with high PD-L1 tumors that were treated with RT + anti-CTLA4 poorly reinvigorated exhausted T cells, did not respond, and rapidly progressed. In mice, adding anti-PD-L1/PD-1 to RT + anti-CTLA4 reinvigorated exhausted T cells, leading to complete responses and immunity across multiple cancer types. The extent of T cell exhaustion/reinvigoration predicts response and can be assessed through peripheral blood. Conclusions: Resistance to RT + anti-CTLA4 results from depression in the CD8/Treg ratio due to elevated tumor PD-L1 and persistent T cell exhaustion. Both clinical and pre-clinical data suggest that the combination of RT, anti-CTLA4, and anti-PDL1 is a rational and non-redundant approach to improving resistance, response, and immunity to melanoma and other cancer types. Citation Format: Andrew J. Rech, Christina Twyman, Amit Maity, Ramesh Rengan, Kristen E. Pauken, Erietta Stelekati, Bihui Xu, Hannah Dada, Pamela M. Odorizzi, Ramin D. Herati, Kathleen D. Mansfield, Dana Patsch, Ravi Amaravadi, Lynn Schuchter, Hemant Ishwaran, Rosemarie Mick, Daniel Pryma, George Xu, Michael Feldman, Tara C. Gangadhar, Stephen Hahn, E. John Wherry, Andy J. Minn, Robert H. Vonderheide. Radiation and dual PD-L1 and CTLA4 checkpoint blockade non-redundantly improves tumor resistance, response, and immunity. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A52.