B16-BL6 Melanoma Cells Research Articles

Pivotal role of oxidative stress in tumor metastasis under diabetic conditions in mice

Diabetic patients are reported to have a high incidence and mortality of cancer, but little is known about the linkage. In this study, we investigated whether high oxidative stress is involved in the acceleration of tumor metastasis in diabetic mice. Murine melanoma B16-BL6 cells stably labeled with firefly luciferase (B16-BL6/Luc) were inoculated into the tail vein of streptozotocin (STZ)-treated or untreated mice. A luciferase assay demonstrated that tumor cells were present largely in the lung of untreated mice, whereas large numbers of tumor cells were detected in both the lung and liver of STZ-treated mice. Repeated injections of polyethylene glycol-conjugated catalase (PEG-catalase), a long-circulating derivative, reduced the elevated fasting blood glucose levels and plasma lipoperoxide levels of STZ-treated mice, but had no significant effects on these parameters in untreated mice. In addition, the injections significantly reduced the number of tumor cells in the lung and liver in both untreated and STZ-treated mice. Culture of B16-BL6/Luc cells in medium containing over 45mg/dl glucose hardly affected the proliferation of the cells, whereas the addition of plasma of STZ-treated mice to the medium significantly increased the number of cells. Plasma samples of STZ-treated mice receiving PEG-catalase exhibited no such effect on proliferation. These findings indicate that a hyperglycemia-induced increase in oxidative stress is involved in the acceleration of tumor metastasis, and the removal of systemic hydrogen peroxide by PEG-catalase can inhibit the progression of diabetic conditions and tumor metastasis in diabetes.

Abstract 4716: Chronic alcohol consumption activates the LiTAF pathway in melanoma-bearing mice.

Abstract We previously reported that chronic alcohol consumption increases myeloid-derived suppressor cells (MDSC) and compromises melanoma-specific CD8+ T cells in B16BL6 melanoma-bearing mice; however, the underlying mechanism has not been elucidated. Activation of LPS-induced TNF-α factor (LiTAF) gene leads to up-regulation in the expression of various cytokines, chemokines and growth factors such as TNF-α, IL-1β, and VEGF, which are inducers of MDSC. Alcohol consumption is associated with the leakage of LPS into the blood. However, it is not known whether alcohol consumption modulates the expression of LiTAF. Herein, we examined the effects of alcohol and subcutaneous inoculation of melanoma in mice on the expression of LiTAF and its downstream cytokines, chemokines and growth factors. Female C57BL/6 mice were given 20% (w/v) alcohol for three to six months followed by subcutaneous inoculation of 2x105 B16BL6 melanoma cells. Two weeks after tumor inoculation, the expression of LiTAF and related cytokine genes in the spleen were examined by RT-PCR. Chronic alcohol consumption dramatically up regulated the expression of LiTAF in the spleen of melanoma-bearing mice; however, expression was almost undetectable in the spleen of corresponding water-drinking mice. The expression of IL-1β, IL-10, TNF-α, CCL5, and VEGF-β was also significantly up regulated by alcohol. These results indicate that chronic alcohol consumption activates the LiTAF pathway and up regulates the expression of the downstream cytokines, chemokines and growth factors. The up regulation of these cytokines could contribute to the induction of MDSC and inhibition on CD8+ T cells. (Supported by funds received from the Summer Undergraduate Research Fellowship (SURF) Program, from NIH grants R01 AA07193 and K05AA017149, and from funds provided for medical and biological research by the State of Washington Initiative Measure No. 171). Citation Format: Andrew Marron, Hui Zhang, Zhaohui Zhu, Gary G. Meadows. Chronic alcohol consumption activates the LiTAF pathway in melanoma-bearing mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4716. doi:10.1158/1538-7445.AM2013-4716

Visualization and in vivo tracking of the exosomes of murine melanoma B16-BL6 cells in mice after intravenous injection

The development of exosomes as delivery vehicles requires understanding how and where exogenously administered exosomes are distributed in vivo. In the present study, we designed a fusion protein consisting of Gaussia luciferase and a truncated lactadherin, gLuc-lactadherin, and constructed a plasmid expressing the fusion protein. B16-BL6 murine melanoma cells were transfected with the plasmid, and exosomes released from the cells were collected by ultracentrifugation. Strong luciferase activity was detected in the fraction containing exosomes, indicating their efficient labeling with gLuc-lactadherin. Then, the labeled B16-BL6 exosomes were intravenously injected into mice, and their tissue distribution was evaluated. Pharmacokinetic analysis of the exosome blood concentration–time profile revealed that B16-BL6 exosomes disappeared very quickly from the blood circulation with a half-life of approximately 2min. Little luciferase activity was detected in the serum at 4h after exosome injection, suggesting rapid clearance of B16-BL6 exosomes in vivo. Moreover, sequential in vivo imaging revealed that the B16-BL6 exosome-derived signals distributed first to the liver and then to the lungs. These results indicate that gLuc-lactadherin labeling is useful for tracing exosomes in vivo and that B16-BL6 exosomes are rapidly cleared from the blood circulation after systemic administration.

효소(Protopectinase) 처리한 대두가 세포내 멜라닌 생성에 미치는 영향

본 연구에서는 효소처리 한 대두추출물의 항산화 및 미백효과를 확인하기 위하여 DPPH 및 hydroxyl radical 포착활성을 측정하였고, 멜라닌 생성의 첫 단계인 tyrosinase 활성, mouse melanoma B16BL6 세포 생존율 및 TRP-1, TRP-2 발현 저해활성을 측정하였다. 또한 기존의 기계적 마쇄 가공한 대두추출물과 효소처리 가공한 대두추출물의 항산화 및 미백효과를 비교 조사하였다. 효소처리 한 대두추출물의 DPPH radical 소거능과 hydroxyl radical 소거능이 마쇄처리 한 추출물보다 높았으며, 특히 효소처리군이 마쇄군에 비하여 20% 이상 높은 항산화력을 나타내었다. 또한 마쇄군에 비하여 효소처리군이 tyrosinase와 TRP-1, TRP-2의 더 높은 활성 억제능을 보였다. 이는 대두가 B16BL6 melanoma 세포의 tyrosinase 단백질의 활성을 저해시킴으로써 멜라닌 생성을 억제하는데 효과적임을 의미하는 동시에 효소처리에 의하여 대두의 영양소의 손실이 더 적었음을 나타낸다. 결론적으로 효소처리에 의한 대두 추출물은 항산화 활성과 미백 효과가 우수하여 기능성 화장품의 천연 소재로서 활용가능성이 매우 높은 것으로 사료된다.

Codelivery of zoledronic acid and double-stranded RNA from core-shell nanoparticles

BackgroundZoledronic acid, an inhibitor of osteoclast-mediated bone resorption, has been shown to have both direct and indirect antitumor activity. However, its use in extraskeletal malignancy is limited due to rapid uptake and accumulation within bone. Polyinosinic acid-polycytidylic acid [poly (I:C)] is a synthetic double-stranded RNA with direct antitumor cytotoxicity if it can be delivered to tumor cells intracellularly.MethodsCationic lipid-coated calcium phosphate nanoparticles (LCP) were developed to enable intracellular codelivery of zoledronic acid and poly (I:C). LCP codelivering zoledronic acid and poly (I:C) were prepared using an ethanol injection method. Briefly, the ethanol solution of lipids was rapidly injected into newly formed calcium phosphate crystals containing poly (I:C) and zoledronic acid, and the mixture was then sonicated briefly to form LCP. The LCP were fully characterized for mean diameter size and zeta potential, efficiency in loading zoledronic acid, cytotoxic effect in a B16BL6 melanoma cell line in vitro, and antitumor effect in B16BL6 melanoma-bearing mice.ResultsLCP with a mean diameter around 200 nm and a narrow size distribution (polydispersity index 0.17) and high zoledronic acid encapsulation efficiency (94%) were achieved. LCP loaded with zoledronic acid and poly (I:C) had significantly greater antitumor activity than the free drugs in the B16BL6 melanoma cell line (P < 0.05). Furthermore, codelivery of zoledronic acid and poly (I:C) by LCP had higher cytotoxicity than delivering poly (I:C) alone by LCP (P < 0.05), indicating a synergism between zoledronic acid and poly (I:C). Finally, the antitumor study in melanoma-bearing mice also demonstrated synergism between zoledronic acid and poly (I:C) codelivered by LCP.ConclusionCationic lipid-coated calcium phosphate nanoparticles constructed for codelivery of zoledronic acid and double-stranded RNA poly (I:C) had better antitumor activity both in vitro and in vivo. Future preclinical development of LCP encapsulating zoledronic acid and poly (I:C) for the treatment of human cancer is under way.

Anti-metastatic effect of polysaccharide isolated from Colocasia esculenta is exerted through immunostimulation

In the present study, an edible corm of the plant Colocasia esculenta, commonly known as Taro was extracted with cold water (4˚C). Finally, 10.44 g (1.04%) of the crude polysaccharide (Taro-0) was obtained from Taro. The purified active compound (Taro-4-I) was isolated using DEAE-Sepharose FF and Sephadex G-100. The anti-complementary activity of Taro-4-I (57.3±4.5%) was similar to that of polysaccharide K (used as the positive control). The molecular weight of Taro-4-I was 200 kDa and it was a polysaccharide composed of 64.4% neutral sugars and 35.6% uronic acid. Taro-4-I activated the complement system through the classical and alternative pathways. The treatment of peritoneal macrophages with Taro-4-I significantly increased the production of interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) in a dose-dependent manner. However, IL-12 production showed maximal activity at 56 µg/ml and subsequently decreased. Splenocytes obtained from mice which were administered Taro-4-I intravenously showed a higher toxicity to Yac-1 cells compared to those obtained from untreated mice in a effector‑to‑target (E/T) ratio-dependent manner. The group treated with 50 µg/ml Taro-4-I showed a significantly increased toxicity to Yac-1 cells compared to the group treated with 500 µg/ml Taro-4-I. The administration of Taro-4-I significantly inhibited the lung metastasis of B16BL6 melanoma cells. However, the group treated with 50 µg/mouse Taro-4-I had a significantly lower number of tumors compared to the group injected with 500 µg/mouse Taro-4-I.

Naturally Occurring Nanoparticles from Arthrobotrys oligospora as a Potential Immunostimulatory and Antitumor Agent

AbstractArthrobotrys oligospora, a representative flesh eater in the fungal kingdom, is a potential source for natural‐based biomaterials due to the presence of specialized 3D adhesive traps that can capture, penetrate, and digest free‐living nematodes in diverse environments. The purpose of this study is to discover novel nanoparticles that occur naturally in A. oligospora and to exploit its potential biomedical applications. A new culture method, fungal sitting drop culture method, is established in order to monitor the growth of A. oligospora in situ, and observe the nanoparticle production without interfering or contamination from the solid media. Abundant spherical nanoparticles secreted from the fungus are first revealed by scanning electron microscopy and atomic force microscopy. They have an average size of 360–370 nm, with a zeta potential of –33 mV at pH 6.0. Further analyses reveal that there is ≈28 μg of glycosaminoglycan and ≈550 μg of protein per mg of nanoparticles. Interestingly, the nanoparticles significantly induce TNF‐α secretion in RAW264.7mouse macrophages, indicating a potential immunostimulatory effect. The nanoparticles themselves are also found slightly cytotoxic to mouse melanoma B16BL6 and human lung cancer A549 cells, and show a synergistic cytotoxic effect upon conjugation with doxorubicin against both cells. This study proposes a new approach for producing novel organic nanoparticles secreted from microorganisms under controlled conditions. The findings here also highlight the potential roles of the naturally occurring nanoparticles from A. oligospora as an immunostimulatory and antitumor agent for cancer immunochemotherapy.

Ror2-Src signaling in metastasis of mouse melanoma cells is inhibited by NRAGE

The receptor tyrosine kinase (RTK) Ror2 plays important roles in developmental morphogenesis and mediates the filopodia formation in Wnt5a-induced cell migration. However, the function of Ror2 in noncanonical Wnt signaling resulting in cancer metastasis is largely unknown. Here, we show that Ror2 expression is higher in the highly metastatic murine B16-BL6 melanoma cells than in the low metastatic variant B16 cells. Overexpression of Ror2 increases the metastasis ability of B16 cells, and knockdown of Ror2 reduces the migration ability of B16-BL6 cells. Furthermore, the inhibition of Src kinase activity is critical for the Ror2-mediated cell migration upon Wnt5a treatment. The C-terminus of Ror2, which is deleted in brachydactyly type B (BDB), is essential for the mutual interaction with the SH1 domain of Src. Intriguingly, the Neurotrophin receptor-interacting MAGE homologue (NRAGE), which, as we previously reported, can remodel the cellular skeleton and inhibit cell-cell adhesion and metastasis of melanoma and pancreatic cancer, sharply blocks the interaction between Src and Ror2 and inhibits Ror2-mediated B16 cell migration by decreasing the activity of Src and focal adhesion kinase (FAK). Our data show that Ror2 is a potential factor in the tumorigenesis and metastasis in a Src-dependent manner that is negatively regulated by NRAGE.

Early detection and longitudinal monitoring of experimental primary and disseminated melanoma using [18F]ICF01006, a highly promising melanoma PET tracer

Here, we report a new and rapid radiosynthesis of (18)F-N-[2-(diethylamino)ethyl]-6-fluoro-pyridine-3-carboxamide ([(18)F]ICF01006), a molecule with a high specificity for melanotic tissue, and its evaluation in a murine model for early specific detection of pigmented primary and disseminated melanoma. [(18)F]ICF01006 was synthesized using a new one-step bromine-for-fluorine nucleophilic heteroaromatic substitution. Melanoma models were induced by subcutaneous (primary tumour) or intravenous (lung colonies) injection of B16BL6 melanoma cells in C57BL/6J mice. The relevance and sensitivity of positron emission tomography (PET) imaging using [(18)F]ICF01006 were evaluated at different stages of tumoural growth and compared to (18)F-fluorodeoxyglucose ([(18)F]FDG). The fully automated radiosynthesis of [(18)F]ICF01006 led to a radiochemical yield of 61 % and a radiochemical purity >99 % (specific activity 70-80 GBq/μmol; total synthesis time 42 min). Tumours were visualized before they were palpable as early as 1 h post-injection with [(18)F]ICF01006 tumoural uptake of 1.64 ± 0.57, 3.40 ± 1.47 and 11.44 ± 2.67 percentage of injected dose per gram of tissue (%ID/g) at days 3, 5 and 14, respectively. [(18)F]ICF01006 PET imaging also allowed detection of melanoma pulmonary colonies from day 9 after tumour cell inoculation, with a lung radiotracer accumulation correlated with melanoma invasion. At day 21, radioactivity uptake in lungs reached a value of 5.23 ± 2.08 %ID/g (versus 0.41 ± 0.90 %ID/g in control mice). In the two models, comparison with [(18)F]FDG showed that both radiotracers were able to detect melanoma lesions, but [(18)F]ICF01006 was superior in terms of contrast and specificity. Our promising results provide further preclinical data, reinforcing the excellent potential of [(18)F]ICF01006 PET imaging for early specific diagnosis and follow-up of melanin-positive disseminated melanoma.

Preparation of Curcumin-Loaded Liposomes and Evaluation of Their Skin Permeation and Pharmacodynamics

This study aimed to investigate the in vitro skin permeation and in vivo antineoplastic effect of curcumin by using liposomes as the transdermal drug-delivery system. Soybean phospholipids (SPC), egg yolk phospholipids (EPC), and hydrogenated soybean phospholipids (HSPC) were selected for the preparation of different kinds of phospholipids composed of curcumin-loaded liposomes: C-SPC-L (curcumin-loaded SPC liposomes), C-EPC-L (curcumin-loaded EPC liposomes), and C-HSPC-L (curcumin-loaded HSPC liposomes). The physical properties of different lipsomes were investigated as follows: photon correlation spectroscopy revealed that the average particle sizes of the three types of curcumin-loaded liposomes were 82.37 ± 2.19 nm (C-SPC-L), 83.13 ± 4.89 nm (C-EPC-L), and 92.42 ± 4.56 nm (C-HSPC-L), respectively. The encapsulation efficiency values were found to be 82.32 ± 3.91%, 81.59 ± 2.38%, and 80.77 ± 4.12%, respectively. An in vitro skin penetration study indicated that C-SPC-L most significantly promoted drug permeation and deposition followed by C-EPC-L, C-HSPC-L, and curcumin solution. Moreover, C-SPC-L displayed the greatest ability of all loaded liposomes to inhibit the growth of B16BL6 melanoma cells. Therefore, the C-SPC-L were chosen for further pharmacodynamic evaluation. A significant effect on antimelanoma activity was observed with C-SPC-L, as compared to treatment with curcumin solution in vivo. These results suggest that C-SPC-L would be a promising transdermal carrier for curcumin in cancer treatment.

SBF-1, a synthetic steroidal glycoside, inhibits melanoma growth and metastasis through blocking interaction between PDK1 and AKT3

In the present study, we demonstrate that SBF-1, a synthetic steroidal glycoside, has a strong antitumor activity against melanoma cells in vitro and in vivo. SBF-1 induced cell cycle arrest with a reduced expression of various cell cycle related proteins in B16BL6 melanoma cells without causing apoptosis. SBF-1 dramatically inhibited kinase activity of 3-phosphoinositide dependent protein kinase 1 (PDK1) and thus down-regulated phosphorylation of protein kinase B (AKT). Among three known isoforms of AKT, PDK1 only interacted with AKT3 in B16BL6 melanoma cells, and SBF-1 almost completely blocked this interaction. In addition, adhesion to fibronectin and expression of integrin α4 were significantly reduced in a concentration-dependent manner. Knockdown of AKT3 resulted in the decrease in integrin α4 expression and cell adhesion. Moreover, SBF-1 inhibited the growth of melanoma xenografts and down-regulated the phosphorylation of AKT in vivo. In a mouse model of spontaneous metastasis, SBF-1 at very low doses of 1 and 3μg/kg enormously inhibited melanoma metastasis into draining popliteal lymph nodes. Taken together, this study shows a small molecular compound SBF-1 with a very strong anti-melanoma activity both in vitro and in vivo. Its mechanism underlying such antitumor effect is related to the blockage of the interaction between PDK1 and AKT3.

Formulation, characterization, and evaluation of in vitro skin permeation and in vivo pharmacodynamics of surface-charged tripterine-loaded nanostructured lipid carriers

BackgroundNanostructured lipid carriers (NLCs) are attractive materials for topical drug delivery, and in a previous study, we demonstrated that NLCs loaded with tripterine enhance its deposition. However, the surface charge of nanoparticles influences percutaneous drug penetration. Therefore, we aimed to evaluate the influence of the surface charge of NLCs on in vitro skin permeation and in vivo pharmacodynamics of tripterine and optimize tripterine- loaded NLCs for the treatment of skin diseases.MethodsDifferent solid and liquid matrices were selected to prepare cationic, anionic, and neutral NLCs by the solvent evaporation method. The in vitro studies were evaluated by using Franz diffusion cells. The effect of surface-charged NLCs on cellular uptake was appraised across HaCaT and B16BL6 cells. The in vitro and in vivo anticancer activity of surface-charged NLCs was evaluated in B16BL6 cells and melanoma-bearing mice, respectively.ResultsThe average particle sizes of the cationic, anionic, and neutral NLCs were 90.2 ± 9.7, 87.8 ± 7.4, and 84.5 ± 10.2 nm, respectively; their encapsulation efficiencies were 64.3% ± 5.1%, 67.8% ± 4.4%, and 72.5% ± 4.9%, respectively. In vitro studies showed delayed tripterine release, and the order of skin permeation was cationic NLCs > anionic NLCs > neutral NLCs. Further, in vitro cytotoxicity studies showed that the cationic NLCs had the highest (P < 0.05) inhibition ratio in B16BL6 (melanoma) cells. Moreover, in vivo pharmacodynamic experiments in melanoma-bearing mice indicated that the cationic NLCs had significantly higher (P < 0.05) antimelanoma efficacy than the anionic and neutral NLCs.ConclusionThe surface charge of NLCs has a great influence on the skin permeation and pharmacodynamics of tripterine. Cationic tripterine-loaded NLCs could enhance the percutaneous penetration and antimelanoma efficacy of tripterine and offer several advantages over tripterine alone. Therefore, they are promising carriers of tripterine for topical antimelanoma therapy.

ENHANCED BIOLOGICAL ACTIVITIES OF LAMINARIN DEGRADED BY GAMMA-RAY IRRADIATION

ABSTRACT In this study, we investigated the antioxidant and melanin synthesis inhibitory properties of gamma-irradiated laminarin. Antioxidant activities as measured by ferric-reducing antioxidant potential values and 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity were found to be greater in irradiated laminarin than in nonirradiated laminarin. However, nonirradiated and irradiated laminarins did not exhibit significant differences in β-carotene bleaching assays. Moreover, inhibitory activity against melanin synthesis in B16BL6 melanoma cells was increased by gamma-irradiated laminarin. The content of reducing sugars in laminarin was found to increase with increased irradiation doses. These results and the molecular weight measured by gel permeation chromatography indicate that laminarin is degraded to a low molecular weight form by the scission of glycosidic bonds. Overall, low molecular weight laminarin may have beneficial biological properties, and gamma irradiation may be an effective method of producing low molecular weight laminarin. PRACTICAL APPLICATIONS Laminarin is a type of storage polysaccharide derived from seaweed. Because of its indigestibility in the upper gastrointestinal tract, laminarin is considered as a dietary fiber. Other beneficial biological properties have been attributed to laminarin, but studies have shown these properties to be ineffective. Therefore, in this study, laminarin was degraded by gamma irradiation to enhance the effects of these biological properties. We observed that gamma-irradiated laminarin had higher antioxidative and melanin synthesis inhibitory activities than nonirradiated laminarin did. The results of this study suggest that laminarin may provide dietary benefits beyond its use as an effective dietary fiber.

Host-derived MMP-13 exhibits a protective role in lung metastasis of melanoma cells by local endostatin production

Background:Although matrix metalloproteinases (MMPs) are implicated in tumourigenesis and cancer progression, the role of MMP-13 in melanoma cell metastases is poorly understood.Methods:Lung metastases of mouse melanoma B16BL6 cells were analysed in MMP-13 knockout (KO) and wild-type (WT) mice after intravenous injection. The mRNA and protein expression of MMP-13 in lung tissues was analysed by RT–PCR, real-time PCR, immunoblotting and immunohistochemistry. The expression of SDF-1α, CXCR4 and endostatin, and effects of endostatin to cultured melanoma cells and lung metastases were also studied.Results:Lung metastases of B16BL6 cells were significantly higher by 2.5–5.7-fold in MMP-13 KO mice than in WT mice. The expression of MMP-13 in WT mouse lung tissue was stimulated on day 1 after intravenous injection of the melanoma cells and MMP-13 was immunolocalised to vascular endothelial cells in the lungs. Endostatin formation, but not degradation of SDF-1α, in the lung tissue was associated with reduced lung metastasis in WT mice. Endostatin significantly inhibited migration of B16BL6 cells in monolayer wounding assay and remarkably suppressed Matrigel invasion and transendothelial invasion of the cells. In addition, lung metastases of melanoma cells in MMP-13 KO mice were reduced by intraperitoneal administration of endostatin.Conclusion:Our results suggest that MMP-13 is overproduced by endothelial cells in the lungs with melanoma cells and has a protective role in lung metastasis by local generation of endostatin.

Knockdown of proteolipid protein 2 or focal adhesion kinase with an artificial microRNA reduces growth and metastasis of B16BL6 melanoma cells

Proteolipid protein 2 (PLP2) promotes the metastasis of B16F10 cells in an experimental metastasis model. However, the effect of PLP2 on spontaneous metastasis has yet to be demonstrated, and whether PLP2 may become a new therapeutic target for malignant tumors is as yet unknown. In this study, PLP2 or focal adhesion kinase (FAK) microRNA-based short hairpin RNAs (miRNAs) were used as target molecules to specifically reduce the expression of PLP2 or FAK in B16BL6 cells. In vitro, the knockdown of PLP2 or FAK significantly inhibited cell proliferation, adhesion, migration and invasion. In a spontaneous metastatic tumor model using a footpad injection, the knockdown of PLP2 or FAK markedly inhibited the proliferation of the primary tumor and prevented tumor cells from invading the popliteal lymph nodes. The results indicate that downregulation of PLP2 or FAK may improve outcomes of malignant tumor therapy.

Adipocytes Promote B16BL6 Melanoma Cell Invasion and the Epithelial-to-Mesenchymal Transition

Metastatic melanoma is one of the most deadly and evasive types of cancer. On average, cancer patients with metastatic melanoma survive only 6-9months after diagnosis. Epidemiological and animal studies suggest that obesity increases the metastatic ability of malignant melanoma, though the mechanism is not known. In the present studies, we assessed the ability of 3T3L1 adipocytes to modulate B16BL6 melanoma cell invasion and the Epithelial-to-Mesenchymal Transition (EMT). For this purpose, we induced the differentiation of 3T3L1 fibroblasts to adipocytes. Then, we collected the cell culture media from both fibroblasts and adipocytes and determined their effect on the invasive ability and EMT gene expression of B16BL6 melanoma cells. Results show that adipocyte media increased that ability of B16BL6 cells to invade. The higher invasive ability of B16BL6 melanoma cells was associated with increased expression of EMT genes such as Snai1, MMP9, Twist, and Vimentin. Additionally, the expression of the cell-to-cell adhesion protein E-cadherin and the metastasis suppressor gene Kiss1 were down-regulated in these B16BL6 cells. Also, adipocytes had high levels of the pro-inflammatory cytokine Interleukin 6 (IL-6). Treatment of B16BL6 cells with IL-6 elicited similar effects as the adipocyte media; IL-6 promoted the invasive ability of B16BL6 melanoma cells, increased the expression of Snai1, and decreased Kiss1 expression. IL-6 neutralization, however, did not have a visible effect on adipocyte media-induced invasion and snai1 staining. In summary, adipocytes may increase the invasive ability of B16BL6 melanoma cells by promoting EMT and decreasing the expression of genes such as E-cadherin and Kiss1.

Ascidian dermatan sulfates attenuate metastasis, inflammation and thrombosis by inhibition of P‐selectin

Cancer-associated thrombosis and enduring inflammation are strongly associated with cancer progression and metastasis. Heparin is the mostly clinically used anticoagulant/antithrombotic drug, and has recently been shown to exhibit antimetastatic and anti-inflammatory activities that are linked to inhibition of P-selectin and/or L-selectin. P-selectin-mediated platelet-tumor cell and tumor cell-endothelium interactions facilitate the initial steps of metastasis. The aim of the present study was to determine the capacity of dermatan sulfates to inhibit P-selectin and to test their potential to affect thrombosis, inflammation and metastasis in respective experimental mouse models. Two dermatan sulfates isolated from the ascidians Styela plicata and Phallusia nigra, composed of the same disaccharide core structure (IdoA2-GalNAc)(n) , but sulfated at carbon 4 or 6 of the GalNAc, respectively, have opposed heparin cofactor II (HCII) activities and are potent inhibitors of P-selectin. The ascidian dermatan sulfates effectively attenuated metastasis of both MC-38 colon carcinoma and B16-BL6 melanoma cells and the infiltration of inflammatory cells in a thioglycollate peritonitis mouse model. Moreover, both glycosaminoglycans reduced thrombus size in an FeCl(3) -induced arterial thrombosis model, irrespective of their HCII activities. The analysis of arterial thrombi demonstrated markedly reduced platelet deposition after dermatan sulfate treatment, suggesting that the glycosaminoglycan inhibited P-selectin and thereby the binding of activated platelets during thrombus formation. Collectively, these findings provide evidence that specific inhibition of P-selectin represents a potential therapeutic target in thrombosis, inflammation and metastasis, and that ascidian dermatan sulfates may serve as antiselectin agents.

Ob/ob serum promotes a mesenchymal cell phenotype in B16BL6 melanoma cells

In 2009, malignant melanoma was responsible for approximately 9,000 deaths in the US. These deaths are often associated with aggressive metastasis to secondary sites such as the lungs. Epidemiological and animal studies suggest that obesity is a risk factor for melanoma. Others have shown that B16BL6 melanoma cells metastasize more aggressively in obese ob/ob than in lean mice. However, the mechanism by which obesity promotes B16BL6 melanoma metastasis in ob/ob mice has not been identified. In the present study, we used serum obtained from control and ob/ob leptin-deficient obese mice to determine if obese serum increases the aggressive phenotype of melanoma cells. Results showed that ob/ob serum has higher levels of resistin, insulin, tPAI1, IL-6, TNF-α, and MCP-1 compared to control serum. We showed that ob/ob serum increases the invasive ability of B16BL6 melanomas. To further determine the mechanism by which ob/ob serum increases the invasive ability of melanomas, we determined the effect of ob/ob and control serum on genes associated with the epithelial-to-mesenchymal transition (EMT). Cancer cells with a mesenchymal phenotype have a higher metastatic ability. Snai1 and Twist are genes that are strongly associated with EMT and metastasis of melanomas. Our results showed that ob/ob serum increases the expression of Snai1 and Twist. Moreover, ob/ob serum increased matrix metalloproteast 9 (MMP9) activity and decreased the expression of E-cadherin and the metastasis suppressor gene Kiss1. In summary, results suggest that obesity may increase the metastatic ability of melanoma by promoting a mesenchymal cell phenotype.

짚신나물 물 추출물의 항산화 활성 및 미백효과에 관한 연구

This study was performed to assess the antioxidant activities and whitening effects of Agrimonia pilosa Ledeb on melanin synthesis. The whitening effects of Agrimonia pilosa Ledeb water extracts were examined by in vitro mushroom tyrosinase assay and B16BL6 melanoma cells. We assessed inhibitory effect of Agrimonia pilosa Ledeb water extract on expression of melanogenic enzyme proteins including tyrosinase, tyrosinase-related protein 1 (TRP-1) and tyrosinaserelated protein 2 (TRP-2) in B16BL6 cells. Inhibitory effect of Agrimonia pilosa Ledeb onto free radical generation was determined by measuring DPPH and hydroxyl radical scavenging activitie. Our results indicated that Agrimonia pilosa Ledeb water extract effectively inhibited free radical generation. In DPPH and hydroxy radical scavenging activity, Agrimonia pilosa Ledeb water extract had a potent anti-oxidant activity in a dose-dependent manner. They significantly inhibited tyrosinase activity in vitro and in B16BL6 melanoma cells. Also, Agrimonia pilosa Ledeb suppressed the expression of tyrosinase in B16BL6 melanoma cells. These results show that Agrimonia pilosa Ledeb inhibited melanin production on the melanogenesis. The underlying mechanism of Agrimonia pilosa Ledeb on whitening activity may be due to the inhibition of tyrosinase activity. We suggest that Agrimonia pilosa Ledeb may be useful as new natural active ingredients for antioxidant and whitening cosmetics.

Resveratrol modulates the malignant properties of cutaneous melanoma through changes in the activation and attenuation of the antiapoptotic protooncogenic protein Akt/PKB

Resveratrol, a nontoxic natural product, exhibits multifaceted biological effects including antimutagenic and anticancer properties. We examined the effect of resveratrol on the expression and activation of Akt/protein kinase B and its impact on melanoma cell migration and invasiveness. We also explored the use of resveratrol as an antimalignant treatment option against skin melanoma in mouse models of the disease. Akt expression and activity were determined by a combination of real-time PCR and western blot analysis. Cell lines stably expressing Akt or a dominant negative variant were used to further establish the role of Akt during the response to resveratrol. Wound healing and transwell assays were used as in-vitro correlates of melanoma cell migration and invasiveness. The efficacy of resveratrol in the treatment of melanoma was assessed in two syngeneic mouse models. Resveratrol downregulated and inactivated Akt in B16F10 and B16BL6 melanoma cells. Resveratrol also inhibited the migratory and invasive properties of these highly malignant cells. The reduction of cell migration and invasion, however, was reversed in cell lines overexpressing Akt or after cotreatment with pharmacological inhibitors that blocked Akt degradation. Dominant-negative Akt cells were more sensitive to resveratrol and had diminished migratory properties. Oral treatment with resveratrol reduced primary tumor volume, Akt expression, and the propensity for metastasis in syngeneic mouse models of melanoma. These results suggest that resveratrol can reduce the malignant properties of highly invasive melanoma cells by inactivating Akt. The nontoxic targeting of Akt by resveratrol makes it an attractive treatment option for melanoma.