Macrophages, IL-10, and nitric oxide increase, induced by hyperglycemic conditions, impact the development of murine melanoma B16F10-Nex2.

Epidemiological studies show a strong correlation between diabetes and the increased risk of developing different cancers, including melanoma. In the present study, we investigated the impact of a streptozotocin (STZ)-induced hyperglycemic environment on B16F10-Nex2 murine melanoma development. Hyperglycemic male C57Bl/6 mice showed increased subcutaneous tumor development, partially inhibited by metformin. Tumors showed increased infiltrating macrophages, and augmented IL-10 and nitric oxide (NO) concentrations. In vivo neutralization of IL-10, NO synthase inhibition, and depletion of macrophages reduced tumor development. STZ-treated TLR4 KO animals showed delayed tumor development; the transfer of hyperglycemic C57Bl/6 macrophages to TLR4 KO reversed this effect. Increased concentrations of IL-10 present in tumor homogenates of hyperglycemic mice induced a higher number of pre-angiogenic structures in vitro, and B16F10-Nex2 cells incubated with different glucose concentrations in vitro produced increased levels of IL-10. In summary, our findings show that a hyperglycemic environment stimulates murine melanoma B16F10-Nex2 primary tumor growth, and this effect is dependent on tumor cell stimulation, increased numbers of macrophages, and augmented IL-10 and NO concentrations. These findings show the involvement of tumor cells and other components of the tumor microenvironment in the development of subcutaneous melanoma under hyperglycemic conditions, defining novel targets for melanoma control in diabetic patients.

Bradykinin promotes murine melanoma cell migration and invasion through endogenous production of superoxide and nitric oxide.

Spatial confinement and temporal regulation of signaling by nitric oxide (NO) and reactive oxygen species (ROS) occurs in cancer cells. Signaling mediated by NO and ROS was investigated in two sub clones of the murine melanoma B16F10-Nex2 cell line, Nex10C and Nex8H treated or not with bradykinin (BK). The sub clone Nex10C, similar to primary site cells, has a low capacity for colonizing the lungs, whereas the sub clone Nex8H, similar to metastatic cells, corresponds to a highly invasive melanoma. BK-treated Nex10C cells exhibited a transient increase in NO and an inhibition in basal O2- levels. Inhibition of endogenous NO production by l-NAME resulted in detectable levels of O2-. l-NAME promoted Rac1 activation and enhanced Rac1-PI3K association. l-NAME in the absence of BK resulted in Nex10C cell migration and invasion, suggesting that NO is a negative regulator of O2- mediated cell migration and cell invasion. BK-treated Nex8H cells sustained endogenous NO production through the activation of NOS3. NO activated Rac1 and promoted Rac1-PI3K association. NO stimulated cell migration and cell invasion through a signaling axis involving Ras, Rac1 and PI3K. In conclusion, a role for O2- and NO as positive regulators of Rac1-PI3K signaling associated with cell migration and cell invasion is proposed respectively for Nex10C and Nex8H murine melanoma cells.

Antiangiogenic activity of the penicillin derivative TAP7f in melanoma.

Previously , we demonstrated that the non-antibiotic penicillin derivative TAP7f inhibited melanoma metastasis in vitro and in vivo through the downregulation of ß-catenin and integrin αVß3. As angiogenesis is required for tumor growth and metastasis, we decided to explore the possible antiangiogenic effect of TAP7f. We found that TAP7f inhibited proliferation, migration, tube formation, and actin cytoskeleton organization of human endothelial cells. In a gel plug assay, an in vivo model for angiogenesis, TAP7f also blocked vascular formation induced by fibroblast growth factor 2. Furthermore, when murine B16-F10 melanoma cells pre-treated with TAP7f were injected intradermally in mice, we observed a decrease in the number and thickness of the capillaries surrounding the tumor. Additionally, TAP7f downregulated vascular endothelial growth factor (VEGF) and platelet-derived growth factor-B (PDGF-B) expression in B16-F10 cells and VEGF receptor expression in HMEC-1 endothelial cells. When the antitumor effect of TAP7f was studied in C57BL/6 J mice challenged with B16-F10 melanoma cells, a significant reduction of tumor growth was observed. Furthermore, a decreased expression of VEGF, PDGF-B, and the endothelial cell marker CD34 was observed in tumors from TAP7f-treated mice. Together, our results suggest that the antiangiogenic activity of TAP7f contributes to its antitumor and antimetastatic action and positions this penicillin derivative as an alternative or complementary agent for the treatment of melanoma. KEY MESSAGES: • TAP7f inhibits proliferation, migration, tube formation, and actin cytoskeleton organization of endothelial cells. • TAP7f downregulates VEGF receptor expression in endothelial cells. • TAP7f downregulates VEGF and PDGF expression in melanoma cells. • TAP7f inhibits angiogenesis in vivo.

Bacitracin is a non-competitive inhibitor of porcine M1 family neutral and glutamyl aminopeptidases.

Membrane alanyl and glutamyl aminopeptidases (APN and APA, respectively) are established targets for the development of biomedical tools in human pathologies. APN overexpression correlates with the progression of tumours, including melanoma. Bacitracin, widely used as a topical antibiotic, inhibits subtilisin-like serine peptidases and disulphide isomerases. In the present contribution, we demonstrate that bacitracin is a non-competitive α = 1 and α < 1 inhibitor of porcine kidney APN and APA, respectively, with Ki values in the micromolar range. To test a potential application of this result, we assayed the effect of bacitracin on murine melanoma MB16F10 cell line viability. We demonstrated the cell line expresses an APN-like activity inhibited by bacitracin and bestatin. Additionally, we identified a cytotoxic effect of bacitracin. Further experiments are required to understand in depth the mechanisms of action of bacitracin on melanoma cells. They will clarify the therapeutic potential of bacitracin for melanoma treatment.

In Vitro Antitumor Potential of Sulfated Polysaccharides from Seaweed Caulerpa cupressoides var. flabellata.

Seaweeds are important source of bioactive compounds, including sulfated polysaccharides (SP). Because of their structural heterogeneity, these compounds are promising sources of anticancer compounds. SP from brown and red seaweeds have shown antimelanoma activity in different in vitro and in vivo models. However, SP from green seaweed are still poorly evaluated. Therefore, SP were extracted from the green alga Caulerpa cupressoides var. flabellata, and their antiproliferative, anti-migratory, and inhibitory effect on melanin production on B16-F10 melanoma cells was evaluated. Cell assays, including flow cytometry, demonstrated that SP (100-1000 µg mL-1) are non-cytotoxic, do not induce apoptosis or necrosis, and do not interfere with cell cycle. However, SP (1000 µg mL-1) were found to significantly inhibit cell colony formation (80-90%), cell migration (40-75%), and melanin production (~ 20%). In summary, these results showed that SP inhibited important melanoma development events without cytotoxicity effects, suggesting that C. cupressoides may be an important source of SP with antitumor properties.

A Penicillin Derivative Exerts an Anti-Metastatic Activity in Melanoma Cells Through the Downregulation of Integrin αvß3 and Wnt/ß-Catenin Pathway.

The synthetic triazolylpeptidyl penicillin derivative, named TAP7f, has been previously characterized as an effective antitumor agent in vitro and in vivo against B16-F0 melanoma cells. In this study, we investigated the anti-metastatic potential of this compound on highly metastatic murine B16-F10 and human A375 melanoma cells. We found that TAP7f inhibited cell adhesion, migration and invasion in a dose-dependent manner. Additionally, we demonstrated that TAP7f downregulated integrin αvß3 expression and Wnt/ß-catenin pathway, a signaling cascade commonly related to tumor invasion and metastasis. Thus, TAP7f reduced both the enzymatic activity and the expression levels of matrix-metalloproteinases-2 and -9 in a time dependent manner. Moreover, TAP7f inhibited the expression of the transcription factor Snail and the mesenchymal markers vimentin, and N-cadherin, and up-regulated the expression of the epithelial marker E-cadherin, suggesting that the penicillin derivative affects epithelial-mesenchymal transition. Results obtained in vitro were supported by those obtained in a B16-F10-bearing mice metastatic model, that showed a significant TAP7f inhibition of lung metastasis. These findings suggest the potential of TAP7f as a chemotherapeutic agent for the treatment of metastatic melanoma.

The Influence of Some Axial Ligands on Ruthenium-Phthalocyanine Complexes: Chemical, Photochemical, and Photobiological Properties.

This work presents a new procedure to synthesize ruthenium-phthalocyanine complexes and uses diverse spectroscopic techniques to characterize trans-[RuCl(Pc)DMSO] (I) (Pc = phthalocyanine) and trans-[Ru(Pc)(4-ampy)2] (II) (4-ampy = 4-aminopyridine). The triplet excited-state lifetimes of (I) measured by nanosecond transient absorption showed that two processes occurred, one around 15 ns and the other around 3.8 µs. Axial ligands seemed to affect the singlet oxygen quantum yield. Yields of 0.62 and 0.14 were achieved for (I) and (II), respectively. The lower value obtained for (II) probably resulted from secondary reactions of singlet oxygen in the presence of the ruthenium complex. We also investigate how axial ligands in the ruthenium-phthalocyanine complexes affect their photo-bioactivity in B16F10 murine melanoma cells. In the case of (I) at 1 µmol/L, photosensitization with 5.95 J/cm2 provided B16F10 cell viability of 6%, showing that (I) was more active than (II) at the same concentration. Furthermore, (II) was detected intracellularly in B16F10 cell extracts. The behavior of the evaluated ruthenium-phthalocyanine complexes point to the potential use of (I) as a metal-based drug in clinical therapy. Changes in axial ligands can modulate the photosensitizer activity of the ruthenium phthalocyanine complexes.

Antitumor effect of depsidones from lichens on tumor cell lines and experimental murine melanoma

Abstract Lichens have exhibited numerous biological activities, including growth inhibition of tumor cells. This study evaluated the antiproliferative activity of hypostictic and salazinic acids against tumor cell lines (B16-F10, PC-03, MCF7, HT-29, HEP-G2, K562 and 786-0) by the SRB assay in vitro and antitumor activity in experimental murine melanoma in vivo. Activation of caspase-3 was quantified by flow cytometry. The murine experimental melanoma model B16-F10 was used in BALB/c mice for evaluation of antitumor activity. Hypostictic acid showed significant antiproliferative activity in K562 cells (GI50 2.20 µM), B16-F10 (GI50 13.78 µM) and 786-0 (GI50 14.24 µM), whereas salazinic acid was more active against K562 cells (GI50 64.36 µM), HT-29 (GI50 67.91 µM) and B16-F10 (GI5078.64 µM). Quantification of capase-3 revealed that the test compounds did not increase the expression of that enzyme. In the in vivo antitumor evaluation in B16-F10 melanoma, the isolated compounds inhibited tumor growth in relation to weight and volume. Hypostictic acid (16.7 mg/kg) inhibited 72% and salazinic acid 88% of tumor volume (p < 0.05). The results indicated that, both in the in vitro and in vivo models, the compounds evaluated showed antiproliferative and antitumor activities.

The biochemical characterization, stabilization studies and the antiproliferative effect of bromelain against B16F10 murine melanoma cells.

The current study aims to extract bromelain from different parts (stem, crown, peels, pulp and leaves) of Ananas comosus var. comosus AGB 772; to determine of optimum pH and temperature; to test bromelain stability in disodium EDTA and sodium benzoate, and to investigate its pharmacological activity on B16F10 murine melanoma cells in vitro. The highest enzymatic activity was found in bromelain extracted from the pulp and peel. The optimum bromelain pH among all studied pineapple parts was 6.0. The optimum temperature was above 50 °C in all bromelain extracts. The fluorescence analysis confirmed the stability of bromelain in the presence of EDTA and sodium benzoate. Bromelain was pharmacologically active against B16F10 melanoma cells and it was possible verifying approximately 100% inhibition of tumor cell proliferation in vitro. Since bromelain activity was found in different parts of pineapple plants, pineapple residues from the food industry may be used for bromelain extraction.

Antiproliferative effect of Canavalia brasiliensis lectin on B16F10 cells.

Lectins are proteins or glycoproteins with the ability to link to carbohydrates at the cell surface in a specific and reversible manner. Studies have showed that lectins are demonstrate immunomodulatory and antitumor agents. This study aims to evaluate the effect of lectin extracted from the seeds of Canavalia brasiliensis (ConBr) on murine melanoma B16F10 cells by analyzing cell viability, apoptosis index, cell migration, production of cytokines and nitric oxide (NO). Results showed that ConBr was able to reduce cell viability and thwart apoptosis, which could be observed by decrease in cell migration. ConBr also induced NO and IL-12 synthesis. Altogether, these data demonstrate the potential of ConBr as a therapeutic agent for melanoma.

FTY720 induces apoptosis in B16F10-NEX2 murine melanoma cells, limits metastatic development in vivo, and modulates the immune system

OBJECTIVE: Available chemotherapy presents poor control over the development of metastatic melanoma. FTY720 is a compound already approved by the Food and Drug Administration for the treatment of patients with multiple sclerosis. It has also been observed that FTY720 inhibits tumor growth in vivo (experimental models) and in vitro (animal and human tumor cells). The aim of this study was to evaluate the effects of FTY720 on a metastatic melanoma model and in tumor cell lines. METHODS: We analyzed FTY720 efficacy in vivo in a syngeneic murine metastatic melanoma model, in which we injected tumor cells intravenously into C57BL/6 mice and then treated the mice orally with the compound for 7 days. We also treated mice and human tumor cell lines with FTY720 in vitro, and cell viability and death pathways were analyzed. RESULTS: FTY720 treatment limited metastatic melanoma growth in vivo and promoted a dose-dependent decrease in the viability of murine and human tumor cells in vitro. Melanoma cells treated with FTY720 exhibited characteristics of programmed cell death, reactive oxygen species generation, and increased β-catenin expression. In addition, FTY720 treatment resulted in an immunomodulatory effect in vivo by decreasing the percentage of Foxp3+ cells, without interfering with CD8+ T cells or lymphocyte-producing interferon-gamma. CONCLUSION: Further studies are needed using FTY720 as a monotherapy or in combined therapy, as different types of cancer cells would require a variety of signaling pathways to be extinguished. .