In vitro attenuation of classic metastatic melanoma­related features by highly diluted natural complexes: Molecular and functional analyses.

Metastasis is responsible for the majority of deaths among patients with malignant melanoma. Despite recent advances, the majority of current and modern therapies are ineffective and/or financially unfeasible. Thus, in this study, we investigated two low­cost highly­diluted natural complexes (HDNCs) that have been shown to be effective against malignant melanoma in a murine model in vivo. The aim of this study was to determine the mechanisms through which these HDNCs directly affect melanoma cells, either alone or in an artificial tumor microenvironment, suppressing the metastatic phenotype, thus explaining previous in vivo effects. For this purpose, HDNC in vitro treatments of B16­F10 melanoma cells, alone or in co­culture with Balb/3T3 fibroblasts, were carried out. Molecular biology techniques and standard functional assays were used to assess the changes in molecule expression and in cell behaviors related to the metastatic phenotype. Melanoma progression features were found to be regulated by HDNCs. Molecules related to cell adhesion (N­cadherin, ß1­integrin and CD44), and migration, extracellular matrix remodeling and angiogenesis were modulated. The cell migratory, invasive and clonogenic capacities were reduced by the HDNCs. No loss of cell proliferation or viability were observed. On the whole, the findings of this study indicate that HDNCs directly reprogram, molecularly and functionally, melanoma cells in vitro, modulating their metastatic phenotype. Such findings are likely to be responsible for the attenuation of tumor growth and lung colonization previously observed in vivo.

Differential effects of Zincum metallicum on cell models.

INTRODUCTION: Zinc is an essential trace element necessary for life. Traditional and complementary medicines use zinc-based formulations to treat different classes of diseases. Basic research on homeopathic preparations of zinc are rare and there are a few published clinical cases describing its effects on patients. The use of cell-based models in drug screening is a reliable source of evidence. METHODS: We sought to investigate experimental end-points using cell-based models to determine the effects of dilutions of Zincum metallicum prepared according to the Brazilian Homeopathic Pharmacopoeia. Murine RAW 264.7 macrophages and melanoma B16-F10 cell lines were cultured according to standard procedures. Cells were treated with either 5c, 6c or 30c Zincum metallicum and control cells with its respective vehicle (5c, 6c, or 30c Lactose). Macrophage activation by CD54 immunolabeling and intracellular reactive oxygen species (ROS) using DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) were detected by flow cytometry. Phagocytic capacity (endocytic index) was quantified by light microscopy. Features of melanoma cells were analyzed by colorimetric assays to determine melanin content and cell proliferation rate. All obtained data were submitted to normality test followed by statistical analysis. RESULTS: Zincum metallicum 6c shifted high ROS-producing macrophages to a low ROS-producing phenotype. Macrophage CD54 expression was increased by Zincum metallicum 5c. No changes in endocytic index were observed. Melanoma cells were not affected by any treatment we tested. CONCLUSIONS: Differing responses and non-linearity were found on macrophages challenged with Zincum metallicum at high dilutions. No changes in melanoma cells were observed. Customised assays using target cells can be useful to investigate high-dilution effects. Other cell types and conditions should be explored.

Inhalation therapy with M1 inhibits experimental melanoma development and metastases in mice.

BACKGROUND: M1 is a homeopathic medicine with immunostimulatory properties used mainly by cancer patients to complement current therapies. Metastatic melanoma is a skin-originated form of cancer without a single therapy able to produce high rate and sustained responses, which attracts the use of complementary therapies such as M1. However, M1's anti-melanoma effects remain to be pre-clinically demonstrated. Therefore in the present work, we utilized a pulmonary metastatic melanoma model and a subcutaneous melanoma growth model to investigate the potential benefits of treatment with M1. METHODS: C57BL/6 mice were injected intravenously or subcutaneously with B16F10 mouse melanoma cells. After 24 h, mice were treated with either M1 or vehicle (water) for 14 days, euthanized and harvested for multi-parameter pulmonary and tumor analyses. RESULTS: Mice treated with M1 had significantly lower tumor burden in the lungs and subcutaneous tissue than control mice. Furthermore, tumors were impaired in proliferation and tumor related angiogenesis by the inhibition of myeloid derived suppressor cells (MDSC) positive for angiotensin II type 1 receptor (AT1R). CONCLUSION: Altogether these data suggest M1 is an efficient candidate for melanoma therapy to be considered for future clinic studies as this study is the first supporting the idea that melanoma patients may benefit with the treatment. The treatment with M1 provides advantages considering the highly-diluted properties and a cost effective alternative to costly chemotherapeutic approaches with, if any, lower toxicity.

Effect of pegylated phosphatidylserine-containing liposomes in experimental chronic arthritis.

BACKGROUND: Phosphatidylserine-containing liposomes (PSL) have been shown to reduce inflammation in experimental models of acute arthritis, by mimicking the apoptotic process. The aim of this study was to evaluate the effect of pegylated PSL (PEG-PSL) on chronic inflammation of collagen induced arthritis (CIA) in DBA/1J mice. METHODS: CIA was induced in 24 DBA/1J mice (n = 6/group), which were divided into control (0.9 % saline) or treated with PEG-PSL (5, 10 and 15 mg/kg/day, subcutaneously for 20 days). Clinical score, limb histology and measurement of cytokines in knee joints of animals by ELISA and cytometric bead array (CBA) were evaluated. The in vitro study employed macrophage cultures stimulated with 100 ng/ml of LPS plus 10 ng/ml of PMA and treated with 100 µM PEG-PSL. RESULTS: Resolution of the disease in vivo and the inflammatory process in vitro were not observed. PEG-PSL, in doses of 10 and 15 mg/kg, were not shown to reduce the score of the disease in animals, whereas with the dose of 5 mg/kg, the animals did not show the advanced stage of the disease when compared to the controls. The PEG- PSL 5, 10 and 15 mg/kg treatment groups did not show significant reduction of TNF-α, IL-1ß, IL-6, IL-2 and IFN-γ when compared to the controls. Disease incidence and animal weights were not affected by treatment. Regarding the paw histology, PEG-PSL did not yield any reductions in the infiltrating mononuclear, synovial hyperplasia, extension of pannus formation, synovial fibrosis, erosion of cartilage, bone erosion or cartilage degradation. The concentration of 100 µM of PEG-PSL has not been shown to reduce inflammation induced by LPS/PMA in the in vitro study. Treated groups did not show any reduction in inflammatory cytokines in the knee joints of animals affected by the disease compared to the control, although there were higher concentrations of TGF-ß1 in all experimental groups. CONCLUSION: The experimental model showed an expression of severe arthritis after the booster. TGF-ß1 as well other pro inflammatory cytokines were presented in high concentrations in all groups. PEG-PSL had no impact on the clinical score, the histopathology from tibial-tarsal joints or the production of cytokines in the knee joints. Other alternatives such as dosage, route of administration, and as an adjunct to a drug already on the market, should be evaluated to support the use of PEG-PSL as a new therapeutic tool in inflammatory diseases.

Tratamento in vivo com M8, uma solução complexa altamente diluída, reduziu a malignidade de um modelo de melanoma de camundongo / In vivo treatment with M8, a highly diluted tinctures complex, reduced the malignancy of a mouse melanoma model

Background: Cancer is a class of disease responsible for 13% of death cause worldwide. Among all types of cancers, one of the most aggressive and with the highest death rate is melanoma. It is highly metastatic and current treatments with chemotherapeutic drugs do not yield satisfactory results. Therefore, the interest on new therapeutics for cancer treatment has been increasing on research. Highly diluted tinctures (HDT) are intended to enhance immune system responses resulting in reduced frequency of various diseases, and often present no risk of serious side-effects due to its low toxicity. Previous results have demonstrated in vitro inhibition of invasion ability and in vivo anti-metastatic potential of B16F10 lung metastasis model after mice treatment with M8 inhalation.Conclusion: Even though further investigation are necessary to elucidate the mechanisms of action of M8 treatment there is an indication that these highly diluted tinctures could be a promising therapy to treat metastatic melanoma.(AU)

Introdução: O Câncer é uma classe de doenças responsáveis por 13% das causas de mortes no mundo todo. Entre todos os tipos de cânceres, um dos mais agressivos e com maior índice de mortalidade é o melanoma. Ele é altamente metastático, e os tratamentos atuais com drogas quimioterapeuticas não geram resultados satisfatórios. Portanto, o interesse em novos agentes terapeuticos para o tratamento do câncer tem aumentado na pesquisa. Soluções altamente diluídas (CAD) são destinadas à aumentar a resposta do sistema imunológico resultando em menores frequencias de várias doenças, e também não apresentam riscos de graves efeitos colaterais, devido à sua toxicidade reduzida. Resultados anteriores demostraram a inibição in vitro da habilidade de invasão e potencial antimetastático in vivo do modelo de metástase pulmonar da B16F10, após o tratamento dos camundongos pela inalação do M8.Conclusão: Mais pesquisas são necessárias para esclarecer os mecanismos de ação do tratamento com M8. Entretanto, há um indicativo de que soluções altamente diluídas podem ser terapias coadjuvantes para o tratamento do melanoma metastático.(AU)