GcMAF Abstracts 2

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Effects of oxaliplatin and oleic acid Gc-protein-derived macrophage-activating factor on murine and human microglia.
            (Branca et al., 2015) Download
The biological properties and characteristics of microglia in rodents have been widely described, but little is known about these features in human microglia. Several murine microglial cell lines are used to investigate neurodegenerative and neuroinflammatory conditions; however, the extrapolation of the results to human conditions is frequently met with criticism because of the possibility of species-specific differences. This study compares the effects of oxaliplatin and of oleic acid Gc-protein-derived macrophage-activating factor (OA-GcMAF) on two microglial cell lines, murine BV-2 cells and human C13NJ cells. Cell viability, cAMP levels, microglial activation, and vascular endothelial growth factor (VEGF) expression were evaluated. Our data demonstrate that oxaliplatin induced a significant decrease in cell viability in BV-2 and in C13NJ cells and that this effect was not reversed with OA-GcMAF treatment. The signal transduction pathway involving cAMP/VEGF was activated after treatment with oxaliplatin and/or OA-GcMAF in both cell lines. OA-GcMAF induced a significant increase in microglia activation, as evidenced by the expression of the B7-2 protein, in BV-2 as well as in C13NJ cells that was not associated with a concomitant increase in cell number. Furthermore, the effects of oxaliplatin and OA-GcMAF on coculture morphology and apoptosis were evaluated. Oxaliplatin-induced cell damage and apoptosis were nearly completely reversed by OA-GcMAF treatment in both BV-2/SH-SY5Y and C13NJ/SH-SY5Y cocultures. Our data show that murine and human microglia share common signal transduction pathways and activation mechanisms, suggesting that the murine BV-2 cell line may represent an excellent model for studying human microglia. (c) 2015 Wiley Periodicals, Inc.

Effects of vitamin D(3)-binding protein-derived macrophage activating factor (GcMAF) on angiogenesis.
            (Kanda et al., 2002) Download
BACKGROUND: The vitamin D(3)-binding protein (Gc protein)-derived macrophage activating factor (GcMAF) activates tumoricidal macrophages against a variety of cancers indiscriminately. We investigated whether GcMAF also acts as an antiangiogenic factor on endothelial cells. METHODS: The effects of GcMAF on angiogenic growth factor-induced cell proliferation, chemotaxis, and tube formation were examined in vitro by using cultured endothelial cells (murine IBE cells, porcine PAE cells, and human umbilical vein endothelial cells [HUVECs]) and in vivo by using a mouse cornea micropocket assay. Blocking monoclonal antibodies to CD36, a receptor for the antiangiogenic factor thrombospondin-1, which is also a possible receptor for GcMAF, were used to investigate the mechanism of GcMAF action. RESULTS: GcMAF inhibited the endothelial cell proliferation, chemotaxis, and tube formation that were all stimulated by fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor-A, or angiopoietin 2. FGF-2-induced neovascularization in murine cornea was also inhibited by GcMAF. Monoclonal antibodies against murine and human CD36 receptor blocked the antiangiogenic action of GcMAF on the angiogenic factor stimulation of endothelial cell chemotaxis. CONCLUSIONS: In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.

Effect of paricalcitol and GcMAF on angiogenesis and human peripheral blood mononuclear cell proliferation and signaling.
            (Pacini et al., 2012) Download
BACKGROUND: In addition to its role in calcium homeostasis and bone mineralization, vitamin D is involved in immune defence, cardiovascular function, inflammation and angiogenesis, and these pleiotropic effects are of interested in the treatment of chronic kidney disease. Here we investigated the effects of paricalcitol, a nonhypercalcemic vitamin D analogue, on human peripheral blood mononuclear cell proliferation and signaling, and on angiogenesis. These effects were compared with those of a known inhibitor of angiogenesis pertaining to the vitamin D axis, the vitamin D-binding protein-derived Gc-macrophage activating factor (GcMAF). METHODS: Since the effects of vitamin D receptor agonists are associated with polymorphisms of the gene coding for the receptor, we measured the effects of both compounds on mononuclear cells harvested from subjects harboring different BsmI polymorphisms. RESULTS: Paricalcitol inhibited mononuclear cell viability with the bb genotype showing the highest effect. GcMAF, on the contrary, stimulated cell proliferation, with the bb genotype showing the highest stimulatory effect. Both compounds stimulated 3'-5'-cyclic adenosine monophosphate formation in mononuclear cells with the highest effect on the bb genotype. Paricalcitol and GcMAF inhibited the angiogenesis induced by proinflammatory prostaglandin E1. CONCLUSIONS: Polymorphisms of the vitamin D receptor gene, known to be associated with the highest responses to vitamin D receptor agonists, are also associated with the highest responses to GcMAF. These results highlight the role of the vitamin D axis in chronic kidney disease, an axis which includes vitamin D, its receptor and vitamin D-binding protein-derived GcMAF.


Glycosylation status of vitamin D binding protein in cancer patients.
            (Rehder et al., 2009) Download
On the basis of the results of activity studies, previous reports have suggested that vitamin D binding protein (DBP) is significantly or even completely deglycosylated in cancer patients, eliminating the molecular precursor of the immunologically important Gc macrophage activating factor (GcMAF), a glycosidase-derived product of DBP. The purpose of this investigation was to directly determine the relative degree of O-linked trisaccharide glycosylation of serum-derived DBP in human breast, colorectal, pancreatic, and prostate cancer patients. Results obtained by electrospray ionization-based mass spectrometric immunoassay showed that there was no significant depletion of DBP trisaccharide glycosylation in the 56 cancer patients examined relative to healthy controls. These results suggest that alternative hypotheses regarding the molecular and/or structural origins of GcMAF must be considered to explain the relative inability of cancer patient serum to activate macrophages.

Oleic Acid, deglycosylated vitamin D-binding protein, nitric oxide: a molecular triad made lethal to cancer.
            (Ruggiero et al., 2014) Download
BACKGROUND: Oleic Acid (OA) has been shown to have anticancer properties mediated by interaction with proteins such as alpha-lactalbumin and lactoferrins. Therefore, we synthesized complexes of OA and Gc protein-derived macrophage activating factor (GcMAF) that inhibits per se cancer cell proliferation and metastatic potential. We hypothesised that OA-GcMAF complexes could exploit the anticancer properties of both OA and GcMAF in a synergistic manner. We postulated that the stimulating effects of GcMAF on macrophages might lead to release of nitric oxide (NO). PATIENTS AND METHODS: Patients with advanced cancer were treated at the Immuno Biotech Treatment Centre with OA-GcMAF-based integrative immunotherapy in combination with a low-carbohydrate, high-protein diet, fermented milk products containing naturally-produced GcMAF, Vitamin D3, omega-3 fatty acids and low-dose acetylsalicylic acid. RESULTS: Measuring the tumour by ultrasonographic techniques, we observed a decrease of tumour volume of about 25%. CONCLUSION: These observations demonstrate that OA, GcMAF and NO can be properly combined and specifically delivered to advanced cancer patients with significant effects on immune system stimulation and tumour volume reduction avoiding harmful side-effects.


 

The in vitro GcMAF effects on endocannabinoid system transcriptionomics, receptor formation, and cell activity of autism-derived macrophages.
            (Siniscalco et al., 2014) Download
BACKGROUND: Immune system dysregulation is well-recognized in autism and thought to be part of the etiology of this disorder. The endocannabinoid system is a key regulator of the immune system via the cannabinoid receptor type 2 (CB2R) which is highly expressed on macrophages and microglial cells. We have previously published significant differences in peripheral blood mononuclear cell CB2R gene expression in the autism population. The use of the Gc protein-derived Macrophage Activating Factor (GcMAF), an endogenous glycosylated vitamin D binding protein responsible for macrophage cell activation has demonstrated positive effects in the treatment of autistic children. In this current study, we investigated the in vitro effects of GcMAF treatment on the endocannabinoid system gene expression, as well as cellular activation in blood monocyte-derived macrophages (BMDMs) from autistic patients compared to age-matched healthy developing controls. METHODS: To achieve these goals, we used biomolecular, biochemical and immunocytochemical methods. RESULTS: GcMAF treatment was able to normalize the observed differences in dysregulated gene expression of the endocannabinoid system of the autism group. GcMAF also down-regulated the over-activation of BMDMs from autistic children. CONCLUSIONS: This study presents the first observations of GcMAF effects on the transcriptionomics of the endocannabinoid system and expression of CB2R protein. These data point to a potential nexus between endocannabinoids, vitamin D and its transporter proteins, and the immune dysregulations observed with autism.

A novel role for a major component of the vitamin D axis: vitamin D binding protein-derived macrophage activating factor induces human breast cancer cell apoptosis through stimulation of macrophages.
            (Thyer et al., 2013) Download
The role of vitamin D in maintaining health appears greater than originally thought, and the concept of the vitamin D axis underlines the complexity of the biological events controlled by biologically active vitamin D (1,25(OH)(2)D3), its two binding proteins that are the vitamin D receptor (VDR) and the vitamin D-binding protein-derived macrophage activating factor (GcMAF). In this study we demonstrate that GcMAF stimulates macrophages, which in turn attack human breast cancer cells, induce their apoptosis and eventually phagocytize them. These results are consistent with the observation that macrophages infiltrated implanted tumors in mice after GcMAF injections. In addition, we hypothesize that the last 23 hydrophobic amino acids of VDR, located at the inner part of the plasma membrane, interact with the first 23 hydrophobic amino acids of the GcMAF located at the external part of the plasma membrane. This allows 1,25(OH)(2)D3 and oleic acid to become sandwiched between the two vitamin D-binding proteins, thus postulating a novel molecular mode of interaction between GcMAF and VDR. Taken together, these results support and reinforce the hypothesis that GcMAF has multiple biological activities that could be responsible for its anti-cancer effects, possibly through molecular interaction with the VDR that in turn is responsible for a multitude of non-genomic as well as genomic effects.

Clinical experience of cancer immunotherapy integrated with oleic acid complexed with de-glycosylated vitamin d binding protein
            (Ward et al., 2014) Download
Proteins highly represented in milk such as ?-lactalbumin and lactoferrin bind Oleic Acid (OA) to form complexes with selective anti-tumor activity. A protein present in milk, colostrum and blood, vitamin D binding protein is the precursor of a potent Macrophage Activating Factor (GcMAF) and in analogy with other OA-protein complexes, we proposed that OA-GcMAF could demonstrate a greater immunotherapeutic activity than that of GcMAF alone. We describe a preliminary experience treating patients with advanced cancers, often labelled as incurable with an integrative immunotherapy centred on OA-GcMAF.


Abstracts Of The Ninth International Conference Of Anticancer Research, Anticancer Res October 2014 34 (10) 5761-6257
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Marco Ruggiero, Jacopo J.V. Branca, David Noakes, Massimo Gulisano, Gabriele Morucci, Lynda Thyer, and Stefania Pacini. GLYCOSYLATED Oleic Acid/Vitamin D-Binding Protein Suppresses Her2 Oncogene Expression In Human Breast Cancer. Anticancer Res. 34(10): 5845-5847, 2014.

Lynda Thyer, Jacopo J.V. Branca, Margit Taubmann. Clinical Experience Of Immunotherapy Based On Oleic Acid Bound To Glycosylated Vitamin D-Binding Protein In Localised And Metastatic Adenocarcinoma Of The Pancreas. Anticancer Res. 34(10): 5847-5849, 2014.

Jacopo J.V. Branca, Stefania Pacini and Marco Ruggiero. FOCUSSED Transcranial Ultrasounds: Application To The Delivery Of Glycosylated Oleic Acid/Vitamin D-Binding Protein To Brain Tumours And Metastases. Anticancer Res. 34(10): 5844-5845, 2014.

Rodney Smith, Emma Ward, Jacopo J.V. Branca, Gabriele Morucci and Stefania Pacini. The Effect Of Gcmaf Complexed With Oleic Acid On Multiple Myeloma Cultures. Anticancer Res. 34(10): 6175-6177, 2014.

 

 


References

Branca, JJ, et al. (2015), ‘Effects of oxaliplatin and oleic acid Gc-protein-derived macrophage-activating factor on murine and human microglia.’, J Neurosci Res, PubMedID: 25782915
Kanda, S, et al. (2002), ‘Effects of vitamin D(3)-binding protein-derived macrophage activating factor (GcMAF) on angiogenesis.’, J Natl Cancer Inst, 94 (17), 1311-19. PubMedID: 12208896
Pacini, S, et al. (2012), ‘Effect of paricalcitol and GcMAF on angiogenesis and human peripheral blood mononuclear cell proliferation and signaling.’, J Nephrol, 25 (4), 577-81. PubMedID: 21956771
Rehder, DS, RW Nelson, and CR Borges (2009), ‘Glycosylation status of vitamin D binding protein in cancer patients.’, Protein Sci, 18 (10), 2036-42. PubMedID: 19642159
Ruggiero, M, et al. (2014), ‘Oleic Acid, deglycosylated vitamin D-binding protein, nitric oxide: a molecular triad made lethal to cancer.’, Anticancer Res, 34 (7), 3569-78. PubMedID: 24982371
Siniscalco, D, et al. (2014), ‘The in vitro GcMAF effects on endocannabinoid system transcriptionomics, receptor formation, and cell activity of autism-derived macrophages.’, J Neuroinflammation, 11 78. PubMedID: 24739187
Thyer, L, et al. (2013), ‘A novel role for a major component of the vitamin D axis: vitamin D binding protein-derived macrophage activating factor induces human breast cancer cell apoptosis through stimulation of macrophages.’, Nutrients, 5 (7), 2577-89. PubMedID: 23857228
Ward, E, et al. (2014), ‘Clinical experience of cancer immunotherapy integrated with oleic acid complexed with de-glycosylated vitamin d binding protein’, Am J Immunol, 10 (1), 23-32. PubMedID: