Alpha-N-acetylgalactosaminidase levels in cancer patients are affected by Vitamin D binding protein-derived macrophage activating factor

It is well assessed that alpha-N-acetylgalactosaminidase (nagalase) accumulates in serum of cancer patients and is responsible for deglycosylation of vitamin D binding protein, which is the precursor of vitamin D binding protein-derived macrophage activating factor (GcMAF), eventually leading to immunodeficiency in advanced cancer patients. The increase in nagalase activity in cancer patients is due to the fact that cancer cells release nagalase, and nagalase activity reflects tumour burden, aggressiveness and progression of the disease up to the point that determination of nagalase activity is a non-invasive way of evaluation of cancer severity. Here we report the observation of a series of clinical cases describing the results obtained administering GcMAF to patients with diverse types of cancers. In all cases, GcMAF treatment was initiated at late stages of tumour progression. The response to GcMAF was robust. All patients (n= 20) presented with nagalase levels well above the threshold of normal values (2.84+0.26 nM/min/mg). All patients, but one, showed significant decrease of nagalase levels following GcMAF weekly injections (1.59+0.17. p<0.01). Nagalase decrease was associated with improved clinical conditions. No adverse side effects were reported. The observation reported here confirm and extend the results presented in [1] and open the way to further studies aimed at assessing the precise role and indications for GcMAF in the immunotherapy of cancer

Vitamin D binding protein-derived macrophage activating factor stimulates proliferation and signalling in a human neuronal cell line

Vitamin D (vitD), vitD binding protein-derived macrophage activating factor (DBP-MAF), and vitD receptor (VDR) are essential for adult neurogenesis [1], and this effect could be responsible for the recently reported effects of DBP-MAF on autism spectrum disorders (ASD) [2]. In order to test this hypothesis, we challenged a human neuronal cell line (SH-SY5Y, IZSLER) with DBP-MAF (Immuno Biotech), and we studied cAMP formation (cAMP EIA kit, Abnova), cell proliferation (MTT assay, Sigma Aldrich), apoptosis (human caspase 3 act, Invitrogen) and cell morphology. SH-SY5Y cells represent a validated in vitro model of human neurons in neurodegenerative diseases [3]. DBP-MAF induced rapid (15 min) formation of cAMP in a dose-dependent manner (0.4-40 ng/ml) as well as increase in cell proliferation at 24-48 and 72 h. Cell morphology was consistent with neurogenesis and an increase in the number of cells with high synthetic activity was observed. No apoptosis following DBP-MAF treatment was observed. Our results open the way to exploit these newly described effects to treat neurodegenerative disorders from Parkinson’s and Alzheimer’s diseases to Myalgic Encephalomyelitis and ASD

EFFECTS OF GC-MACROPHAGE ACTIVATING FACTOR IN HUMAN NEURONS; IMPLICATIONS FOR TREATMENT OF CHRONIC FATIGUE SYNDROME

ABSTRACT Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease of multifactorial aetiology characterized by immune system dysfunction, widespread inflammation, multisystemic neuropathology and persistent pain. Given the central role of the immune system in the pathogenesis of the syndrome, we studied the effects of a potent modulator of the immune system in in vitro and in vivo models that could help clarifying its role and indications in ME/CFS treatment. To this end, we studied the effects of vitamin D-binding protein-derived macrophage activating factor (also designated as GcMacrophage Activating Factor or (GcMAF)) on human neuronal cells (SH-SY5Y) and on the persistent pain induced by osteoarticular damage in rats. GcMAF at pM concentration increased neuronal cell viability and metabolism through increased mitochondrial enzyme activity. These effects were accompanied by cAMP formation and by morphological changes that were representative of neuronal differentiation. We hypothesize that these effects are to be ascribed to the interconnection between the GcMAF and Vitamin D Receptor (VDR) signalling pathways. The results presented here confirm at the experimental level the therapeutic effects of GcMAF in ME/CFS and elucidate the mechanisms of action through which GcMAF might be responsible for such therapeutic effects

EFFECTS OF GC-MACROPHAGE ACTIVATING FACTOR IN HUMAN NEURONS; IMPLICATIONS FOR TREATMENT OF CHRONIC FATIGUE SYNDROME

ABSTRACT Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease of multifactorial aetiology characterized by immune system dysfunction, widespread inflammation, multisystemic neuropathology and persistent pain. Given the central role of the immune system in the pathogenesis of the syndrome, we studied the effects of a potent modulator of the immune system in in vitro and in vivo models that could help clarifying its role and indications in ME/CFS treatment. To this end, we studied the effects of vitamin D-binding protein-derived macrophage activating factor (also designated as GcMacrophage Activating Factor or (GcMAF)) on human neuronal cells (SH-SY5Y) and on the persistent pain induced by osteoarticular damage in rats. GcMAF at pM concentration increased neuronal cell viability and metabolism through increased mitochondrial enzyme activity. These effects were accompanied by cAMP formation and by morphological changes that were representative of neuronal differentiation. We hypothesize that these effects are to be ascribed to the interconnection between the GcMAF and Vitamin D Receptor (VDR) signalling pathways. The results presented here confirm at the experimental level the therapeutic effects of GcMAF in ME/CFS and elucidate the mechanisms of action through which GcMAF might be responsible for such therapeutic effects

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

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 al1ows 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

Glycosylated oleic acid/vitamin dbinding protein suppresses her2 oncogene expression in human breast cancer

A woman was diagnosed with mammary adenocarcinoma in the right breast in 1985 at the age of 37, followed by quadrantectomy, lymphadenectomy and irradiation. In 1999, an adenocarcinoma was diagnosed in the left breast, followed by ample resection and anti-oestrogen receptor treatment for 6 years. In April 2014, an infiltrating adenocarcinoma was diagnosed in the right breast that had been operated in 1985. Pre-operative biopsy showed weak positivity for progesterone receptor (PgR, 10%, score 2+). With the goal of boosting her immune system during the 3 weeks preceding surgery, glycosylated oleic acid/vitamin D-binding protein (OAGcMAF) was administered by subcutaneous injections, nebulisation and with a fermented milk product rich in OAGcMAF. No drug was administered in the 3 weeks preceding surgery, nor had the patient received any treatment for the previous 8 years. Following right mastectomy, analysis of the surgical specimen showed no positivity for HER2 expression (negative, score 0) and significant increase in positivity of PgR, from 10% of positivity and a score of 2+ (Figure 1). After 3 weeks of OAGcMAF treatment and subsequent mastectomy, analysis of the surgical specimen showed no positivity for HER2 expression (negative, score 0; Figure 1), thus indicating complete suppression of oncogene expression. Study of the expression of progesterone receptor (PgR, clone 1E2) was consistent with such a reversal of the neoplastic phenotype. PgR expression in the biopsy was low (<1%), a finding consistent with poor differentiation and aggressiveness. However, in the surgical specimen taken after the 3 weeks of treatment with OAGcMAF, PgR expression was significantly increased to 20% (Figure). The selectivity of these effects was confirmed by a study of the expression of Ki67 and estrogen receptor (30% and 90%, respectively) that did not show any change following OA-GcMAF treatment (Figure). Discussion: These results demonstrate that OA-GcMAF, administered by subcutaneous injections, aerosol or in a functional food product, suppressed the expression of HER2, an oncogene which plays a key role in the aetiology, invasive progression and metastasis in breast cancer. This effect was paralleled by increase of PgR expression, thus indicating that OA-GcMAF treatment induced healthy differentiation of cancer cells. We hypothesize that these multifaceted effects on the regulation of gene expression in human breast cancer are due to the peculiar association of OA with GcMAF that is an association between two molecules endowed with anticancer properties. In fact, OA has been shown to down-regulate HER2 expression in cancer cell lines (9) and we demonstrated that GcMAF inhibits human breast cancer cell proliferation and reverts their malignant phenotype (10). We hypothesize that OA-GcMAF, but not OA or GcMAF taken singularly, interacts with the HER2 protein through hydrophobic interaction between the amino-terminal of GcMAF and the extracellular region of HER2, and between the OA-binding region of GcMAF and the plasma membrane (4). In fact, in the stretch of aminoacids between position 17-46 of GcMAF, and position 243-273 of HER2, there is a high density of hydrophobic aminoacids that may favour selective binding. Whatever the case, these results indicate that the effects of OAGcMAF in cancer are due to a multiplicity of actions that involve suppression of oncogene expression

GC protein-derived macrophage-activating factor decreases α- N

α-N-acetylgalactosaminidase (nagalase) accumulates in the serum of cancer patients and its activity correlates with tumor burden, aggressiveness and clinical disease progression. The administration of GC protein-derived macrophage-activating factor (GcMAF) to cancer patients with elevated levels of nagalase has been associated with a decrease of serum nagalase activity and with significant clinical benefits. Here, we report the results of the administration of GcMAF to a heterogeneous cohort of patients with histologically diverse, advanced neoplasms, generally considered as “incurable” diseases. In most cases, GcMAF therapy was initiated at late stages of tumor progression. As this is an open-label, non-controlled, retrospective analysis, caution must be employed when establishing cause-effect relationships between the administration GcMAF and disease outcome. However, the response to GcMAF was generally robust and some trends emerged. All patients (n = 20) presented with elevated serum nagalase activity, well above normal values. All patients but one showed a significant decrease of serum nagalase activity upon weekly GcMAF injections. Decreased nagalase activity was associated with improved clinical conditions and no adverse side effects were reported. The observations reported here confirm and extend previous results and pave the way to further studies aimed at assessing the precise role and indications for GcMAF-based anticancer immunotherapy

Oleic Acid, Deglycosylated Vitamin D-Binding Protein, Nitric Oxide: A Molecular Triad Made Lethal to Cancer

Oleic Acid (OA) has been shown to have anticancer properties mediated by interaction with proteins such as α-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 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. Measuring the tumour by ultrasonographic techniques, we observed a decrease of tumour volume of about 25%. 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