N-Acetylgalactosamine-glycosylated vitamin D binding protein (GalNAc-DBP) is known to stimulate macrophages and to directly inhibit angiogenesis and cancer cell growth in vitro. In this study, we evaluated the direct effect of GalNAc-DBP complexed with oleic acid on human multiple myeloma and Hodgkin’s lymphoma cell lines (KMS-12-BM and L540 respectively) as well as the effect, on the same cell lines, of human spleen macrophages (CRL9853) previously activated by GalNAc-DBP complexed with oleic acid. Cell viability and living cell number were evaluated respectively by Tetrazolium dye cell viability assay and by Trypan Blue staining. Interactions between activated macrophages and cancer cells were studied by time lapse photography. Our results show that GalNAc-DBP complexed with oleic acid inhibits the proliferation of myeloma and Hodgkin’s lymphoma cells in a dose-dependent manner. Furthermore, GalNAc-DBP complexed with oleic acid activates human spleen macrophages which in turn phagocytise cancer cells. In addition, we observed that GalNAc-DBP complexed with oleic acid activates human spleen macrophages in vivo as documented by color-doppler ultrasonography of the spleen. These results demonstrate that GalNAc-DBP complexed with oleic acid has a double effect on myeloma and Hodgkin’s lymphoma cancer cells: a direct inhibition of their proliferation and viability and, at the same time, an efficient macrophage activation leading to a significant depletion of cancer cell population. Taken together, these results suggest that GalNAc-DBP complexed with oleic acid may prove effective in the integrative immunotherapy of multiple myeloma and Hodgkin’s lymphoma.

BACKGROUND: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 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.

Deglycosylated vitamin D-binding protein-derived macrophage-activating factor (GcMAF) is known to be a strong immune stimulatory natural molecule. Data in literature demonstrate that GcMAF has a direct role in decreasing cell proliferation of different cancer cell lines. In this study we evaluate the direct effect of GcMAF complexed with oleic acid (OA-GcMAF) on human multiple myeloma cells (KMS-12BM), as well as the effect on the same cell line of human macrophages (CRL9853) previously activated by OA-GcMAF. Cell viability and living cell number were evaluated respectively by tetrazolium dye cell viability assay and by Trypan blue staining. Interactions between activated macrophages and myeloma cells were studied by time lapse photography. Our results show that OA-GcMAF decreases the cell viability of KMS-12-BM with a dose-dependent pathway. Furthermore OA-GcMAF activates human macrophages, which in turn phagocytise myeloma cancer cells. OA-GcMAF confirms its double effect on cancer cells: a direct inhibition of their viability and, at the same time, an efficient macrophage activation leading to a significant depletion of cancer cell population. Introduction: In recent years the interest of vitamin D-binding protein-derived macrophage-activating factor (GcMAF) as a potent immunotherapeutic agent has increased. The GcMAF has been shown to be effective in stimulating murine macrophages in vitro to phagocytose human breast carcinoma cultures (1, 2), as well as inhibiting the growth of prostate cancer cells (3). It has also been the agent that has been referenced as reducing tumour burden in several clinical approaches (4, 5). In previous studies GcMAF has been used to stimulate Raw 264.7 cells (murine macrophage cell line) that were observed in vitro to phagacytose MCF-7 cells (human breast carcinoma). In this study we demonstrate the effect of GcMAF stabilized with oleic acid (OA-GcMAF) directly on KMS-12-BM multiple myeloma cells and on co-culture of stimulated human macrophages (CRL9853) and KMS-12-BM. Materials and Methods: Cell lines: KMS-12-BM: human multiple myeloma cell line was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and cultured in RPMI 1640 supplemented with 10% FBS and 2mM L-Glutamine (Life Technologies, Paisley, UK). Cultures were passaged every 3-4 days. CRL9853: human spleen macrophage was purchased from ATCC (American Type Culture Collection, Teddington, UK) and routinely cultured in IMDM supplemented with 10% FBS and 2mM L-Glutamine (Life Technologies). Cultures were passaged every 3-4 days. Prior to assay, CRL9853 cells were activated culturing them for 72h in the presence of OAGcMAF at a concentration 100ng/ml in complete medium. Stimuli: OA-GcMAF, commercially available, was prepared at Immuno Biotech Ltd. (Guernsey, Channel Island) with a proprietary procedure previously described (6). Cell viability Abstracts of the 9th International Conference of Anticancer Research, 6-10 October 2014, Porto Carras, Sithonia, Greece 6175 Figure 1. KMS-12-BM cell viability assay and Trypan Blue viable cell counts. (A) Results for (8 pM) demonstrate a slight decrease on cell proliferation which becomes significantly greater at (800 pM) indicating a direct effect on cell viability. (B) The decrease in viable cell number as the OA-GcMAF concentration increases corroborating the observed cell viability assay data. Results are expressed as mean value±S.E.M. for five replicates. (*p<0.01). assay: Cell viability was evaluated by the reduction of a tetrazolium salt (WST-8) as an index of cell dehydrogenases’ activity. KMS-12-BM cells were seeded into a 96-well plate at a density of 3x104 cells/well in their appropriate starvation medium (without FBS). After incubation for 24h the cell line was treated for 24h with the following different concentrations of OA-GcMAF ([8-80-800 pM). At the end of the treatment, the medium was replaced with 100μl of fresh starvation medium plus 10μl of WST-8. The 96-well plate was incubated for 3h at 37 C and the optical density (O.D.) was directly measured at A450nm by Multiscan FC photometer (ThermoScientific, Milano, Italy). Cell counting – Trypan blue assay: To corroborate the results obtained by cell viability assay, a viable cell count was performed. Briefly, KMS-12-BM cells were plated into a 6-well plate at a density of 2x105 cells/well in starvation medium. After 24h incubation, human multiple myeloma cells were treated with OA-GcMAF at the increasing concentrations (8-80-800 pM) for 24h. At the end of the treatment, a volume of cell suspension was collected and the viable cell number was counted by Trypan Blue staining. Video-time lapse photography: KMS-12-BM cells were seeded into a 24-well plate at a density of 1x106 cells/well with a 1ml volume. The cells were allowed to settle for a minimum of 2h prior to the addition of the OA-GcMAF-activated CRL9853 macrophages. The staging mat was set at a temperature of 37 C and allowed to equilibrate prior to placement of the 24-well plate. The OA-GcMAF-activated CRL9853 macrophages were added to a final concentration of 5x105 cells per well in 1ml. HEPES (Fisher Scientific, Loughborough, UK) was added to each well to provide a final concentration of 25mM to stabilize the culture pH. Once activated macrophages and the HEPES were added, the 24-well plate was observed microscopically and an image selected. An initial frame was taken and a timelapse film initiated. A frame was taken every 3 minutes until filming was stopped. Results: Cell viability assay: Cell viability (Figure 1), evaluated both by tetrazolium dye cell assay (A) and by Trypan blue staining (B), decreased when KMS-12-BM cells were treated with increasing concentrations of OAGcMAF. In particular, when cells were treated with OAGcMAF (800 pM) a significant reduction (p<0.01) in cell ANTICANCER RESEARCH 34: 5761-6258 (2014) 6176 Figure 2. Selected frame shots over time of KMS-12-BM cells in the presence of OA-GCMAF-stimulated CRL9853 human macrophages. (A) KMS-12-BM suspension culture typical growth pattern. (B) KMS-12-BM cells 24h post addition of OA-GcMAF activated human macrophages. CRL9853 cultures as large masses of suspension cells and the dense collection of cells can be observed sitting on the KMS-12-BM suspension cells. The OA-GcMAF-activated macrophages are extremely motile under observation and move around clearing the KMS-12-BM cells. During the phagocytosis smaller dense “clusters” of cells break off and scavenge the KMS-12-BM cells quite rapidly. (C) KMS-12-BM cells 48h post addition of OA-GcMAF-activated human macrophages. CRL9853 cells now have a few large cell mass “clusters” and many smaller “clusters” as can be observed (red arrows). The number of KMS-12-BM cells has been decreased and the clearance of cells can be easily observed. (D) KMS-12BM cells 72h post addition of OA-GcMAF-activated human macrophages CRL9853. OA-GcMAF-activated CRL9853 cells are still extremely active with the phagocytotic activity and are clearing the KMS-12-BM cells from the culture. This image shows the smaller clusters of cells as indicated by the red arrows. The clusters have become smaller and rapid moving seeking out and destroying the remaining KMS-12-BM cells. viability was observed in comparison to the untreated control cells. Discussion: It has been shown that Gc-MAF activates Raw 264.7 murine macrophages to phagocytose and destroy MCF-7 human breast carcinoma cells (1). An identical effect has been recorded for the first time with the stimulation of human macrophages CRL9853 on KMS-12-BM cell line . The OA-GcMAF-stimulated human macrophages seek out surround and phagocytose the myeloma cell lines destroying them. This demonstrates that the CRL9853 behave as postulated against KMS-12BM cell line. In addition the effect of increased cell death in the presence of OA-GcMAF alone as indicated by direct evaluation of viable cell counts and viability assay also provides supports to the previous data generated (1). This study provides some inferred evidence to support the in vivo clinical data that has recently been published (6) providing some insight into the method of potential tumour removal by stimulated macrophages. In conclusion OA-GcMAF has demonstrated two major effects: a direct decrease of KMS-12BM cell viability and an efficient activation of human macrophages, which become able to phagocytose and destroy the myeloma cells. The studies will be expanded further to encompass additional cancer cell lines. 1 Pacini S et al: Effects of vitamin D-binding protein-derived macrophage-activating factor on human breast cancer cells. Anticancer Res, 2012; 32(1):45-52. 2 Thyer L et al: 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 2013; 5(7):2577-2589. 3 Gregory KJ et al: Vitamin D binding protein-macrophage activating factor directly inhibits proliferation, migration, and uPAR expression of prostate cancer cells. PLoS One 2010; 5(10):e13428. 4 Thyer L et al: Therapeutic effects of highly purified deglycosylated GcMAF in the immunotherapy of patients with chronic diseases. Am J Immunol 2013; 9(3):78-84. 5 Yamamoto Net al: Immunotherapy for prostate cancer with gc protein derived macrophage-activating factor OAGcMAF. Trans Oncol 2008; 1:65-72 6 Ruggerio M et al: Oleic acid, deglycosylated vitamin Dbinding protein, nitric oxide: a molecular triad made lethal to cancer. Anticancer Res 2014; 34: 3569-3578. 605 SENESCENCE LINKED INFLAMMATION, REPLICATION STRESS, FRAGILE SITES AND DNA METHYLATION CHANGES IN SPORADIC BREAST AND PROSTATE CANCER.

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.

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.

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.