The effect of Gcmaf complexed with oleic acid on multiple myeloma cultures

Abstract: 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-12- BM), 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 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