Branca, Jacopo Junio Valerio

David, Noakes

Gulisano, Massimo

Lynda, Thyer

Morucci, Gabriele

Pacini, Stefania

Ruggiero, Marco

English

Florence Research

ultrasound could then be directed toward the anatomicalregions where the lesion is located. Most regions of the braincan be accessed through the temporal acoustic window, withthe exception of distal areas of the frontal and occipital lobes.Focussed ultrasounds are known to transiently increase thepermeability of the BBB (3) and, therefore, allow passage ofOA-GcMAF that is a relatively lipophilic molecule thanks tothe presence of oleic acid. The procedure lasts 5-15 min,causes no discomfort or side effects and can be repeatedcontinuously or in cycles. The procedure can be safelyperformed before, during or after other anti-neoplastictreatments.1 Ruggiero M et al: Oleic Acid, deglycosylated vitamin d-binding protein, nitric oxide: a molecular triad made lethalto cancer. Anticancer Res 34(7): 3569-3578, 2014.2 Bradstreet JJ et al: A new methodology of viewing extra-axial fluid and cortical abnormalities in children with autismvia transcranial ultrasonography. Front Hum Neurosci 7:934, 2014.3 Burgess A et al: Drug delivery across the blood-brain barrierusing focused ultrasound. Expert Opin Drug Deliv1 1(5):711-721, 2014.85GLYCOSYLATED OLEIC ACID/VITAMIN D-BINDING PROTEIN SUPPRESSES HER2 ONCOGENEEXPRESSION IN HUMAN BREAST CANCERMarco Ruggiero1,3, Jacopo J.V. Branca2, David Noakes3,Massimo Gulisano2, Gabriele Morucci2, Lynda Thyer3 and Stefania Pacini2Departments of 1Experimental and Clinical BiomedicalSciences and 2Experimental and Clinical MedicineUniversity of Firenze, Firenze, Italy;3Immuno Biotech Ltd; Guernsey, Channel Islands, UKA woman was diagnosed with mammary adenocarcinoma inthe right breast in 1985 at the age of 37, followed byquadrantectomy, lymphadenectomy and irradiation. In 1999, anadenocarcinoma was diagnosed in the left breast, followed byample resection and anti-oestrogen receptor treatment for 6years. In April 2014, an infiltrating adenocarcinoma wasdiagnosed in the right breast that had been operated in 1985.Pre-operative biopsy showed weak positivity for progesteronereceptor (PgR, <1%) and high positivity for the oncogeneHER2 (>10%, score 2+). With the goal of boosting herimmune system during the 3 weeks preceding surgery,glycosylated oleic acid/vitamin D-binding protein (OA-GcMAF) was administered by subcutaneous injections,nebulisation and with a fermented milk product rich in OA-GcMAF. No drug was administered in the 3 weeks precedingsurgery, nor had the patient received any treatment for theprevious 8 years. Following right mastectomy, analysis of thesurgical specimen showed no positivity for HER2 expression(negative, score 0) and significant increase in positivity of PgR,from <1% to 20%. These results indicate that OA-GcMAFtreatment suppressed oncogene expression and induceddifferentiation of cancer cells. Introduction: The healthyproperties of oleic acid (OA) in breast cancer have been knownfor centuries (1) and recent evidences suggest that theseproperties are amplified by association of OA with proteinssuch as α-lactalbumin and lactoferrins. These proteins formOA-protein complexes that exhibit highly selective anti-tumouractivity in vitro and in vivo (2). We recently demonstrated thatalso a serum protein with the capability to bind OA showsanticancer effects; this is the glycosylated vitamin D-bindingprotein also known as Gc-protein-derived macrophageactivating factor or GcMAF (3). This protein binds both OAand vitamin D and exerts its immune-stimulating andanticancer effects through cross-talk with the vitamin Dreceptor (4). Here we report a clinical observation suggestingthat OA-GcMAF, that is GcMAF-complexed with OA,suppresses the expression of a major oncogene involved inhuman breast cancer that is the human epidermal growth factorreceptor 2 (HER2). Patients and Methods: A woman wasdiagnosed with mammary adenocarcinoma in the right breast in1985 at the age of 37, followed by quadrantectomy,lymphadenectomy and irradiation. In 1999, an adenocarcinomawas diagnosed in the left breast, followed by ample resectionand anti-oestrogen receptor treatment for 6 years. In April2014, an infiltrating adenocarcinoma was diagnosed in the rightbreast that had been operated in 1985. With the goal ofboosting her immune system during the 3 weeks elapsingbetween biopsy and programmed surgery, OA-GcMAF(Goleic®, Immuno Biotech Ltd.) was administered bysubcutaneous injections (880 ng) and nebulisation (880 ng) asindicated in (3). The patient followed a nutritional regime basedon a low carbohydrate, high protein diet (5). To this end, thepatient was provided with food containing only 2%carbohydrates (Le Gamberi Foods, Forlì, Italy), and withessential aminoacids (Master Aminoacid Pattern®, dr. reinwaldhealthcare gmbh, Schwarzenbruck, Germany) (6). The patientwas also provided with a fermented milk product containingcolostrum and microorganisms known to produce OA-GcMAFfrom the Gc-protein present in milk (Bravo Probiotic®, LesAlpes, Wellington, NZ). No drug was administered or wasprogrammed in the 3 weeks preceding surgery, nor had thepatient received any treatment for the previous 8 years. Theanalyses on HER2 and other gene expression on the biopsy andsurgical specimens were performed by the laboratory of theUniversity Hospital of Careggi of the Italian Public HealthService, in Firenze, Italy. Analyses were performed accordingto the European standards of quality (UNI EN ISO 9001:2008)and were examined and countersigned by four differentprofessionals. The original documents are conserved in thearchives of the Department of Biomedicine of the CareggiAbstracts of the 9th International Conference of Anticancer Research, 6-10 October 2014, Porto Carras, Sithonia, Greece5845Hospital (Molecular Diagnostic and Pathologic Histology). Thepatient gave the informed consent to the treatment as well asto this description of her results. Surgery was performed at theDivision of General Surgery n. 2 of the University Hospital ofCareggi. Results: Amplification or overexpression of HER2plays an important role in the development and progression ofbreast cancer and has become an important biomarker andtarget of therapy (7) since it is strongly associated withincreased disease recurrence and a poor prognosis (8).Consistent with the aggressive nature of the cancer in thispatient, pre-operative biopsy on four specimens collected underultrasound guidance showed significant positivity for HER2assessed by the polyclonal antibody A 0485 with >10% ofpositivity and a score of 2+ (Figure 1). After 3 weeks of OA-GcMAF treatment and subsequent mastectomy, analysis of thesurgical specimen showed no positivity for HER2 expression(negative, score 0; Figure 1), thus indicating completesuppression of oncogene expression. Study of the expressionof progesterone receptor (PgR, clone 1E2) was consistent withsuch a reversal of the neoplastic phenotype. PgR expression inthe biopsy was low (<1%), a finding consistent with poordifferentiation and aggressiveness. However, in the surgicalspecimen taken after the 3 weeks of treatment with OA-GcMAF, PgR expression was significantly increased to 20%(Figure). The selectivity of these effects was confirmed by astudy of the expression of Ki67 and estrogen receptor (30%and 90%, respectively) that did not show any change followingOA-GcMAF treatment (Figure). Discussion: These resultsdemonstrate that OA-GcMAF, administered by subcutaneousinjections, aerosol or in a functional food product, suppressedthe expression of HER2, an oncogene which plays a key role inthe aetiology, invasive progression and metastasis in breastcancer. This effect was paralleled by increase of PgRexpression, thus indicating that OA-GcMAF treatment inducedhealthy differentiation of cancer cells. We hypothesize thatthese multifaceted effects on the regulation of gene expressionin human breast cancer are due to the peculiar association ofOA with GcMAF that is an association between two moleculesendowed with anticancer properties. In fact, OA has beenshown to down-regulate HER2 expression in cancer cell lines(9) and we demonstrated that GcMAF inhibits human breastcancer cell proliferation and reverts their malignant phenotype(10). We hypothesize that OA-GcMAF, but not OA or GcMAFtaken singularly, interacts with the HER2 protein throughhydrophobic interaction between the amino-terminal ofGcMAF and the extracellular region of HER2, and between theOA-binding region of GcMAF and the plasma membrane (4).In fact, in the stretch of aminoacids between position 17-46 ofGcMAF, and position 243-273 of HER2, there is a high densityof hydrophobic aminoacids that may favour selective binding.Whatever the case, these results indicate that the effects of OA-GcMAF in cancer are due to a multiplicity of actions thatinvolve suppression of oncogene expression.Figure. Level of expression of HER2, progesterone receptor(PgR), Ki67 protein and estrogen receptor (ER). “Before”, indicates level of expression observed on the biopsysamples obtained 3 weeks before surgery and before anytreatment. “After”, indicates level of expression observed onsurgical specimens after OA-GcMAF treatment. Level ofexpression is expressed as percentages as in the originalreports. The axis on the left (0-50%) refers to HER2, PgR andKi67. The axis on the right (0-100%) refers only to ER. Theactual levels were: HER2, before: >10%, score 2. HER2, after:negative, score 0. PgR, before: <1%. PgR, after: 20%. Ki67before and after: 30%. ER, before and after: 90%. 1 Colomer R et al: Giacomo Castelvetro’s salads. Anti-HER2oncogene nutraceuticals since the 17th century? Clin TranslOncol 10(1): 30-34, 2008.2 Fang B et al: Bovine lactoferrin binds oleic acid to form ananti-tumor complex similar to HAMLET. Biochim BiophysActa 1841(4): 535-543, 2014.3 Ward E et al: Clinical experience of cancer immunotherapyintegrated with oleic acid complexed with de-glycosylatedvitamin d binding protein. Am J Immunol 10(1): 23-32,2014.4 Thyer L et al: A novel role for a major component of thevitamin D axis: vitamin D binding protein-derivedmacrophage activating factor induces human breast cancercell apoptosis through stimulation of macrophages. Nutrients5(7): 2577-2589, 2013.5 Ho VW et al: A low carbohydrate, high protein diet slowstumor growth and prevents cancer initiation. Cancer Res71(13): 4484-4493, 2011.6 Lucà-Moretti M et al: Master Amino acid Pattern assubstitute for dietary proteins during a weight-loss diet toachieve the body’s nitrogen balance equilibrium withessentially no calories. Adv Ther 20(5): 282-291, 2003.7 Mitri Z et al: The HER2 Receptor in Breast Cancer:Pathophysiology, Clinical Use, and New Advances inANTICANCER RESEARCH 34: 5761-6258 (2014)5846Therapy. Chemother Res Pract 2012: 743193, 2012.8 Tan M et al: Molecular mechanisms of erbB2-mediatedbreast cancer chemoresistance. Adv Exp Med Biol 608: 119-129, 2007.9 Carrillo C et al: Antitumor effect of oleic acid; mechanismsof action: a review. Nutr Hosp 27(6): 1860-1865, 2012.10 Pacini S et al: Effects of vitamin D-binding protein-derivedmacrophage-activating factor on human breast cancer cells.Anticancer Res 32(1): 45-52, 2012.86CLINICAL EXPERIENCE OF IMMUNOTHERAPYBASED ON OLEIC ACID BOUND TOGLYCOSYLATED VITAMIN D-BINDING PROTEIN IN LOCALISED AND METASTATICADENOCARCINOMA OF THE PANCREASLynda Thyer1, Jacopo J.V. Branca2, Margit Taubmann31Macro Innovations Ltd, CB4 0DS Cambridge, UK;2Department of Experimental and Clinical Medicine,University of Firenze, 50134 Firenze, Italy;3Naturheilzentrum, D-95444 Bayreuth, Germany(jacopo.branca@libero.it) Adenocarcinoma of the pancreas still carries a dramaticallypoor prognosis and the survival rate for this disease has notimproved substantially in the past 40 years. Therefore, newtreatment options are urgently needed and this need motivatesoncologists to search for novel approaches such asimmunotherapy. Here we report two clinical cases successfullytreated with an integrative immunotherapeutic approach basedon oleic acid bound to glycosylated vitamin D-binding protein(OA-GcMAF). Considering that immune suppression inducedby pancreatic cancer is one of the main causes for resistanceto chemotherapy and targeted therapy, this novelimmunotherapeutic approach has the potential forrevolutionising the field of pancreatic adenocarcinomatreatment. Introduction: Adenocarcinoma of the pancreas is,after colorectal cancer, the second most common digestivecancer in the USA where it represents the fourth leading causeof cancer-related death in both genders. In Europe,adenocarcinoma of the pancreas is the sixth most frequentcancer and is predicted to become the fourth cause of cancerdeath in both sexes in due course in the European Union (1, 2).Patients with pancreatic adenocarcinoma have an especiallypoor prognosis with a 5-year survival rate of <1% and amedian survival of 4-6 months. Patients with a metastaticdisease are usually treated with chemotherapy that is minimallyeffective (3). It has been demonstrated that pancreatic cancer-induced immune suppression is the main cause for thisdramatically poor prognosis and, therefore, it has beenproposed that immunotherapies may be particularly effectivein this type of cancer (4). We recently demonstrated thatimmunotherapy based on oleic acid (OA) bound toglycosylated vitamin D-binding protein (OA-GcMAF) iseffective in a variety of cancers (5); here we describe twoclinical cases demonstrating that OA-GcMAF has therapeuticefficacy also in pancreatic adenocarcinomas. Patients andMethods: Patients with adenocarcinoma of the pancreas weretreated with OA-GcMAF-based integrative immunotherapyaccording to the “Good practice in prescribing and managingmedicines and devices” effective February 2013. Theapproaches described below, aimed at strengthening theimmune system and reducing tumour growth, are consideredcomplementary to other anti-neoplastic therapeutic procedures.OA-GcMAF complexes (GOleic) were prepared in-house atImmuno Biotech Ltd as previously described (5). The protocolfor pancreatic adenocarcinoma was the following: OA-GcMAF(880 ng/day) was administered by subcutaneous injections inproximity of the inguinal lymphnodes (440 ng in each side)under ultrasound guidance. OA-GcMAF (880 ng) was alsoadministered daily by nebulisation (880 ng dissolved in 5 mlsaline). Suppositories containing 200 ng OA-GcMAF wereadministered daily. The total amount of daily OA-GcMAF was1960 ng, an amount consistent with the procedure described byNonaka et al. (6). Patients were provided with supplementationof vitamin D3, 20.000 IU per day, and they were taught todrink at least 2 litres of water per day. Patients followed anutritional regime based on a diet very low in carbohydrates,and high in proteins (7). This regimen includedsupplementation with essential aminoacids (Master AminoacidPattern, dr. reinwald healthcare gmbh, Schwarzenbruck,Germany). Considering that probiotics are efficientimmunopotentiators and have a role in cancer prevention (8),patients were provided with a probiotic fermented milk productcontaining colostrum and microorganisms known to producenatural OA-GcMAF during the fermentation process (BravoProbiotic, Les Alpes, Wellington, NZ). Finally, considering therole of low-dose acetylsalicylic acid in cancer prevention (9),patients were provided with 100 mg of such a principle perday. Results: Clinical case #1. A 58-year-old lady wasdiagnosed with pancreatic adenocarcinoma localised in thebody of the pancreas. The lesion could be detected byultrasonography and it appeared as a roundish mass of 0.924ml of calculated volume (Figure 1A). After two weeks oftreatment as described above, the calculated volume of themass was reduced to 0.517 ml that is about 39% (Figure 1B).The patient did not report any side effect: her body weight andlean mass increased during the two weeks of treatment.Clinical case #2. A 73-year-old man was diagnosed withmetastatic pancreatic adenocarcinoma. Previous CT scans hadevidenced peritoneal metastases. The patient was overweightand presented with an insulin-dependent diabetes. Due to themorphological constitution of the patient, the primarypancreatic lesion could not be evidenced by ultrasonography.However, a hypo-echoic roundish mass in the abdomen,Abstracts of the 9th International Conference of Anticancer Research, 6-10 October 2014, Porto Carras, Sithonia, Greece5847

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

https://flore.unifi.it/bitstream/2158/918539/1/85.Glycosylated%20oleic%20acid-vitamin%20d-binding%20protein%20suppresses%20her2%20oncogene%20expression%20in%20human%20breast%20cancer.pdf

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

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