Tumours

Tumours are one of the major causes of death worldwide and also one of the greatest fears of our time. Despite consumption of red meat being linked to an increase in development of some types of tumours, on the other hand there are encouraging experimental findings on following a ketogenic diet as an aid in treating some types of tumours. This is hardly surprising if we consider that the ketogenic diet, if followed correctly, simulates some aspects of fasting which has been effective in reducing the toxicity of chemotherapy. Furthermore, some tumours preferentially use glucose as an energy source and therefore the constant low blood glucose levels present during a ketogenic diet could have a role in controlling the growth of tumour masses.

Key references:

Chu-Shore CJ, Thiele EA. Tumour growth in patients with tuberous sclerosis complex on the ketogenic diet. Brain & development. 2010; 32 (4): 318-322.

Klement RJ, Kammerer U. Is there a role for carbohydrate restriction in the treatment and prevention of cancer? Nutrition & metabolism. 2011; 8: 75.

Lee C, Raffaghello L, Longo VD. Starvation, detoxification, and multidrug resistance in cancer therapy. Drug resistance updates: reviews and commentaries in antimicrobial and anticancer chemotherapy. Feb-Apr 2012; 15 (1-2): 114-122.

Maurer GD, Brucker DP, Bahr O, et al. Differential utilization of ketone bodies by neurons and glioma cell lines: a rationale for ketogenic diet as experimental glioma therapy. BMC Cancer. 2011; 11: 315.

Scheck AC, Abdelwahab MG, Fenton KE, P. Stafford The ketogenic diet for the treatment of glioma: Insights from genetic profiling. Epilepsy research. 2011.

Schmidt M, N Pfetzer, Schwab M, Strauss I, Kammerer U. Effects of a ketogenic diet on the quality of life in 16 patients with advanced cancer: A pilot trial. Nutrition & metabolism. 2011; 8 (1): 54.

Seyfried BT, Kiebish M, J Marsh, P. Mukherjee Targeting energy metabolism in brain cancer through calorie restriction and the ketogenic diet. Journal of cancer research and therapeutics. 2009; 5 Suppl 1: S7-15.

Seyfried TN, Marsh J, Shelton LM, LC Huysentruyt, Mukherjee P. Is the restricted ketogenic diet a viable alternative to the standard of care for managing malignant brain cancer? Epilepsy research. 2011.

Translated text:

American Journal of Immunology 10 (1): 23-32, 2014

ISSN: 1553-619X

©2014 Science Publication

doi:10.3844/ajisp.2014.23.32 Published Online 10 (1) 2014 (http://www.thescipub.com/aji.toc)

 

CLINICAL EXPERIENCE OF CANCER IMMUNOTHERAPY INTEGRATED WITH OLEIC ACID COMPLEXED WITH DE-GLYCOSYLATED VITAMIN D BINDING PROTEIN

 

1Emma Ward, 1Rodney Smith, 2Jacopo J.V. Branca,

3David Noakes, 2Gabriele Morucci and 1Lynda Thyer

1Macro Innovations Ltd, Cambridge, UK

2Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy

3Immuno Biotech Ltd, Guernsey, Channel Islands

 

Received: 26/02/2014; Revised: 07/03/2014; Accepted: 08/03/2014

 

Abstract

 

Proteins highly represented in milk such as α-lactalbumin and lactoferrin bind with Oleic Acid (OA) to form complexes with selective anti-tumour 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. Patients with advanced cancer were treated at the Immuno Biotech Treatment Centre with OA-GcMAF-based integrative immunotherapy in combination with a very low carbohydrate, high protein diet, fermented milk products containing naturally produced GcMAF, vitamin D3 and low-dose acetylsalicylic acid. When the primary tumour or a metastasis could be measured by ultrasonographic techniques, we observed, on average, a decrease of tumour volume of approximately 25% in a week. We also observed a consistent increase in splenic blood flow that was interpreted in the context of generalised immune system activation and allowed us to assess the degree of responsiveness of the individual patient. The results reported here are consistent with the results previously described in the experimental animal harbouring a human hepatocellular carcinoma as well as with the results reported for neoadjuvant chemotherapy. OA-protein complexes are bound to play a leading role in cancer therapy thanks to selectivity of anti-tumoural effects, absence of any side effects, safety and oral availability. We hypothesise that OA-GcMAF, combines the known anticancer effects OA-protein complexes with the well-established immune stimulating effects of GcMAF.

 

Keywords: GcMAF, Oleic Acid, Advanced Cancer, Integrative Immunotherapy, Ultrasonography

 

1. INTRODUCTION

 

It is well assessed that proteins highly represented in milk such as α-lactalbumin and lactoferrin can bind Oleic Acid (OA) to form complexes, which exhibit highly selective anti-tumour activity in vitro and in vivo (Fang et al., 2013). Recent observations seem to indicate that OA plays the key role in the tumouricidal action of these protein-OA complexes (Hoque et al., 2013).

Study of the structural features of such tumouricidal protein-OA complexes demonstrated that all these complexes exhibit a common feature that is a tendency toward protein oligomeristion. Since OA-induced oligomerisation has been reported for other proteins as well, it was hypothesised that this phenomenon may be inherent to many proteins (Nemashkalova et al., 2013).

Some of the milk proteins forming OA-protein tumouricidal complexes, such as lactoferrins, are potent modulators of the immune system (Vogel, 2012), thus lending credit to the hypothesis that the immune system is involved in the anticancer effects of OA-protein complexes.

In addition to lactoferrins, another immunogenic protein highly represented in milk, as well as in colostrum and blood, is the vitamin D binding protein. This is the precursor of a very potent macrophage activating factor that derives from its selective deglycosylation. Since vitamin D binding protein is also termed Gc-globulin, this macrophage activating factor is known as Gc-globulin-derived Macrophage Activating Factor (GcMAF) (for review on vitamin D binding protein and GcMAF, Ruggiero and Pacini, 2011).

The potent immunotherapeutic effects of GcMAF in human tumours have been demonstrated since 2007 in a variety of cancers ranging from the most common breast and prostate cancers to the less frequent oligodendroglioma (Yamamoto et al., 2008a; 2008b; 2008c; Inui et al., 2013; Thyer et al., 2013a; 2013b).

Therefore, in analogy with other OA-protein complexes, we postulated that OA-GcMAF complexes could show an anti-tumour activity even greater than that of GcMAF alone. Since OA-protein complexes and GcMAF show no evidence of toxicity (Ho et al., 2012; Bradstreet et al., 2012), these complexes were selected as part of an integrative immunotherapeutic approach to advanced cancers in the context of the so-called compassionate approach. In this study we describe the preliminary experiences of treating patients with advanced cancers, some of them considered as “incurable, with an integrative immunotherapy centred on OA-GcMAF.

 

2. PATIENTS AND METHODS

 

At the Immuno Biotech Treatment Centre, patients with advanced late stage 4 cancer are currently being treated with an OA-GcMAF-based integrative immunotherapy. OA-GcMAF is used in combination with a very low carbohydrate, high protein diet that is known to slow tumour growth and prevent cancer initiation (Ho et al., 2011), fermented milk products containing naturally produced GcMAF, high vitamin D3 supplementation (Den Hollander et al., 2013) and low-dose acetylsalicylic acid (Thun et al., 2012). All of these approaches aim to strengthen and activate the immune system and can be considered complementary and not alternative to other anti-neoplastic therapeutic procedures that the patients may want to take into consideration.


2.1. OA-GcMAF

 

OA-GcMAF complexes were prepared “in-house at Immuno Biotech with a proprietary procedure. Briefly, GcMAF was purified according to the procedure previously described (Yamamoto et al., 2008b). Vitamin D binding protein was isolated from purified human serum obtained from the American Red Cross, using either 25-hydroxyvitamin D3-sepharose high affinity chromatography or actin-agarose affinity chromatography. The bound material was eluted and then further processed by incubation with three immobilised enzymes as described (Bradstreet et al., 2012). The resulting GcMAF was filter sterilised. The protein content and concentration was assayed using standard Bradford protein assay methods (Bradford, 1976). Purity was assessed by SDS-PAGE and Western Blot analysis performed after each step of the preparation procedure; only one band of the expected molecular weight was visible (Smith et al., 2013). At the end of the production process, GcMAF was checked for sterility in-house and externally by independent laboratories. Its safety and biological activity were tested in monocytes, human breast cancer cells and chick embryo chorionallantoic membrane (Pacini et al., 2011; Pacini et al., 2012; Thyer et al., 2013c). Highly purified OA (molecular weight, 282.46; molecular formula, C18 H34 O2; Acros Organics, Geel, Belgium) was complexed with GcMAF in accordance with the molecular structure described in Thyer et al. (2013c). The optimal conditions for the preparation of the complexes were established according to the principles described in Knyazeva et al. (2008).

Because of the complexing of the protein with the fatty acid hydrophobic moiety together with the well-known properties of OA as an absorption enhancer, OA-GcMAF complexes could also be administered sublingually (Cui et al., 2005; Sakata et al., 2011), as an aerosol with a common nebuliser (Lu et al., 2011), or as suppositories (Goto et al., 1991).

 

2.2. Assessment of OA-GcMAF Induced Immunotherapeutic Effects

 

Macrophage activation in vivo could be assessed by monitoring the patients blood pressure before and after OA-GcMAF administration. Thus, it is well known that activated macrophages release nitric oxide a compound that causes vasodilatation and seems to be responsible for some of the anti-cancer properties of activated macrophages (Stuehr and Nathan, 1989; Nathan and Hibbs, 1991; Hiroi et al., 2013). It has been consistently observed that a small but significant decrease in both systolic and diastolic blood pressure, e.g. from 137/84 to 122/71. In our experience, the administration of OA-GcMAF (880 ng dissolved in 5 ml saline) with a nebuliser resulted in the most rapid decrease of blood pressure with the effects clearly appreciable 1 min after the end of the nebulisation. In order to have another assessment of immune stimulation, the splenic blood flow has been monitored with an ultrasound system (MyLab25Gold, Esaote, Genoa, Italy) using the echo-colour-doppler technique. An increase in the peripheral splenic blood flow has been consistently observed after OAGcMAF administration and it persisted for several days (Fig. 1A e 1B).( fig. 1A e 1B).

 

2.3. Integrative Cancer Immunotherapy with OA-GcMAF

 

The standard protocol of our integrative cancer immunotherapy is as follows: OA-GcMAF (880 ng) has been administered daily using the route of administration that was regarded as most suitable for each patient e.g., in patients with lung cancer or metastases, the preference was to administer OA-GcMAF with a nebuliser. In patients with liver cancer or metastases, the preference was through suppositories. In other cases, the intramuscular route as originally proposed by Yamamoto et al. (2008a) was the preferred route.

 

2.4. Other Complementary Integrative Approaches

 

In order to exploit the synergistic anti-cancer properties of vitamin D3 and bearing in mind that GcMAF constitutes part of the vitamin D axis (Thyer et al., 2013c), patients have taken a nutritional supplementation of vitamin D3 at 20,000 IU per day (Den Hollander et al., 2013) with monitoring the blood levels of such a vitamin. Patients are taught to drink at least 2 litres of water (or other liquids such as herbal teas) per day. Patients have followed a nutritional regime based on the recent scientific observations demonstrating that a very low carbohydrate, high protein diet slows tumour growth and prevents cancer initiation (Ho et al., 2011) . In order to favour the compliance to this type of diet, non-glucidic food containing only 2% of carbohydrates and a relatively high protein content (Le Gamberi Foods, Forlì, Italy) has been provided.

The patients weight and muscle mass is monitored and they are taught the strategies to decrease the Prognostic Inflammatory Nutritional Index (PINI) score is upheld as well as strategies to avoid Cancer Anorexia Cachexia Syndrome (CACS) according to Fabris et al. (2012). The supplementation with amino acids (Master Aminoacid Patter, International Nutrition Research Center, Coral Gables, FL, USA) is also intended for this scope (Luc-Moretti et al., 2003).

tumours

Fig. 1. Increased splenic blood flow following administration of OA-GcMAF. Oleic Acid (OA) complexed with vitamin D binding protein-derived Macrophage Activating Factor (OA-GcMAF, Goleic; Immuno Biotech Ltd, Guernsey, Channel Islands), 880 ng, dissolved in 5 mL saline, was administered by nebulisation. Splenic blood flow was assessed by echo-colour-doppler ultrasonographic technique. Panel A: basal, i.e., before any administration of OA-GcMAF. Panel B: 48 hours after OA-GcMAF administration. The increase in peripheral splenic blood flow is evident. This picture is representative of several others that gave qualitatively identical results.

 

 

Finally, considering the well assessed role of low-dose acetylsalicylic acid in cancer prevention (Thorat and Cuzick, 2013), patients have been receiving a 100 mg dose per day.

 

3. RESULTS

 

At the end of October 2013, patients with cancer started treatment at the Immuno Biotech Treatment Centre using the OA-GcMAF integrative approach described above. By the end of February 2014, immuno biotech had treated 30 patients. When the primary tumour or the metastasis could be accurately measured by ultrasonographic techniques, it has been observed, on average, a decrease of tumour volume of approximately 25% in a week. Although this reduction may appear dramatic, it is fully consistent with the results reported by Nonaka et al. (2012), who observed a 97% volume reduction of human hepatocellular carcinoma after 3 weeks of subcutaneous injection with GcMAF. It is also consistent with the results reported for neoadjuvant chemotherapy (Partridge et al., 2005).

Among the patients observed at the Immuno Biotech Treatment Centre, we present several cases here, each as a representative of common cancers for whom this integrative immunotherapy was remarkably effective. To our knowledge, this is the first example of actual images of tumour volume reduction following GcMAF immunotherapy. In effect, so far the effectiveness of immunotherapy of cancer with GcMAF has relied upon non-specific markers such as serum α-Nacetylgalactosaminidase levels (Yamamoto et al., 2008a; 2008b; 2008c; Thyer et al., 2013a) or anecdotic reports (Inui et al., 2013; Thyer et al., 2013b).

 

Patient 1

 

A 63-year-old woman was diagnosed with colon cancer in 1997. A CT scan performed in April 2013 had confirmed metastases in lung and liver. The scan of the liver reported a distortion of the superior liver suspected due to pleural carcinomatosis (diaphragmatic invasion probable) and invasion of the liver surface also possible. After 2 weeks of integrative immunotherapy where OA-GcMAF was administered by alternating nebulisation and intramuscular injections, the lesion at the level of the liver, measured with ultrasonography showed a decrease of calculated volume from 13 to 6.6 mL  ( Figg. 2A e B).

 

Patient 2

 

A 62-year-old woman was diagnosed with multiple myeloma. After 4 weeks of integrative immunotherapy where OA-GcMAF was administered by intramuscular injections, the monocytes almost doubled (from 0.39×109 /L to 0.70×109 /L). This finding is consistent with the original observation that GcMAF stimulates the bone marrow production of the progenitor cells for extensive mitogenesis and activation of macrophages, thus rebuilding the immune system (Yamamoto and Naraparaju, 1998). The stimulation of the immune system in this case could also be determined by the increase of lymphocytes that were abnormally low before the integrative immunotherapy (0.81×109 /L) and had returned to normal level in afterward (1.59×109 /L). The total number of white blood cells returned to normal level (5.0×109 /L) from previously low levels (3.3×109 /L). Also the modest increase of platelets (from 187 to 201×109 /L) and of red blood cells (from 3.31 to 3.48×1012/L) has to be interpreted as a consequence of successful macrophage activation. Thus, that it has been known for a long time that nitric oxide released by activated macrophages stimulates bone marrow haematopoiesis (Iversen et al., 1994; Cokić and Schechter, 2008). In addition to multiple myeloma and independently of this condition, this patient had simple breast cysts that could be visualised by ultrasonography. It has been determined that a significant decrease in the major diameter of one of these cysts, that was taken as being representative of the others. The diameter decreased from 5.4 to 3.8 mm (Fig. 3A and B). It is well assessed that breast cysts, although non-malignant, develop when an overgrowth of glands and connective tissue block milk ducts, causing them to dilate and fill with fluid (Guinebretire et al., 2005). Therefore, the observed effects of the integrative immunotherapy on the size of this breast cyst seem to indicate that this approach could be effective in controlling abnormal cell growth even when this is not frankly malignant.

 

Patient 3

 

A 43-year-old woman was diagnosed with metastatic breast cancer to bone and liver in February 2013. After 1 week of integrative immunotherapy where OA-GcMAF was administered by localised subcutaneous injections, the primary tumour at the level of the breast, measured with ultrasonography showed a decrease of calculated volume from 1.8 to 1.3 mL ( Figg. 4A e B). This case was representative of four other cases of breast cancer that qualitatively gave similar results.

 

Patient 4

 

A 60-year-old man was diagnosed with metastatic thyroid cancer to bone and soft tissue. After 4 weeks of integrative immunotherapy where OA-GcMAF was administered by alternating nebulisation and localised subcutaneous injections, one of the bone metastases taken as representative, measured with ultrasonography showed a decrease of its thickness from 43.3 to 37.3 mm ( Figg. 5A e B).

tumours

Fig. 2. Ultrasonography of liver metastasis. A 63-year-old woman was diagnosed with colon cancer in 1997. A CT scan performed in April 2013 had confirmed metastases in lung and liver. After 2 weeks of integrative immunotherapy where OA-GcMAF (880 ng/day) was administered by alternating nebulisation and intramuscular injections, the lesion at the level of the liver showed a decrease of calculated volume from 13 ml (panel A) to 6.6 mL (panel B). This corresponds to approximately a 50% overall reduction.

tumours

Fig. 3. Ultrasonography of breast cyst. A 62-year-old woman was diagnosed with multiple myeloma. After 4 weeks of integrative immunotherapy where OA-GcMAF (880 ng/day) was administered by intramuscular injections, the diameter of one benign breast cyst decreased from 5.4 mm (panel A) to 3.8 mm (panel B). This corresponds to approximately a 65% overall reduction.

tumours

Fig. 4. Ultrasonography of breast cancer. A 43-year-old woman was diagnosed with metastatic breast cancer to bone and liver in February 2013. After 1 week of integrative immunotherapy where OA-GcMAF (880 ng/day) was administered by localised subcutaneous injections, the primary tumour at the level of the breast, measured with ultrasonography, showed a decrease of calculated volume from 1.8 mL (panel A) to 1.3 mL (panel B). This corresponds to approximately a 28% overall reduction. This case was representative of four other cases of breast cancer that qualitatively gave similar results.

 

Patient 5

 

A 78-year-old man was diagnosed with metastatic renal carcinoma After 2 weeks of integrative immunotherapy where OA-GcMAF was administered by alternating nebulisation and intramuscular injections, one of the kidney lesions taken as representative, measured with ultrasonography, showed a decrease of its diameter from 33.7 to 29.8 mm  ( Figg. 6A e B).

 

Patient 6

 

A 36-year-old man was diagnosed with metastatic melanoma. After 1 week of integrative immunotherapy where OA-GcMAF was administered by alternating nebulisation and intramuscular injections, one of the lesions in the abdomen, interpreted as coalescent metastatic peritoneal nodes, measured with ultrasonography, showed a decrease of its length from 52.1 to 48.6 mm ( Figg. 7A e B).

 

Patient 7

 

A 55-year-old woman was diagnosed with metastatic adenocarcinoma of the colon. After 3 weeks of integrative immunotherapy where OA-GcMAF was administered by alternating nebulisation, intramuscular injections and suppositories, one of the lesions in the abdomen, interpreted as one of the metastases previously described, measured with ultrasonography, showed a decrease of its length from 76.8 to 70.1 mm  ( Figg. 8A e B).

tuomori

Fig. 5.Ultrasonography of bone metastasis. A 60-year-old man was diagnosed with metastatic thyroid cancer to bone and soft tissue. After 4 weeks of integrative immunotherapy where OA-GcMAF (880 ng/day) was administered by alternating nebulisation and localised subcutaneous injections. One of the bone metastases taken as representative, measured with ultrasonography, showed a decrease of its thickness from 43.3 mm (panel A) to 37.3 mm (panel B). This corresponds to an approximate 14% linear diameter reduction. In this case the volume could not be fully extrapolated since the mass showed an irregular shape.

tumours

Fig 6. Ultrasonography of kidney metastasis A 78-year-old man was diagnosed with metastatic renal carcinoma After 2 weeks of integrative immunotherapy where OA-GcMAF (880 ng/day) was administered by alternating nebulisation and intramuscular injections, one of the kidney lesions taken as representative, measured with ultrasonography, showed a decrease of its diameter from 33.7 mm (panel A) to 29.8 mm (panel B). This corresponds to approximately a 31% overall reduction.

tumours

Fig. 7. Ultrasonography of abdominal nodes. A 36-year-old man was diagnosed with metastatic melanoma. After 1 week of integrative immunotherapy where OA-GcMAF (880 ng/day) was administered by alternating nebulisation and intramuscular injections, one of the lesions in the abdomen, interpreted as coalescent metastatic peritoneal nodes, measured with ultrasonography, showed a decrease of its length from 52.1 mm (panel A) to 48.6 mm (panel B). This corresponds to an approximate 7% linear diameter reduction. In this case the volume could not be fully extrapolated since the mass showed an irregular elongated shape

tumours

Fig. 8. Ultrasonography of abdominal metastasis. A 55-year-old woman was diagnosed with metastatic adenocarcinoma of the colon. After 3 weeks of integrative immunotherapy where OA-GcMAF (880 ng/day) was administered by alternating nebulisation, intramuscular injections and suppositories, one of the lesions in the abdomen, interpreted as one of the metastases previously described, measured with ultrasonography, showed a decrease of its length from 76.8 mm (panel A) to 70.1 mm  ( Figg. 8A e B). This corresponds to an approximate 9% linear diameter reduction. In this case the volume could not be fully extrapolated since the mass showed an irregular elongated shape

 

4. DISCUSSION

 

OA-protein complexes are bound to play a leading role in cancer therapy (Smith, 2013) thanks to the selectivity of anti-tumoural effects, absence of side effects, safety and oral availability.

The synthesis of OA-GcMAF, combines the known anticancer effects of OA-protein complexes with the anticancer and immune stimulating effects of GcMAF. In fact, it is well assessed that GcMAF, in addition to activating tumouricidal macrophages (Thyer et al., 2013c), directly inhibits cancer cell proliferation (Gregory et al., 2010; Pacini et al., 2012) as well as cancer cell-induced neo-angiogenesis.

On account of the natural absorption enhancing activity of OA, the complexed OA-GcMAF represents the physiological assembly of naturally occurring GcMAF. This new formulation allows GcMAF to be administered through a variety of more desirable routes that eliminate a major obstacles to its widespread use. This feature favours a treatment tailored to the individual patients characteristics, with the possibility of delivering the OAGcMAF to the sites where it can best exert its action.

 

Table 1. Showing the decrease in tumour volumes (as calculated by ultrasonography measurements), with the types of cancer treated and the no of weeks treatment at the point of measurement

tumours

 

We are well aware that the clinical cases reported here are heterogeneous and describe patients with different types and different stages of cancer. In all cases, patients have undergone a variety of conventional and complementary therapies. In all cases, supplementary immunotherapy was started in the final stages of tumour progression, as it is understandable that conventional therapies were preferred in the earlier stages. Also, since this is a retrospective, uncontrolled, open-label type analysis, caution should be used while attributing cause and effect for any outcome of the treatment. However, the response to integrative immunotherapy was strong (Table 1) and, although any statistical analysis is inappropriate in such a heterogeneous collection of clinical histories, the absence of side effects and clinical improvement supported by objective evidence clearly emerge.

 

5. CONCLUSION

 

To our knowledge, the ultrasound images presented here represent the first direct evidence that shows an anti-tumour effect of GcMAF in humans (in this case OA - GcMAF). There was a significant reduction in tumour size (Table 1) and are fully in accordance with the actual images of tumour shrinkage indicated by Nonaka et al. (2012), who reported the effects of GcMAF in human cells with human hepatocellular carcinoma, transplanted into SCID mice. The ultrasound images also show a substantial increase in the flow of splenic blood which must be interpreted in the context of generalised activation of the immune system and allows an almost immediate assessment of the degree of reactivity of the individual patient. Therefore, we suggest that diagnostic imaging rather than measurement of α-n-acetylgalactosaminidase be considered as the main standard for evaluating the effectiveness of GcMAF treatment in cancer patients.

Although almost all patients were in the advanced stage 4, the requirement was that they were well enough to travel and be active enough to tackle the off-site residence during treatment. All these patients showed significant clinical improvements. Patients were then followed up and monitored in more detail at a later date.

 

5.1. Potential conflicts of interest

 

DN is the CEO of Immuno Biotech Ltd (the company which isolates and purifies the GcMAF protein). However, DN has no knowledge of the therapies in use, nor the names of all the patients whose data was analysed. Neither the CEO nor any employee of Immuno Biotech Ltd, had any knowledge of the clinical records and/or patient names in this study.

 

6. REFERENCES

 

(See point 6. REFERENCES of the original English text.)

_______________________________________________________________________________________________________________________________

Translated text:

ANTICANCER RESEARCH 34: 3569-3578 (2014)

 

OLEIC ACID, DEGLYCOSYLATED VITAMIN D-BINDING PROTEIN, NITRIC OXIDE: A MOLECULAR TRIAD MADE LETHAL TO CANCER

 

MARCO RUGGIERO1,4, EMMA WARD2, RODNEY SMITH2, JACOPO J.V. BRANCA3, DAVID NOAKES4, GABRIELE MORUCCI3, MARGIT TAUBMANN5, LYNDA THYER2 AND STEFANIA PACINI3

 

1Department of Experimental and Clinical Biomedical Sciences, University of Florence, Florence, Italy;

2Macro Innovations Ltd, Cambridge, UK;

3Department of Experimental and Clinical Medicine, University of Florence, Florence, Italy;

4Immuno Biotech Ltd, Guernsey, Channel Islands, UK;

5Naturheilzentrum, Bayreuth, Germany

 

 

Abstract. Background: Oleic Acid (OA) has been shown to have anticancer properties mediated by interaction with proteins such as α-lactalbumin and lactoferrins. Therefore, we synthesised 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 OAGcMAF-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.

 

It is well-assessed that Oleic Acid (OA), a recognised fundamental component of healthy diets (1), shows anticancer properties (2) that contribute to the increase in longevity and reduced risk of mortality and morbidity associated with its consumption (1). Although the precise molecular mechanism responsible for its anticancer properties is not completely understood (2), it appears that OA is involved in intracellular calcium signalling associated with the induction of cancer cell apoptosis.

It has been proposed that the anticancer effects of OA are mediated by its interaction with proteins highly represented in biological fluids such as α-lactalbumin and lactoferrins. These proteins bind OA to form OA-protein complexes which exhibit highly selective anti-tumour activity in vitro and in vivo (3). Soon after their identification, these complexes were labelled as HAMLET an acronym that stands for “human α-lactalbumin made lethal to tumour cells, even though further studies demonstrated that α- lactalbumin is not the sole protein forming such complexes. Thus, other proteins, forming complexes with OA, exhibit identical anticancer properties (4). It is now accepted that these OA-protein complexes destroy tumour cells with high selectivity and with no evidence of toxicity for normal tissues, a key feature in the quest for anticancer treatments devoid of toxic side effects.

Study of the structural characteristics of such anticancer OA-protein complexes demonstrated that a common molecular feature is the tendency toward OA-induced protein oligomerisation. Since OA-induced oligomerisation has been reported for a number of proteins in addition to those initially identified in HAMLET, it was hypothesised that this phenomenon may be inherent to many proteins (Nemashkalova et al., 5).

Some of the proteins forming OA-protein anticancer complexes such as α-lactalbumin and lactoferrins are highly represented in milk. These compounds are known to exert powerful stimulatory effects on the immune system (6). These findings provide additional acceptance to the hypothesis that the immune system (Vogel, 2012) is directly involved in the overall anticancer effects of OA-protein complexes.

 

Correspondence to: Jacopo J.V. Branca, Department of Experimental and Clinical Medicine, University of Florence, Largo Brambilla 3, 50134 Florence, Italy. Tel: +39 0552758067, Fax: +39

0554379500, e-mail: jacopo.branca@libero.it

 

Keywords: Oleic acid, nitric oxide, macrophages, human cancer, vitamin D, Gc protein-derived macrophage activating factor

 

 

In addition to α-lactalbumin and lactoferrins, another immunostimulatory protein highly represented in milk, as well as in colostrum and blood, is the vitamin D binding protein. This is the precursor of a very potent macrophage activating factor that derives from its selective deglycosylation. Since vitamin D binding protein is also termed Gc-globulin, this macrophage-activating factor is known as GcMAF (Gc-globulin-derived Macrophage Activating Factor) (for a review on vitamin D binding protein and GcMAF, see Reference 7).

The potent immunotherapeutic effects of GcMAF in human tumours have been demonstrated since 2007 in a variety of cancers ranging from the most common breast and prostate cancers to the less frequent oligodendroglioma (8-14).

Therefore, considering that GcMAF binds OA in a manner identical to that of α-lactalbumin or lactoferrins (15), we hypothesised that OA-GcMAF complexes were endowed with anticancer activity greater than that of OA or GcMAF alone when combining the anticancer properties of GcMAF with those of OA-protein complexes. Since OA-protein complexes and GcMAF show no evidence of toxicity (4, 16), an OA-GcMAF complex was used as part of an integrative immunotherapeutic approach to advanced cancers in the context of the so-called compassionate approach (14).

In the present study we hypothesise that the dramatic effects of OA-GcMAF in cancer patients recently reported (14) are mediated by the production of nitric oxide (NO) by activated macrophages. This identifies a triad of molecular elements, OA, GcMAF and NO, each one endowed with individual anticancer properties, that demonstrate synergistic effects and might open the way to a much improved cancer treatment strategy devoid of harmful side effects.

 

Patients and methods

 

At the Immuno Biotech Treatment Centre, patients with advanced cancer are currently being treated with OA-GcMAF-based integrative immunotherapy. OA-GcMAF complexes are used in combination with the following approaches: (i) A very low carbohydrate, high protein, equicaloric diet that is known to slow tumour growth and prevent cancer initiation (17), (ii) Fermented milk products containing naturally produced GcMAF, (iii) High Vitamin D3 supplementation (18), (iv) Low-dose acetylsalicylic acid (19) and (v) Omega-3 fatty acid supplementation (20).

These approaches, aimed at strengthening the immune system and reducing tumour growth, are considered complementary and not alternative to other anti-neoplastic therapeutic procedures that the patients and their caring health professional may want to take into consideration.

 

Preparation of OA-GcMAF complexes. OA-GcMAF complexes (GOleic) were prepared in-house at Immuno Biotech Ltd, with a proprietary procedure. Briefly, GcMAF was purified according to the procedure previously described (Yamamoto et al., 2008b). Vitamin D-binding protein was isolated from purified human serum obtained from the American Red Cross (2025 E St NW Washington, DC, USA), using either 25- hydroxyvitamin D3-Sepharose high affinity chromatography or actinagarose affinity chromatography. The bound material was eluted and then further processed by incubation with three immobilised enzymes as described (16). The resulting GcMAF was filter sterilised. The protein content and concentration was assayed using standard Bradford protein assay methods (21). Purity was assessed by SDS-PAGE and Western Blot analysis performed after each step of the preparation procedure; only one band of the expected molecular weight was visible (22). At the end of the production process, GcMAF was checked for sterility in-house and externally by independent laboratories. Its safety and biological activity were tested in human monocytes, human breast cancer cells and chick embryo chorionallantoic membrane (15, 23, 24). Highly purified OA (molecular weight, 282.46; molecular formula, C18 H34 O2; Acros Organics, Geel, Belgium) was complexed with GcMAF in accordance with the molecular structures and modelling already described (15). The optimal conditions for the preparation of the complexes were established according to described principles (25).

Due to the complexing of the protein with the fatty acid hydrophobic moiety and in view of the well-known properties of OA as an absorption enhancer, OA-GcMAF complexes could be administered sublingually (26), as an aerosol with a common nebuliser (27), as suppositories (28) or transdermally (29).

 

Assessment of OA-GcMAF-induced immunotherapeutic effects. Macrophage activation in vivo was assessed by monitoring the patients blood pressure and the splenic blood flow before and after OA-GcMAF administration. Thus, it is well known that activated macrophages release NO, a compound that causes vasodilatation and seems to be responsible for some of the anticancer properties. The administration of OA-GcMAF (440-880 ng dissolved in 5 ml saline) with a nebuliser, resulted in rapid decrease of blood pressure with the effects clearly appreciable

1 min after the end of the nebulisation. In order to have another assessment of NO production and immune stimulation, the splenic blood flow was monitored with an ultrasound system (MyLab25Gold, Esaote, Genoa, Italy) using the echo-colour-Doppler technique.

 

Integrative cancer immunotherapy with OA-GcMAF. The standard protocol of the Immuno Biotech Treatment Centre is as follows: OA-GcMAF (880 to 2,000 ng according to the patients needs) is administered daily using the route of administration that is most suitable for each patient For example, in patients with lung cancer or metastases, administration of OA-GcMAF with a nebuliser is preferred. In patients with liver cancer or metastases, administration through suppositories may be used. In other cases, the intramuscular route, as originally proposed by Yamamoto et al. (8), is used.

 

Other complementary integrative approaches. In order to exploit the known anticancer properties of Vitamin D3, and bearing in mind that GcMAF is a component of the vitamin D axis (15), patients are provided with nutritional supplementation of Vitamin D3, 20.000 IU per day (18) monitoring the blood levels of such a vitamin. Patients are taught to drink at least 2 litres of water (or other liquids such as herbal teas) per day.

Patients are instructed to follow a nutritional regime based on the recent observation demonstrating that a low carbohydrate, high protein diet slows tumour growth and prevents cancer initiation (17).. In order to favour the compliance to this type of diet, patients are provided with food containing only 2% of carbohydrates and a ANTICANCER RESEARCH 34: 3569-3578 (2014) 3570 relatively high protein content (Le Gamberi Foods, Forlì, Italy)In addition, in order to exploit the known anticancer properties of OA contained in extra-virgin olive oil (32), patients are provided with extra-virgin olive oil as a conspicuous portion of their daily fat intake.

Patients weight and muscle mass are constantly monitored, and patients are taught the strategies to decrease the Prognostic Inflammatory Nutritional Index (PINI) score as well as the strategies to avoid Cancer Anorexia Cachexia Syndrome (CACS) according to Fabris et al. (33), is used. The supplementation of aminoacids (Master Aminoacid Pattern, Dr. Reinwald Healthcare gmbh, Schwarzenbruck, Germany) is intended for this scope (34). Also the administration of low-dose acetylsalicylic acid and of extra-virgin olive oil are intended to reduce systemic inflammation and, therefore, to reduce the PINI score (35).

In order to exploit the well-known immune stimulating and anticancer effects of probiotic fermented milk products (36), patients are provided with a probiotic fermented milk product containing colostrum and microorganisms known to produce natural GcMAF from milk and colostrum Gc-globulin during the fermentation process (Bravo Probiotic, Les Alpes, Wellington, New Zealand). Finally, considering the well assessed role of low-dose acetylsalicylic acid in cancer prevention (37), patients are provided with 100 mg of such an active principle per day.

 

Results

 

It is well-assessed that OA-GcMAF stimulates macrophages (15) which in turn release NO (30). Considering the impressive effects of NO in pre-clinical models of cancer where it slows tumour growth and enhances the efficacy of both chemotherapy and radiotherapy (38), as a first step we sought to determine whether OA-GcMAF stimulated the release of NO from macrophages in vivo. To this end, we chose to study the variation of systolic and diastolic blood pressure and of the splenic blood flow, following administration of OA-GcMAF (880 ng in 5 ml saline) by nebulisation. According to our hypothesis, OA-GcMAF would stimulate alveolar macrophages that in turn would produce NO, thus lowering blood pressure. OA-GcMAF would then be absorbed into the bloodstream and it would eventually stimulate macrophages in other areas of the body. Considering the huge amount of macrophages residing in the spleen (39), we would expect a massive release of NO by activated macrophages in this organ, with a concomitant increase of blood flow to be measured easily by echo-colour-Doppler.

A subject with mild immunodeficiency due to multiple thyroid nodules (test subject # 1; Figures 1 and 2), self-administered OA-GcMAF (880 ng in 5 ml saline) by nebulisation for about 5 min. About 1 min after the end of nebulisation, blood pressure, taken while the subject was comfortably seated, decreased from 124/85 to 115/74. A qualitatively similar effect was observed in the majority of the subjects who were administered OA-GcMAF by nebulisation, and the average decrease was in the order of 10 mm Hg. We interpreted these results as indirect evidence of NO production by OA-GcMAF-activated alveolar macrophages. In fact, it is well-assessed that NO decreases blood pressure in humans as well as in experimental animal systems (40, 41). Figure 3, shows the ultrasonographic appearance of the spleen of test subject #1 prior to nebulisation; spleen morphology, size and ultrasonographic structure appear normal. The splenic blood flow could be appreciated in correspondence of the hilum of the spleen. Little or no signal could be observed in the parenchyma of the organ. Figure 4 shows the splenic blood flow 5 min (panel A) and 1 hour after nebulisation (panel B). The increase in peripheral splenic blood flow is evident. We interpreted this increase in parenchymal blood flow as a consequence of intra-splenic macrophage activation with the release of NO that in turn was responsible for hilar and peripheral blood vessel dilation. This effect lasted for at least 48 hours. Figure 5, depicts the splenic blood flow 24 hours (panel A) and 48 hours (panel B) after nebulisation. Notice that test subject #1, unlike bona fide patients, did not undergo any other treatment, neither GcMAF-related nor associated with the other complementary strategies described above.

The reliability and reproducibility of the method was assessed based on the patients responsiveness to OA-GcMAF administration. A similar effect, although much slower in its development, was observed in patients where OA-GcMAF was administered by localised subcutaneous injections. Figure 6 shows the increase in splenic blood flow observed after 7 days of treatment in a patient with metastatic breast cancer and retrosternal node involvement, where the OA-GcMAF (1.320 ng/day) was administered by localised subcutaneous injections in the abdominal wall (epigastrium, 880 ng) and the axilla (440 ng).

Activation of alveolar and splenic macrophages by OAGcMAF administration with consequent release of NO was associated with significant decrease in tumour volume in all cases where the primary tumour, metastases or lymphnodes could be measured by ultrasonographic techniques. On average, we observed a decrease of tumour volume of about 25% in a week (14). Although this reduction may appear dramatic, it is fully consistent with the results reported by Nonaka et al. (42) who observed a 97% volume reduction of human hepatocellular carcinoma after 3 weeks of subcutaneous injection with GcMAF. It is also consistent with the results reported for neo-adjuvant chemotherapy (43). These results add support to the hypothesis that OA, GcMAF and NO, molecules endowed with individual anticancer properties targeting different aspects of neoplastic growth, provide an indication for a synergistic effect when administered in such a way to exploit their physiological characteristics.

Among the cases observed at the Immuno Biotech Treatment Centre, we present the cases of two patients, each representative of common cancers, for whom the integrative immunotherapy based on OA, GcMAF and NO, was

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Figure 1. Ultrasonographic findings of test subject #1: thyroid ultrasonography, axial scan of the lower right lobe. A. Lightly-protruding solid nodule of 1.17 cm in the anterior part of the lower lobe. The internal ultrasonographic structure is finely inhomogeneous and hypo-reflecting in comparison with the surrounding normal thyroid tissue. The margins are well-defined. B. Colour-Doppler ultrasonography of the same nodule. The blood vessels are located around the solid nodule in a typical “basket-like appearance. C. A smaller, prevalently liquid (hypo-reflecting), lesion (0.7 cm) with another small solid nodule in the vicinity (white arrow). Inside the hypo-reflecting lesion, an area of protruding solid tissue can be noticed. D. Colour-Doppler ultrasonography of the same lesion. The blood vessels are located around the two lesions

 

 

remarkably effective. To our knowledge, this is one of the few examples of actual images of tumour volume reduction following GcMAF immunotherapy. Thus, so far the effectiveness of immunotherapy of cancer with GcMAF has relied upon non-specific markers such as serum α-Nacetylgalactosaminidase levels (8-10, 12) or anecdotal reports (11, 13).

 

Patient 1. A 56-year-old man was diagnosed with metastatic squamous cell carcinoma, previously treated with several cycles of radiation therapy and chemotherapy. After a period of remission, several new metastases were detected, including one in the soft tissues of the right abdominal wall. In addition, the patient showed difficulty in breathing, probably because of bronchi-obstructing metastases that prevented ventilation of his apical right lobe. Because of these symptoms, OA-GcMAF (880 ng/day) was administered with a nebuliser containing 440 ng of the active principle dissolved in 5 ml of saline, and with localised subcutaneous injections (440 ng) in the right abdominal wall in proximity of the subcutaneous lesion indicated by the patient and easily recognisable by ultrasonography. The patient did not report any adverse effects following these ways of administration that had been chosen with his informed consent. Ultrasonography of the lesion in the right abdominal wall was performed to assess modifications.

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Figure 2. Ultrasonographic findings of test subject #1: thyroid ultrasonography, longitudinal scan of the lower left lobe. A. A massive mixed (solid and cystic) protruding nodule substituting for most of the lobe can be observed. The anterior-posterior diameter is about 3 cm, whereas the longitudinal diameter was greater than the length of the probe (4 cm). The structure is grossly inhomogeneous with cystic areas and hypo-reflecting areas alternated with iso- or hyper-reflecting areas. The margins bordering the residual normal thyroid tissue observable on the right of the figure (white arrow, cranial portion of the left lobe) appear well defined. The hypo-reflecting border might indicate an area of oedema surrounding the nodule. B. Colour-Doppler ultrasonography of the same nodule. The blood vessels are located around and inside the nodule. It is worth noticing that few or no blood vessels can be observed in the hypo-reflecting areas (indicated by the dotted white circles) suggesting the presence of metabolically inactive tumour tissue.

 

 

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Figure 3. Ultrasonographic appearance of the spleen of test subject #1 before OA-GcMAF administration. Panel A: Morphology, size and ultrasonographic structure of the spleen appear normal. Panel B: The splenic blood flow could be appreciated in correspondence of the hilum of the spleen. Little or no signal could be observed in the parenchyma of the organ.

 

 

Bearing in mind that measurements taken on ultrasonographic images may be affected by a number of variables, preliminary evidence appears to indicate that this lesions, taken as representative, showed a reduction of its calculated volume from 0.30 to 0.22 ml (possibly 27%) after 5 days of treatment (Figure 7). The patient reported a subjective improvement of breathing and ventilation in the apical right lobe was improved as well.

 

Patient 2.A 62-year-old woman was diagnosed with extensive breast cancer with widespread axillar lymph node involvement. The patient had elected not to undergo surgery or any other conventional treatment since the initial diagnosis. Because the size of the primary tumour was larger than the probe (4 cm), we opted for measuring the coalescent axillary nodes in order to monitor a response to OA-GcMAF. OA-GcMAF (880 ng/day) was administered with a nebuliser

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Figure 4. Immediate increase of splenic blood flow following administration of OA-GcMAF by nebulisation in test subject #1. Splenic blood flow was assessed by an echo-colour-Doppler ultrasonographic technique. Panel A: Splenic blood flow 5 min after the end of nebulisation with OA-GcMAF. Panel B: Splenic blood flow 1 hour after the end of nebulisation. The increase in peripheral splenic blood flow is evident.

 

 

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Figure 5. Sustained increase of splenic blood flow following administration of OA-GcMAF by nebulisation in test subject #1. Splenic blood flow was assessed by echo-colour-doppler ultrasonographic technique after administration of OA-GcMAF. Panel A: Splenic blood flow 24 hours after the end of nebulisation. Panel B: Splenic blood flow 48 hours after the end of nebulisation. The increase in parenchymal blood flow appears to be persistent.

 

 

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Figure 6. Increase of splenic blood flow following administration of OA-GcMAF by localised subcutaneous injection in a cancer patient. Splenic blood flow was assessed after administration of OA-GcMAF by an echo-colour-Doppler ultrasonographic technique. Panel A: Splenic blood before administration. Panel B: Splenic blood flow after 7 days of administration. The increase in parenchymal blood flow is evident.

 

 

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Figure 7. Ultrasonography of soft tissue metastasis before and after OA-GcMAF administration. Patient 1. A 56-year-old man was diagnosed with metastatic squamous cell carcinoma, previously treated with several cycles of radiation therapy and chemotherapy. After a period of remission, several new metastases were detected, including one in the soft tissues of the right abdominal wall. Panel A: Ultrasonographic appearance of the lesion before OA-GcMAF administration. Panel B: Ultrasonographic appearance of the lesion after 4 consecutive days of OA-GcMAF administration. An approximate 27% volume reduction can be appreciated.

 

 

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Figure 8. Ultrasonography of the axillary lymph nodes with cancer before and after administration of OA-GcMAF. Patient #2: A woman of 62 years was diagnosed with breast cancer extensively spread in the axillary lymph nodes. The coalescing ipsilateral axillary nodes were measured. After 4 consecutive days of OA-GcMAF treatment, the diameter of the two axillary coalescent nodes was reduced from 3.90 (panel A, that is, before treatment) to 3.46 cm (panel B, that is, after 4 days of treatment). In terms of the theoretical volume calculated, it was assumed that the lesion was spherical and this corresponds to a reduction of about 28%.

 

 

containing 440 ng of active ingredient dissolved in 5 ml of saline solution and localised subcutaneous injections (440 ng) in the armpit, 1 cm distal to the nodules identified via ultrasonography. The patient did not report any adverse effects due to these routes of administration which were chosen with her informed consent. Figure 8 shows that after 4 days of treatment the diameter of the two coalescent axillary nodes was reduced from 3.90 to 3.46 cm. In terms of the theoretical volume calculated, it was assumed that the lesion was spherical and this corresponds to a reduction of about 28%.

 

Discussion

 

OA, GcMAF and nitric oxide molecules are equipped with multiple biological effects that can be exploited in the strategies of cancer treatment. The effect of selective tumour killer complexes of OA protein such as HAMLET is attributed to OA which, when delivered into tumour cells because of its association with proteins, activates the apoptotic metabolism of tumour cells. These include interference with the expression of oncogenes (Ras, erbB-2 and c-Myc) and with the glycolytic (2, 4) enzyme. In this scenario, the protein is primarily, if not solely, a vehicle for transporting OA within the cancer cell, where the fatty acid can target the cytoplasmic membrane, cytoskeleton, mitochondria, proteasomes, lysosomes and the nucleus (4). In addition, a recent study has shown that the biological activities of the OA-α-lactalbumin complex reside in the fatty acid. Moreover, the α-lactalbumin group does not have a lethal effect on the tumour itself, but simply acts as a solubilising agent for OA (44).

Therefore, the synthesis of the OA-GcMAF complex could be an advantage of HAMLET, because GcMAF, unlike α - lactalbumin, has a direct effect on cancer cells. In particular, thanks to our work and that of other groups, it was shown that GcMAF inhibits proliferation and metastatic potential of cancer cells (24, 45). In other words, the OA-GcMAF complex could exploit the anticancer properties of OA and GcMAF, possibly synergistically. In this scenario, OA would favour the binding of the complex to the cytoplasmic membrane, as already demonstrated (15) and the internalisation of the multi-molecular complex of (VDR) OA-GcMAF-vitamin D receptor. Once inside the cell, and OA GcMAF would steer their respective pathways, signalling and inducing rapid apoptosis of cancer cells.

In fact, we have recently observed that the OA-GcMAF complex is about 200 times more powerful than GcMAF alone in the induction of apoptosis of in-vitro breast cancer cell (Thyer L, et al., Personal communication test data submitted to the XVIII International Meeting of the European Society of Gynecologic Oncology, Liverpool, United Kingdom, October 2013), probably because of this synergistic effect.

If this hypothesis is correct, it could help explain the direct effects of the OA-GcMAF complexes on cancer cells; the stimulatory effect of GcMAF on macrophages may be responsible for the synthesis and release of a third anti-cancer molecule, i.e. nitric oxide. Thus, we have shown that the effects of GcMAF are mediated by the VDR (15, 46), and VDR activation leads to the synthesis and release of nitric oxide by macrophages (47). In our observation, the synthesis and release of nitric oxide after administration of OA-GcMAF could be attributed to the decrease in blood pressure and increase in blood flow to the spleen (see "Results").

The anti tumour effects of nitric oxide have been known for many years (30) and it seems that OA and nitric oxide share the common goal of destroying tumour cells. Thus, nitric oxide suppresses cellular respiration and DNA synthesis and induces apoptosis of cancer cells by activating the caspase family of proteases, a feature which is common with OA (2, 48). More interesting from the point of view of the clinical effects are convergence of OA and nitric oxide on the glycolytic path of cancer cells (49, 2).

In agreement with the hypothesis of glycolysis as a primary target of OA and no anti-tumour effects, it has been shown that the deprivation of glucose sensitises tumour cells to cell death induced by HAMLET (50). In fact, this observation was the base of one of the principles of integrative cancer treatment described above, which is a nutritional plan low in carbohydrates. In fact, we have reflected on the fact that a diet low in carbohydrates not only deprives the cancer cells of their only source of energy (17), but also makes them more sensitive to the anticancer effects of OA and nitric oxide.

In conclusion, the observations presented in this study, along with the results of recent molecular modelling (15), show that OA, GcMAF and nitric oxide, individual molecules with anticancer properties, can be successfully combined and specifically administered to patients with significantly advanced cancer to study the effects on the stimulation of the immune system and reduction in tumour volume. In fact, with the exception of 40 patients accepted as per the criteria of Immuno Biotech Treatment Centre, mostly with advanced stage 4 tumours, all other patients showed significant comparable clinical improvements. These observations may revolutionise the field of cancer treatment, as it appears that the proposed anti-cancer strategy is completely free of any harmful side effects.

 

Potential conflicts of interest

 

DN is the CEO of Immuno Biotech Ltd (the company which isolates and purifies the GcMAF protein). However, DN has no knowledge of the therapies in use, nor the names of all the patients whose data was analysed. Neither the CEO nor any employee of Immuno Biotech Ltd, had any knowledge of the clinical records and/or patient names in this study. MR is the chief scientific adviser of Immuno Biotech Ltd. and is a professor of molecular biology that has contributed to the interpretation of molecular modelling and the described results.

 

Awards

 

Stefania Pacini has received grants from the University of Florence and the PRIN 2009 project.

 

Reference

 

(See the References in the original English text.)

 

Received: 23 April 2014

Revised: 26 May 2014

Accepted: 27 May 2014

IXth International Conference of Anticancer Research

Translated text:

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