2005] Dietary restriction reduces angiogenesis and growth in an orthotopic mouse brain tumour model. Dietary restriction reduces angiogenesis and growth in an orthotopic mouse brain tumour model. Dietary restriction reduces angiogenesis and growth in an orthotopic mouse brain tumour model. Role of acetoacetyl-CoA synthetase in acetoacetate utilization by tumor cells. Cancer Biochem Biophys.

Many current therapies for malignant brain tumors fail to provide long-term management because they ineffectively target tumor cells while negatively impacting the health and vitality of normal brain cells. In contrast to brain tumor cells, which lack metabolic flexibility and are largely dependent on glucose for growth and survival, normal brain cells can metabolize both glucose and ketone bodies for energy. Adult mice were implanted orthotopically with the malignant brain tumors and KetoCal&x000ae; was administered to the mice in either unrestricted amounts or in restricted amounts to reduce total caloric intake according to the manufacturers recommendation for children with refractory epilepsy.

KetoCal&x000ae; administered in restricted amounts significantly decreased the intracerebral growth of the CT-2A and U87-MG tumors by about 65% and 35%, respectively, and significantly enhanced health and survival relative to that of the control groups receiving the standard low fat/high carbohydrate diet. The restricted KetoCal&x000ae; diet reduced plasma glucose levels while elevating plasma ketone body (&x003b2;-hydroxybutyrate) levels.

Tumor microvessel density was less in the calorically restricted KetoCal&x000ae; groups than in the calorically unrestricted control groups. The results indicate that KetoCal&x000ae; has anti-tumor and anti-angiogenic effects in experimental mouse and human brain tumors when administered in restricted amounts. The therapeutic effect of KetoCal&x000ae; for brain cancer management was due largely to the reduction of total caloric content, which reduces circulating glucose required for rapid tumor growth.

A dependency on glucose for energy together with defects in ketone body metabolism largely account for why the brain tumors grow minimally on either a ketogenic-restricted diet or on a standard-restricted diet. Genes for ketone body metabolism should be useful for screening brain tumors that could be targeted with calorically restricted high fat/low carbohydrate ketogenic diets.

Note. References. Background Malignant brain cancer persists as a major disease of morbidity and mortality in adults and is the second leading cause of cancer death in children [ 1 | 4 ]. Many current therapies for malignant brain tumors are ineffective in providing long-term management because they focus on the defects of the tumor cells at the expense of the health and vitality of normal brain cells [ 5 |

7 ]. We previously showed that caloric restriction (CR) is anti-angiogenic, anti-inflammatory, and pro-apoptotic in the experimental mouse (CT-2A astrocytoma) and the human (U87-MG malignant glioma) brain tumors [ 8 | 11 ]. CR targets tumor cells by reducing circulating glucose levels and glycolysis, which tumor cells need for survival, and by elevating ketone bodies, which provide normal brain cells with an alternative fuel to glucose [ 5 , 9 , 11 ].

We previously used linear regression analysis to show that blood glucose levels could predict CT-2A growth as well as insulin-like growth factor 1 (IGF-1) levels, which influences tumor angiogenesis [ 8 , 9 ].

21 ]. These enzymes become critical when neurons and glia transition to ketone bodies in order to maintain energy balance under conditions of reduced glucose availability.

Mice Male mice (10&x02013;12 weeks of age) were used for the studies and were provided with food under either restricted or unrestricted conditions (as below).

The syngeneic mouse brain tumor CT-2A, was originally produced by implantation of a chemical carcinogen, 20-methylcholanthrene, into the cerebral cortex of B6 mice and was characterized as an anaplastic astrocytoma [ 37 , 38 ].

Initiation of tumors from intact tumor pieces is preferable to initiation from cultured cells, since the tumor tissue contains an already established microenvironment that facilitates rapid tumor growth [ 41 ]. The flanks of recipient mice were shaved in order to facilitate tumor detection and growth assessment. The mice were group housed prior to the initiation of the experiment and were then individually housed in plastic shoebox cages one day before tumor implantation.

All mice received PROLAB chow (Agway Inc., NY) prior to the experiment. This is a standard high carbohydrate mouse chow diet (SD) and contains a balance of mouse nutritional ingredients. According to the manufacturers specification, this diet delivers 4.4 Kcal/g gross energy, where fat, carbohydrate, protein, and fiber comprised 55 g, 520 g, 225 g, and 45 g/Kg of the diet, respectively. The KetoCal&x000ae; ketogenic diet was obtained as a gift from Nutricia North America (Rockville, MD, formally SHS International, Inc.).

KetoCal&x000ae; is a nutritionally complete ketogenic formula and, according to the manufacturers specification, delivers 7.2 Kcal/g gross energy where fat, carbohydrate, protein, and fiber comprised 720 g, 30 g, 150 g, and 0 g/Kg of the diet, respectively. There are also minor differences between the two diets for the content (g/kg of diet) of amino acids, vitamins, minerals and trace elements.

The diet has a ketogenic ratio (fats: proteins + carbohydrates) of 4:1 and the fat was derived from soybean-oil. The KetoCal&x000ae; diet was fed to the mice in paste form (water: KetoCal&x000ae;; 1:2) within the cage using procedures as we previously described [ 18 ]. A comparison of the nutritional composition of the SD and the KetoCal&x000ae; diet is presented in Table 1 | The animal room was maintained at 22 &x000b1; 1&x000b0;C, and cotton nesting pads were provided for additional warmth.

All intracerebral (i.c.) tumor-bearing B6 and SCID mice were maintained on the SD for three days following tumor implantation (day 0, Figure &x200B;Figure1) 1 ) and were then randomly assigned (arrow, Figure &x200B;Figure1) 1 ) to one of three diet groups that received either: 1) the standard diet fed ad libitum, or unrestricted (SD-UR), 2) the KetoCal&x000ae; diet fed ad libitum, or unrestricted (KC-UR), 3) the KC diet restricted to reduce body weight by approximately 20% of the original body weight at day 3 (KC-R).

The dietary treatments were continued for 8 days for tumor weight analysis. For the survival analysis, the dietary treatment was initiated 7 days after subcutaneous implantation and continued until the tumors reached 2.5 cm3| Tumor growth and survival analysis Intracerebral tumor growth was analyzed directly by measuring total tumor wet weight. Tumors were dissected from normal appearing brain tissue and were weighed.

Tumor samples were fixed in 10% neutral buffered formalin (Sigma, St. Louis, MO) and embedded in paraffin.

. Tumor sections were incubated with trypsin at 37&x000b0;C for 30 min after deparaffinization, rehydration, and washing as we described recently [ 10 , 11 ]. Briefly, the sections were quenched with 0.3% H2O2-methanol for 30 min and then blocked with 10% normal goat serum in phosphate buffer plus albumin (PBA) that contained 100 ml of 0.01 M phosphate and 0.9% sodium chloride (pH 7.4) with 1.0 g of bovine serum albumin and 0.1 ml of Tween 20.

The sections were treated with rabbit polyclonal antibody directed against human factor VIII-related antigen (Dako Corp., Carpinteria, CA; 1:100 dilution with PBA) followed by a biotinylated anti-rabbit IgG at 1:100 dilution (Vector Laboratories, Inc., Burlingame, CA). The sections were then treated with avidin-biotin complex followed by 3,3'-diaminobenzidine as substrate for staining according to the manufacturers directions (Vectastain Elite ABC kit; Vector Laboratories, Inc.).

The sections were then rinsed three times with 0.01 M phosphate buffer with 0.9% NaCl. Sections were counterstained with methyl green and mounted. Corresponding tissue sections without primary antibody served as negative controls. Microvessel density was quantified by examining areas of vascular hot spots in high power fields (hpf, 200 &x000d7;) as previously described [ 50 ] with some modifications [ 51 ].

Briefly, sections were scanned at 40 &x000d7; and at 100 &x000d7; for the localization of vascular hot spots.

KetoCal&x000ae; could be administered to patients with brain tumors at medical centers or clinics currently using the ketogenic diet for managing epilepsy. Based on our findings in mice and those of Nebeling and co-workers in humans, initiation of randomized clinical trials are warranted to determine whether KetoCal&x000ae; is effective for the long-term management of malignant brain cancer and possibly other glycolysis dependent cancers [ 78 ].

This is especially pertinent to patients with glioblastoma multiforme, an aggressive brain tumor type for which few effective therapeutic options are available. While KetoCal&x000ae; was formulated for managing childhood seizures, it is likely that new KD formulations can be designed with nutritional and caloric compositions more appropriate for managing brain tumors [ 5 , 78 ].

Lowry JK, Snyder JJ, Lowry PW. Brain tumors in the elderly: recent trends in a Minnesota cohort study. Arch Neurol. 1998;55 :922?928. doi: 10.1001/archneur.55.7.922. [ PubMed ] [ Cross Ref ]. Kaiser J. No meeting of minds on childhood cancer. Science.

. Bai H, Seyfried TN. Influence of ganglioside GM3 and high density lipoprotein (HDL) on the cohesion of mouse brain tumor cells. J Lipid Res. 1997;38 :160?172. [ PubMed ]. Ecsedy JA, Yohe HC, Bergeron AJ, Seyfried TN. Tumor-infiltrating macrophages contribute to the glycosphinglipid composition of brain tumors.

Expression of mouse sialic acid on gangliosides of a human glioma grown as a xenograft in SCID mice. J Neurochem. 1999;73 :254?259. doi: 10.1046/j.1471-4159.1999.0730254.x. [ PubMed ] [ Cross Ref ]. Malik A, Afaq F, Sarfaraz S, Adhami VM, Syed DN, Mukhtar H. Pomegranate fruit juice for chemoprevention and chemotherapy of prostate cancer. Proc Natl Acad Sci USA. 2005;102 :14813?14818. doi: 10.1073/pnas.0505870102. [ PMC free article ] [ PubMed ] [ Cross Ref ].

Tomayko MM, Reynolds CP. Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol.

Br J Cancer. 1983;47 :293?297. [ PMC free article ] [ PubMed ]. Fredericks M, Ramsey RB. 3-Oxo acid coenzyme A transferase activity in brain and tumors of the nervous system. J Neurochem. 1978;31 :1529?1531. doi: 10.1111/j.1471-4159.1978.tb06581.x. [ PubMed ] [ Cross Ref ].

The Metabolism of Tumours.

Implementing a ketogenic diet based on medium-chain triglyceride oil in pediatric patients with cancer. J Am Diet Assoc. 1995;95 :693?697. doi: 10.1016/S0002-8223(95)00189-1. [ PubMed ] [ Cross Ref ]. Stewart JW, Koehler K, Jackson W, Hawley J, Wang W, Au A, Myers R, Birt DF. Prevention of mouse skin tumor promotion by dietary energy restriction requires an intact adrenal gland and glucocorticoid supplementation restores inhibition.

Carcinogenesis. 2005;26 :1077?1084. doi: 10.1093/carcin/bgi051. [ PubMed ] [ Cross Ref ].

2002;23 :707?717. doi: 10.1016/S0197-4580(02)00017-9. [ PubMed ] [ Cross Ref ]. Tisdale MJ, Brennan RA, Fearon KC.

Low-glycemic-index treatment: a liberalized ketogenic diet for treatment of intractable epilepsy.

. Nutr Metab (Lond). 2005 Oct 21; 2():30. [Nutr Metab (Lond). 2005] Profile of daily life in children with brain tumors: an assessment of health-related quality of life. J Clin Oncol. 2005 Aug 20; 23(24):5493-500. [J Clin Oncol. 2005] Role of glucose and ketone bodies in the metabolic control of experimental brain cancer. . Br J Cancer.

Review Ketones: metabolism's ugly duckling. Nutr Rev. 2003 Oct; 61(10):327-41 [Nutr Rev. 2003]. Role of acetoacetyl-CoA synthetase in acetoacetate utilization by tumor cells. Cancer Biochem Biophys. [Cancer Biochem Biophys.

Effects of a ketogenic diet on tumor metabolism and nutritional status in pediatric oncology patients: two case reports. . J Am Coll Nutr. 1995 Apr; 14(2):202-8. [J Am Coll Nutr. 1995]. Role of glucose and ketone bodies in the metabolic control of experimental brain cancer. Br J Cancer. 2003 Oct 6; 89(7):1375-82. [Br J Cancer. 2003]. Caloric restriction in C57BL/6J mice mimics therapeutic fasting in humans.

. Lipids Health Dis. 2006 May 18; 5():13. [Lipids Health Dis. 2006]. The ketogenic diet: an effective medical therapy with side effects. J Child Neurol. 2001 Sep; 16(9):633-5 [J Child Neurol. 2001]. See more articles cited in this paragraph ..

. Mol Chem Neuropathol. 1992 Oct; 17(2):147-67. [Mol Chem Neuropathol.

1993] Long term culture of normal and neoplastic human glia. . Acta Pathol Microbiol Scand. 1968; 74(4):465-86. [Acta Pathol Microbiol Scand. 1968] Expression of mouse sialic acid on gangliosides of a human glioma grown as a xenograft in SCID mice. J Neurochem. 1999 Jul; 73(1):254-9 [J Neurochem. 1999]. See more articles cited in this paragraph N -butyldeoxynojirimycin reduces growth and ganglioside content of experimental mouse brain tumours.

Br J Cancer.

. Nutr Metab (Lond). 2004 Oct 19; 1(1):11. [Nutr Metab (Lond). 2004]. See more articles cited in this paragraph .. Pomegranate fruit juice for chemoprevention and chemotherapy of prostate cancer.

2005] Determination of subcutaneous tumor size in athymic (nude) mice. Cancer Chemother Pharmacol. [Cancer Chemother Pharmacol. 1989]. See more articles cited in this paragraph Enzymic determination of D(-)-beta-hydroxybutyric acid and acetoacetic acid in blood. Biochem J. 1962 Jan; 82():90-6. [Biochem J. 1962]. See more articles cited in this paragraph .. Antiangiogenic and proapoptotic effects of dietary restriction on experimental mouse and human brain tumors.

Clin Cancer Res. 2004 Aug 15; 10(16):5622-9 [Clin Cancer Res. 2004]. Dietary restriction reduces angiogenesis and growth in an orthotopic mouse brain tumour model. Br J Cancer. 2002] Tumor angiogenesis and metastasis--correlation in invasive breast carcinoma. . N Engl J Med. 1991 Jan 3; 324(1):1-8. [N Engl J Med. 1991]. Energy intake and prostate tumor growth, angiogenesis, and vascular endothelial growth factor expression.

J Natl Cancer Inst. 1999 Mar 17; 91(6):512-23 [J Natl Cancer Inst. 1999]. See more articles cited in this paragraph Gene-linked shift in ganglioside distribution influences growth and vascularity in a mouse astrocytoma. J Neurochem. 2006 Sep; 98(6):1973-84 [J Neurochem. 2006]. See more articles cited in this paragraph .. Antiangiogenic and proapoptotic effects of dietary restriction on experimental mouse and human brain tumors.

Clin Cancer Res. 2004 Aug 15; 10(16):5622-9 [Clin Cancer Res. 2004]. Br J Cancer.

2005] Dietary restriction reduces angiogenesis and growth in an orthotopic mouse brain tumour model. [Br J Cancer. Br J Cancer. [Br J Cancer. 2003] Management of multifactorial idiopathic epilepsy in EL mice with caloric restriction and the ketogenic diet: role of glucose and ketone bodies.

Dietary restriction reduces angiogenesis and growth in an orthotopic mouse brain tumour model. Br J Cancer. [Br J Cancer. 2002] Energy intake and prostate tumor growth, angiogenesis, and vascular endothelial growth factor expression.

2007] Antiangiogenic and proapoptotic effects of dietary restriction on experimental mouse and human brain tumors.

Br J Cancer. [Br J Cancer.

Dietary restriction reduces angiogenesis and growth in an orthotopic mouse brain tumour model. [Br J Cancer. 2002]. Targeting energy metabolism in brain cancer: review and hypothesis. . Nutr Metab (Lond). 2005 Oct 21; 2():30. [Nutr Metab (Lond). 2005] Review The therapeutic implications of ketone bodies: the effects of ketone bodies in pathological conditions: ketosis, ketogenic diet, redox states, insulin resistance, and mitochondrial metabolism.

Role of acetoacetyl-CoA synthetase in acetoacetate utilization by tumor cells. Cancer Biochem Biophys. [Cancer Biochem Biophys.

The inhibition of malignant cell growth by ketone bodies.

1979] Ketone-body metabolism in tumour-bearing rats. Biochem J. [Biochem J. 1986].

Late-occurring stroke among long-term survivors of childhood leukemia and brain tumors: a report from the Childhood Cancer Survivor Study. J Clin Oncol.

1995] Early- and late-onset complications of the ketogenic diet for intractable epilepsy.

2004] Prevention of mouse skin tumor promotion by dietary energy restriction requires an intact adrenal gland and glucocorticoid supplementation restores inhibition.

Human Genome. Mouse Genome.