We found that a moderate DR of 30–40% significantly reduced angiogenesis and growth of the CT-2A experimental mouse astrocytoma. Moreover, DR enhanced CT-2A cell apoptosis without effecting cell proliferation. Previous studies showed that moderate DR could reduce the growth of histologically diverse non-neural tumours (Rous, 1914
; Tannenbaum, 1959
; Kritchevsky, 1999a
; Mukherjee et al, 1999a
). Our studies are the first to document this phenomenon in a brain tumour model and suggest that brain tumours may be especially vulnerable to the growth-inhibitory effects of DR. It will be important to document the extent to which DR reduces angiogenesis and growth in other brain tumour models.
Despite a 12% reduction in body weight, the DR-fed mice were more active and healthy than the AL fed mice. Keenan and co-workers recently suggested that the AL feeding of sedentary rodents is a form of over feeding that can produce adverse health effects (Keenan et al, 1999
). Our results support this contention since CT-2A tumour angiogenesis and growth was significantly greater in mice under AL feeding than under DR.
We found that angiogenic biomarkers may be useful for evaluating the influence of energy intake and nutrition on the growth and progression of experimental brain cancer. Moderate DR significantly reduced microvessel density, increased the apoptotic index, but had little effect on the PCNA labelling index in the CT-2A brain tumour. Other investigators have also reported that antiangiogenic growth factors and cytokines can reduce tumour microvessel density, increase apoptosis, but have little effect on cell proliferation (Holmgren et al, 1995
; O'Reilly et al, 1996
; Tanaka et al, 1997
; Beecken et al, 2001
). Our results therefore support previous findings that DR produces a pattern of biomarker changes similar to the changes seen following the implementation of antiangiogenic therapies (Mukherjee et al, 1999a
The mechanisms by which DR reduced CT-2A tumour angiogenesis and growth are not yet clear, but may involve effects on both the tumour cells and the tumour-associated host cells. It is documented that human and experimental gliomas are dependent on glycolysis for energy (Mies et al, 1990
; Ikezaki et al, 1992
; Oudard et al, 1997
), and that DR-induced caloric restriction reduces glycolytic energy and down-regulates glycolytic gene expression (Lee et al, 2000
; Cao et al, 2001
; Greene et al, 2001
). Additionally, the DR-induced down regulation of glycolysis should also reduce the level of pyruvic acid, a glycolytic end product with angiogenic activity (Lee et al, 2001
Glucose is used exclusively for adult brain energy metabolism under normal physiological conditions, but the brain will metabolise ketone bodies for energy when blood glucose levels decrease as during fasting or DR (Clarke and Sokoloff, 1999
; Greene et al, 2001
). Since ketone bodies are metabolised directly to acetyl-CoA in the mitochondria, they bypass cytoplasmic glycolysis and provide energy directly through the Krebs cycle (Nehlig and Pereira de Vasconcelos, 1993
; Clarke and Sokoloff, 1999
). We recently showed that DR produces ketosis in epileptic mice and that the degree of ketosis is inversely proportional to blood glucose levels (Greene et al, 2001
). Further studies will be needed to determine if reduced glycolytic energy and elevated ketosis underlie the antiangiogenic and growth inhibitory effects of DR.
In addition to possible effects on energy metabolism, DR may also reduce CT-2A angiogenesis and growth through effects on tumour associated host cells. The progression of human and experimental brain tumours is dependent to a large extent on the proangiogenic and inflammatory properties of activated glia and macrophages (Seyfried, 2001
). Indeed, the degree of tumour angiogenesis and malignancy is generally correlated with the number and activation state of tumour-associated macrophages and microglia (Wood and Morantz, 1979
; Roggendorf et al, 1996
; Nishie et al, 1999
; Polverini, 1999
; Badie and Schartner, 2000
). Recent studies also indicate that moderate DR reduces brain inflammation associated with ageing and neurodegeneration (Duan et al, 2001
; Lee et al, 2000
). Furthermore, dietary energy restriction can elevate glucocorticoid hormone that could further reduce tumour inflammation and growth through down regulation of stress-activated protein kinase pathways (Birt et al, 1999
). Hence, DR may reduce CT-2A progression through a global down-regulation of inflammatory and angiogenic properties of the tumour microenvironment.
We also found that DR caused a noticeable reduction in the number and the dilation of blood vessels in the in vivo Matrigel model of angiogenesis indicating that DR can reduce angiogenesis both within and outside of the central nervous system. It is possible that DR reduces the inflammatory properties of tumour-associated host cells and thereby shifts tumour-host cell interactions from a proangiogenic to an antiangiogenic state. Studies are planned to test these possibilities.
Our findings may have relevance to those in vivo
studies where food intake and body weight are reduced in conjunction with anticancer therapies or with cancer cachexia. Reduction of energy intake as a covariable of anorexic anticancer therapies may confound interpretation of results (Ranes et al, 2001
). It would be important therefore to control for the antitumour effects of dietary reduction in the preclinical evaluation of new cancer drugs. Weight loss associated with cancer cachexia differs from weight loss associated with anorexia (reduction in food intake) since cachexia can occur without anorexia and is produced from factors released by the tumour (Tisdale, 2001
). Although appearing counterintuitive, we suggest that DR may antagonise cachexia by reducing tumour size and therby reducing levels of procachexic factors.
Although DR is recognised as a preventative measure for carcinogenisis, it is clear from our findings on the the CT-2A brain tumour that DR is not a preventative intervention since all of the tumours implanted grew despite the 7 day DR pretreatment period. The DR-induced inhibition of CT-2A angiogenesis and growth suggests that DR retards tumour progression. Whether DR would also increase the survival time of CT-2A-tumour bearing mice is not clear. Survival studies are difficult with this rapidly growing brain tumour model since the tumour will grow through the implantation burr hole and then subcutaneously over the skull as we previously described for the EPEN model (Seyfried et al, 1987
). This relieves intracranial pressure and artificially extends longevity. In humans with malignant brain tumours, it is the intracranial pressure that usually leads to morbidity.
In summary, we have demonstrated that DR alone can reduce angiogenesis and growth in an experimental mouse brain tumour. Moreover, the antitumour action of DR likely operates through multiple effects on the tumour cells and on the tumour associated host cells. We contend that our experimental protocol may have therapeutic potential for recurrent human gliomas since the time of surgical tumour resection in humans would be comparable to the time of tumour transplantation in mice. In other words, implementation of DR in the clinic could be most effective immediately following tumour removal and may delay tumour recurrence. Because DR is easy to administer and is devoid of adverse side effects, our preclinical studies suggest that DR or caloric restriction may have efficacy as a non-invasive therapy for recurrent malignant brain cancers.