Since the initial discoveries in the 1940s (38
), CR has been well recognized for its ability to reduce the incidence and attenuate the growth of several types of tumors. In fact, CR is the most widely studied and most potent, broadly acting dietary intervention for preventing carcinogenesis in a variety of experimental models (8
). Much evidence supports the link between CR, reduced levels of oxidative stress, and inhibition of carcinogenesis, suggesting that reduced oxidative stress could be a major contributor to CR's anti-tumor effects (19
). However, the effects of CR on brain tumors and on their underlying regulatory mechanisms remain unclear.
Because of the lack of in vivo
studies investigating the role of oxidative stress and CR in brain tumors, to our knowledge this study is the first to demonstrate the overall effects of CR on oxidative stress and brain tumors in their natural state. Of particular importance is that our study makes use of the ENU-induced glioma model, a well-established in vivo
model that has many advantages: (1) an extremely high rate (100%) of multiple tumor induction per brain; (2) continuous profile and time course of tumor progression have been well documented; (3) immature glia are initiated simultaneously in utero
, allowing for evaluation of CR's effect on tumor promotion/progression by comparing tumor number/size; (4) previous studies suggest a possible role for oxidative stress in nitrosoamine-induced carcinogenesis; and (5) the brain consumes approximately 10% of the total oxygen required by the body, yet the effect of this high oxygen consumption on oxidative stress and brain tumor formation has not been extensively examined. Using this well-suited in vivo
brain tumor model (24
), our experiments demonstrated that CR reduces the incidence and growth of gliomas and that CR's anti-tumor effects are associated with reduced levels of oxidative stress, reduced cell proliferation, increased apoptosis, decreased formation of glycated end products, decreased presence of HO-1 and Trx1, and decreased levels of HIF-1α in brain tissues.
The development of tumors, especially chemically induced tumors, is considered a multistage process that can be divided into three distinct stages, i.e. initiation, promotion, and progression (49
). In the ENU-induced glioma model, initiation of tumors is controlled by the injection of ENU at day 15 of gestation, but monitoring the incidence and growth of tumors over time can provide insight into the effects of CR on promotion and progression. Thus, the observed reduction in the number and size of tumors in CR rats compared to AL rats at 8 months of age suggests that CR may intervene in both the promotion and progression of carcinogenesis in ENU-induced brain tumors, as it appears to attenuate the growth of existing tumors as well as prolong or prevent tumor onset.
A large number of studies indicate that chronic disease states, including cancer, are associated with increased oxidative stress (50
). Oxidative stress causes damage to critical cellular biomacromolecules including lipids, proteins, and DNA, which in many cases is shown to be mutagenic. Thus, documenting the accumulation of oxidized biomacromolecules provides an index of cellular oxidative damage that may contribute to malignant cellular transformation (18
). Because CR is shown to suppress oxidative stress and thereby reduce oxidative damage to various biomacromolecules (54
), CR's anti-tumor effects may be a result of decreased accumulation of oxidative damage to tissues.
Lipids are particularly vulnerable to free radical attack because of their relatively high level of unsaturation compared with other biological molecules. In addition, lipid peroxidation products have a relatively long half-life and can diffuse through membranes, making them capable of damaging other biomacromolecules and affecting cellular homeostasis (55
). MDA and HNE are major lipid peroxidation products, and HNE is highly toxic and readily reacts with critical biomolecules (56
). The presence of MDA and HNE observed in ENU-induced brain tumors indicates that oxidative damage involved the lipid component of tumor tissue, and that the extent of lipid damage appeared to decrease with attenuated tumor growth in CR rats.
Protein oxidation could also play important roles in the pathophysiology of carcinogenesis. Oxidation of proteins can cause structural modifications resulting in changes in enzyme activity and signaling pathways. In this study, we assessed oxidative damage to proteins by measuring the presence and accumulation of nitrotyrosine in tumors because it is a major product formed when protein is exposed to reactive nitrogen oxides (57
). In addition, tyrosine nitration can alter protein and enzyme function (58
), thereby potentially affecting cellular homeostasis. The staining pattern of nitrotyrosine was similar to that of the lipid peroxidation products; i.e. there was a significant decrease in nitrotyrosine in the tumors of CR rats that appeared to be associated with a reduction in tumor size and number. Oxidative damage of proteins and lipids appears to be concurrent, suggesting that CR's anti-tumor effects may be associated with decreased oxidative damage to both lipids and proteins. Thus, reduced oxidative stress may be a potential underlying mechanism of CR's anti-tumor effects; however, the downstream consequences of reduced oxidative stress, e.g. redox sensitive signaling pathways, remain to be examined.
We also used immunohistochemistry to determine the distribution of HO-1, one of two isoforms of heme oxygenase, an enzyme that is well known to be responsive to cellular stress levels. HO-1 produces biliverdin, which is subsequently converted to bilirubin by the action of biliverdin reductase, and both biliverdin and bilirubin are potent antioxidants that may contribute to protection against oxidative stress (60
). Substantial evidence indicates that many cancers overexpress HO-1 (63
) and that it may be beneficial for tumor growth because it promotes angiogenesis (64
) and limits apoptosis (62
). Another protein that protects cells through its antioxidant properties is thioredoxin (Trx). Trx is overexpressed in many types of cancer and has been linked to cancer cell proliferation rates and cancer progression (65
). Like HO-1, Trx provides antioxidant protection to cells, but it does so by acting as a hydrogen donor for enzymes involved in reductive reactions and providing protection against oxidative stress (66
). Trx also functions as a physiological inhibitor of apoptosis signal-regulating kinase 1 (ASK1) (67
). Tumors of CR rats showed a significant decrease in the number of PCNA positive cells and an increase in ssDNA compared to tumors of AL rats at 8 months of age. Tumors of CR rats also showed a significant decrease in HO-1 and Trx levels compared to tumor tissues from the brains of AL rats. Thus, reduced levels of HO-1 and Trx in the tumors of CR rats may have contributed to the reduction in cell proliferation and the increase in apoptosis, which could attenuate tumor growth.
We also examined the effects of CR on glycation and the formation of advanced glycation end products (AGEs) in tumors. AGEs have been implicated in a number of diseases (69
), and the majority of work has shown AGEs to be related to oxidative stress (70
). We measured MG (35
), an intermediate formed during AGE production, and CML (36
), an advanced glycation end product, as indices of protein glycation and AGE formation in ENU-induced brain tumors. We found that MG and CML levels were significantly lower in the tumors of CR rats compared to AL rats, suggesting that brain tumors of CR rats were subject to decreased AGE formation and a decrease in the associated production of reactive oxygen intermediates, which could be important in limiting tumor development.
Hypoxia-inducible factor-1 is a heterodimeric protein that consists of two proteins, one being HIF-1α. HIF-1α has been significantly associated with cancer (72
), and it activates the transcription of various genes that are involved in carcinogenesis, including those regulating angiogenesis, cell proliferation, and metastasis (72
). Because it is redox-sensitive, we believed that HIF-1α might be related to oxidative stress in tumorigenesis. ENU-treated brain tissues of CR rats showed a significant decrease in the levels of HIF-1α that was similar to the decrease in oxidative stress exhibited in tumors of CR rats. This suggests that CR decreases HIF-1α levels, but the effects of CR and oxidative stress on the regulation of HIF-1α have yet to be defined. It is also important to note that because HIF-1α regulates angiogenesis, proliferation, and metastasis, which are essential for tumor growth, decreasing HIF-1α activity could be a direct way of limiting tumor growth. Thus, continuing to investigate the effects of CR on HIF-1α levels in brain tumors is of clear importance.
Using the ENU-induced glioma model, this series of experiments demonstrated that the anti-tumor effects of CR were associated with decreased oxidative stress, decreased glycation and AGE formation, decreased presence of HO-1 and Trx1, decreased cell proliferation, increased apoptosis, and decreased levels of HIF-1α activity. Because CR has been shown to reduce levels of growth factors, such as IGF-I, reduced levels of growth factors and/or energy intake could also play important roles in CR's anti-tumor effects (75
). However, we believe reduced oxidative stress could play more important roles in the anti-tumor effects of CR because rats on CR diets typically exhibit food intake per gram of body weight as well as a metabolic rate per lean body mass similar to AL rats (76
). Based on our results, the ENU-induced glioma model would appear to be an excellent experimental model to continue to investigate the effects of CR and the role of oxidative stress and its underlying mechanisms in the development and pathophysiology of gliomas. Further exploration of CR's effects on cellular processes, such as changes in the intracellular signal transduction system and in proto-oncogenes, could provide more insight into the pathophysiology and development of a therapeutic treatment to attenuate the growth and development of gliomas.