We chose to study the effect of a mixture of nutrients on MNU-induced mammary tumors in the Sprague–Dawley rat model because the histologic structure of mammary gland tumors in this animal closely resembles that of human mammary tumors. Induction of mammary carcinomas by MNU in female rats is one of the most frequently used animal models for the investigation of breast carcinogenesis and mammary tumor treatment [7–9]. In contrast to mouse lesions, which are primarily alveolar, rat mammary tumors are predominantly ductal, as are human ones , and the most highly malignant rat tumors share some features with human intraductal and infiltrating ductal carcinomas . It has been reported that the MNU model has several advantages, such as reliability of tumor induction, organ site specificity, tumor of ductal origin and predominantly carcinomatous histopathologic characterization, and the ability to examine tumor initiation and promotion processes . Generally MNU-induced mammary carcinomas are aggressive and locally invasive.
The results of the present study demonstrate significant inhibition of mammary tumor incidence and multiplicity in Sprague–Dawley female rats by supplementation with 0.5% of the nutrient mixture (which contains ascorbic acid, lysine, proline, and epigallocatechin gallate). Furthermore, rats that consumed the nutrient supplemented diet exhibited decreased growth of MNU-induced mammary tumors and tumor burden compared with rats fed control diets. The numerous tumors in the control rats not only were larger but also had characteristics diagnostic of adenocarcinoma, including increased mitotic index and prominent angiogenesis, in contrast to the few, small adenomas with low mitotic index found in the nutrient treated rats.
Although the mechanism underlying the reduced tumor size in tumor bearing rats was not identified in this experiment, these findings are consistent with our previous in vitro studies that demonstrated significant inhibition of angiogenic and invasive parameters in human breast cancer cell lines MDA MB-231 and MCF-7. Expression of vascular endothelial growth factor, MMP secretion, and matrix invasion by these breast cancer cells were dramatically inhibited in a dose-dependent manner by the combined effect of the nutrients in this mixture . Matrix invasion can be controlled by inhibition of MMP expression, as well as by increasing connective tissue strength and stability, contributing to the 'encapsulation' of the tumor. Optimization of synthesis and structure of collagen fibrils depends upon hydroxylation of proline and lysine residues in collagen fibers. It is well known that ascorbic acid is essential for the hydroxylation of these amino acids and that it regulates collagen synthesis at the transcriptional level.
Inhibitory and chemopreventive effects in malignant cell lines of some of the individual nutrients composing the NS have been reported in both clinical and experimental studies. Ascorbic acid has been shown to have growth inhibitory and antineoplastic activities in human mammary tumor bearing mice . In addition, low levels of ascorbic acid have been reported in cancer patients [13–15]. Green tea extract is another potent anticancer agent that has been reported to have a growth inhibitory effect against certain human cancer cell lines, especially breast cancer [16–18]. For example, both in vitro and animal studies of the effect of green tea extract on breast cancer revealed suppressed xenograft size and tumor vessel density and suppression of cell proliferation .
Furthermore, studies conducted before the clinical onset of breast cancer found that increased green tea consumption was associated with improved prognosis in stage I and II breast cancers, as well as decreased numbers of axillary lymph node metastases in premenopausal women, suggesting significant chemopreventative potential . Utilizing the Sprague–Dawley rat model, researchers showed that EGCG reduced tumor burden and number of invasive tumors, and drastically increased the mean latency to the initial tumor in mammary tumor bearing rats .
Our previous in vitro studies demonstrated that the anticancer effect of a mixture of ascorbic acid, proline, lysine, and EGCG on several cancer cell lines in tissue culture studies was greater than that of the individual nutrients . Furthermore, in contrast to chemotherapy, which causes indiscriminate cellular and ECM damage, previous studies showed that cell morphology was not affected even at the highest concentrations of this nutrient mixture, demonstrating that this formulation is safe to cells.