Modulation of N-methyl-N-nitrosourea induced mammary tumors in Sprague–Dawley rats by combination of lysine, proline, arginine, ascorbic acid and green tea extract

Breast Cancer Research Vol7No3 Modulation of N-methyl-N-nitrosourea induced mammary tumors in Sprague-Dawley rats by combination of lysine, proline, arginine, ascorbic acid and green tea extract M Waheed Roomi 0 Nusrath W Roomi 0 Vadim Ivanov 0 Tatiana Kalinovsky 0 Aleksandra Niedzwiecki 0 Matthias Rath 0 0 Matthias Rath Research, Cancer Division , Santa Clara, California , USA Introduction The limited ability of current treatments to control metastasis and the proposed antitumor properties of specific nutrients prompted us to examine the effect of a specific formulation (nutrient supplement [NS]) of lysine, proline, arginine, ascorbic acid, and green tea extract in vivo on the development of N-methyl-N-nitrosourea (MNU)-induced mammary tumors in rats. Methods A single intraperitoneal dose of MNU was injected into each of 20 female Sprague-Dawley rats (aged 50 days) to induce tumors. Two weeks after MNU treatment, a time by which the animals had recovered from MNU-induced toxicity, the rats were divided into two groups. Rats in group 1 (n = 10) were fed Purina chow diet, whereas those in group 2 (n = 10) were fed the same diet supplemented with 0.5% NS. After a further 24 weeks, the rats were killed and tumors were excised and processed. antitumor effect; mammary tumors; N-methyl-N-nitrosourea; Sprague-Dawley rats - Introduction Breast cancer is the most prevalent cancer in women worldwide, excluding nonmelanoma skin cancer, and is the second leading cause of cancer deaths in women (following lung cancer) [1]. Once metastasis has occurred, the survival rate is drastically reduced to a median of 23 years; therapy is then aimed at controlling symptoms, prolonging survival and improving quality of life [2]. Unfortunately, the diagnostic criteria currently used to stage breast cancer often yield inaccurate findings with regard to metastasis. Analyses of bone marrow samples (not a routine procedure) have revealed the presence of disseminated cells in up to 40% of primary breast cancer patients without any clinical or histopathologic signs of metastasis. Circulating breast cancer cells in bone marrow are indicative of metastasis to such sites as bone, lung, and liver [3]. Cancer cells form tumors and spread by degrading the extracellular matrix (ECM) through various matrix metalloproteinases (MMPs). The activity of these enzymes correlates with the aggressiveness of tumor growth and the invasiveness of the cancer. Rath and Pauling [4] postulated that nutrients such as lysine and ascorbic acid could act as natural inhibitors of ECM proteolysis, and as such they have ECM = extracellular matrix; EGCG = epigallocatechin-3-gallate; MMP = matrix metalloproteinase; MNU = N-methyl-N-nitrosourea; NS = nutrient supplement. the potential to inhibit tumor growth and expansion. These nutrients may exert their antitumor effect through inhibiting MMPs and strengthening the connective tissue surrounding cancer cells (a 'tumor encapsulating' effect). Additionally, it has been suggested that, through inhibition of hyaluronidase, ascorbic acid can prevent metastases by preventing degradation of the ground substance surrounding the tumor. In a previous study [5] we demonstrated the antiproliferative and anti-invasive potential of a specific formulation (nutrient supplement [NS]) of lysine, ascorbic acid, proline, and green tea extract on human breast cancer (MDA-MB 231), colon cell cancer (HCT 116), and melanoma (A2058) cell lines. NS also suppressed the growth of these tumors, without any adverse effects, in nude mice. In the present study we investigated the inhibitory effect of NS in vivo on development of N-methyl-N-nitrosourea (MNU)induced mammary tumors in rats. Methods Animals On arrival at our laboratory, 40-day-old pathogen free female SpragueDawley rats (Simonsen Laboratories, Gilroy, CA, USA) were housed in solid bottom cages with corncob bedding, at 22C and 50% humidity, with a 12hour lightdark cycle. The rats had free access to water and Purina rat chow diet. All animals were cared for in accordance with institutional guidelines for the care and use of experimental animals. Experiments At day 50, all rats (n = 20) received a single dose of MNU 50 mg/kg intraperitoneally. (MNU, reagent grade, was obtained from Sigma, St. Louis, MO, USA). Two weeks after MNU treatment, a time by which the animals had recovered from MNU-induced toxicity, the rats were divided into two groups. Rats in group 1 (n = 10) were fed a pellet Purina chow diet (Purina Mills, Richmond, IN, USA), whereas those in group 2 (n = 10) were fed a pellet diet custom prepared by Purina containing the same diet but supplemented with 0.5% of the NS. Body weight and diet consumption of the rats were monitored every week. Beginning 6 weeks after MNU administration, the rats were palpated every week for evidence of tumors. Dimensions (length width) of the tumors were measured using a digital caliper, and the tumor burden was calculated using the following formula: 0.5 length width. Twenty-four weeks later the rats were killed by carbon dioxide asphyxiation and skinned; tumors were excised and a detailed necropsy was performed on each rat. Location, weight, and dimensions of excised mammary tumors were recorded. Tumors were processed for histologic examination, using criteria described by Russo and coworkers [6]. Briefly, histopathologic criteria used to determine malignancy were loss of tubularalveolar pattern of the normal mammary gland; presence of large epithelial cells with increased nuclearcytoplasmic ratio; stromal response by fibrosis and inflammatory cell infiltration; and necrosis and hemorrhage. Tumor incidence and multiplicity for control and supplemented rats Control group (n = 10) Supplemented group (n = 10) 1 Tumor 2 Tumors 3 Tumors 4 Tumors 5 Tumors Total number of Tumors 19 Mean tumor burden per rat and tumor burden per group Control group (n = 10; 19 tumors) Supplemented group (n = 10; six tumors) Total tumor burden per group Mean tumor burden per rat The mean tumor burden per rat is expressed as mean standard deviation. The differences between groups were significant (P < 0.0001). Total tumor weight per group Mean tumor weight per rat Mean individual tumor weight P = 0.002 P = 0.002 Mean tumor weight per rat and per group Control group (n = 10; 19 tumors) Supplemented group (n = 10; six tumors) Mean growth of control and supplemented rats Control group (n = 10) Supplemented group (n = 10) Composition of the nutrient supplement Stock solution of the NS (total weight 4.2 g) is composed of the following: 700 mg vitamin C (as ascorbic acid and as magnesium, calcium, and palmitate ascorbate), 1000 mg Llysine, 750 mg L-proline, 500 mg L-arginine, 200 mg Nacetyl cysteine, 1000 mg standardized green tea extract, 30 mg selenium, 2 mg copper, and 1 mg manganese. Green tea extract, derived from green tea leaves, was obtained from US Pharma Lab (Newark, NJ, USA). The certificate of analysis (...truncated)