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

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. RESULTS: NS reduced the incidence of MNU-induced mammary tumors and the number of tumors by 68.4%, and the tumor burden by 60.5%. The inhibitory effect of NS was also reflected by decreased tumor weight; the tumor weights per rat and per group were decreased by 41% and 78%, respectively. In addition, 30% of the control rats developed ulcerated tumors, in contrast to 10% in the nutrient supplemented rats. CONCLUSION: These findings suggest that the specific formulation of lysine, proline, arginine, ascorbic acid, and green tea extract tested significantly reduces the incidence and growth of MNU-induced mammary tumors, and therefore has strong potential as a useful therapeutic regimen for inhibiting breast cancer development

Abstract 2260: Modulation of P-glycoprotein expression by a novel nutrient mixture in multidrug-resistant human uterine sarcoma cell line MES-SA/Dx5 but not in drug-sensitive MES-SA cell line

Abstract Introduction: We have characterized a nutrient mixture containing lysine, proline, ascorbic acid and green tea extract as a novel antineoplastic agent with a broad spectrum of antitumor activity against a number of cancer cell lines. Objective: We investigated the effect of NM on modulation of P-glycoprotein (Pgp) in the drug-resistant human uterine sarcoma cell line MES-SA/Dx5 and compared it with the effect on drug-sensitive cell line MES-SA. In addition we also studied the effect of NM on MMP expression and Rhodamine-123 accumulation and efflux. Material and Methods: Human drug insensitive uterine sarcoma cell line MES-SA/Dx5 and drug sensitive cell line MES-SA (ATCC) were grown in RPMI 1640 medium supplemented with fetal bovine serum and antibiotics. At near confluence, the cells were tested with NM at 0, 50, 100, 250, 500 and 1000 mcg/ml, in triplicate at each dose. Cell proliferation was evaluated by MTT assay, MMPs by gelatinase zymography, and Pgp expression by Western blot and immunodetection using FITC-conjugated antibody and rhodamine-123 (Rh-123) accumulation and efflux assays. Results: NM exhibited antiproliferative effect on MES-SA/Dx5, by 20% at 50 and 100 mcg/ml and by 40% at 25, 500 and 1000 mcg/ml, whereas in MES-SA cell line, NM showed dose-response toxicity of 40% at 50 and 30% at 1000 mcg/ml. In both cell lines, zymography demonstrated a band corresponding to MMP-2 in normal cells and MMP-9 with PMA treatment. Both MMPs showed dose-response inhibition by NM. NM treatment also showed diminished dose-dependent Pgp expression by MES-SA/Dx5 cell line by Western blot and by immunodetection, whereas MES-SA did not exhibit Pgp by Western blot or by immunostaining. NM enhanced the accumulation and efflux of Pgp substrate Rh-123 in MES-SA/Dx5 uterine sarcoma cell line but not in the drug-sensitive cell line MES-SA. Conclusions: In summary, this study demonstrated that Pgp is modulated by NM, which may be an attractive potential agent for therapeutic use in cancer treatment Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2260.</jats:p

Abstract 2348: Suppression of metastasis of intratesticular inoculation of B16FO melanoma cells by a novel nutrient mixture in male athymic nude mice

Abstract Testicular cancer (TC) is rare, but still represents one of the most common diseases in young men between the ages of 20-45. However, men of any age can develop this disease. The incidence in Caucasians is greater than in African Americans. Risk factors include undescended testis, Klinefelter syndrome, and HIV positive patients. If left untreated, it is almost certainly fatal. Metastasis is the major cause of cancer death. The most common place for TC to spread in the body is to the lung. In this investigation, we studied the effect of a novel nutrient mixture (NM) containing ascorbic acid, amino acids and green tea extract that has been shown to exhibit anti-cancer activity, on inhibition of B16FO melanoma cells inoculated intratesticularly. Male athymic mice (n=12), 10-12 weeks of age, were inoculated with half a million B16FO melanoma cells in 100 µL of PBS into the right testis; the left testis was left untreated. After inoculation, the mice were randomly divided into two groups. Group A (n=6) was fed a regular mouse chow diet, while the mice in Group B (n=6) were fed the same diet but supplemented with 0.5% NM. Four weeks later the mice were sacrificed and the abdominal cavity was opened. Mice in the control group (Group A) exhibited extensive metastasis in the peritoneal cavity, which was totally masked by B16FO melanoma cells. The testis was severely enlarged and replaced by invading malignant melanoma cells. The remaining testicular tissue was represented by necrotic seminiferous tubules. The capsular region of the testis was severely infiltrated with a population of mixed cells. In contrast, in the NM fed group (Group B), there was no evidence of peritoneal metastasis, but the testes were enlarged. Seminiferous tubules in the area of invasion showed evidence of degeneration. In all groups, there was no metastasis to liver, kidney and spleen. However, severe lung metastasis was observed in the control group (2 out of 6) and mild in the test group (2 out of 6). In conclusion, these results suggest that NM has potential to suppress tumor metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2348. doi:1538-7445.AM2012-2348</jats:p

Micronutrient Synergy in the Fight against Hepatocellular Carcinoma

The incidence of hepatocellular carcinoma (HCC), once thought to be a rare tumor in North America, has rapidly increased in recent years in the United States. Current treatment modalities to halt the progression of this disease are only marginally effective. The mainstay treatment is liver transplantation, which is often confronted with donor shortage. Invasion, metastasis and recurrence contribute to the high mortality rate of this disease. Matrix metalloproteinases (MMPs) that degrade the extracellular matrix (ECM) have been associated with the progression, invasion and metastasis of the disease. We have developed strategies to strengthen the ECM collagen and inhibit MMPs through micronutrients such as lysine, proline and ascorbic acid. Addition of epigallocatechin gallate or green tea extract to these micronutrients synergistically enhanced anti-carcinogenic activity in HepG2 cells. Addition of certain other micronutrients, such as N-acetylcysteine, selenium, copper and zinc (NM) synergistically enhanced the anticancer activity of the mixture in a model of hepatocellular carcinoma using HepG2 cells. In vitro studies using HepG2 demonstrated that NM was very effective in inhibiting cell proliferation (by MTT assay), MMPs secretion (by gelatinase zymography), cell invasion (through Matrigel) and induction of apoptosis (by live green caspase). In addition, NM was shown to down-regulate urokinase plasminogen activator (by fibrin zymography) and up-regulate tissue inhibitors of metalloproteinases (by reverse zymography) in another HCC cell line, SK-Hep-1. MMP-2 and MMP-9 activities were further modulated by phorbol 12-myristate 13-acetate (PMA) induction and inhibited by NM. In previous studies, NM inhibited Sk-Hep-1 xenografts in nude mice and also inhibited hepatic metastasis of B16FO melanoma cells. Our results suggest that NM is an excellent candidate for therapeutic use in the treatment HCC by inhibiting critical parameters in cancer development and progression, such as proliferation, invasion and metastasis, and by inducing apoptosis

Chemopreventive Effect of a Novel Nutrient Mixture on Lung Tumorigenesis Induced by Urethane in Male A/J Mice

Aims and background Lung cancer, a leading cause of cancer death, is associated with exposure to inhalation carcinogens, most commonly those found in tobacco smoke. We investigated the in vivo effect of dietary supplementation with a nutrient mixture containing lysine, proline, arginine, ascorbic acid, green tea extract, N-acetyl cysteine, selenium, copper and manganese on the development of urethane-induced lung tumors in male A/J mice. Methods After one week of isolation, seven-week-old male A/J mice (n = 25) weighing 17–19 g were randomly divided into three groups: group A (n = 5), group B (n = 10), and group C (n = 10). Mice in groups B and C were each given a single intraperitoneal injection of urethane (1 mg/g body weight) in saline, whereas group A mice received an injection of saline alone. Groups A and B were fed a regular diet, whereas group C was fed the same diet supplemented with 0.5% nutrient mixture. After 20 weeks, mice were sacrificed, lungs were excised and weighed, and tumors were counted and processed for histology. Results Urethane-challenged mice developed tumors. However, the mean number of tumors and the mean lung weights in the mice on the supplemented diet were significantly reduced, by 49% (P &lt;0.0001) and 18% (P = 0.0025), respectively, compared to mice on the control diet. We observed neither significant differences in body weight gains nor in diet consumption among the mice. Pulmonary lesions were morphologically similar for both the groups (adenomas), but lesions were smaller in the test group. Conclusions The results suggest that nutrient mixture has inhibitory potential on the development of mouse lung tumors induced by urethane </jats:sec