Free radical theory of aging: an update: increasing the functional life span.
Aging is the progressive accumulation of diverse, deleterious changes with time that increase the chance of disease and death. The basic chemical process underlying aging was first advanced by the free radical theory of aging (FRTA) in 1954: the reaction of active free radicals, normally produced in the organisms, with cellular constituents initiates the changes associated with aging. The involvement of free radicals in aging is related to their key role in the origin and evolution of life. Aging changes are commonly attributed to development, genetic defects, the environment, disease, and an inborn aging process (IAP). The latter produces aging changes at an exponentially increasing rate with age, becoming the major risk factor for disease and death for humans after the age of 28 years in the developed countries. In them the IAP limits human average life expectancy at birth (ALE-B)—a rough measure of the healthy life span—to about 85 years; few reach 100 years and only one is known to have lived to 122 years. In these countries, improvements in living conditions (ILC) have gradually raised ALE-Bs to 76-79 years, 6-9 years less than the limit imposed by aging, with no change in the maximum life span (MLS). The extensive studies based on the FRTA hold promise that ALE-B and the MLS can be extended, the ALE-B possibly by a few years, and the MLS somewhat less.
Ann N Y Acad Sci. 2006 May;1067:10-21
Vitamin C deficiency increases the lung pathology of influenza virus-infected gulo-/- mice.
This study was designed to determine the effects of vitamin C deficiency on the immune response to infection with influenza virus. l-Gulono-gamma-lactone oxidase gene-inactivated mice (gulo-/- mice) require vitamin C supplementation for survival. Five-wk-old male and female gulo-/- mice were provided water or water containing 1.67 mmol/L vitamin C for 3 wk before inoculation with influenza A/Bangkok/1/79. There were no differences in lung influenza virus titers between vitamin C-adequate and -deficient mice; however, lung pathology in the vitamin C-deficient mice was greater at 1 and 3 d after infection but less at d 7 compared with vitamin C-adequate mice. Male vitamin C-deficient mice had higher expression of mRNA for regulated upon activation normal T expressed and secreted (RANTES), IL-1beta, and TNF-alpha in the lungs at d 1 after infection compared with male controls. However, at d 3 after infection, male vitamin C-deficient mice had less expression of mRNA for RANTES, monocyte chemotactic protein-1 (MCP-1), and IL-12 compared with male controls. None of these differences were observed in female mice. Vitamin C-deficient male mice also had greater nuclear factor-kappaB activation as early as 1 d after infection compared with male controls. These data suggest that vitamin C is required for an adequate immune response in limiting lung pathology after influenza virus infection.
J Nutr. 2006 Oct;136(10):2611-6
A pilot clinical study of continuous intravenous ascorbate in terminal cancer patients.
Case studies suggest that vitamin C, given intravenously at doses of 10-100 grams/day can improve patient well being and in some cases, reduce tumor size. While ascorbate is generally considered safe, clinical data on high intravenous doses is limited. Twenty-four late stage terminal cancer patients were given continuous infusions of 150 to 710 mg/kg/day for up to eight weeks. Blood chemistry and blood count profiles were obtained at roughly one-week intervals while patient health, adverse events and tumor progression were monitored. The majority of patients were vitamin C deficient prior to treatment. Intravenous infusions increased plasma ascorbate concentrations to a mean of 1.1 mM. The most common adverse events reported were nausea, edema, and dry mouth or skin; and these were generally minor. Two Grade 3 adverse events ‘possibly related’ to the agent were reported: one patient with a history of renal calculi developed a kidney stone after thirteen days of treatment and another patient experienced hypokalemia after six weeks of treatment. White blood cell counts were stable while hemoglobin and hematocrit levels dropped slightly during treatment, consistent with trends observed prior to therapy. Blood creatinine, BUN, glucose, and uric acid concentrations decreased or remained stable during therapy, suggesting that ascorbate infusions did not adversely affect renal function. One patient had stable disease and continued the treatment for forty-eight weeks. These data suggest that intravenous vitamin C therapy for cancer is relatively safe, provided the patient does not have a history of kidney stone formation.
P R Health Sci J. 2005 Dec;24(4):269-76
Suppression of human cervical cancer cell lines Hela and DoTc2 4510 by a mixture of lysine, proline, ascorbic acid, and green tea extract.
Cervical cancer, the second most common cancer in women, once metastasized, leads to poor prognosis. We investigated the antitumor effect of a nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid, and green tea extract on human cervical cancer cells Hela (CCL-2) and DoTc2 4510 by measuring cell proliferation (MTT assay), modulation of matrix metalloproteinases (MMP)-2 and MMP-9) expression (gelatinase zymography), and cancer cell invasive potential (Matrigel). NM showed significant antiproliferative effect on CCL-2 and DoTc2 4510 cancer cells. The NM inhibited CCL-2 expression of MMP-2 and MMP-9 in a dose-dependent fashion, with virtual total inhibition of MMP-2 at 1000 microg/mL and MMP-9 at 500 microg/mL NM. Untreated DoTc2 4510 cells showed MMP-9 expression, which was enhanced with phorbol 12-myristate 13-acetate treatment. NM inhibited MMP-9 expression in a dose-dependent fashion, with virtual inhibition at 500 microg/mL. Invasion of human cervical cancer cells CCL-2 and DoTc2 4510 through Matrigel decreased in a dose-dependent fashion, with 100% inhibition at 500 microg/mL NM (P < 0.0001) and 1000 microg/mL NM (P < 0.0001), respectively. Our results suggest that the mixture of lysine, proline, arginine, ascorbic acid, and green tea extract has potential in the treatment of cervical cancer by inhibiting critical steps in cancer development and spread.
Int J Gynecol Cancer. 2006 May-Jun;16(3):1241-7
Inhibition of malignant mesothelioma cell matrix metalloproteinase production and invasion by a novel nutrient mixture.
Malignant mesothelioma (MM), an asbestos-associated cancer with no known cure, is a highly aggressive tumor causing profound morbidity and nearly universal mortality. Extracellular matrix (ECM) matrix metalloproteinases (MMPs) produced by tumor and stromal cells play a key role in tumor invasion and metastasis. Prevention of ECM degradation by MMP inhibition has been shown to be a promising therapeutic approach to inhibition of cancer development. Based on reported anticancer properties, the authors investigated the effect of a mixture (NM) containing lysine, proline, ascorbic acid, and green tea extract on MM cell line MSTO-211 H proliferation (by [MTT] [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay), MMP secretion (by gelatinase zymography), invasion (through Matrigel), and morphology (by hematoxylin and eosin [H&E] staining). MMP-2 and phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 secretion were inhibited by NM in a dose-dependent fashion, with virtual total inhibition at 500 microg/ml NM. Invasion through Matrigel was inhibited at 50, 100, and 500 microg/ml by 27%, 36%, and 100%, respectively. NM was not toxic to the MM cell line, and H&E staining did not indicate any changes at and below 100 microg/ml concentration. In conclusion, NM significantly inhibited MM cell MMP secretion and invasion-both important parameters for cancer prevention, suggesting NM is an effective treatment strategy for MM.
Exp Lung Res. 2006 Mar-Apr;32(3-4):69-79
Antitumor effect of ascorbic acid, lysine, proline, arginine, and green tea extract on bladder cancer cell line T-24.
AIMS: Bladder cancer, the fourth highest incident cancer in men and tenth in women, is associated with a high rate of recurrence, even when treated in situ, and prognosis is poor once the cancer metastasizes to distant sites. Based on anticancer properties, we investigated the effect of a mixture of lysine, proline, arginine, ascorbic acid, and green tea extract on human bladder cancer cells T-24 by measuring: proliferation, matrix metalloproteinase (MMP) expression, and cancer cell invasive potential. METHODS: Human bladder cancer cells T-24 (ATCC) were grown in McCoy medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 mg/mL) in 24-well tissue culture plates. At near confluence, the cells were treated with the nutrient mixture dissolved in media and tested at 0, 10, 50, 100, 500, and 1000 microg/mL in triplicate at each dose. Cells were also treated with PMA 200 ng/mL to study enhanced MMP-9 activity. Cell proliferation was evaluated by MTT assay, MMP activity by gelatinase zymography, and invasion through Matrigel. RESULTS: Nutrient mixture inhibited the T-24 cell secretion of MMP-2 and -9, with virtual total inhibition of MMP-2 at 500 microg/mL and MMP-9 at 100 microg/mL. The nutrient mixture significantly reduced the invasion of human bladder cancer cells T-24 through Matrigel in a dose-dependent fashion, with 95% inhibition at 500 microg/mL and 100% at 1000 microg/mL nutrient mixture (P < 0.001). CONCLUSION: Our results suggest that our nutrient mixture is an excellent candidate for therapeutic use in the treatment of bladder cancer, by inhibiting critical steps in cancer development and spread, such as MMP secretion and invasion.
Int J Urol. 2006 Apr;13(4):415-9
In vivo and in vitro antitumor effect of ascorbic acid, lysine, proline, arginine, and green tea extract on human fibrosarcoma cells HT-1080.
Current treatment of fibrosarcoma, an aggressive cancer of the connective tissue, is generally associated with poor prognosis. Matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), and constituents of the extracellular matrix (ECM), such as fibronectin, play a critical role in angiogenesis and underlie neoplastic invasion and metastasis. This and anticancer properties of lysine, proline, arginine, ascorbic acid, and green tea extract (NM) prompted us to investigate the effect of these nutrients in vitro on human fibrosarcoma cells HT-1080 by measuring cell proliferation, modulation of MMP-2 and MMP-9, and invasive potential. In vivo, we studied the growth of human fibrosarcoma HT-1080 cells in athymic nude mice and the expression of MMPs and VEGF. Cell proliferation was evaluated by MTT assay, MMP expression by gelatinase zymography, and invasion through Matrigel and migration by scratch assay. Tumors were excised, weighed, and processed for histology in both the control and nutrient-supplemented groups. Results showed NM inhibited the growth and reduced the size of tumors in nude mice; decreased MMP-9 and VEGF secretion was found in the supplemented group tissues. NM inhibited invasion through Matrigel and migration with total inhibition at 1,000 microg/mL. These results offer promise in the therapeutic use of the nutrient mixture of lysine, proline, arginine, ascorbic acid, and green tea extract tested in the treatment of fibrosarcoma.
Med Oncol. 2006;23(1):105-11
Inhibition of matrix metalloproteinase-2 secretion and invasion by human ovarian cancer cell line SK-OV-3 with lysine, proline, arginine, ascorbic acid and green tea extract.
AIMS: Based on the poor prognosis associated with ovarian cancer and reported anticancer properties of specific nutrients, we investigated the effect of a nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid and epigallocatechin gallate on human ovarian cancer cells SK-OV-3 by measuring: cell proliferation, modulation of matrix metalloproteinase (MMP)-2 and -9 expression, and cancer cell invasive potential. METHODS: Cell proliferation was evaluated by MTT assay, MMP activity by gelatinase zymography, and invasion through Matrigel. RESULTS: Human ovarian cancer cell growth was not significantly affected by the NM. Zymography demonstrated inhibition of MMP-2 secretion in a dose-dependent fashion with virtual total inhibition at 50 microg/mL NM concentration. Invasion of human ovarian cancer cells through Matrigel decreased in a dose-dependent fashion, with 90% inhibition at 500 microg/mL NM and 100% inhibition at 1000 microg/mL NM (P < 0.0001). CONCLUSION: The combination of lysine, proline, arginine, ascorbic acid and green tea extract tested inhibited critical steps in cancer development and spread, such as MMP expression and invasion, indicating its potential as a treatment modality against ovarian cancer.
J Obstet Gynaecol Res. 2006 Apr;32(2):148-54
In vivo and in vitro antitumor effect of ascorbic acid, lysine, proline and green tea extract on human melanoma cell line A2058.
BACKGROUND: Melanoma, a very serious form of skin cancer, causes the most skin cancer-related deaths, due to metastasis. Structural changes in the extracellular matrix (ECM) are necessary for cell migration during tissue remodeling. MMPs, VEGF, Ki-67 (proliferative protein) and constituents of ECM play a critical role in angiogenesis, and are crucial in neoplastic invasion and metastasis. MATERIALS AND METHODS: The effect of a diet (NM) containing lysine, proline, arginine, ascorbic acid and green tea extract on the growth of tumors induced by implanting human melanoma A2058 cells in athymic nude mice was examined and, also, on the expression of MMPs, VEGF and Ki-67 in these tumors. The effect of NM in vitro on the melanoma A2058 cell line was tested by measuring: cell proliferation by the MTT assay, expression of MMPs by gelatinase zymography and invasion through Matrigel. RESULTS: Nutrient supplementation strongly suppressed the growth of tumors (by 57%)without adverse effects in nude mice. Histological studies supported these findings by showing inhibition of MMP-9 and VEGF secretion and mitotic index. In vitro, NM inhibited melanoma cell growth by 64% at 500 microg/ml and Matrigel invasion by 95% at 100 microg/ml NM. CONCLUSION: These results suggest that NM may have a therapeutic potential in melanoma.
In Vivo. 2006 Jan-Feb;20(1):25-32
Inhibitory effect of a mixture containing ascorbic acid, lysine, proline and green tea extract on critical parameters in angiogenesis.
Degradation of extracellular matrix (ECM) is a hallmark of tumor invasion, metastasis and angiogenesis. Based on the Rath multitargeted approach to cancer using natural substances to control ECM stability and enhancing its strength, we developed a novel formulation (NM) of lysine, proline, ascorbic acid and green tea extract that has shown significant anti-cancer activity against a number of cancer cell lines. The aim of the present study was to determine whether NM exhibits anti-angiogenic and anti-metastatic effects using in vitro and in vivo experimental models. Angiogenesis was measured using a chorioallantoic membrane (CAM) assay in chick embryos and bFGF-induced vessel growth in C57BL/6J female mice. To determine the in vivo effect of NM on the tumor xenograft growth, male nude mice were inoculated with 3 x 10(6) MNNG-HOS cells. Control mice were fed a mouse chow diet, while the test group was fed a mouse chow diet supplemented with 0.5% NM for 4 weeks. In vitro studies on cell proliferation (MTT assay), MMP expression (zymography) and Matrigel invasion were conducted on human osteosarcoma U2OS, maintained in McCoy medium, supplemented with 10% FBS, penicillin and streptomycin in 24-well tissue culture plates and tested with NM at 0, 10, 50, 100, 500, and 1000 microg/ml in triplicate at each dose. NM at 250 microg/ml caused a significant (p<0.05) reduction in bFGF-induced angiogenesis in CAM. NM inhibited tumor growth of osteosarcoma MNNG-HOS cell xenografts in nude mice by 53%; furthermore, tumors in NM-treated mice were less vascular and expressed lower levels of VEGF and MMP-9 immunohistochemically than tumors in the control group. In addition, NM exhibited a dose-dependent inhibition of osteosarcoma U2OS cell proliferation (up to 60% at 1000 microg/ml), MMP-2 and -9 expression (with virtual total inhibition at 500 microg/ml NM), and invasion through Matrigel (with total inhibition at 100 microg/ml NM). Moreover, NM decreased U2OS cell expression of VEGF, angiopoietin-2, bFGF, PDGF and TGFbeta-1. These results together with our earlier findings suggest that NM is a relatively non-toxic formulation, which inhibits growth, invasion, metastasis, and angiogenesis of tumor cells.
Oncol Rep. 2005 Oct;14(4):807-15
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.
Breast Cancer Res. 2005;7(3):R291-5. Epub 2005 Jan 31
In vitro and in vivo antitumorigenic activity of a mixture of lysine, proline, ascorbic acid, and green tea extract on human breast cancer lines MDA-MB-231 and MCF-7.
Current treatments are generally ineffective once breast cancer has metastasized; median survival is reduced to 2-3 yr. Previous research studies demonstrating potent synergistic antitumor activity of lysine, proline, ascorbic acid, and epigallocatechin gallate prompted us to investigate the in vivo inhibitory effect of a nutrient mixture containing lysine, proline, arginine, ascorbic acid, and epigallocatechin gallate (NM) on the growth of human cancer xenografts in female athymic nude mice. Five to six week old female mice were inoculated with 3x106 breast cancer cells MDA-MB-231. After injection, the mice were randomly divided into two groups A and B; group A was fed a regular diet and group B with the regular diet supplemented with 0.5% of the nutrient mixture (NM). Four weeks later, the mice were sacrificed, and their tumors were excised, weighed, and processed for histology. We also tested the effect of NM in vitro on estrogen-receptor positive (ER+) MCF-7 and estrogen-receptor negative (ER-) MDA-MB-231 breast cancer cell lines by measuring: cell proliferation by MTT assay, expression of MMPs by gelatinase zymography, invasion through Matrigel, and VEGF by ELISA. MCF-7 cells were also treated with estradiol to study enhanced invasion and expression of MMPs and VEGF. Results showed that NM inhibited the growth and reduced the size of tumors in female nude mice by 27%. Furthermore, histological evaluation revealed increased mitotic index, MMP-9 and VEGF secretion, and PAS material (mucin) in the control group tissues. In vitro studies showed NM inhibited MDA-MB-231 cell growth by 34% at 500 microg/mL and MCF-7 cell growth by 18% at 1000 microg/mL. Invasion of MDA-MB-231 through Matrigel was inhibited by 50%, 60%, and 95% by 10, 50, and 100 microg/mL of NM, respectively. The results of this study demonstrated that the nutrient mixture tested significantly suppressed tumor growth of breast cancer cells in female athymic nude mice and significantly inhibited MMP expression, angiogenesis, and invasion in breast cancer cells, in vitro, offering promise for therapeutic use in the treatment of breast cancer.
Med Oncol. 2005;22(2):129-38.
Antitumor effect of a combination of lysine, proline, arginine, ascorbic acid, and green tea extract on pancreatic cancer cell line MIA PaCa-2.
BACKGROUND: Current treatment of pancreatic cancer is generally associated with poor prognosis, even if diagnosed early, owing to its aggressive rate of metastasis and non-responsiveness to chemotherapy and radiotherapy. Matrix metalloproteinases (MMPs) have received much attention in recent years for their role in various malignancies, and have been implicated in tumor invasion, metastasis, and angiogenesis. AIM OF STUDY: Reported antitumor properties of ascorbic acid, lysine, proline, and green tea extract prompted us to investigate the effect of a combination of lysine, proline, arginine, ascorbic acid, and green tea extract on pancreatic cancer cell line MIA PaCa-2 for viability, MMP expression, invasion, and morphology. METHODS: Viability was evaluated based on cell proliferation by MTT assay and MMP expression in condition media by gelatinase zymography. Invasion through Matrigel was assayed and morphology was observed by hematoxylin and eosin (H+E)staining. Data was analyzed by independent sample “t” test. RESULTS: The nutrient mixture (NM) did not inhibit cell proliferation at 10 microg/mL and exhibited a dose-dependent antiproliferative effect with maximum inhibition of 38% over the control at 1000 microg/mL. Zymography demonstrated production of only MMP-9, which showed a dose-dependent decreased expression that was abolished at 100 microg/mL of NM. Invasion through Matrigel was inhibited at 10, 50, 100, and 500 microg/mL by 66%, 66%, 87% and 100%, respectively. H&E staining did not indicate changes even at the highest concentration of NM. CONCLUSION: Our results suggest that the formulation of green tea extract, lysine, proline, and ascorbic acid, tested as a promising adjunct to standard treatment of pancreatic cancer, by inhibiting MMP expression and invasion without toxic effects important parameters in cancer metastasis.
Int J Gastrointest Cancer. 2005;35(2):97-102
In vivo antitumor effect of ascorbic acid, lysine, proline and green tea extract on human prostate cancer PC-3 xenografts in nude mice: evaluation of tumor growth and immunohistochemistry.
BACKGROUND: Matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), Ki 67 (proliferative protein) and constituents of ECM play a critical role in angiogenesis, and are crucial in neoplastic invasion and metastasis. Based on the antitumor properties of certain nutrients, we investigated the effect of a diet containing lysine, proline, arginine, ascorbic acid and green tea extract on the growth of tumors induced by implanting human prostate cancer PC-3 cells in athymic nude mice and on the expression of MMPs, VEGF, Ki 67 and fibronectin in these tumors, as well as the production of mucin (by PAS staining). MATERIALS AND METHODS: Male nude mice (n =12) were inoculated with 3x10(6) prostate cancer PC-3 cells and randomly divided into two groups; Group A was fed a regular diet and Group B was fed a regular diet supplemented with 0.5% of the nutrient mixture (NM). Four weeks later, tumors were excised, weighed and processed for histology. RESULTS: The results showed inhibition of tumor growth in Group B. Histological studies revealed inhibition of MMP-9 and VEGF secretion and mitosis in Group B tissues. CONCLUSION: Nutrient supplementation strongly suppressed the growth of tumors without any adverse effects in nude mice, suggesting strong potential as an anticancer agent.
In Vivo. 2005 Jan-Feb;19(1):179-83