Repression of matrix metalloproteinases and inhibition of cell invasion by a nutrient mixture, containing ascorbic acid, lysine, proline, and green tea extract on human fanconi anemia fibroblast cell lines

Aim: Fanconi Anemia, an autosomal recessive disorder, is characterized by chromosomal abnormality leading to birth defects, progressive bone marrow failure, and a high probability of developing malignancy at an early age. Head and neck squamous cell carcinoma and myeloid leukemia are the major causes of cancer related morbidity and mortality in Fanconi anemia patients. Me­thods: We investigated the effect of a nutrient mixture on Fanconi Anemia human fibroblast cell lines FA‐A:PD20 and FA‐A:PD220 on matrix metalloproteinase expression, invasion, cell proliferation, morphology and apoptosis. The cell lines were grown in a modified Dulbecco’s Eagle medium and at near confluence were treated with the nutrient mixture at increasing doses: 0; 10; 50; 100; 500; 1000 µg/ml. The cells were also treated with PMA to induce MMP-9 expression. Results: Zymography demonstrated MMP‐2 and PMA‐induced MMP‐9 activity. The nutrient mixture inhibited expression of both, MMP-2 and MMP-9, in a dose dependent manner with virtually total inhibition observed at 500 µg/ml. Matrigel invasion was inhibited in both cells lines; with 100% inhibition for FA-A:PD20 at 500 µg/ml and 100% inhibition of FA-A:P220 cells at 100 µg/ml. H&E staining did not indicate any change in cell morphology and causes apoptosis at higher doses. Conclusion: Our data demonstrated that the nutrient mixture inhibited matrix metalloproteinase expression, invasion and induced apoptosis, the important parameters for cancer prevention. The results suggest that the nutrient mixture may have therapeutic potential in Fanconi Anemia associated neoplasia

Nutrient mixture inhibits in vitro and in vivo growth of human acute promyelocytic leukemia HL-60 cells

Aim: Untreated acute promyelocytic leukemia is the most malignant form of acute leukemias, with median survival of less than one month. We investigated in vitro and in vivo synergistic effects of a nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea extract, on acute promyelocytic leukemia HL-60 cells. Methods: In vitro, the HL-60 cells were cultured and exposed to NM at doses 0–1000 μg/ml. Cell viability was assessed by Trypan blue dye exclusion test, matrix metalloproteinases (MMP) expression by gelatinase zymography, invasion through Matrigel and apoptosis by live green Poly Caspase Detection Kit. In vivo studies were carried out in athymic nude mice subcutaneously inoculated with HL-60 cells. Results: In vitro, NM exhibited a dose dependent reduction in cells viability. Zymography revealed matrix MMP-2 and phorbol 12-myristate 13-acetate induced MMP-9 expression. NM inhibited expression of both MMP in a dose dependent manner. Similar step-wise reduction in the Matrigel invasion by HL-60 cells was also observed by this combination with incremental doses. Gradually increasing doses of NM induced significant apoptosis in HL-60 cells. In vivo, NM inhibited tumor growth by 50%. Conclusion: The results indicate that NM significantly suppresses tumor growth, decreases cell viability, inhibits MMP expression, Matrigel invasion and induces apoptosis in HL-60 cells

In vivo and in vitro antitumor effects of nutrient mixture in murine leukemia cell line P-388

Aim: Leukemia is characterized by uncontrolled marrow cell proliferation and metastatic foci. We investigated the antitumor potential of a nutrient mixture on malignant leukemia P-388 cells. Methods: The nutrient mixture containing lysine, proline, ascorbic acid, green tea extract and other nutrients is formulated to target key pathways in cancer progression. The cells were treated with the mixture, and tested at doses 0, 10, 50, 100, 500 and 1000 μg/ml in triplicates. The effects were evaluated by cell proliferation, Matrigel invasion, cell morphology and apoptosis. The in vivo effect was measured in male nude mice (n = 12) inoculated with P-388 cells. After randomly dividing in two groups, each group was fed regular and the nutrient mixture supplemented diet and the mice were sacrificed after four weeks. Results: The nutrient mixture decreased P-388 cell proliferation at 500 and 1000 μg/ml. Only 10% cells were viable at 1000 μg/ml. Matrigel invasion was significantly inhibited in a dose dependent manner with virtually total inhibition at 1000 μg/ml. Cell morphological features notably changed with dose increase to 1000 μg/ml. Analysis of apoptotic cells on live green caspase kit exhibited gradual increase with the increasing dose of the nutrient mixture, and at 1000 μg/ml 92% of P-388 cells were in late apoptosis. Tumors in the group of mice supplemented with the nutrient mixture had 50% lower weight compared to the tumors in control group (p = 0.0105). Histopathologically, both the groups of tumors were similar, yet size of tumors in the group treated with the nutrient mixture was considerably smaller. Conclusion: These results indicate that the nutrient mixture exhibited significant action against multiple targets in P-388 leukemia and may have potential in human leukemia

In vivo and In vitro effect of a nutrient mixture on human hepatocarcinoma cell line SK-HEP-1

Long-term survival of patients with hepatocellular carcinoma (HCC), a common cancer worldwide, remains poor, due to metastasis and recurrence. Aim: To investigate the effect of a novel nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea extract on human HCC cell line Sk-Hep-1 In vivo and In vitro. Methods: After one week of isolation, 5–6 week old male athymic nude mice were inoculated with 3 x 106 SK-Hep-1 cells subcutaneously and randomly divided into two groups; group A was fed a regular diet and group B a regular diet supplemented with 0.5% 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 SK-Hep-1 cells, measuring cell proliferation by MTT assay, invasion through Matrigel, apoptosis by green caspase detection kit, MMP secretion by zymography, and morphology by H&E staining. Results: NM inhibited tumor weight and burden of SK-Hep-1 xenografts by 42% and 33% respectively. In vitro, NM exhibited 33% toxicity over the control at 500 and 1000 μg/ml concentration. Zymography demonstrated MMP-2 and MMP-9 secretion which was inhibited by NM in a dose dependent fashion, with virtual total inhibition at 1000 μg/ml. Invasion through Matrigel was inhibited at 100, 500 and 1000 μg/ml by 53%, 83% and 100% respectively. NM induced slight apoptosis at 100 μg/ml, and profound apoptosis at 500 μg/ml and 1000 μg/ml concentration. Conclusions: These results suggest that NM has therapeutic potential in treatment of HCC

Antiangiogenic properties of a nutrient mixture in a model of hemangioma

The pathogenesis of hemangiomas is still largely unknown and the current therapy, such as systemic corticosteroid, vincristine, and interferon-alpha, is toxic and remains unsatisfactory. A nutrient mixture (NM) containing lysine, proline, ascorbic acid and green tea extract has shown significant anti-angiogenic and anti-tumor effect against a number of cancer cell lines. Aim: Using a mouse hemangioendothelioma model, we investigated the efficacy of NM. We also tested the effect of NM in vitro, evaluating cell viability, MMP secretion, invasion, morphology and apoptosis. Methods: Athymic nude mice, 5–6 weeks old, were inoculated with 3 x106 EOMA cells subcutaneously and randomly divided into two groups; group A was fed a regular diet and group B — a regular diet supplemented with 0.5% 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. Results: NM inhibited the growth of tumors by 50%. In vitro, NM exhibited dose response cytotoxicity with 10%, 30% and 55% at 10, 100 and 1000 μg/ml. Invasion through Matrigel was inhibited at 50, 100 and 500 μg/ml by 25%, 30% and 100% respectively. NM induced dose-dependent apoptosis of EOMA cells. Conclusions: These results suggest that NM may have therapeutic potential in treating infantile hemangioendotheliomas and, perhaps, other cutaneous vascular tumors

Antineoplastic effects of nutrient mixture on Raji and Jurkat t cells: the two highly aggressive non hodgkin’s lymphoma cell lines

Non-Hodgkin lymphomas incidence has increased more than 70% in last 25 years. Aggressiveness, higher relapse rate, and treatment complications pose significant barriers. Decreased food intake and side effects of treatments make cancer patients vulnerable to deficiency of essential nutrients such as vitamin C, lysine, and proline leading to the formation of weak extra cellular matrix susceptible to easy breakdown by matrix metalloproteinase enzymes. Inhibition of these enzymes has shown promise in stopping metastasis. Aim: In this study, we investigated the effects of a specific nutrient mixture, containing ascorbic acid, lysine, proline, green tea extract among others, in most aggressive forms of non-Hodgkin’s lymphoma — Burkitt’s lymphoma, and T-cell lymphoma — using Raji and Jurkat cells respectively. Methods: Nutrient mixture (NM) doses of 0, 10, 50, 100, 500, 1000 μg/ml, were used to study effects on cell proliferation, expression of matrix metalloproteinase, Matrigel™ invasion and apoptosis. Results: The results demonstrated that the dose response toxicity of the nutrient mixture on Raji cells gradually increased with increasing concentration. The nutrient mixture was non-toxic to Jurkat cells, however exhibited anti-proliferative properties at higher concentrations. Zymography demonstrated, NM had a significant inhibitory effect on matrix metalloproteinase-9 expression with total inhibition at 1000 μg/ml for Raji cells and at 500 μg/ml for Jurkat cells. The NM at 100 μg/ml completely inhibited Matrigel ™ invasion for Raji cells, and at 1000 μg/ml for Jurkat cells. After the NM challenge virtually all Raji and Jurkat cells exposed to 1000 μg/ml were in late apoptosis. Conclusion: Considering the lack of treatment options and continually increasing incidence, NM could be further explored for its therapeutic potential in Burkitt’s lymphoma and T-cell lymphoma

In vitro inhibition of matrix metalloproteinases, invasion and growth of Fanconi anemia human FANCA and FANCC lymphoblasts by nutrient mixture

Aim: Fanconi anemia is a rare genetic disorder with high propensity for development of cancers, such as aplastic anemia, leukemia and head and neck cancers. Collagen digesting matrix metalloproteinase (MMP) enzymes have been implicated in for their role in various malignancies and to promote metastasis. Biological agents that prevent extracellular matrix digestion by the MMPs have been shown to be promising therapeutic approaches to cancer. In this study, we investigated effects of a nutrient mixture (NM) containing, ascorbic acid, lysine, proline and green tea extract, on human FANCA and FANCC lymphoblasts for viability, MMP secretion and invasion. Methods: Human FANCA lymphoblasts GM13022 and HCS536 were challenged with NM at concentration range within 10–1000 µg/ml. Cell toxicity was assessed by Trypan blue dye exclusion test. Invasion was evaluated through Matrigel and gelatinase zymography for MMP activity. Results: NM was toxic in dose dependent mode to HCS536 cells but not to GM13022 cells. GM13022 cells but not HCS536 cells exhibited MMP-9 secretion, which was inhibited by NM. Matrigel invasion was inhibited in HCS536 cells at 100 and 500 µg/ml by 27% and 93%, respectively. In GM13022 cells, the NM showed completely blocked Matrigel invasion at 500 µg/ml. Conclusion: NM inhibited MMP secretion and Matrigel invasion in FANCA and inhibited invasion and induced toxicity in FANCC lymphoblasts. These results suggest that the NM may have therapeutic potential in Fanconi anemia associated neoplasia. Key Words: Fanconi anemia, nutrient mixture, MMP, Matrigel invasion

Ascorbate depletion increases growth and metastasis of melanoma cells in vitamin C deficient mice

Aim: Our main objective was to determine the effect of ascorbate supplementation in mice unable to synthesize ascorbic acid (gulo KO) when challenged with murine B16FO cancer cells. Methods: Gulo KO female mice 36–40 weeks of age were deprived of or maintained on ascorbate in food and water for 4 weeks prior to subcutaneous injection of 2.5×106 B16FO murine melanoma cells in the right flank of mice. A control group of wild type mice were also injected with the melanoma cells and maintained on a regular murine diet. Mice were continued on their respective diets for another 2 weeks after injection. The mice were then sacrificed, blood was drawn and their tumors were measured, excised and processed for histology. Results: Mean weight of animals decreased significantly (30%, p < 0.0001) in the ascorbate-restricted group but increased slightly, but insignificantly, in the ascorbate-supplemented group. The mean tumor weight in ascorbate supplemented mice was significantly reduced (by 64%, p = 0.004) compared to tumor weight in ascorbate-deprived gulo mice. The mean tumor weight of wild type mice did not differ significantly from the ascorbate-supplemented mice. Gulo KO mice supplemented with ascorbate developed smaller tumors with more collagen encapsulation and fibrous capsule interdigitation, while gulo KO mice deprived of ascorbate hosted large tumors with poorly defined borders, showing more necrosis and mitosis. Ascorbate supplementation of gulo KO mice resulted in profoundly decreased serum inflammatory cytokine IL-6 (90% decrease, p = 0.04) and IL-1β (62% decrease) compared to the levels in gulo KO mice deprived of ascorbate. Conclusion: Ascorbate supplementation modulated tumor growth and inflammatory cytokine secretion as well as enhanced encapsulation of tumors in scorbutic mice

Down regulation of u-PA by a nutrient mixture in hemangioma (EOMA) cells by inducing caspase-dependent apoptosis

Hemangiomas are the most common congenital vascular and benign tumor in infants and children. Most hemangiomas do not cause major symptoms to require intervention, however, the larger hemangiomas have tendency to bleed and may require surgical removal. Experimental studies have demonstrated the role of urokinase plasminogen activator (u-PA), especially cell surface u-PA, as an initiator of extra-cellular matrix proteolysis and associated tumor cell invasion. Aim: To examine, whether the antitumor effects of a specific nutrient mixture are due to induction of apoptosis by inhibition of u-PA. Materials and Methods: A nutrient mixture containing lysine, proline, ascorbic acid, and green tea extract which has showed anticancer activity against a number of cancer cell lines was used as an experimental composition. EOMA cells were grown in appropriate media with antibiotics in 24-well tissue culture plates. At near confluence, the cells were treated with nutrition mixture at 10, 100, 1000 µg/ml in triplicate. Analysis of u-PA activity was carried out by fibrin zymography. Morphological changes and caspase activation associated with apoptosis induction was checked by H&E staining and Live Green caspase assay, respectively. Apoptosis inducing anticancer drug camptothecin (10 µM) was used as positive control. Results: The nutrition mixture exhibited dose response toxicity with maximum toxicity 55% (p < 0.001) at 1000 µg/ml. EOMA cells expressed u-PA, which was inhibited by nutrition mixture in a dose-dependent manner. The caspase analysis revealed a dose dependent increase in apoptosis of EOMA hemangioma cells, with an increasing apoptosis observed at 100 μg/ml, and maximum at 1000 μg/ml. Cells treated with nutrition mixture showed significantly more apoptotic changes than the control or camptothecin-treated cells. Conclusion: These results suggest that NM may induce apoptosis of hemangioma cells in vitro thus warranting further investigation. Key Words: nutrient mixture, urokinase plasminogen activators, hemangioma, EOMA, caspase, apoptosis