BACKGROUND:Brain tumors are highly aggressive tumors characterized by secretions of high levels of matrix metalloproteinase-2 and -9, leading to tumor growth, invasion and metastasis by digesting the basement membrane and extracellular matrix components. We previously demonstrated the effectiveness of a nutrient mixture (NM) containing ascorbic acid, lysine, proline, and green tea extract in vitro: on activity of urokinase plasminogen activator, matrix metalloproteinases and TIMPs in various human glioblastoma (LN-18, T-98G and A-172) cell lines and on glioblastoma A-172 cell proliferation and Matrigel invasion.

AIM:Our main objective in this study was to investigate the effect of the NM in vivo on human glioblastoma U-87 MG cell line.

MATERIALS AND METHODS:Athymic male nude mice inoculated with 3·10(6) U-87 MG cells subcutaneously and were fed a regular diet or a regular diet supplemented with 0.5% NM. Four weeks later, the mice were sacrificed, the tumors were weighed and measured. The samples were studied histologically.

RESULTS:NM inhibited tumor weight and tumor burden by 53% (p = 0.015) and 48% (p = 0.010), respectively.

CONCLUSIONS:These results suggest the therapeutic potential of NM as an adjuvant in the treatment of glioblastoma.

Ovarian cancer is the deadliest gynecological malignancy in women, and fifth leading cause of death. Despite advances made in chemotherapy and surgery, the average time of clinical remission is approximately 2 years and the 5-year survival rate is 45%. Thus, there is an urgent need for the development of a novel therapeutic approach to ovarian cancer treatment. We investigated the effect of a specific nutrient mixture (EPQ) containing ascorbic acid, lysine, proline, green tea extract, and quercetin on human ovarian cancer cell A-2780 in vivo and in vitro. Athymic female nude mice (n = 12) were all inoculated intraperitoneally (IP) with 2× 10⁶ cells in 0.1 mL of phosphate buffered saline (PBS) and randomly divided into two groups. Upon injection, the Control group (n = 6) was fed a regular diet and the EPQ group (n = 6) a regular diet supplemented with 0.5% EPQ. Four weeks later, the mice were sacrificed and tumors that developedin the ovary were excised, weighed, and processed for histology. Lungs were inspected for metastasis. In vitro, A-2780 cells were cultured in Dulbecco modified Eagle medium supplemented with 10% FBS and antibiotics. At near confluence, cells were treated with EPQ in triplicate at concentrations between 0 and 1000 μg/mL. Cell proliferation was measured via MTT assay, MMP-9 secretion via gelatinase zymography, invasion through Matrigel and morphology via hematoxylin and eosin (H& E) staining. All Control mice developed large ovarian tumors, whereas 5 out of 6 mice in the EPQ group developed no tumors, and one, a small tumor. Control mice also showed lung metastasis in 6 out of 6 mice, while no lung metastasis was evident in EPQ mice. Zymography demonstrated only MMP-9 expression, which EPQ inhibited in a dose-dependent fashion, with virtual total block at 250μg/mL concentration. EPQ significantly inhibited invasion through Matrigel with total block at 250 μg/mL concentration. MTT showed dose-dependent inhibition of cell proliferation with EPQ, and H& E staining showed no morphological changes below 500μg/mL EPQ. These results suggest that EPQ has therapeutic potential in the treatment of ovarian cancer by significantly suppressing ovarian tumor incidence and growth and lung metastasis, and by inhibiting MMP-9 secretion and invasion of A-2780 ovarian cancer cells.

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 microg/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 microg/ml for Raji cells and at 500 microg/ml for Jurkat cells. The NM at 100 microg/ml completely inhibited Matrigel invasion for Raji cells, and at 1000 microg/ml for Jurkat cells. After the NM challenge virtually all Raji and Jurkat cells exposed to 1000 microg/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.

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.

The effects of a novel nutrient formulation Epican Forte (EF) were evaluated on proliferation and induction of apoptosis using non-cytotoxic concentrations against HTLV-1 positive (HuT-102&C91-PL) and negative (CEM&Jurkat) cells. EF showed anti-proliferative effect as determined by MTT assay and TGF mRNA protein expression using RT-PCR. EF resulted in the down-regulation of TGF-alpha and an up-regulation in TGF-beta2. EF caused a significant increase in apoptotic cells in the preG1 phase. These results were confirmed using Cell Death ELISA and Annexin V-FITC. Induction of apoptosis was caused by an up-regulation of p53, p21 and Bax protein levels and a down-regulation of Bcl-2alpha protein expression level.

Cervical cancer is one of the most commonly diagnosed cancers and a significant cause of mortality in women worldwide. Although cervical cancer is fully treatable in the early stages, once it has metastasized, patient outcome is poor. The objective of the present study was to investigate the effect of dietary supplementation with a nutrient mixture (NM) containing lysine, ascorbic acid, proline, green tea extract and other micronutrients on the expression of extracellular matrix (ECM) proteins in HeLa cell xenografts in nude female mice. After housing for 1 week, female athymic nude mice between 5 and 6 weeks of age (n=12) were inoculated subcutaneously with 3×10(6) HeLa cells in phosphate-buffered saline and Matrigel and randomly divided into two groups. These were the control group, in which the mice were fed with regular mouse chow, and the NM group, in which the mice were fed with the regular diet supplemented with 0.5% NM (w/w). After 4 weeks, thetumors were excised and processed for histology. Tumor growth was evaluated and the tumors were stained for the ECM proteins collagen I, collagen IV, fibronectin, laminin, periodic acid-Schiff (PAS) and elastin. NM strongly inhibited (by 59%, P=0.001) the growth of HeLa xenografts in nude mice. Tumors from control mice exhibited little to no collagen I expression either internally or in the fibrous capsule, while tumors from the NM group expressed collagen I in the fibrous capsule and within the tumor. Tumors from the control group showed diffuse cytoplasmic and capsular collagen IV with abundant nucleated cells. NM treatment substantially increased collagen IV production and induced a dense fibrous network of collagen IV with chambers that surrounded live nucleated cells and large amounts of necrotic cell debris. Tumors from the mice fed with the NM exhibited a well-defined border of fibronectin in the capsule and intense areas of staining internally whereas control group tumors showed less overall fibronectin with sporadic internal staining and little in the fibrous capsule. Although laminin appeared abundantly in control and NM-treated tumors, the NM group tumors exhibited a chamber-like network of laminin internally. Tumors from the control group exhibited internal areas of intense PAS staining, whereas tumors from the NM-treated group exhibited a more uniform diffuse pattern of PAS staining. In conclusion, NM supplementation of HeLa xenograft-bearing female nude mice demonstrated a potent inhibition of tumor growth and enhancement of ECM proteins, suggesting the therapeutic value of this specific nutrient complex in the treatment of cervical cancer.

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.

UNLABELLED: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 10(6) 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 1,000 microg/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 microg/ml. Invasion through Matrigel was inhibited at 100, 500 and 1,000 microg/ml by 53%, 83% and 100% respectively. NM induced slight apoptosis at 100 microg/ml, and profound apoptosis at 500 microg/ml and 1000 microg/ml concentration.

CONCLUSIONS:These results suggest that NM has therapeutic potential in treatment of HCC.

Corneal neovascularization is a significant, sight-threatening complication of many ocular surface disorders. Various growth factors and proteinases are involved in corneal neovascularization. The data supporting a causal role for vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMPs) are extensive. Inhibition of VEGF and MMPs is a main strategy for treating corneal neovascularization. Several findings have shown that corneal neovascularization can be reduced by using anti-VEGF and anti-MMPs agents. Efficacy of a nutrient mixture (NM) containing lysine, proline, ascorbic acid, and green tea extract has been demonstrated for reducing VEGF and MMPs secretion by various cells. Moreover, NM can inhibit endothelial cell migration and capillary tube formation. We herein note that topical application of NM is potentially useful for inhibiting corneal neovascularization and restoration of corneal clarity. Further investigations in animal models are needed to place NM alongside corneal neovascularization therapeutics.

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.

Objective:Healing of bone fractures is a prolonged process that can be affected by nutrition. Our objective was to critically evaluate the effect of supplementation with an essential nutrient complex, containing ascorbic acid, lysine, proline, and vitamin B6 on healing time of tibial fractures.

Design:Random double-blind placebo-controlled study

Setting: Dr. Jamdar Hospital, Jabalpur, India

Subjects and Intervention:113 patients with unilateral displaced closed or grade I open tibial fractures were randomized to receive either standard care with placebo or with supplementation with an essential nutrient complex containing ascorbic acid, lysine, proline, and vitamin B6. Qualifying patients, on admission to the study, were clinically examined, radiographs of the affected limbs taken, fractures reduced under anesthesia, and above knee plaster casts applied. Radiographs were taken at each follow-up visit to confirm reduced alignment of fracture and proper callus formation.
Primary Outcome Measure: The primary outcome measure was the number of weeks required for fracture to be healed. Healing was defined as absence of abnormal mobility at fracture site clinically, absence of pain elicited by stressing the fracture or by walking, and radiographic confirmation of callus formation.

Results:Data analysis demonstrated reduced fracture-healing time associated with experimental supplementation. For PP analysis group, fracture healing time in 75% of the supplemented group of patients (N=21) was 17 weeks or less and 19 weeks or less in 75% of the placebo group patients (N=36). The percentage of patients with fractures healing in 10 weeks or less was 33.3% for the supplemented group and 11.1% for the placebo group. However, the difference in healing time between the two groups did not reach statistical significance.

Conclusion:

Results showed encouraging trends that fracture-healing time is reduced by supplementation with an essential nutrient complex containing ascorbic acid, lysine, proline, and vitamin B6. In addition, the nutrient supplemented participants reported improved feeling of well-being with use of the supplement.